SEARCH · bioRxiv Science

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

bioRxiv2016

Tunable synthetic transcription factors assembled from TALE-like repeat elements

Introduction Introduction Methods Results Discussion SI References TALE (Transcriptional activator like effector) repeat arrays are a popular form of programmable DNA binding domain. Reprogrammed TALE transcription factors are referred to as dTALEs (designer-TALEs), and are used within the field of synthetic biology1 as well as fundamental research2.\n\nNaturally TALEs are pathogenicity factors secreted by Xanthomonas spp. into host plant cells to bind specific promoters and manipulate host transcription3. Sequence specific DNA binding is conferred by the TALE repeat array. Natural TALE repeat arrays are formed of 10-30 repeats4, each of which pairs with one DNA base. Collectively the repeats ...

Bioengineering↗

bioRxiv2016

Paperfuge: An ultra-low cost, hand-powered centrifuge inspired by the mechanics of a whirligig toy

Sample preparation, including separation of plasma from whole blood or isolation of parasites, is an unmet challenge in many point of care (POC) diagnostics and requires centrifugation as the first key step. From the context of global health applications, commercial centrifuges are expensive, bulky and electricity-powered, leading to a critical bottle-neck in the development of decentralized, electricity-free POC diagnostic devices. By uncovering the fundamental mechanics of an ancient whirligig toy (3300 B.C.E), we design an ultra-low cost (20 cents), light-weight (2 g), human-powered centrifuge that is made out of paper (\"paperfuge\"). To push the operating limits of this unconventional centrifuge, we present an experimentally-validated theoretical model that describes the paperfuge as a non-linear, non-conservative oscillator system. We use this model to inform our design process, achieving speeds of 125,000 rpm and equivalent centrifugal forces of 30,000 g, with theoretical limits predicting one million rpm. We harness these speeds to separate pure plasma in less than 1.5 minutes and isolate malaria parasites in 15 minutes from whole human blood. By expanding the materials used, we implement centrifugal microfluidics using PDMS, plastic and 3D-printed devices, ultimately opening up new opportunities for electricity-free POC diagnostics, especially in resource-poor settings.

Bioengineering↗

bioRxiv2016

Decellularised Human Liver is too Heterogeneous for Designing a Generic ECM-mimic Hepatic Scaffold

Decellularised human livers are considered the perfect ECM (extracellular matrix) surrogate because both 3-dimensional architecture and biological features of the hepatic microenvironment are thought to be preserved. However, donor human livers are in chronically short supply, both for transplantation or as decellularised scaffolds, and will become even scarcer as life expectancy increases. It is hence of interest to determine the structural and biochemical properties of human hepatic ECM to derive design criteria for engineering bio-mimetic scaffolds. The intention of this work was to obtain quantitative design specifications for fabricating scaffolds for hepatic tissue engineering using human livers as a template. To this end, hepatic samples from 5 human donors were decellularised using a protocol shown to reproducibly conserve matrix composition and micro-structure in porcine livers. The decellularisation outcome was evaluated through histological and quantitative image analyses to evaluate cell removal, protein and glycosaminoglycan content per unit area. Applying the same decellularisation protocol to human liver samples obtained from 5 different donors yielded 5 different outcomes. Only 1 liver out of 5 was completely decellularised, while the other 4 showed different levels of remaining cells. Moreover, protein and glycosaminoglycan content per unit area after decellularisation were also found to be donor-dependent. The donor-to-donor variability of human livers thus precludes their use as templates for engineering a generic 'one-size fits all' ECM-mimic hepatic scaffold.

Bioengineering↗

bioRxiv2016

An Electrochemistry Study of Cryoelectrolysis in Frozen Physiological Saline

This is the first quantitative study on the fundamental physical and electrochemical processes that occur during cryoelectrolysis. Cryoelectrolysis is a new minimally invasive tissue ablation surgical technique that combines the processes of electrolysis and solid/liquid phase transformation (freezing). We measured the pH front propagation and the changes in resistance in a tissue simulant made of physiological saline gel with a pH dye as a function of the sample temperature in the high subzero range above the eutectic. Results demonstrate that effective electrolysis can occur in a high subzero freezing milieu and that the propagation of the pH front is only weakly dependent on temperature. These observations are consistent with a mechanism involving ionic movement through the concentrated saline solution channels between ice crystals at subfreezing temperatures above the eutectic. The Joule heating in these microchannel may cause local microscopic melting, the observed weak dependence of pH front propagation on temperature, and the large changes in resistance with time. In addition, we observed that the pH front propagation from the anode is more rapid than from the cathode. The explanation is the electro-osmotic flow from the cathode to the anode. The findings in this paper may be of fundamental value for designing future cryoelectrolytic ablation surgery protocols.

Bioengineering↗

bioRxiv2016

Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells.

Patient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modelling and drug discovery. However, the processes of reprogramming, maintenance and differentiation are labour intensive and subject to inter-technician variability. To address these issues, we established and optimised protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60 positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.

Bioengineering↗

bioRxiv2016

QTL-guided metabolic engineering of a complex trait

Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness of Saccharomyces cerevisiae during fermentation of a Miscanthus x giganteus plant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic Bulk Reciprocal Hemizygosity analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this \"genetic blueprint\", and using a dual-guide Cas9-based method to efficiently perform multi-kilobase locus replacements, we engineered an S288C strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait in S. cerevisiae, as well as other organisms.

Bioengineering↗

bioRxiv2016

Feasibility Study of an Optical Caustic Plasmonic Light Scattering Sensor for Human Serum Anti-Dengue Protein E Antibody Detection

Antibody detection and accurate diagnosis of tropical diseases is essential to help prevent the spread of disease. However, most detection methods lack cost-effectiveness and field-portability, which are essential features for achieving diagnosis in a timely manner in developing countries. To address this problem, transparent 3D printed sample chambers with a total volume of 700 microliters and an oblate spheroid shape were fabricated to measure green light scattering of gold nanoparticles using an optical caustic focus to detect antibodies. Scattering signals from 90 degree scattering of 20, 40, 50, 60, 80, 100, and 200 nm gold nanoparticles using a green laser and standard quartz cuvette were compared to the scattering signals from a green LED light source with an oblate spheroid sample chamber and to Mie theory by fitting the data to a logistic curve. The change in signal from 60 nm to 120 nm decreased in the order of Mie Theory > Optical Caustic scattering > standard laser 90 degree scattering. These results suggested that conjugating 60 nm gold nanoparticles with Dengue Protein E and using an optical caustic system to detect plasmonic light scattering would result in a sensitive test for detecting human antibodies against Dengue Protein E in serum. To explore this possibility, we studied the light scattering response of protein E conjugated gold nanoparticles exposed to different concentrations of anti-protein E antibody, and posteriorly via a feasibility study consisting of 10 human serum samples using a modified dot blot protocol and a handheld optical caustic-based sensor device. The overall agreement between the benchtop light scattering and dot blot results and the handheld optical caustic sensor suggest that the new sensor concept shows promise to detect gold nanoparticle aggregation caused by the presence of the antibody using a homogeneous assay. Further testing and protocol optimization is needed in order to draw conclusions on the positive predictive and negative predictive values for this new testing system.

bioengineering↗

bioRxiv2016

Cell-to-cell Modelling of the inter-phase between Atrial and Sinoatrial Anisotropic Heterogeneous Nets

AO_SCPCAPBSTRACTC_SCPCAPThe transition between Sinoatrial cells and Atrial cells in the Heart is not fully undefirst ood. Here we focus in cell-to-cell mathematical models involving typical Sinoatrial cells and Atrial cells connected with experimentally observed conductance values exclusively. We are interested mainly in the geometry of the microstructure of the conduction paths in the Sinoatrial Node. We show with some models that appropriate source-sink relationships between Atrial and Sinoatrial cells may occur according to certain geometric arrangements.

bioengineering↗

bioRxiv2016

Electroceutical disinfection strategies impair the motility of pathogenic Pseudomonas aeruginosa and Escherichia coli

Electrotaxis or galvanotaxis refers to the migration pattern of cells induced in response to electrical potential. Although it has been extensively studied in mammalian cells, electrotaxis has not been explored in detail in bacterial cells; information regarding the impact of current on pathogenic bacteria is severely lacking. Therefore, we designed a series of single and multi-cue experiments to assess the impact of varying currents on bacterial motility dynamics in pathogenic multi-drug resistant (MDR) strains of Pseudomonas aeruginosa and Escherichia coli using a microfluidic platform. Motility plays key roles in bacterial migration and the colonization of surfaces during the formation of biofilms, which are inherently recalcitrant to removal and resistant to traditional disinfection strategies (e.g. antibiotics). Use of the microfluidic platform allows for exposure to current, which can be supplied at a range that is biocidal to bacteria, yet physiologically safe in humans (single cue). This system also allows for multi-cue experiments where acetic acid, a relatively safe compound with anti-fouling/antimicrobial properties, can be combined with current to enhance disinfection. These strategies may offer substantial therapeutic benefits, specifically for the treatment of biofilm infections, such as those found in the wound environment. Furthermore, microfluidic systems have been successfully used to model the unique microfluidic dynamics present in the wound environment, suggesting that these investigations could be extended to more complex biological systems. Our results showed that the application of current in combination with acetic acid has profound inhibitory effects on MDR strains of P. aeruginosa and E. coli, even with brief applications. Specifically, E. coli motility dynamics and cell survival were significantly impaired starting at a concentration of 125 A DC and 0.31% acetic acid, while P. aeruginosa was impaired at 70 A and 0.31% acetic acid. As these strains are relevant wound pathogens, it is likely that this strategy would be effective against similar strains in vivo and could represent a new approach to hasten wound healing.

bioengineering↗

bioRxiv2016

Summary of muscle parameters for Hill-based muscle modeling in the human lower limb

A summary is presented of five mechanical parameters from human lower limb skeletal muscles critical for Hill-based muscle modeling: the optimal fiber length, the fiber pennation angle, the physiological cross-sectional area (PCSA), the unloaded tendon length, and the fast-twitch fiber fraction. The data presented are drawn from a total of 29 publications including human cadaver studies, in vivo imaging studies of live humans, musculoskeletal modeling studies, and combinations of these methods. Where possible, parameter values were adjusted from the referenced data to present them with consistent definitions (normalization of measured fiber lengths to optimal sarcomere length, and calculation of PCSA as the ratio of fiber volume to fiber length). It is seen that within a specific muscle, optimal fiber lengths are fairly consistent between studies, pennation angles and PCSAs vary widely between studies, and data for unloaded tendon length are comparatively sparse. Few studies have reported fiber type fractions for a large number of muscles. Guidelines for implementing these parameter values in muscle modeling and musculoskeletal modeling are suggested.\n\nUpdate HistoryO_LIDecember 2, 2016: original submission\nC_LIO_LIDecember 3, 2016: discussion on maximum isometric force and specific tension added\nC_LIO_LIDecember 6, 2016: minor edits for typos, clarity, and missing references\nC_LIO_LIDecember 7, 2016: Tirrell et al. (2012) study added\nC_LI

bioengineering↗

bioRxiv2016

Fast and Stable Signal Deconvolution via Compressible State-Space Models

Abstract-Objective:Common biological measurements are in the form of noisy convolutions of signals of interest with possibly unknown and transient blurring kernels. Examples include EEG and calcium imaging data. Thus, signal deconvolution of these measurements is crucial in understanding the underlying biological processes. The objective of this paper is to develop fast and stable solutions for signal deconvolution from noisy, blurred and undersampled data, where the signals are in the form of discrete events distributed in time and space. Methods: We introduce compressible state-space models as a framework to model and estimate such discrete events. These state-space models admit abrupt changes in the states and have a convergent transition matrix, and are coupled with compressive linear measurements. We consider a dynamic compressive sensing optimization problem and develop a fast solution, using two nested Expectation Maximization algorithms, to jointly estimate the states as well as their transition matrices. Under suitable sparsity assumptions on the dynamics, we prove optimal stability guarantees for the recovery of the states and present a method for the identification of the underlying discrete events with precise confidence bounds. Results: We present simulation studies as well as application to calcium deconvolution and sleep spindle detection, which verify our theoretical results and show significant improvement over existing techniques. Conclusion: Our results show that by explicitly modeling the dynamics of the underlying signals, it is possible to construct signal deconvolution solutions that are scalable, statistically robust, and achieve high temporal resolution. Significance: Our proposed methodology provides a framework for modeling and deconvolution of noisy, blurred, and undersampled measurements in a fast and stable fashion, with potential application to a wide range of biological data.

bioengineering↗

bioRxiv2016

Single-channel in-Ear-EEG predicts the focus of auditory attention to concurrent tone streams and mixed speech

Conventional, multi-channel scalp electroencephalography (EEG) allows the identification of the attended speaker in concurrent-listening (\"cocktail party\") scenarios. This implies that EEG might provide valuable information to complement hearing aids with some form of EEG and to install a level of neuro-feedback. To investigate whether a listeners attentional focus can be predicted from single-channel hearing-aid-compatible EEG configurations, we recorded EEG from three electrodes inside the ear canal (\"in-Ear-EEG\") and additionally from 64 electrodes on the scalp. In two different, concurrent listening tasks, participants (n = 7) were fitted with individualized in-Ear-EEG pieces and were either asked to attend to one of two dichotically-presented, concurrent tone streams or to one of two diotically-presented, concurrent audiobooks. A forward encoding model was trained to predict the EEG response at single EEG channels. We found that all individual participants attentional focus could be predicted from single-channel EEG response recorded from short-distance configurations consisting only of a single in-Ear-EEG electrode and an adjacent scalp-EEG electrode. The responses to attended and ignored stimuli reveal differences consistent across subjects. In sum, our findings show that the EEG response from a single-channel, hearing-aid-compatible configuration provides valuable information to identify a listeners focus of attention.

bioengineering↗

bioRxiv2017

The leading-edge vortex of swift wings

1Recent investigations on the aerodynamics of natural fliers have illuminated the significance of the Leading-Edge Vortex (LEV) for lift generation in a variety of flight conditions. A well documented example of an LEV is that generated by aircraft with highly swept, delta shaped wings. While the wing aerodynamics of a manoeuvring aircraft, a bird gliding and a bird in flapping flight vary significantly, it is believed that this existing knowledge will serve to add understanding to the complex aerodynamics of natural fliers. In this investigation, the wing of a common swift Apus apus is simplified to a model with swept wings and a sharp leading-edge, making it readily comparable to a model delta shaped wing of the same leading-edge geometry. Particle image velocimetry provides an understanding of the effect of the tapering swift wing on LEV development and stability, compared with the delta wing model. For the first time a dual LEV is recorded on a swift shaped wing, where it is found across all tested conditions. It is shown that the span-wise location of LEV breakdown is governed by the local chord rather than Reynolds number or angle of attack. These findings suggest that the common swift is able to generate a dual LEV while gliding, potentially delaying vortex breakdown by exploiting other features non explored here, such as wing twist and flexibility. It is further suggested that the vortex system could be used to damp loading fluctuations, reducing energy expenditure, rather than for lift augmentation.

bioengineering↗

bioRxiv2017

An Adaptive Geometric Search Algorithm for Macromolecular Scaffold Selection

A wide variety of protein and peptidomimetic design tasks require matching functional three-dimensional motifs to potential oligomeric scaffolds. Enzyme design, for example, aims to graft active-site patterns typically consisting of 3 to 15 residues onto new protein surfaces. Identifying suitable proteins capable of scaffolding such active-site engraftment requires costly searches to identify protein folds that can provide the correct positioning of side chains to host the desired active site. Other examples of biodesign tasks that require simpler fast exact geometric searches of potential side chain positioning include mimicking binding hotspots, design of metal binding clusters and the design of modular hydrogen binding networks for specificity. In these applications the speed and scaling of geometric search limits downstream design to small patterns. Here we present an adaptive algorithm to searching for side chain take-off angles compatible with an arbitrarily specified functional pattern that enjoys substantive performance improvements over previous methods. We demonstrate this method in both genetically encoded (protein) and synthetic (peptidomimetic) design scenarios. Examples of using this method with the Rosetta framework for protein design are provided but our implementation is compatible with multiple protein design frameworks and is freely available as a set of python scripts (https://github.com/JiangTian/adaptive-geometric-search-for-protein-design).

bioengineering↗

bioRxiv2017

Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared to the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces ~4-fold more total protein. However the majority of these polypeptides are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ~2/3rd of that measured using the S30 crude extract system. Both of the two systems suffer from low ribosome recycling efficiency when translating proteins from 90 kD to 220 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination prior to energy depletion increased Fluc protein yield by ~1.5 to ~2-fold, while accumulation of inorganic phosphate contributes to reaction termination. Additionally, while adding EF-P to PURE reduced total protein translated, it also increased the fraction of active product and reduced partial translated product probably by slowing down the translation process. Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion.

bioengineering↗

bioRxiv2017

Experimental evolution reveals a novel avenue to release catabolite repression via mutations in XylR

Microbial production of fuels and chemicals from lignocellulosic biomass provides promising bio-renewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocel-lulosic biomass materials are glucose and xylose. While industrial Escherichia coli strains efficiently utilize glucose, their ability to utilize xylose is often repressed in the presence of glucose. Here we independently evolved three E. coli strains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into the E. coli D-lactate producer TG114, 94 % of a glucose-xylose mixture (50 g L-1 each) was utilized in mineral salt media that led to a 50 % increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in different E. coli hosts, including wild-type, suggesting its potential wide application in industrial E. coli biocatalysts.

bioengineering↗

bioRxiv2017

Computer-Assisted Engineering of Hyperstable Fibroblast Growth Factor 2

Fibroblast growth factors (FGFs) play numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability in vivo and in vitro. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function.

bioengineering↗

bioRxiv2017

Shortwave Infrared Fluorescence Imaging with the Clinically Approved Near-Infrared Dye Indocyanine Green

Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave infrared region (SWIR, 1-2 m) promises higher contrast, sensitivity, and penetration depths compared to conventional visible and near-infrared (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, due in part to the absence of FDA-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Despite the fact that their emission reaches a maximum in the NIR, these dyes can be imaged non-invasively in vivo in the SWIR spectral region, even beyond 1500 nm. We demonstrate real-time fluorescence angiography at wavelengths beyond 1300 nm using ICG at clinically relevant doses. Furthermore, we show tumortargeted SWIR imaging with trastuzumab labeled with IRDye 800CW, a NIR dye currently being tested in multiple phase II clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide (InGaAs) SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications.

bioengineering↗