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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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Comparison of the 3D-IR-BTFE method and the conventional method in the head MRI contrast

MRI using gadolinium contrast media is useful in diagnosis; however, nephrogenic systemic fibrosis is a serious side effect of gadolinium exposure. Moreover, it turns out that gadolinium deposits in the brain. This has escalated the necessity for a suitable method to use gadolinium contrast media. I developed a new imaging method that had excellent contrast. This study examined the usefulness of that new imaging method and found the method is highly effective.

bioengineering

A Noninvasive Molecular Clock for Fetal Development Predicts Gestational Age and Preterm Delivery

We performed a high time-resolution, longitudinal study of normal pregnancy development by measuring cell-free RNA (cfRNA) in blood from women during each week of pregnancy. Analysis of tissue-specific transcripts in these samples enabled us to follow fetal and placental development with high resolution and sensitivity, and also to detect gene-specific responses of the maternal immune system to pregnancy. We established a \"clock\" for normal pregnancy development and enabled a direct molecular approach to determine expected delivery dates with comparable accuracy to ultrasound, creating the basis for a portable, inexpensive fetal dating method. We also identified a related gene set that accurately discriminated women at risk for spontaneous preterm delivery up to two months in advance of labor, forming the basis of a potential screening test for risk of preterm delivery.

bioengineering

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using near-infrared dyes

1We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We first show that fast and precise in vitro quantification of NIR fluorophores lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation. We apply this approach to the study of binding equilibria by Forster resonant energy transfer (FRET), using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend our demonstration to dynamic imaging of the pharmacokinetics of transferrin binding to the transferrin receptor in live mice, elucidating the kinetic of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from non-specific binding. Our method, implemented in a freely-available software package, has all the advantages of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging, and could be extended to applications as diverse as image guided-surgery or optical tomography.

bioengineering

The Improvement Of TSP Fertilizer Production & Quality

Results obtained showed that some essential manufacturing factors needs to be respected throughout the production loop. These factors will help obtaining a slurry exits with the desired chemical characteristics of P2O5SE (33%), Al (10%), P2O5SE + Citrate (34.5%), H2O (22%), P2O5total (39%)). It is necessary to keep a temperature within 100 {+/-} 5 {degrees} C., with a vapor pressure of 6 to 7 bars, an average density of H3PO4 (1470 g/l) and the solid rate in the feed acid less than 2%. After the reaction phase, the slurry is conveyed to the granulator where it is mixed with the recycled product at a recycling rate (RT) close to 3 and a K ratio between 0.1 and 0.2. This parameter is very important to be monitored to ensure a Good penetration of heat into the next phase in a relatively short time. During the drying process, the product humidity at the outlet must be in the range of 3.5% to 5% to allow the reaction to continue during the developing period. At the exit of the drying tube, the product is in very variable grains diameter (D), the objective of the following step is to extract the grain size range between 2 and 4 mm. To do so we changed the mesh size of the lower webs to 3/50 and 3.2 / 30 mm, which reduces the quantity of particle size less than 2%. Thus, the particles of diameter more than 3.15 mm must be continuously controlled to reach 64%. This portion will ensure conformity of the product for D50 and for the two intervals [2-3.15 mm] and [3.15-4 mm]. In the Storage Hall the reaction continues, the unconverted phosphate will be attacked by the unreacted H3PO4, and according to a monitoring of the evolution of the chemical composition of the final product (TSP). The required ripening time is limited to 21 days. During this study, we were able to identify and modify a number of parameters in different production phases to improve the quality of TSP according to AFCOME, knowing that each phase of the process is a client of the previous phase and supplier of the next one.

bioengineering

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast

BackgroundGamma valerolactone (GVL) is a promising technology for degradation of biomass for biofuel production; however, GVL has adverse toxicity effects on fermentative microbes. Using a combination of chemical genomics and chemical proteomics we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast.\n\nResultsChemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate phenolic acids to vinyl derivatives, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain.\n\nConclusionsWe found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol content in cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates

bioengineering

Squid Ring Teeth Coated Mesh Improves Abdominal Wall Repair

BackgroundHernia repair is a common surgical procedure with mesh often used. Current mesh materials have a high incidence of repair failures, due to poor tissue integration, and complications such as seroma and pain. Polypropylene (PP) mesh is the standard material in hernia repair secondary to its material durability; however, failures still approach 15%. In this first time animal study, we hypothesized that squid ring teeth (SRT), a biologically-derived high strength protein, coated polypropylene (SRT-PP) mesh, would offer enhanced tissue integration and strength compared to standard PP mesh, while proving biocompatibility for in vivo use.\n\nMaterials and methodsPolypropylene mesh was coated with SRT. Mechanical properties and cell proliferation studies of the composite mesh were performed in vitro. Rats underwent inlay mesh implantation in an anterior abdominal wall defect model. Repair was assessed clinically and radiographically, with integration evaluated by histology and mechanical testing.\n\nResultsCell proliferation was enhanced on SRT-PP composite mesh. This was corroborated by abdominal wall histology, dramatically diminished cranio-caudal mesh contraction, improved strength testing, and higher tissue failure strain following in vivo implantation. There was no increase in complications with SRT, with regard to seroma or visceral adhesion. No foreign body reactions were noted on liver histology.\n\nConclusionSRT-PP mesh showed better tissue integration than PP mesh. SRT is a high strength protein that is applied as a coating to augment mesh-tissue integration leading to improvements in abdominal wall stability with potential to reduce re-intervention for failures.

bioengineering

Large protein complex production using the SmartBac System - Strategies and Applications

Recent revolution of cryo-electron microscopy has opened a new door to solve high-resolution structures of macromolecule complexes without crystallization while how to efficiently obtain homogenous macromolecule complex sample is therefore becoming a bottleneck. Here we report SmartBac, an easy and versatile system for constructing large-sized transfer plasmids used to generate recombinant baculoviruses that express large multiprotein complexes in insect cells. The SmartBac system integrates the univector plasmid-fusion system, Gibson assembly method and polyprotein strategy to construct the final transfer plasmids. The fluorescent proteins are designed to be co-expressed with recombinant proteins to monitor transfection and expression efficiencies. A scheme of screening an optimal tagged subunit for effective purification is provided. Six large multiprotein complexes including the human exocyst complex and dynactin complex were successfully expressed, suggesting a great potential of SmartBac for its wide application in the future structural biology study.

bioengineering

Complementary, High-throughput Methods for Creating Multi-dimensional, PEG-based Biomaterials

Tunable biomaterials that mimic selected features of the extracellular matrix (ECM), such as its stiffness, protein composition, and dimensionality, are increasingly popular for studying how cells sense and respond to ECM cues. In the field, there exists a significant trade-off for how complex and how well these biomaterials represent the in vivo microenvironment, versus how easy they are to make and how adaptable they are to automated fabrication techniques. To address this need to integrate more complex biomaterials design with high-throughput screening approaches, we present several methods to fabricate synthetic biomaterials in 96-well plates and demonstrate that they can be adapted to semi-automated liquid handling robotics. These platforms include 1) glass bottom plates with covalently attached ECM proteins, and 2) hydrogels with tunable stiffness and protein composition with either cells seeded on the surface, or 3) laden within the three-dimensional hydrogel matrix. This study includes proof-of-concept results demonstrating control over breast cancer cell line phenotypes via these ECM cues in a semi-automated fashion. We foresee the use of these methods as a mechanism to bridge the gap between high-throughput cell-matrix screening and engineered ECM-mimicking biomaterials.

bioengineering

Wide-field dynamic monitoring of immune cell trafficking in murine models of glioblastoma

Leukocyte populations, especially tumor-associated macrophages (TAMs), are capable of mediating both anti- and pro-tumor processes and play significant roles in the tumor microenvironment. Moreover, TAMs have been shown to exert substantial influence on the efficacy of various cancer immunotherapy treatment strategies. Laboratory investigation into the behavior of TAMs has been limited by a lack of methods capable of resolving the in vivo distribution and dynamics of this cell population across wide fields of view. Recent studies have employed magnetic resonance imaging and intravital microscopy in conjunction with nanoparticle labeling methods to detect TAMs and observe their responses to therapeutic agents. Here we describe a novel method to enable high-resolution, wide-field, longitudinal imaging of leukocytes based on contrast-enhanced Speckle-Modulating Optical Coherence Tomography (SM-OCT), which substantially reduces imaging noise. We were able to specifically label TAMs and activated microglia in vivo with large gold nanorod contrast agents (LGNRs) in an orthotopic murine glioblastoma model. After labeling, we demonstrated near real-time tracking of leukocyte migration and distribution within the tumors. The intrinsic resolution, imaging depth, and sensitivity of this method may facilitate detailed studies of the fundamental behaviors of TAMs in vivo, including their intratumoral distribution heterogeneity and the roles they play in modulating cancer proliferation. In future studies, the method described herein may also provide the necessary means to characterize TAM responses to immunotherapeutic regimens in a range of solid tumors.

bioengineering

Closed-loop feedback control for microfluidic systems through automated capacitive fluid height sensing

Precise fluid height sensing in open-channel microfluidics has long been a desirable feature for a wide range of applications. However, performing accurate measurements of the fluid level in small-scale reservoirs (<1mL) has proven to be an elusive goal, especially if direct fluid-sensor contact needs to be avoided. In particular, gravity-driven systems used in several microfluidic applications to establish pressure gradients and impose flow remain open-loop and largely unmonitored due to these sensing limitations. Here we present an optimized self-shielded coplanar capacitive sensor design and automated control system to provide submillimeter fluid-height resolution (~250 m) and control of small-scale open reservoirs without the need for direct fluid contact. Results from testing and validation of our optimized sensor and system also suggest that accurate fluid height information can be used to robustly characterize, calibrate and dynamically control a range of microfluidic systems with complex pumping mechanisms, even in cell culture conditions. Capacitive sensing technology provides a scalable and cost-effective way to enable continuous monitoring and closed-loop feedback control of fluid volumes in small-scale gravity-dominated wells in a variety of microfluidic applications.

bioengineering

Multi-scale periodicities in the functional brain networks of patients with epilepsy and their effect on seizure detection

The task of automated epileptic seizure detection and prediction by using non-invasive measurements such as scalp EEG signals or invasive, intracranial recordings, has been at the heart of epilepsy studies for at least three decades. By far, the most common approach for tackling this problem is to examine short-length recordings around the occurrence of a seizure - normally ranging between several seconds and up to a few minutes before and after the epileptic event - and identify any significant changes that occur before or during the event. An inherent assumption in these studies is the presence of a relatively constant EEG activity in the interictal period, which is presumably interrupted by the occurrence of a seizure. Here, we examine this assumption by using long-duration scalp EEG data (ranging between 21 and 94 hours) in patients with epilepsy, based on which we construct functional brain networks. Our results suggest that not only these networks vary over time, but they do so in a periodic fashion, exhibiting multiple peaks at periods ranging between around one and 24 hours. The effects of seizure onset on the functional brain network properties were found to be considerably smaller in magnitude compared to the changes due to the inherent periodic cycles of these networks. Importantly, the properties of the identified network periodic components (instantaneous phase, particularly that of short-term periodicities around 3 and 5 h) were found to be strongly correlated to seizure onset. These correlations were found to be largely absent between EEG signal periodicities and seizure onset, suggesting that higher specificity may be achieved by using network-based metrics. In turn, this suggests that to achieve more robust seizure detection and/or prediction, the evolution of the underlying longer term functional brain network periodic variations should be taken into account.\n\nHighlights- We have examined the long-term characteristics of EEG functional brain networks and their correlations to seizure onset\n\n- We show periodicities over multiple time scales in network summative properties (degree, efficiency, clustering coefficient)\n\n- We also show that, in addition to average network properties, similar periodicities exist in network topology using a novel measure based on the graph edit distance, suggesting that specific connectivity patterns recur over time\n\n- These periodic patterns were preserved when we corrected for the effects of volume conduction and were found to be of much larger magnitude compared to seizure-induced modulations\n\n- For the first time to our knowledge, we demonstrate that seizure onset occurs preferentially at specific phases of network periodic components that were consistently observed across subjects, particularly for shorter periodicities (around 3 and 5 hours)\n\n- These correlations between the phase of network periodic components and seizure onset were nearly absent when examining univariate properties (EEG signal power), suggesting that network-based measures are more tightly coupled with seizure onset compared to EEG signal-based measures\n\n- Our findings suggest that seizure detection and prediction algorithms may benefit significantly by taking into account longer-term variations in brain network properties\n\n- As we show strong evidence that shorter network-based periodicities (3-5 hours) are tightly coupled with seizure onset, our results pave the way for further investigation into the pathophysiology of seizure generation mechanisms beyond the well-known effects of circadian rhythms

bioengineering

A method to keep the excitation light sheet in focus in selective plane illumination microscopy

Keeping the excitation light sheet in focus is critical in selective plane illumination microscopy (SPIM) to ensure its 3D imaging ability. Unfortunately, an effective method that can be used in SPIM on general biological specimens to find the axial position of the excitation light sheet and keep it in focus is barely available. Here, we present a method to solve the problem. We investigate its mechanism and demonstrate its performance on a lattice light sheet microscope.

bioengineering

Single-molecule fluorescent amplification of RNA using clampFISH probes

Non-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present click-amplifying FISH (clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and extremely high-gain (>400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that the modular design of clampFISH probes enables multiplexing, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH works in tissue samples.

bioengineering

Computational Design of Asymmetric Three-dimensional RNA Structures and Machines.

The emerging field of RNA nanotechnology seeks to create nanoscale 3D machines by repurposing natural RNA modules, but successes have been limited to symmetric assemblies of single repeating motifs. We present RNAMake, a suite that automates design of RNA molecules with complex 3D folds. We first challenged RNAMake with the paradigmatic problem of aligning a tetraloop and sequence-distal receptor, previously only solved via symmetry. Single-nucleotide-resolution chemical mapping, native gel electrophoresis, and solution x-ray scattering confirmed that 11 of the 16 miniTTR designs successfully achieved clothespin-like folds. A 2.55 [A] diffraction-resolution crystal structure of one design verified formation of the target asymmetric nanostructure, with large sections achieving near-atomic accuracy (< 2.0 [A]). Finally, RNAMake designed asymmetric segments to tether the 16S and 23S rRNAs together into a synthetic singlestranded ribosome that remains uncleaved by ribonucleases and supports life in Escherichia coli, a challenge previously requiring several rounds of trial-and-error.

bioengineering

Photocatalytic disinfection of surfaces with copper doped TiO2 nanotube coatings illuminated by ceiling mounted fluorescent lights

High economic burden is associated with foodborne illnesses. Different disinfection methods are therefore employed in food processing industry; such as use of ultraviolet light or usage of surfaces with copper-containing alloys. However, all the disinfection methods currently in use have some shortcomings. Here we show that copper doped TiO2 nanotubes deposited on existing surfaces and illuminated with ceiling mounted fluorescent lights or additional low power light emitting diodes can be employed for an economical and permanent disinfection of surfaces.\n\nWe deposited the nanotubes on various surfaces: polyethylene terephatlate, polystyrene, and aluminum oxide, where they could withstand repeated washings with neutral, alkaline or acidic medium. Here we show that the polymer surfaces coated with the nanotubes and innoculated with 107 bacteria, illuminated with ceiling mounted fluorescent lights retard the growth of Listeria Innocua by up to 99% in seven hours of exposure to the fluorescent lights, compared to a control surface. The disinfection properties of the surfaces depend mainly on the temperature difference of the surface and the dew point, where for maximum effectiveness of the photocatalytic effect the difference should be at least 2.5 degrees celsius.\n\nUsage of one dimensional nanomaterials, such as TiO2 nanotubes, offers a promising low cost alternative to current disinfection methods, since illumination of surfaces with common fluorescent lights is sufficient to photo-excite the nanotubes, which sequentially produce microbicidal hydroxyl radicals. Future use of such surfaces with antibacterial nano-coating and resulting sterilizing effect holds promise for such materials to be used in different environments or in better control of critical control points in food production as well as an improved biosecurity during the food manufacturing process.

bioengineering

In Vitro Study Of Dentin Hypersensitivity Treated By Whitlockite Glass-Ceramics

The aim of this study is to evaluate the potential of a bioactive glass based on the 3CaO.P2O5-SiO2MgO-system and its glassceramics containing whitlockite on the remineralization of dentin as a possible treatment to dentin hypersensitivity. For that, 40 third molar human teeth were artificially demineralized and randomly distributed in 4 groups (n = 10): G1 - Negative Control (no treatment), G2 - Positive Control (treated by Bioglass(R) 45S5), G3 - BG (treated by bioactive glass based on 3CaO.P2O5-SiO2-MgOsystem), and G4 - BGC (treated by bioactive whitlockite glass-ceramics). After treatment, the samples were emerged in artificial saliva and stored for 7 days in a controlled temperature of 37{o}C. After that, scanning electron microscopy (SEM) and atomic force microscopy (AFM) was used to evaluate samples morphology. The analysis confirmed the formation of hydroxyapatite on the surface of all the biomaterials studied, that in the dentine specimens treated by bioactive glass and whitlockite glas-ceramic most of the dentinal tubules were completely occluded.

bioengineering

Label-free non-invasive quantitative measurement of lipid contents in individual microalgal cells using refractive index tomography

Microalgae are promising candidates for biofuel production due to their high lipid content. To facilitate utilization of the microalgae for biofuel, rapid quantification of the lipid contents in microalgae is necessary. However, conventional methods based on the chemical extraction of lipids require a time-consuming destructive extraction process. Here, we demonstrate label-free, non-invasive, rapid quantification of the lipid contents in individual micro-algal cells measuring the three-dimensional refractive index tomograms. We measure three-dimensional refractive index distributions within Nannochloropsis oculata cells and find that lipid droplets are identifiable in tomograms by their high refractive index. In addition, we alter N. oculata under nitrogen deficiency by measuring the volume, lipid weight, and dry cell weight of individual cells. Characterization of individual cells allows correlative analysis between the lipid content and size of individual cells.

bioengineering

Real-time halo correction in phase contrast imaging

As a label-free, nondestructive method, phase contrast is by far the most popular microscopy technique for routine inspection of cell cultures. Yet, features of interest such as extensions near cell bodies are often obscured by a glow, which came to be known as the halo. Advances in modeling image formation have shown that this artifact is due to the limited spatial coherence of the illumination. Yet, the same incoherent illumination is responsible for superior sensitivity to fine details in the phase contrast geometry. Thus, there exists a trade-off between high-detail (incoherent) and low-detail (coherent) imaging systems. In this work, we propose a method to break this dichotomy, by carefully mixing corrected low-frequency and high-frequency data in a way that eliminates the edge effect. Specifically, our technique is able to remove halo artifacts at video rates, requiring no manual interaction or a priori point spread function measurements. To validate our approach, we imaged standard spherical beads, sperm cells, tissue slices, and red blood cells. We demonstrate the real-time operation with a time evolution study of adherent neuron cultures whose neurites are revealed by our halo correction. We show that with our novel technique, we can quantify cell growth in large populations, without the need for thresholds and calibration.

bioengineering