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Results for “Bioengineering”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 307 records · Page 17Linked to original sources

bioRxiv2018

A robot-based gait training therapy for pediatric population with Cerebral Palsy: goal setting, proposal and preliminary clinical implementation

BACKGROUNDThe use of robotic trainers has increased with the aim of improving gait function in patients with limitations. Nevertheless, there is an absence of studies that deeply describe detailed guidelines of how to correctly implement robot-based treatments for gait rehabilitation. This contribution proposes an accurate robot-based training program for gait rehabilitation of pediatric population with Cerebral Palsy (CP).\n\nMETHODSThe program is focused on the achievement of some specifications defined by the International Classification of Functioning, Disability and Health framework, Children and Youth version (ICF-CY). It is framed on 16 non-consecutive sessions where motor control, strength and power exercises of lower limbs are performed in parallel with a postural control strategy. A clinical evaluation with four pediatric patients with CP using the CPWalker robotic platform is presented.\n\nRESULTSThe preliminary evaluation with patients with CP shows improvements in several aspects as strength (74.03{+/-}40.20%), mean velocity (21.46{+/-}33.79%), step length (17.95{+/-}20.45%) or gait performance (e.g. 18.88{+/-}14.31% in Gross Motor Function Measure-88 items, E and D dimensions).\n\nCONCLUSIONSThe improvements achieved in the short term show the importance of working strength and power functions meanwhile over-ground training with postural control. This research could serve as preliminary support for future clinical implementations in any robotic device.\n\nTRIAL REGISTRATIONThe study was carried out with the number R-0032/12 from Local Ethical Committee of the Hospital Infantil Nino Jesus. Public trial registration: ISRCTN18254257. Registered 23 March 2017, retrospectively registered.

bioengineering↗

bioRxiv2018

Sub-voxel light-sheet microscopy for high-resolution, high-throughput volumetric imaging of large biomedical specimens

A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). Here, we report a sub-voxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular-resolution. A non-axial, continuous scanning strategy is used to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then by adopting a sub-voxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and iteratively recovers a 3-D image with improved resolution throughout the sample. This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times, with high acquisition speeds of gigavoxels per minute. As demonstrated by quick reconstruction (minutes to hours) of various samples, e.g., 3-D cultured cells, an intact mouse heart, mouse brain, and live zebrafish embryo, the SLSM method presents a high-throughput way to circumvent the tradeoff between intoto mapping of large-scale tissue (>100 mm3) and isotropic imaging of single-cell (~1-m resolution). It also eliminates the need of complicated mechanical stitching or precisely modulated illumination, using a simple light-sheet setup and fast graphics-processing-unit (GPU)-based computation to achieve high-throughput, high-resolution 3-D microscopy, which could be tailored for a wide range of biomedical applications in pathology, histology, neuroscience, etc.

bioengineering↗

bioRxiv2018

Deep Learning personalised, closed-loop Brain-Computer Interfaces for multi-way classification

Exoskeletons and robotic devices are for many motor disabled people the only way to interact with their envi-ronment. Our lab previously developed a gaze guided assistive robotic system for grasping. It is well known that the same natural task can require different interactions described by different dynamical systems that would require different robotic controllers and their selection by the user in a self paced way. Therefore, we investigated different ways to achieve transitions between multiple states, finding that eye blinks were the most reliable to transition from off to control modes (binary classification) compared to voice and electromyography. In this paper be expanded on this work by investigating brain signals as sources for control mode switching. We developed a Brain Computer Interface (BCI) that allows users to switch between four control modes in self paced way in real time. Since the system is devised to be used in domestic environments in a user friendly way, we selected non-invasive electroencephalographic (EEG) signals and convolutional neural networks (ConvNets), known by their capability to find the optimal features for a classification task, which we hypothesised would add flexibility to the system in terms of which mental activities the user could perform to control it. We tested our system using the Cybathlon BrainRunners computer game, which represents all the challenges inherent to real time control. Our preliminary results show that an efficient architecture (SmallNet) composed by a convolutional layer, a fully connected layer and a sigmoid classification layer, is able to classify 4 mental activities that the user chose to perform. For his preferred mental activities, we run and validated the system online and retrained the system using online collected EEG data. We achieved 47, 6% accuracy in online operation in the 4-way classification task. In particular we found that models trained with online collected data predicted better the behaviour of the system in real time suggesting, as a side note, that similar (ConvNets based) offline classifying methods present in literature might find a decay in performance when applied online. To the best of our knowledge this is the first time such an architecture is tested in an online operation task. While compared to our previous method relying on blinks with this one we reduced in less than half (1.6 times) the accuracy but increased by 2 the amount of states among which we can transit, bringing the opportunity for finer control of specific subtasks composing natural grasping in a self paced way.

bioengineering↗

bioRxiv2018

Wavelet for binocular vision modeling

In the present study, binocular vision properties were modeled using a single elementary wavelet. Opponent responses (ON-OFF) appeared in the first stages of the neural coding in the retina. This property was assumed to build an adequate wavelet showing a positive part (On) and a negative part (OFF). We have examined the experimental orientation and position disparity given by Bishop. We assumed that the theoretical position disparity was given by a combination of two wavelets for a given orientation disparity {beta}. A change in {beta} implied a change in the magnitude of one of the wavelets and consequently a change in the wavelets combination. There was a close match between the theoretical and experimental position disparity curves according to the changes in orientation disparity.

bioengineering↗

bioRxiv2018

Super-multiplexed fluorescence microscopy via photostability contrast

Many areas of biological research rely heavily on fluorescence microscopy to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously with spectrally distinct fluorescent labels. A high degree of multiplexing is desirable as it affords the experiment greater information content, speeding up research timelines. Multiplexing can be increased by imaging a larger number of spectral channels, however, the wide emission spectra of most fluorophores limits multiplexing to four or five labels in standard fluorescence microscopes. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins, and show that the latter are particularly well suited to our method as their bleaching is often reversible. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.

bioengineering↗

bioRxiv2018

Laminin heparin-binding peptides promiscuously bind growth factors and enhance diabetic wound healing

Laminin, as a key component of the basement membrane extracellular matrix (ECM), regulates tissue morphogenesis. We show that multiple laminin isoforms promiscuously bind to growth factors (GFs) with high affinity, through their heparin binding domains (HBDs) located in the a chain LG domains. Interestingly, these domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. We next explore application of these multifunctional laminin HBDs in skin healing in the type 2 diabetic mouse. We demonstrate that covalent incorporation of laminin HBDs into fibrin matrix enables the slow-release of GFs. Incorporation of the 33043-3067 laminin HBD significantly enhances in vivo wound-healing efficacy of vascular endothelial cell growth factor (VEGF)-A165 and platelet-derived growth factor (PDGF)-BB, under conditions where the GFs alone in fibrin are inefficacious. This laminin HBD peptide may be clinically useful by improving biomaterials as both GF reservoirs and cell scaffolds, leading to effective tissue regeneration.

bioengineering↗

bioRxiv2018

Myoelectric Prosthesis Control: Improving Internal Model Strength and Performance Using Augmented Feedback

Myoelectric prosthetic devices are commonly used to help upper limb amputees perform activities of daily living, however amputees still lack the sensory feedback required to facilitate reliable and precise control. Augmented feedback may play an important role in affecting both short-term performance, through real-time regulation, and long-term performance, through the development of stronger internal models. In this work, we investigate the potential tradeoff between controllers that enable better short-term performance and those that provide sufficient feedback to develop a strong internal model. We hypothesize that augmented feedback may be used to mitigate this tradeoff, ultimately improving both short and long-term control. We used psychometric measures to assess the internal model developed while using a filtered myoelectric controller with augmented audio feedback, imitating classification-based control but with augmented regression-based feedback. In addition, we evaluated the short-term performance using a multi degree-of-freedom constrained-time target acquisition task. Results obtained from 24 able-bodied subjects show that an augmented feedback control strategy using audio cues enables the development of a stronger internal model than the filtered control with filtered feedback, and significantly better path efficiency than both raw and filtered control strategies. These results suggest that the use of augmented feedback control strategies may improve both short-term and long-term performance.

bioengineering↗

bioRxiv2018

Adaptive and Wireless Recordings of Electrophysiological Signals during Concurrent Magnetic Resonance Imaging

Strong electromagnetic fields that occur during functional magnetic resonance imaging (fMRI) presents a challenging environment for concurrent electrophysiological recordings. Here, we present a miniaturized, wireless platform - \"MR-Link\" (Multimodal Recording Link) that provides a hardware solution for simultaneous electrophysiological and fMRI signal acquisition. The device detects the changes in the electromagnetic field during fMRI to synchronize amplification and sampling of electrophysiological signals with minimal artifacts. It wirelessly transmits the recorded data at a frequency detectable by the MR-receiver coil. The transmitted data is readily separable from MRI in the frequency domain. To demonstrate its efficacy, we used this device to record electrocardiograms and somatosensory evoked potential during concurrent fMRI scans. The device minimized the fMRI-induced artifacts in electrophysiological data and wirelessly transmitted the data back to the receiver coil without compromising fMRI signal quality. The device is compact (22 mm dia., 2gms) and can be placed within the MR-bore to precisely synchronize with fMRI. Therefore, MR-Link offers an inexpensive system by eliminating the need for amplifiers with a high dynamic range, high-speed sampling, additional storage or synchronization hardware for electrophysiological signal acquisition. It is expected to enable a broader range of applications of simultaneous fMRI and electrophysiology in animals and humans.

bioengineering↗

bioRxiv2018

Metabolic pairing of aerobic and anaerobic production in a one-pot batch cultivation

BackgroundThe versatility of microbial metabolic pathways enables their utilization in vast number of applications. However, the electron and carbon recovery rates, essentially constrained by limitations of cell energetics, are often too low in terms of process feasibility. Cocultivation of divergent microbial species in a single process broadens the metabolic landscape and thus, the possibilities for more complete carbon and energy utilization.\n\nResultsIn this study, we integrated the metabolisms of two bacteria, an obligate anaerobe Clostridium butyricum and an obligate aerobe Acinetobacter baylyi ADP1. In the process, a glucose-negative mutant of A. baylyi ADP1 first deoxidized the culture allowing C. butyricum to grow and produce hydrogen from glucose. In the next phase, ADP1 produced long chain alkyl esters utilizing the by-products of C. butyricum, namely acetate and butyrate.\n\nConclusionsThe cocultivation of strictly anaerobic and aerobic bacteria allowed the production of both hydrogen gas and long-chain alkyl esters in a simple one-pot batch process. The study demonstrates the potential of metabolic pairing using designed microbial consortia for optimal electron and carbon recovery.

bioengineering↗

bioRxiv2018

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. These challenges are amplified when the enzymes are expressed at single copy from the chromosome. We have explored these issues using 4-hydroxybenzoate monooxygenase homologs heterologously expressed in Escherichia coli. Initial chromosomal enzyme expression was insufficient to support consistent growth with 4-hydroxybenzoate. Experimental evolution identified mutations that improved pathway activity. One set of mutations was common between homologs, while a second class of mutations was homolog-specific. Ultimately, we were able to identify a set of mutations that provided sufficient activity for growth with 4-hydroxybenzoate while maintaining or improving growth with protocatechuate. These findings demonstrate the potential for flexible, scalable chromosomal pathway engineering, as well as the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

bioengineering↗

bioRxiv2018

Single day construction of multi-gene circuits with 3G assembly

The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems take different approaches to accelerate DNA construction. We introduce a hybrid method, Golden Gate-Gibson (3G), that takes advantage of modular part libraries introduced by type IIS restriction enzyme systems and isothermal assembly s ability to build large DNA constructs in single pot reactions. Our method is highly efficient and rapid, facilitating construction of entire multi-gene circuits in a single day. Additionally, 3G allows generation of variant libraries enabling efficient screening of different possible circuit constructions. We characterize the efficiency and accuracy of 3G assembly for various construct sizes, and demonstrate 3G by characterizing variants of an inducible cell-lysis circuit.

bioengineering↗

bioRxiv2018

Engineering Lipid Droplet Assembly Mechanisms for Improved Triacylglycerols Accumulation in S. cerevisiae

Production of triacylglycerols (TAGs) through microbial fermentation is an emerging alternative to plant and animal-derived sources. The yeast Saccharomyces cerevisiae is a preferred organism for industrial use but has natively a very poor capacity of TAG production and storage. Here, we engineered S. cerevisiae for accumulation of high TAG levels through the use of structural and physiological factors that influence assembly and biogenesis of lipid droplets. First, human and fungal perilipin genes were expressed, increasing TAG content by up to 36% when expressing the human perilipin gene PLIN3. Secondly, expression of the FIT2 homologue YFT2 resulted in a 26% increase in TAG content. Lastly, the genes ERD1 and PMR1 were deleted in order to induce an ER stress response and stimulate lipid droplet formation, increasing TAG content by 72% for {Delta}erd1, with an additive effect for both YFT2 and PLIN3 expression. These new approaches were implemented in previously engineered strains that carry high flux of fatty acid biosynthesis and conversion of acyl-CoA into TAG, resulting in improvements of up to 138% over those high-producing strains without any substantial growth effects or abnormal cell morphology. We find that these approaches are not only a major advancement in engineering S. cerevisiae for TAG production, but also highlight the importance of lipid droplet dynamics for high lipid accumulation in yeast.

bioengineering↗

bioRxiv2018

Multiplexed enrichment and genomic profiling of peripheral immune cell subsets on a microfluidic chip

The human immune system consists of many specialized cell subsets that simultaneously carry out a diverse range of functions using overlapping pathways and signals. Subset-specific immune profiling can resolve immune activity in autoimmune disease, cancer immunity, and infectious disease that may not be discoverable or detectable in analyses of crude blood samples. The activity of specific subsets may help predict the course of disease and response to therapy in certain patient populations. Here, we present a low-input microfluidic system for sorting immune cells into subsets and profiling their cellular states by gene expression analysis using full-length RNA-seq. Our system is robust and has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings. We validate the devices technical performance by benchmarking its subset enrichment and genomic profiling performance against standard protocols. We make the added value of subset-resolved profiling over crude samples clear through ex vivo experiments that show subset-specific stimulated responses. Finally, we demonstrate the scalability of our device by profiling four immune cell subsets in blood from systemic lupus erythematosus (SLE) patients and matched controls enrolled in a clinical study. The results from our initial cohort confirm the role of type I interferons in lupus pathogenesis and further show that the canonical interferon signature for SLE is prominent in B cells, demonstrating the ability of our integrated analytical platform to identify cell-specific disease signatures.

bioengineering↗

bioRxiv2018

Mutation in DsbA signal sequence hampers the SRP mechanism: A new strategy to combat virulence factor

Disulphide bond (Dsb) protein, characterized as an important virulence factor in gram negative bacteria. In this study, amino acid mutations in DsbA signal sequence (ss) and its effect on translocation of recombinant Ovine growth hormone (rOGH) was observed. Eight constructs were designed on the basis of increased hydrophobicity and showed that hydrophobicity and specificity of amino acid plays a crucial role in translocation of rOGH. Two DsbAss with the same hydropathy (1.539), one had alteration at -13 and second at -11 position; alanine (Ala) to isoleucine respectively were designed. The former DsbAss translocated rOGH from membrane to cytoplasmic fraction in E. coli as confirmed by SDS-PAGE, Western blot and molecular modelling analysis. MD simulations and binding free energy calculations evidenced that, altering Ala changed the orientation of signal peptide in the Ffh-M domain binding groove and hampered the process of translocation while change at position -11 pointed it outward. We hypothesize, amino acid and position of mutations in DsbAss can hinder the translocation process of signal recognition particle system, thus affecting the virulence of bacteria.

bioengineering↗

bioRxiv2018

Regulation of Decellularized Tissue Remodeling via Scaffold-Mediated Lentiviral Delivery in Anatomically-Shaped Osteochondral Constructs

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-{beta}3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-{beta}3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological, in vitro, joint, organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface.

bioengineering↗

bioRxiv2018

Viral-mediated optical stimulation of peripheral motor nerves in non-human primates

ObjectiveReanimation of muscles paralyzed by disease states such as spinal cord injury remains a much sought after therapeutic goal of neuroprosthetic research. Optogenetic stimulation of peripheral motor nerves expressing light-sensitive opsins is a promising approach to muscle reanimation that may overcome several drawbacks of traditional methods such as functional electrical stimulation (FES). However, the utility of these methods has only been demonstrated in rodents to date, while translation to clinical practice will likely first require demonstration and refinement of these gene therapy techniques in non-human primates.\n\nApproachThree rhesus macaques were injected intramuscularly with either one or both of two optogenetic constructs (AAV6-hSyn-ChR2-eYFP and/or AAV6-hSyn-Chronos-eYFP) to transduce opsin expression in the corresponding nerves. Neuromuscular junctions were targeted for virus delivery using an electrical stimulating injection technique. Functional opsin expression was periodically evaluated up to 13 weeks post-injection by optically stimulating targeted nerves with a 472 nm fiber-coupled laser while recording electromyographic (EMG) responses.\n\nMain ResultsOne monkey demonstrated functional expression of ChR2 at 8 weeks post-injection in each of two injected muscles, while the second monkey briefly exhibited contractions coupled to optical stimulation in a muscle injected with the Chronos construct at 10 weeks. A third monkey injected only in one muscle with the ChR2 construct showed strong optically coupled contractions at 5 [1/2] weeks which then disappeared by 9 weeks. EMG responses to optical stimulation of ChR2-transduced nerves demonstrated graded recruitment relative to both stimulus pulse-width and light intensity, and were able to track stimulus trains up to 16 Hz. In addition, the EMG response to prolonged stimulation showed delayed fatigue over several minutes.\n\nSignificanceThese results demonstrate the feasibility of viral transduction of peripheral motor nerves for functional optical stimulation of motor activity in non-human primates, a variable timeline of opsin expression in a primate model closer to humans, and fundamental EMG response characteristics to optical nerve stimulation. Subsequently, they represent an important step in translating these optogenetic techniques as a clinically viable gene therapy.

bioengineering↗

bioRxiv2018

No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

Introduction Introduction Results and Discussion Conclusion Author Contributions Competing Financial Interests 1. gRNA choices and... 2. Zygote injection 3. DNA extraction from... 4. Amplicon sequencing and... 5. Whole Genome Sequencing 6. Probability of detecting... 7. Variant calling, Trio... 8. Intersection of all... 9. Comparison of SNVs... 10. Validation with PCR... 11. Re-analysis of De... Supplementary Figure Supplementary tables References CRISPR-Cas technologies have transformed genome-editing of experimental organisms and have ...

bioengineering↗

bioRxiv2018

A plug-and-play system for enzyme production at commercially viable levels in fed-batch cultures of Escherichia coli BL21 (DE3)

Commercial exploitation of enzymes in biotransformation necessitates a robust method for enzyme production that yields high enzyme titer. Nitrilases are a family of hydrolases that can transform nitriles to enantiopure carboxylic acids, which are important pharmaceutical intermediates. Here, we report a fed-batch method that uses a defined medium and involves growth under carbon limiting conditions using DO-stat feeding approach combined with an optimized post-induction strategy, yielding high cell densities and maximum levels of active and soluble enzyme. This strategy affords strict control of nutrient feeding and growth rates, and ensures sustained protein synthesis over a longer period. The method was optimized for highest titer of nitrilase reported so far (247 kU/l) using recombinant E. coli expressing the Alcaligenes sp. ECU0401 nitrilase. The fed-batch protocol presented here can also be employed as template to produce a wide variety of enzymes with minimal modification, as demonstrated for alcohol dehydrogenase and formate dehydrogenase.

bioengineering↗