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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 325 records · Page 18Linked to original sources

bioRxiv2018

Combinatorial library of biodegradable polyesters enables delivery of plasmid DNA to polarized human RPE monolayers for retinal gene therapy

AbstractEfficient gene delivery into hard-to-transfect cells is still a challenge despite significant progress in the development of various gene delivery tools. Non-viral and synthetic polymeric nanoparticles offer an array of advantages for gene delivery over the viral vectors and high in demand as they are safe to use, easy to synthesize and highly cell-type specific. Here we demonstrate the use of a high-throughput screening (HTS) platform to screen for biodegradable polymeric nanoparticles (NPs) that can transfect human retinal pigment epithelial (RPE) cells with high efficiency and low toxicity. These NPs can deliver plasmid DNA (pDNA) to RPE monolayers more efficiently compared to the commercially available transfection reagents without interfering the global gene expression profile of RPE cells. In this work, we have established an HTS platform and identified synthetic polymers that can be used for high efficacy non-viral gene delivery to human RPE monolayers, enabling gene loss- and gain-of-function studies of cell signaling and developmental pathways. This platform can be used to identify the optimum polymer, weight-to-weight ratio of polymer to DNA, and the dose of NP for various retinal cell types.

bioengineering↗

bioRxiv2018

ssRNA/DNA-Sensors via Embedded Strand-Displacement Programs in CRISPR/Cas9 Guides

RNA and DNA profiles can help classify a variety of biological states, including disease, metabolism and cell type. In a proof-of-concept study on novel genetically encodable components for detecting single-stranded oligonucleotides, we engineered switchable CRISPR guide RNA (swigRNA) with Cas9 affinity that is conditional on sensing an oligo trigger molecule (trigRNA or trigDNA). RNA- and DNA-sensing swigRNAs that serve as off-to-on and on-to-off switches for Cas9 cleavage were achieved by computational design of hybridization dependencies in the guide. This study highlights functional swigRNA structures that implement toehold-gated strand-displacement with their triggers, and should merit further engineering and kinetic characterization.

bioengineering↗

bioRxiv2018

Engineering bacteria for biogenic synthesis of chalcogenide nanomaterials

Microbes naturally build nanoscale structures, including structures assembled from inorganic materials. Here we combine the natural capabilities of microbes with engineered genetic control circuits to demonstrate the ability to control biological synthesis of chalcogenide nanomaterials in a heterologous host. We transferred reductase genes from both Shewanella sp. ANA-3 and Salmonella enterica serovar Typhimurium into an heterologous host (Escherichia coli) and examined the mechanisms that regulate the properties of biogenic nanomaterials. Expression of arsenic reductase genes and thiosulfate reductase genes in E. coli resulted in the synthesis of arsenic sulfide nanomaterials. In addition to processing the starting materials via redox enzymes, cellular components also nucleated the formation of arsenic sulfide nanomaterials. The shape of the nanomaterial was influenced by the bacterial culture, with the synthetic E. coli strain producing nanospheres and conditioned media or cultures of wild type Shewanella sp. producing nanofibers. The diameter of these nanofibers also depended on the biological context of synthesis. These results demonstrate the potential for biogenic synthesis of nanomaterials with controlled properties by combining the natural capabilities of wild microbes with the tools from synthetic biology.

bioengineering↗

bioRxiv2018

Generation of functional human adipose tissue in mice from primed progenitor cells

AbstarctAdipose tissue is used extensively in reconstructive and regenerative therapies, but transplanted fat often undergoes inflammation and cell death, requiring further revision surgery. We report that functional human adipose tissue can be generated from mesenchymal progenitor cells in-vivo, providing an alternative approach to its therapeutic use. We leveraged previous findings that progenitor cells within the vasculature of human adipose tissue robustly proliferate in 3-dimensional culture under proangiogenic conditions. Implantation of these progenitor cells into immunocompromised mice results in differentiation towards non-adipocyte fates, incapable of generating a distinct tissue structure. However, priming of these progenitor cells in-vitro towards adipogenic differentiation results in formation of functional adipose tissue in-vivo. Mechanistically, priming induces the expression of genes encoding specific extracellular matrix and remodeling proteins, and induces extensive vascularization by host blood vessels. In comparison, grafts from adipose tissue obtained by liposuction undergo poor vascularization, adipocyte death, cyst formation, calcification and inefficient adiponectin secretion. Thus, primed mesenchymal adipose tissue progenitors reveal mechanisms of human adipose tissue development, and have potential to improve outcomes in reconstructive and regenerative medicine.

bioengineering↗

bioRxiv2018

Echo State Networks Ensemble for SSVEP Dynamical Online Detection

BackgroundRecent years have witnessed an increased interest in the use of steady state visual evoked potentials (SSVEPs) in brain computer interfaces (BCI), SSVEP is considered a stationary brain process that appears when gazing at a stimulation light source.\n\nNew MethodsThe complex nature of brain processes advocates for non-linear EEG analysis techniques. In this work we explore the use of an Echo State Networks (ESN) based architecture for dynamical SSVEP detection.\n\nResultsWhen simulating a 6-degrees of freedom BCI system, an information transfer rate of 49bits/min was achieved. Detection accuracy proved to be similar for observation windows ranging from 0.5 to 4 seconds.\n\nComparison with existing methodsSSVEP detection performance has been compared to standard canonical correlation analysis (CCA). CCA achieved a maximum information transfer rate of 21 bits/minute. In this case detection accuracy increased along with the observation window length\n\nConclusionsAccording to here presented results ESN outperforms standard canonical correlation and has proved to require shorter observation time windows. However ESN and CCA approaches delivered diverse classification accuracies at subject level for various stimulation frequencies, proving to be complementary methods. A possible explanation of these results may be the occurrence of evoked responses of different nature, which are then detected by different approaches. While reservoir computing methods are able to detect complex dynamical patterns and/or complex synchronization among EEG channels, CCA exclusively captures stationary patterns. Therefore, the ESN-based approach may be used to extend the definition of steady-state response, considered so far a stationary process.\n\nHighlightsO_LIWe present a novel SSVEP dynamical detection approach based on ESN.\nC_LIO_LIThis is the first time ESNs are applied to SSVEP based BCI systems.\nC_LIO_LIWe provide experimental validation of proposed methodology.\nC_LIO_LIExperimental results indicate non-stationarity in SSVEP patterns.\nC_LI

bioengineering↗

bioRxiv2018

Targeted Drug Delivery by Radiation-Induced Tumor Vascular Modulation

Effective drug delivery is severely restricted by the presence of complex pathophysiological barriers in solid tumors. In human pancreatic adenocarcinoma, mature and hypopermeable tumor blood vessels limit the permeation and penetration of chemo or nanotherapeutics to cancer cells and substantially reduce the treatment efficacy. New, clinically-viable strategies are therefore sought to breach the neoplastic barriers that prevent optimal tumor-specific drug delivery. Here, we present an original idea to boost targeted drug delivery by selectively knocking down the tumor vascular barrier in a poorly permeable human pancreatic cancer model. For the first time, we demonstrate that clinical irradiation (10 Gy, 6 MV) can induce tumor vascular modulation when combined with tumor endothelial-targeting gold nanoparticles. Active disruption of tumor blood vessels by nanoparticle-combined radiotherapy led to increased vessel permeability and improved tumor uptake of two prototypical model nanodrugs: i) a short-circulating nanocarrier with MR-sensitive gadolinium (Gad-NC; 8 kDa; t1/2=1.5 h) and ii) a long-circulating nanocarrier with fluorescence-sensitive NIR dye (FL-NC; 30 kDa; t1/2=25 h). Functional changes in the altered tumor vessel dynamics, measured by relative changes in permeability (Ktrans), flux rate (Kep) and extracellular interstitial volume (Ve) were consistent with the concomitant increase in nanodrug delivery. This combination of radiation-induced antivascular and nanodrug-mediated anti-tumor treatment offers high therapeutic benefit for tumors with pathophysiology that restricts efficient drug delivery.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC=\"FIGDIR/small/268714_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (84K):\norg.highwire.dtl.DTLVardef@a6df8borg.highwire.dtl.DTLVardef@19e3179org.highwire.dtl.DTLVardef@c223aaorg.highwire.dtl.DTLVardef@1b3a08d_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT\n\nC_FIG

bioengineering↗

bioRxiv2018

Relative Patlak Plot for Dynamic PET Parametric Imaging Without the Need for Early-time Input Function

The Patlak graphical method is widely used in parametric imaging for modeling irreversible radiotracer kinetics in dynamic PET. The net influx rate of radiotracer can be determined from the slope of the Patlak plot. The implementation of the standard Patlak method requires the knowledge of full-time input function from the injection time until the scan end time, which presents a challenge for use in the clinic. This paper proposes a new relative Patlak plot method that does not require early-time input function and therefore can be more efficient for parametric imaging. Theoretical analysis proves that the effect of early-time input function is a constant scaling factor on the Patlak slope estimation. Thus, the parametric image of the slope of the relative Patlak plot is related to the parametric image of standard Patlak slope by a global scaling factor. This theoretical finding has been further demonstrated by computer simulation and real patient data. The study indicates that parametric imaging of the relative Patlak slope can be used as a substitute of parametric imaging of standard Patlak slope for certain clinical tasks such as lesion detection and tumor volume segmentation.

bioengineering↗

bioRxiv2018

Partial-Width Injuries of the Rat Rotator Cuff Heal with Fibrosis

PurposeThe purpose of this study was to identify the healing outcomes following a partial-width, full-thickness injury to the rotator cuff tendon-bone attachment and establish if the adult attachment can regenerate the morphology of the healthy attachment.\n\nHypothesisWe hypothesized that a partial-width injury to the attachment would heal via fibrosis and bone remodeling, resulting in increased cellularity and extracellular matrix deposition, reduced bone volume, osteoclast presence and decreased collagen organization compared to shams.\n\nMaterials and MethodsA biopsy punch was used to create a partial-width injury at the center one-third of the rat infraspinatus attachment, and the contralateral limb underwent a sham operation. Rats were sacrificed at 3- and 8-weeks after injury for analyses. Analyses performed at each time-point included cellularity (Hematoxylin & Eosin), ECM deposition (Massons Trichrome), bone volume (micro-computed tomography; microCT), osteoclast activity (Tartrate Resistant Acid Phosphatase; TRAP), and collagen fibril organization (Picrosirius Red). Injured and sham shoulders were compared at both 3- and 8-weeks using paired, two-way ANOVAs with repeated measures and Sidaks correction for multiple comparisons.\n\nResultsCellularity and ECM deposition increased at both 3- and 8-weeks compared to sham contralateral attachments. Bone volume decreased and osteoclast presence increased at both 3- and 8-weeks compared to sham contralateral limbs. Collagen fibril organization was reduced at 3-weeks after injury compared to 3-week sham attachments.\n\nConclusionsThese findings suggest that a partial-width injury to the rotator cuff attachment does not fully regenerate the native structure of the healthy attachment. The injury model healed via scar-like fibrosis and did not propagate into a full-width tear after 8-weeks of healing.

bioengineering↗

bioRxiv2018

An immortalised mesenchymal stem cell line maintains mechano-responsive behaviour and can be used as a reporter of substrate stiffness

The mechanical environment can influence cell behaviour, including changes to transcriptional and proteomic regulation, morphology and, in the case of stem cells, commitment to lineage. However, current tools for characterizing substrates mechanical properties, such as atomic force microscopy (AFM), often do not fully recapitulate the length and time scales over which cells feel substrates. Here, we show that an immortalised, clonal line of human mesenchymal stem cells (MSCs) maintained responsiveness to substrate mechanics observed in primary cells, and could be used as a reporter of stiffness. MSCs were cultured on soft and stiff polyacrylamide hydrogels. In both primary and immortalised MSCs, stiffer substrates promoted increased cell spreading, expression of lamin-A/C and translocation of mechano-sensitive proteins YAP1 and MKL1 to the nucleus. Stiffness was also found to regulate transcriptional markers of lineage. A GFP-YAP / RFP-H2B reporter construct was designed and virally delivered to the immortalised MSCs for in situ detection of substrate stiffness. MSCs with stable expression of the reporter showed GFP-YAP to be increasingly co-localized with nuclear RFP-H2B on stiffer substrates, enabling development of a cellular reporter of substrate stiffness. This will facilitate mechanical characterisation of new materials developed for applications in tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2018

Multimodal on-axis platform for all-optical electrophysiology with near-infrared probes in human stem-cell-derived cardiomyocytes

Combined optogenetic stimulation and optical imaging permits scalable, high-throughput probing of cellular electrophysiology and optimization of stem-cell derived excitable cells, such as neurons and muscle cells. We report a new \"on-axis\" configuration of OptoDyCE, our all-optical platform for studying human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and other cell types, optically driven by Channelrhodopsin2 (ChR2). This solid-state system integrates optogenetic stimulation with temporally-multiplexed simultaneous recording of membrane voltage (Vm) and intracellular calcium ([Ca2+]i) dynamics using a single photodetector. We demonstrate the capacity for combining multiple spectrally-compatible actuators and sensors, including newer high-performance near-infrared (NIR) voltage probes BeRST1 and Di-4-ANBDQBS, to record complex spatiotemporal responses of hiPSC-CMs to drugs in a high-throughput manner.

bioengineering↗

bioRxiv2018

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

We report a high-throughput screening approach to measure Staphylococcus aureus Cas9 (SaCas9) genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporated randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with a 21-nucleotide spacer are most active against off-targets with single and double mismatches, compared to shorter or longer sgRNAs. Using this dataset, we developed an SaCas9 specificity model that performs well in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

bioengineering↗

bioRxiv2018

Novel modular pressure and flow rate balanced microfluidic serial dilution networks on printed circuit boards: Designs, Simulations and Fabrication

Fast, efficient and more importantly accurate serial dilution is a requirement for many chemical and biological microfluidic-based applications. Over the last decade, a large number of microfluidic devices has been proposed, each demonstrating either a different type of dilution technique or complex system architectures based on various flow source combinations. In this work, a novel serial dilution architecture is demonstrated, implemented on a commercially fabricated printed circuit board (PCB). The proposed single layer, stepwise serial diluter comprises an optimised microfluidic network, where identical dilution ratio per stage can be ensured, either by applying equal pressure or equal flow rates at both inlets. The advantages of the proposed serial diluter are twofold. Firstly, it is structured as a modular unit cell, simplifying the required fluid driving mechanism to a single source for both sample and buffer solution. Thus, this unit cell can be seen as a fundamental microfluidic building block, which can form multistage serial dilution cascades, once combined appropriately with itself or other similar unit cells. Secondly, the serial diluter has been fabricated entirely using commercial PCB technologies, allowing the device to be interfaced with standard electronic components, if more complex miniature point-of-care (PoC) systems are desired, where the small footprint and accuracy of the device is of paramount importance.

bioengineering↗

bioRxiv2018

Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging

Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method for rapid and optically efficient 3D microscopy. Initial LSFM designs utilized a static sheet of light, termed selective plane illumination microscopy (SPIM), which exhibited shadowing artifacts and deteriorated contrast due to light scattering. These issues have been addressed, in part, by multidirectional selective plane illumination microscopy (mSPIM), in which rotation of the light sheet is used to mitigate shadowing artifacts, and digital scanned light-sheet microscopy (DSLM), in which confocal line detection is used to reject scattered light. Here we present a simple passive multidirectional digital scanned light-sheet microscopy (mDSLM) architecture that combines the benefits of mSPIM and DSLM. By utilizing an elliptical Gaussian beam with increased angular diversity in the imaging plane, mDSLM provides shadow-free contrast-enhanced imaging of fluorescently labeled samples.\n\nOne Sentence SummaryGlaser et al. describe a light-sheet microscopy architecture that enables passive multidirectional illumination with confocal line detection to enable both uniform fluorescence excitation and contrast-enhanced imaging of fluorescently labeled samples.

bioengineering↗

bioRxiv2018

Optimized breath detection algorithm in Electrical Impedance Tomography

ObjectiveThis paper define a method for optimizing the breath delineation algorithms used in Electrical Impedance Tomography (EIT). In lung EIT the identification of the breath phases is central for generating tidal impedance variation images, subsequent data analysis and clinical evaluation. The optimisation of these algorithms is particularly important in neonatal care since the existing breath detectors developed for adults may give insufficient reliability in neonates due to their very irregular breathing pattern.\n\nApproachOur approach is generic in the sense that it relies on the definition of a gold standard and the associated definition of detector sensitivity and specificity, an optimisation criterion and a set of detector parameters to be investigated. The gold standard has been defined by 11 clinicians with previous experience with EIT and the performance of our approach is described and validated using a neonatal EIT dataset acquired within the EU-funded CRADL project.\n\nMain resultsThree different algorithms are proposed that are improving the breath detector performance by adding conditions on 1) maximum tidal breath rate obtained from zero-crossings of the EIT breathing signal, 2) minimum tidal impedance amplitude and 3) minimum tidal breath rate obtained from Time-Frequency (TF) analysis.\n\nSignificanceBased on the gold standard, the most crucial parameters of the proposed algorithms are optimised by using a simple exhaustive search and a weighted metric defined in connection with the Receiver Operating Characterics (ROC). This provides a practical way to achieve any desirable trade-off between the sensitivity and the specificity of the detectors.

bioengineering↗

bioRxiv2018

Cross-Platform Mechanical Characterization of Lung Tissue

Published data on the mechanical strength and elasticity of lung tissue is widely variable, primarily due to differences in how testing was conducted across individual studies. This makes it extremely difficult to find a benchmark modulus of lung tissue when designing synthetic extracellular matrices (ECMs). To address this issue, we tested tissues from various areas of the lung using multiple characterization techniques, including micro-indentation, small amplitude oscillatory shear (SAOS), uniaxial tension, and cavitation rheology. We report the sample preparation required and data obtainable across these unique but complimentary methods to quantify the modulus of lung tissue. We highlight cavitation rheology as a new method, which can measure the modulus of intact tissue with precise spatial control, and reports a modulus on the length scale of typical tissue heterogeneities. Shear rheology, uniaxial, and indentation testing require heavy sample manipulation and destruction; however, cavitation rheology can be performed in situ across nearly all areas of the lung with minimal preparation. The Youngs modulus of bulk lung tissue using microindentation (1.9{+/-}0.5 kPa), SAOS (3.2{+/-}0.6 kPa), uniaxial testing (3.4{+/-}0.4 kPa), and cavitation rheology (6.1{+/-}1.6 kPa) were within the same order of magnitude, with higher values consistently reported from cavitation, likely due to our ability to keep the tissue intact. Although cavitation rheology does not capture the non-linear strains revealed by uniaxial testing and SAOS, it provides an opportunity to measure mechanical characteristics of lung tissue on a microscale level on intact tissues. Overall, our study demonstrates that each technique has independent benefits, and each technique revealed unique mechanical features of lung tissue that can contribute to a deeper understanding of lung tissue mechanics.

bioengineering↗

bioRxiv2018

Hypoarousal non-stationary ADHD biomarker based on echo-state networks

Attention-Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity and impulsivity. It is one of the most commonly diagnosed neurodevelopmental and psychiatric disorders of childhood and therefore presents a very high prevalence rate. However the high rate of ADHD misdiagnosis makes the discovery of neurophysiological ADHD biomarkers an important clinical challenge. This study proposes a novel non-stationary ADHD biomarker based on Echo State Networks to quantify EEG dynamical changes between low attention/arousal states (resting with eyes closed, or EC) and normal attention/arousal states (resting with eyes open, or EO). Traditionally, EEG biomarkers have revealed an increase in stationary power in the theta band along with a decrease in beta, with these frequencies largely accepted to be altered in the ADHD population. We successfully verify the hypothesis that measured differences between these two conditions are altered in the ADHD population. Statistically significant differences between a group of ADHD subjects and an aged-matched control population were obtained in theta and beta rhythms. Our network discriminates between EO/EC EEG regimes in the ADHDs better than in controls, suggesting that differences in EEG patterns between low and normal arousal/attention states are larger in the ADHD population.

bioengineering↗

bioRxiv2018

High Numerical Aperture Epi-illumination Selective Plane Illumination Microscopy

Selective-plane illumination microscopy (SPIM) provides unparalleled advantages for long-term volumetric imaging of living organisms. In order to achieve high-resolution imaging in common biological sample holders, we designed a high numerical aperture (NA) epi-illumination SPIM (eSPIM) system, which utilizes a single objective and has an identical sample interface as an inverted fluorescence microscope with no additional reflection elements. This system has an effective detection NA of > 1.06. We demonstrated multicolor and fast volumetric imaging of live cells and single-molecule super-resolution microscopy using our system.

bioengineering↗

bioRxiv2018

FITC Conjugated Polycaprolactone-Glycol-Chitosan Nanoparticles Containing The Longwave Emitting Fluorophore IR 820 For In-Vitro Tracking Of Hyperthermia-Induced Cell Death

Cancer theranostic agent IR 820 loses its bioimaging ability once therapy is initiated. At the end of therapy, it becomes difficult to track the cancer cells. To address this, FITC conjugated Polycaprolactone glycol chitosan IR 820 nanoparticles (FITC-PCLGC-IR NPs) has been synthesized for in vitro tracking of hyperthemia induced cell death. Two approaches, namely ex situ and in situ have been pursued FITC conjugation to PCLGC-IR NPs. Further comparisons were made to FITC encapsulated PCLGC-IR NPs in terms of biocompatibility, cellular uptake, photothermal mediated cell death and imaging with respect to laser treatment. We have shown that an 808 nm diode laser treatment did not affect the imaging ability of these NPs whereas cancer. Time scanned fluorescence shows the excellent photostability of this formulation for a maximum of 5 min. The detailed studies of these approaches summarize that FITC conjugation to PCLGC-IR nanoparticles is an effective nano-theranostic solution for image-guided photothermal therapy.

bioengineering↗