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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

An Automated Workflow for Segmenting Single Adult Cardiac Cells from Large-Volume Serial Block-Face Scanning Electron Microscopy Data

This paper presents a new algorithm to automatically segment the myofibrils, mitochondria and nuclei within single adult cardiac cells that are part of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The algorithm only requires a set of manually drawn contours that roughly demarcate the cell boundary at routine slice intervals (every 50th, for example). The algorithm correctly classified pixels within the single cell with 97% accuracy when compared to manual segmentations. One entire cell and the partial volumes of two cells were segmented. Analysis of segmentations within these cells showed that myofibrils and mitochondria occupied 47.5% and 51.6% on average respectively, while the nuclei occupy 0.7% of the cell for which the entire volume was captured in the SBF-SEM dataset. Mitochondria clustering increased at the periphery of the nucleus region and branching points of the cardiac cell. The segmentations also showed high area fraction of mitochondria (up to 70% of the 2D image slice) in the sub-sarcolemmal region, whilst it was closer to 50% in the intermyofibrillar space. We finally demonstrate that our segmentations can be turned into 3D finite element meshes for cardiac cell computational physiology studies. We offer our large dataset and MATLAB implementation of the algorithm for research use at www.github.com/CellSMB/sbfsem-cardiac-cell-segmenter/. We anticipate that this timely tool will be of use to cardiac computational and experimental physiologists alike who study cardiac ultrastructure and its role in heart function.

bioengineering↗

bioRxiv2018

Hemicellulose Degradation and Utilization by a Synthetic Saccharomyces cerevisiae Consortium

Since Saccharomyces cerevisiae does not inherently possess the capability to utilize pentose sugars released from hemicellulose degradation, the degradation and utilization of hemicellulose poses a conundrum to bioethanol production by consolidated bioprocessing (CBP) using S. cerevisiae. In this study, S. cerevisiae was exploited for its ability to degrade xylan, one of the major polysaccharide chains present in hemicellulose. Different hemicellulases from Trichoderma reesei, namely: endoxylanase (Xyn2), {beta}-xylosidase (Bxl1), acetylxylan esterase (Axe1), -D-glucuronidase (Glr1) and -L-arabinofuranosidase (Abf1), were heterologously secreted by S. cerevisiae. A mixture experimental design was adapted to statistically describe the synergistic interactions between the hemicellulases and to determine the optimum formulations for the hydrolysis of xylan substrates. The hydrolytic activities of the hemicellulase mixtures were then improved by displaying the hemicellulases on the yeast surface to serve as whole-cell biocatalysts. The engineered yeast strains displaying hemicellulases were further engineered with xylose-utilization genes to enable abilities of utilizing xylose as a sole carbon source. The resulting consortia were then able to grow and produce ethanol from different xylan substrates.

bioengineering↗

bioRxiv2018

Uncovering novel pathways for enhancing hyaluronan synthesis in recombinant Lactococcus lactis: Genome-scale metabolic modelling and experimental validation

Hyaluronan (HA) is a naturally occurring high-value polysaccharide with important medical applications. HA is commercially produced from pathogenic microbial sources. HA-producing recombinant cell factories that are being developed with GRAS organisms are comparatively less productive than the best natural producers. The metabolism of these recombinant systems needs to be more strategically engineered to achieve significant improvement. Here, we use a genome-scale metabolic network model to account for the entire metabolic network of the cell to predict strategies for improving HA production. We here analyze the metabolic network of Lactococcus lactis adapted to produce HA, and identify non-conventional overexpression and knock-out strategies to enhance HA flux.\n\nTo experimentally validate our predictions, we identify an alternate route for enhancement of HA synthesis, originating from the nucleoside inosine, which has the capacity to function in parallel with the traditionally known route from glucose. Adopting this strategy resulted in a 2.8-fold increase in HA yield. The strategies identified and the experimental results show that the cell is capable of involving a larger subset of metabolic pathways in HA production. Apart from being the first report of the use of a nucleoside to improve HA production, our study shows how experimental results enable model refinement. Overall, we point out that well-constructed genome-scale metabolic models could be very potent tools to derive efficient strategies to improve biosynthesis of important high-value products.

bioengineering↗

bioRxiv2018

Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. Furthermore, we observed a significant improvement in donor fiber dispersion when transplanted MuSCs were delivered in the HAMC hydrogel. Studies to assess primary myoblast and MuSC viability in HAMC culture revealed no differences compared to the media control even when the cells were first ejected through a syringe and needle or exposed to regenerating skeletal muscle extract to mimic the transplantation procedure. However, when we quantified absolute numbers, we found that more cells pass through the syringe and needle when delivered in HAMC. Culture in HAMC also increased the proportion of MuSCs in cell cycle, via a CD44-independent mechanism. An effect on myoblast proliferation was not observed, suggesting a hierarchical effect. Finally, a series of transplant studies indicated that HAMC delivery does not influence passive cell clearance or alter the host immune response, but instead may serve to support in vivo expansion by delaying differentiation following transplant. Therefore, we conclude that MuSC engraftment efficacy is improved by delivering the therapeutic cell population within HAMC.

bioengineering↗

bioRxiv2018

A Reactive Inelasticity Theoretical Framework for modeling Viscoelasticity, Plastic Deformation, and damage in Soft Tissue

Soft tissues are biopolymeric materials, primarily made of collagen and water. These tissues have non-linear, anisotropic, and inelastic mechanical behaviors that are often categorized into viscoelastic behavior, plastic deformation, and damage. While tissues elastic and viscoelastic mechanical properties have been measured for decades, there is no comprehensive theoretical framework for modeling inelastic behaviors of these tissues that is based on their structure. To model the three major inelastic mechanical behaviors of soft tissue we formulated a structurally inspired continuum mechanics framework based on the energy of molecular bonds that break and reform in response to external loading (reactive bonds). In this framework, we employed the theory of internal state variables and kinetics of molecular bonds. The number fraction of bonds, their reference deformation gradient, and damage parameter were used as internal state variables that allowed for consistent modeling of all three of the inelastic behaviors of tissue by using the same sets of constitutive relations. Several numerical examples are provided that address practical problems in tissue mechanics, including the difference between plastic deformation and damage. This model can be used to identify relationships between tissues mechanical response to external loading and its biopolymeric structure.

bioengineering↗

bioRxiv2018

A novel system for online fertility monitoring based on milk progesterone

Timely identification of a cows reproduction status is essential to minimize fertilityrelated losses on dairy farms. This includes optimal estrus detection, pregnancy diagnosis and the timely recognition of early embryonic death and ovarian problems. On farm milk progesterone (P4) analysis could indicate all of these fertility events simultaneously. However, milk P4 measurements are subject to a large variability both in terms of measurement errors and absolute values between cycles. In this study, an innovative monitoring system based on milk P4 using the principles of synergistic control is presented. Instead of using filtering techniques and fixed thresholds, the present system employs an individually updated online model to describe the P4 profile, combined with a statistical process control chart to identify the cows fertility status. The inputs for the latter are the residuals of the online model, corrected for the concentration-dependent variability which is typical for milk P4 measurements. The objective of this paper is to present the developed methodology and give an indication of its use on farm. To this end, the system was validated on the P4 profiles of 38 dairy cows. The positive predictive value for luteolysis followed by estrus was 100%, meaning that the monitoring system picked up all estrous periods identified by the experts. Pregnancy or embryonic mortality was characterized by the absence or detection of luteolysis following an insemination, respectively. For thirteen cows, no luteolysis was detected by the system within the 25-32 days after insemination, indicating pregnancy, which was confirmed later by rectal palpation. It was also shown that the system is able to cope with deviating P4 profiles having prolonged follicular or luteal phases, which may suggest the occurrence of cysts. Future research is recommended for optimizing sampling frequency, prediction of the optimal insemination window and the establishment of rules to detect problems based on deviating P4 patterns.\n\nINTERPRETIVE SUMMARY. Progestrone-based online fertility monitoring. Adriaens.Milk progesterone based monitoring systems can be valuable for indicating fertility events in dairy cows, as the evolution of the milk progesterone levels can be related to acyclicity, estrus, pregnancy, and ovarian problems. We developed a system to translate raw progesterone measurements into clear information for the farmer, based on a mathematical model and a statistical control chart. In this way, a detailed image of a cows reproduction status on-farm can be obtained and fertility-related losses minimized.

bioengineering↗

bioRxiv2018

Linking heat attentions and the moment of LH surge in dairy cows - how close can we get?

Both estrus detection and timely insemination are important factors in optimizing fertility management. The latter is dependent on ovulation time, which is preceded by the LH surge. The performance of an estrus detection system based on activity and based on milk progesterone was evaluated and the timing of the alerts was contrasted against the moment of the LH surge. Activity alerts had a sensitivity of 83% and a positive predictive value of 66%; the LH surge followed average 9.4 {+/-} 16.1 hours later. Using milk progesterone, one can reliably detect luteolysis, which is followed by the LH surge after 62 {+/-} 12 hours.

bioengineering↗

bioRxiv2018

Local Delivery of Stromal Cell-Derived Factor-1α Improves the Pregnancy Rate of Injured Uterus through the Promotion of Endometrial and Vascular Regeneration

Severe infection and mechanical injury of the uterus may lead to infertility and miscarriage. Currently, there is a lack of effective treatment modality for functional repair of uterine injury. To address this clinical challenge, this study aimed to develop a chemotactic composite scaffold by incorporating recombinant human stromal cell-derived factor-1 (rhSDF-1) into a silk fibroin-bacterial cellulose (SF-BC) membrane carrier. A rat model of uterine injury was utilized for this study, which was composed of three groups: blank control, implantation with SF-BC only or SF-BC loaded with rhSDF-1. The tissue regeneration efficacy of the three groups were analyzed and compared. The results showed that SF-BC loaded with rhSDF-1 significantly enhanced endometrial regeneration and arteriogenesis of the injured rat uterus, which led to improved pregnancy outcomes, thus indicating much promise for functional uterine repair and regeneration.

bioengineering↗

bioRxiv2018

High-resolution wide-field human brain tumor margin detection and in vivo murine neuroimaging

Current in vivo neuroimaging techniques provide limited field of view or spatial resolution and often require exogenous contrast. These limitations prohibit detailed structural imaging across wide fields of view and hinder intraoperative tumor margin detection. Here we present a novel neuroimaging technique, speckle-modulating optical coherence tomography (SM-OCT), which allows us to image the brains of live mice and ex vivo human samples with unprecedented resolution and wide field of view using only endogenous contrast. The increased effective resolution provided by speckle elimination reveals white matter fascicles and cortical layer architecture in the brains of live mice. To our knowledge, the data reported herein represents the highest resolution imaging of murine white matter structure achieved in vivo across a wide field of view of several millimeters. When applied to an orthotopic murine glioblastoma xenograft model, SM-OCT readily identifies brain tumor margins with near single-cell resolution. SM-OCT of ex vivo human temporal lobe tissue reveals fine structures including cortical layers and myelinated axons. Finally, when applied to an ex vivo sample of a low-grade glioma resection margin, SM-OCT is able to resolve the brain tumor margin. Based on these findings, SM-OCT represents a novel approach for intraoperative tumor margin detection and in vivo neuroimaging.

bioengineering↗

bioRxiv2018

Selection-free, high frequency genome editing by homologous recombination of human pluripotent stem cells using Cas9 RNP and AAV6

Combination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

bioengineering↗

bioRxiv2018

Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Morphogenesis results from the interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events offer little control over parallel cell type co-emergence and do not offer the capability to selectively perturb gene expression in specific subpopulations of cells. We have developed an in vitro system that can spatiotemporally interrogate cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined the effects of independently knocking down molecular regulators of cortical tension and cell-cell adhesion using inducible CRISPRi: Rho-associated kinase-1 (ROCK1) and E-cadherin (CDH1), respectively. Induced mosaic knockdown of ROCK1 or CDH1 in hiPSC populations resulted in differential patterning events within hiPSC colonies indicative of cell-driven population organization. Patterned colonies retained an epithelial phenotype and nuclear expression of pluripotency markers. Gene expression within each of the mixed populations displayed a transient wave of differential expression with induction of knockdown that stabilized in coordination with intrinsic pattern formation. Mosaic patterning of hiPSCs enables the genetic interrogation of emergent multicellular properties of pluripotent cells, leading to a greater mechanistic understanding of the specific molecular pathways regulating the dynamics of symmetry breaking events that transpire during developmental morphogenesis.\n\nSIGNIFICANCEHuman embryonic development entails a series of multicellular morphogenic events that lead to primitive tissue formation. Attempts to study human morphogenic processes experimentally have been limited due to divergence from model organisms and the inability of current human in vitro models to accurately control the coincident emergence of heterogeneous cell populations in the spatially controlled manner necessary for proper tissue structure. We developed a human induced pluripotent stem cell (iPSC) in vitro model that enables temporal control over the emergence of heterotypic subpopulations of cells. We examined mosaic knockdown of two target molecules to create predictable and robust cell-patterning events within hiPSC colonies. This method allows for dynamic interrogation of intrinsic cell mechanisms that initiate symmetry breaking events and provides direct insight(s) into tissue developmental principles.

bioengineering↗

bioRxiv2018

Using imaging photoplethysmography for heart rate estimation in non-human primates

For humans and for non-human primates heart rate is a reliable indicator of an individuals current physiological state, with applications ranging from health checks to experimental studies of cognitive and emotional state. In humans, changes in the optical properties of the skin tissue correlated with cardiac cycles (imaging photoplethysmogram, iPPG) allow non-contact estimation of heart rate by its proxy, pulse rate. Yet, there is no established simple and non-invasive technique for pulse rate measurements in awake and behaving animals. Using iPPG, we here demonstrate that pulse rate in rhesus monkeys can be accurately estimated from facial videos. We computed iPPGs from seven color facial videos of three awake head-stabilized rhesus monkeys. Pulse rate estimated from iPPGs was in good agreement with reference data from a pulse-oximeter with error of pulse rate estimation below 5% for 82% of all epochs, and below 10% for 98% of the epochs. We conclude that iPPG allows non-invasive and non-contact estimation of pulse rate in non-human primates, which is useful for physiological studies and can be used toward welfare-assessment of non-human primates in research.

bioengineering↗

bioRxiv2018

Natural Gaze Data Driven Wheelchair

Natural eye movements during navigation have long been considered to reflect planning processes and link to users future action intention. We investigate here whether natural eye movements during joystick-based navigation of wheel-chairs follow identifiable patterns that are predictive of joystick actions. To place eye movements in context with driving intentions, we combine our eye tracking with a 3D depth camera system, which allows us to identify which eye movements have the floor as gaze target and distinguish them from other non-navigation related eye movements. We find consistent patterns of eye movements on the floor predictive of steering commands issued by the driver in all subjects. Based on this empirical data we developed two gaze decoders using supervised machine learning techniques and enabled each of these drivers to then steer the wheelchair by imagining they were using a joystick to trigger appropriate natural eye movements via motor imagery. We show that all subjects are able to navigate their wheelchair \"by eye\" learning it within a short time span of minutes. Our work shows that simple gaze-based decoding without need for artificial user interfaces suffices to restore mobility and increasing participation in daily life.

bioengineering↗

bioRxiv2018

Three-dimensional human axon tracts derived from cerebral organoids

Reestablishing cerebral connectivity is a critical part of restoring neuronal network integrity and brain function after trauma, stroke, and neurodegenerative diseases. Creating transplantable axon tracts in the laboratory is a novel strategy for overcoming the common barriers limiting axon regeneration in vivo, including growth-inhibiting factors and the limited outgrowth capacity of mature neurons in the brain. We describe the generation and phenotype of three-dimensional human axon tracts derived from cerebral organoid tissue. These centimeter-long constructs are encased in an agarose shell that permits physical manipulation and are composed of discrete cellular regions spanned by axon tracts and dendrites, mirroring the separation of grey and white matter in the brain. Features of cerebral cortex also are emulated, as evidenced by the presence of neurons with different cortical layer phenotypes. This engineered neural tissue has the translational potential to reconstruct brain circuits by physically replacing discrete cortical neuron populations as well as long-range axon tracts in the brain.\n\neTOC BlurbRestoring axonal connectivity after brain damage is crucial for improving neurological and cognitive function. Cullen, et al. have generated anatomically inspired, three-dimensional human axon tracts projecting from cerebral organoids in a transplantable format that may facilitate the reconstruction of large-scale brain circuits.\n\nHighlightsO_LIA neural tissue engineering approach is applied to human cerebral organoids.\nC_LIO_LIThree-dimensional axon tracts are generated in a transplantable format.\nC_LIO_LIThe growth characteristics of the engineered axons are examined.\nC_LIO_LIThe cellular phenotypes of the organoid tissue and axons are characterized.\nC_LI

bioengineering↗

bioRxiv2018

Vagus Nerve Stimulation Promotes Gastric Emptying by Increasing Pyloric Opening Measured with Magnetic Resonance Imaging

BackgroundVagus nerve stimulation (VNS) is an emerging electroceutical therapy for remedying gastric disorders that are poorly managed by pharmacological treatments and/or dietary changes. Such therapy seems promising since the vagovagal neurocircuitry controlling the enteric nervous system strongly influences gastric functions.\n\nMethodsHere, the modulatory effects of left cervical VNS on gastric emptying in rats was quantified using a 1) feeding protocol in which the animal voluntarily consumed a post-fast, gadolinium-labeled meal and 2) newly developed, robust, sensitive and non-invasive imaging strategy to measure antral motility, pyloric activity and gastric emptying based on contrast-enhanced magnetic resonance imaging (MRI) and computer-assisted image processing pipelines.\n\nKey ResultsVNS significantly accelerated gastric emptying (control vs. VNS: 24.9{+/-}3.5% vs. 40.7{+/-}3.9% of meal emptied per 4hrs, p<0.05). This effect resulted from a greater relaxation of the pyloric sphincter (control vs. VNS: 1.4{+/-}0.2 vs. 2.5{+/-}0.5 mm2 cross-sectional area of lumen, p<0.05), without notable changes in antral contraction amplitude (control vs. VNS: 30.6{+/-}3.0% vs. 32.5{+/-}3.0% occlusion), peristaltic velocity (control vs. VNS: 0.67{+/-}0.03 vs. 0.67{+/-}0.03 mm/s), or frequency (control vs. VNS: 6.3{+/-}0.1 vs. 6.4{+/-}0.2 cpm). The degree to which VNS relaxed the pylorus positively correlated with gastric emptying rate (r = 0.5465, p<0.01).\n\nConclusions & InferencesThe MRI protocol employed in this study is expected to enable advanced preclinical studies to understand stomach pathophysiology and its therapeutics. Results from this study suggest an electroceutical treatment approach for gastric emptying disorders using cervical VNS to control the degree of pyloric sphincter relaxation.\n\nKey PointsO_LIVagus nerve stimulation is emerging as a new electroceutical therapy for treating gastric disorders. However, its underlying mechanism(s) and therapeutic effect(s) remain incompletely understood.\nC_LIO_LIVagus nerve stimulation significantly accelerated gastric emptying by promoting the relaxation of the pyloric sphincter.\nC_LIO_LIMRI offers high spatial and temporal resolution to non-invasively characterize gastric motility and physiology in preclinical animal models.\nC_LI

bioengineering↗

bioRxiv2018

Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

bioengineering↗

bioRxiv2018

Genetically encoded ratiometric indicators for potassium ion

Potassium ion (K+) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K+ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Forster resonance energy transfer (FRET)-based genetically encoded K+ indicator, KIRIN1, constructed by inserting a bacterial cytosolic K+ binding protein (Kbp) between a fluorescent protein (FP) FRET pair, mCerulean3 and cp173Venus. Binding of K+ induces a conformational change in Kbp, resulting in an increase in FRET efficiency. KIRIN1 was able to detect K+ at physiologically relevant concentrations in vitro and is highly selective toward K+ over Na+. We further demonstrated that KIRIN1 allowed real-time imaging of pharmacologically induced depletion of cytosolic K+ in live cells, and KIRIN1 also enabled optical tracing of K+ efflux and reuptake in neurons upon glutamate stimulation in cultured primary neurons. These results demonstrate that KIRIN1 is a valuable tool to detect K+ in vitro and in live cells.

bioengineering↗

bioRxiv2018

EEG-based personal identification: comparison of different functional connectivity metrics

Growing interest is devoted to understanding how brain signals recorded from scalp electroencephalography (EEG) may represent unique fingerprints of individual neural activity. In this context, the present paper aims to investigate the impact of some of the most commonly used techniques to estimate functional connectivity on the ability to unveil personal distinctive patterns of inter-regional interactions. Different metrics, commonly used to estimate functional connectivity and derived centrality measures, were compared in terms of equal error rate. It is widely accepted that each metric carries specific information in respect to the underlying interactions network. Nevertheless, the reason why these metrics convey different subject specific information has not been investigated yet. Experimental results on two publicly available datasets suggest that different functional connectivity metrics define a peculiar subjective profile of connectivity and have different mechanisms to detect subject-specific patterns of inter-channel interactions. It is important to consider the effects that frequency content and spurious connectivity values may play in determining subject-specific characteristics.

bioengineering↗