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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2016

Fast and inexpensive detection of bacterial viability and drug effectiveness through metabolic monitoring

Conventional methods for the detection of bacterial infection such as DNA or immunoassays are either expensive, time consuming, or not definitive; thus may not provide all the information sought by the medical professionals. In particular, it is difficult to obtain information about viability or drug effectiveness, which are crucial to formulate a treatment. Bacterial culture test is the \"gold standard\" because it is inexpensive and does not require extensive sample preparation, and most importantly, provides all the necessary information sought by healthcare professionals, such as bacterial presence, viability and drug effectiveness. These conventional culture methods, however, have a long turnaround time: anywhere between 1 day to 4 weeks. Here, we solve this problem by monitoring the growth of bacteria in thousands of nanowells simultaneously to identify its presence in the sample and its viability, faster. The segmentation of a sample with low bacterial concentration into thousands of nanoliter wells digitizes the samples and increases the effective concentration in those wells that contain bacteria. We monitor the metabolism of aerobic bacteria by using an oxygen sensitive fluorophore, ruthenium tris (2,2-diprydl) dichloride hexahydrate (RTDP) that allows us to monitor the dissolved oxygen concentration in the nanowells. Using E.Coli K12 as a model pathogen, we demonstrate that the detection time of E.coli can be as fast as 35-60 minutes with sample concentrations varying from 104(62 minutes for detection), 106 (42 minutes) and 108 cells/mL (38 minutes). More importantly, we also demonstrate that reducing the well size can reduce the time of detection. Finally we show that drug effectiveness information can be obtained in this format by loading the wells with the drug and monitoring the metabolism of the bacteria. The method that we have developed is low cost, simple, requires minimal sample preparation and can potentially be used with a wide variety of samples in resource poor setting to detect bacterial infections such as Tuberculosis.

Bioengineering↗

bioRxiv2016

Membrane insertion of- and membrane potential sensing by semiconductor voltage nanosensors: feasibility demonstration

We develop membrane voltage nanosensors that are based on inorganic semiconductor nanoparticles. These voltage nanosensors are designed to self-insert into the cell membrane and optically record the membrane potential via the quantum confined Stark effect, with single-particle sensitivity. We present here the approach, design rules, and feasibility proves for this concept. With further improvements, semiconductor nanoparticles could potentially be used to study signals from many neurons in a large field-of-view over a long duration. Moreover, they could potentially report and resolve voltage signals on the nanoscale.

Bioengineering↗

bioRxiv2016

Investigation of the Antimicrobial Activity of Soy Peptides by Developing a High Throughput Drug Screening Assay

BackgroundAntimicrobial resistance is a great concern in the medical community, as well as food industry. Soy peptides were tested against bacterial biofilms for their antimicrobial activity. A high throughput drug screening assay was developed using microfluidic technology, RAMAN spectroscopy, and optical microscopy for rapid screening of antimicrobials and rapid identification of pathogens.\n\nMethodsSynthesized PGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides were tested against Pseudomonas aeruginosa and Listeria monocytogenes using a microdilution assay. Microfluidic technology in combination with Surface Enhanced RAMAN Spectroscopy (SERS) and optical microscopy was used for rapid screening of soy peptides, pathogen identification, and to visualize the impact of selected peptides.\n\nResultsThe PGTAVFK peptide did not significantly affect P. aeruginosa, although it had an inhibitory effect on L. monocytogenes above a concentration of 625 {micro}M. IKAFKEATKVDKVVVLWTA was effective against both P. aeruginosa and L. monocytogenes above a concentration of 37.2 {micro}M. High throughput drug screening assays were able to reduce the screening and bacterial detection time to 4 h. SERS spectra was used to distinguish the two bacterial species.\n\nConclusionsPGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides showed antimicrobial activity against P. aeruginosa and L. monocytogenes. Development of high throughput assays could streamline the drug screening and bacterial detection process. General significance: The results of this study show that the antimicrobial properties, biocompatibility, and biodegradability of soy peptides could possibly make them an alternative to the ineffective antimicrobials and antibiotics currently used in the food and medical fields. High throughput drug screening assays could help hasten pre-clinical trials in the medical field.

Bioengineering↗

bioRxiv2016

Dual immunosensor based on methylene blue-electroadsorbed graphene oxide for rapid detection of influenza virus antigen

Rapid detection of influenza viral infections in the poultry farm is advantageous in several facts such as environmental/personal safety, food-security and socio-economy. Herein, we report the development of an electrochemical based dual sensor platform composed of methylene blue-electroadsorbed graphene oxide nanostructures modified with monoclonal antibodies, H5N1 and H1N1. Bio-functional layers comprised of chitosan and protein-A molecules were implemented at the interface of sensor element and antibodies, which synergistically enriched the bio-activity of immobilized antibodies for the immune complex formation. The differential pulse voltammetric signals resulted from the developed immunosensor platform exhibited a good correlation (R2= 0.9978 for H1N1 and R2=0.9997 for H5N1) for the wide range of target concentrations (25 to 500 pM). Chronoamperometric study also revealed the amplified current sensitivity of the immunoelectrodes even at the picomolar level. The proposed immunosensor design not only provide rapid analytical response time (<1 min) but also provide simplicity in fabrication and instrumentation which paves an attractive platform for on-farm monitoring of viral infections.

Bioengineering↗

bioRxiv2016

Single cell chemotactic responses of Helicobacter pylori to urea in a microfluidic chip

Helicobacter pylori is a spiral-shaped bacterium that grows in the human digestive tract; it infects ~50% of the global population. H. pylori induce inflammation, gastroenteritis, and ulcers, which is associated with significant morbidity and may be linked to stomach cancer in certain individuals. Motility is an essential virulence factor for H. pylori, allowing it to migrate toward and invade the epithelial lining of the stomach to shelter it from the harsh environment of the stomach. H. pylori senses pH gradients and use polar flagella to move towards the epithelium where the pH approaches neutrality. However, its chemotaxis behaviors are incompletely understood. Previous in vitro tests examining the response of H. pylori to chemical gradients have been subjected to substantial limitations. To more accurately mimic/modulate the cellular microenvironment, a nanoporous microfluidic device was used to monitor the real time chemotactic activity of single cell of H. pylori in response to urea. The results showed that microfluidic method is a promising alternative for precisely studying chemotactic behavior of bacteria.

Bioengineering↗

bioRxiv2016

InfoDengue: a nowcasting system for the surveillance of dengue fever transmission

This study describes the development of an integrated dengue alert system (InfoDengue), operating initially in the city of Rio de Janeiro, Brazil. It is a project developed as a partnership between academia and the municipal health secretariat. At the beginning of each epidemiological week, the system captures climate time series, dengue case reporting and activity on a social network. After data pre-processing, including a probabilistic correction of case notification delay, and calculation of dengue's effective reproductive number, indicators of dengue transmission are coded into four dengue situation levels, for each of the city's ten health districts. A risk map is generated to inform the public about the week's level of attention and the evolution of the disease incidence and suggest actions. A report is also sent automatically to the municipality's situation room, containing a detailed presentation of the data and alert levels by health district. The preliminary analysis of InfoDengue in Rio de Janeiro, using historical series from 2011 to 2014 and prospective data from January to December 2015, indicates good degree of confidence and accuracy. The successful experience in the city of Rio de Janeiro is a motivating argument for the expansion of InfoDengue to other cities. After a year in production, InfoDengue has become a unique source of carefully curated data for epidemiological studies, combining epidemological and environmental variables in unprecedented spatial and temporal resolutions.\n\nEthical committee approval: 26910214.7.0000.5240

Bioengineering↗

bioRxiv2016

Long Range Force Transmission in Fibrous Matrices Enabled by Tension-Driven Alignment of Fibers

Cells can sense and respond to mechanical signals over relatively long distances across fibrous extracellular matrices. Recently proposed models suggest that long-range force transmission can be attributed to the nonlinear elasticity or fibrous nature of collagen matrices, yet the mechanism whereby fibers align remains unknown. Moreover, cell shape and anisotropy of cellular contraction are not considered in existing models, although recent experiments have shown that they play crucial roles. Here, we explore all of the key factors that influence long-range force transmission in cell-populated collagen matrices: alignment of collagen fibers, responses to applied force, strain stiffening properties of the aligned fibers, aspect ratios of the cells, and the polarization of cellular contraction. A constitutive law accounting for mechanically-driven collagen fiber reorientation is proposed. We systematically investigate the range of collagen fiber alignment using both finite element simulations and analytical calculations. Our results show that tension-driven collagen fiber alignment plays a crucial role in force transmission. Small critical stretch for fiber alignment, large fiber stiffness and fiber strain-hardening behavior enable long-range interaction. Furthermore, the range of collagen fiber alignment for elliptical cells with polarized contraction is much larger than that for spherical cells with diagonal contraction. A phase diagram showing the range of force transmission as a function of cell shape and polarization and matrix properties is presented. Our results are in good agreement with recent experiments, and highlight the factors that influence long-range force transmission, in particular tension-driven alignment of fibers. Our work has important relevance to biological processes including development, cancer metastasis and wound healing, suggesting conditions whereby cells communicate over long distances.

Bioengineering↗

bioRxiv2016

Ex-STORM: Expansion Single Molecule Nanoscopy

We present critical improvements to recently invented Expansion Microscopy (ExM), which resolve the incompatibility between ExM and single molecule super-resolution microscopy STORM. Specifically, the improved ExM circumvents the massive, 50-100%, bleaching of fluorophores during sample preparations, and preserve the efficiency of enzymatic oxygen scavenging systems in expanded samples. These improvements open up new avenues for Ex-STORM - expanding a sample mechanically, and then visualizing the sample with STORM.

Bioengineering↗

bioRxiv2016

Robust cell tracking in epithelial tissues through identification of maximum common subgraphs

Tracking of cells in live-imaging microscopy videos of epithelial sheets is a powerful tool for investigating fundamental processes in embryonic development. Characterising cell growth, proliferation, intercalation and apoptosis in epithelia helps us to understand how morphogenetic processes such as tissue invagination and extension are locally regulated and controlled. Accurate cell tracking requires correctly resolving cells entering or leaving the field of view between frames, cell neighbour exchanges, cell removals and cell divisions. However, current tracking methods for epithelial sheets are not robust to large morphogenetic deformations and require significant manual interventions. Here, we present a novel algorithm for epithelial cell tracking, exploiting the graph-theoretic concept of a maximum common subgraph to track cells between frames of a video. Our algorithm does not require the adjustment of tissue-specific parameters, and scales in sub-quadratic time with tissue size. It does not rely on precise positional information, permitting large cell movements between frames and enabling tracking in datasets acquired at low temporal resolution due to experimental constraints such as photoxicity. To demonstrate the method, we perform tracking on the Drosophila embryonic epidermis and compare cell-cell rearrangements to previous studies in other tissues. Our implementation is open source and generally applicable to epithelial tissues.

Bioengineering↗

bioRxiv2016

Scalable Production of Human Erythrocytes from Induced Pluripotent Stem Cells

In vitro production of erythrocytes in physiologic numbers from human induced pluripotent stem cells (hiPSCs) holds great promise for improved transfusion medicine and novel cell therapies. We report here, for the first time, a strategy for scalable and xeno-free differentiation of hematopoietic stem/progenitor cells from hiPSCs and subsequent erythrocytes specification, by using stepwise cell culture conditions and by integrating spinner flasks and rocker. This system supported robust and reproducible definitive hematopoietic differentiation of multiple hiPSC lines. We demonstrated an ultra-high yield of up to 4x109 CD235a+ erythrocytes at >98% purity when using a 1-litre spinner flask for suspension culture. Erythrocytes generated from our system can reach a mature stage with red blood cell (RBC) characteristics of enucleation, {beta}-globin protein expression and oxygen-binding ability. The entire process is xeno-free and clinically compliant, allowing future mass production of hiPSC-derived RBCs for transfusion medicine purposes.

Bioengineering↗

bioRxiv2016

Recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs

Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate that homology-dependent repair (HDR) is robust in C. elegans using linear templates with short homologies (~35 bases). Templates with homology to only one side of a double-strand break initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments precisely to DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.

Bioengineering↗

bioRxiv2016

An open-hardware platform for optogenetics and photobiology

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

Bioengineering↗

bioRxiv2016

Intraoperative biomechanics of pedicle screw loosening in the lumbar spine

AbstractPedicle screw loosening has been implicated in recurrent back pain after lumbar spinal fusion, but the degree of loosening has not been systematically quantified in patients. Instrumentation removal is an option for patients with successful arthrodesis, but remains controversial. Here, we quantified pedicle screw loosening by measuring screw insertion and/or removal torque at high statistical power ({beta} = 0.98) in N = 108 patients who experienced pain recurrence despite successful fusion after posterior instrumented lumbar fusion with anterior lumbar interbody fusion (L2-S1). Between implantation and removal, pedicle screw torque was reduced by 58%, indicating significant loosening over time. Loosening was greater in screws with evoked EMG threshold under 11 mA, indicative of screw misplacement. A theoretical stress analysis revealed increased local stresses at the screw interface in pedicles with decreased difference in pedicle thickness and screw diameter. Loosening was greatest in vertebrae at the extremities of the fused segments, but was significantly lower in segments with one level of fusion than in those with two or more.\n\nClinical significanceThese data indicate that pedicle screws can loosen significantly in patients with recurrent back pain and warrant further research into methods to reduce the incidence of screw loosening and to understand the risks and potential benefits of instrumentation removal.

Bioengineering↗

bioRxiv2016

3D-Printed Autonomous Capillaric Circuits

Capillaric circuits (CCs) are advanced capillary microfluidic devices that move liquids in complex pre-programmed sequences without external pumps and valves-relying instead on microfluidic control elements powered by capillary forces. CCs were thought to require high-precision micro-scale features manufactured by photolithography in a cleanroom, which is slow and expensive. Here we present rapidly and inexpensively 3D-printed autonomous CCs. Molds for CCs were fabricated with a benchtop 3D-printer, Poly(dimethylsiloxane) replicas were made, and fluidic functionality was verified with aqueous solutions. We established design rules for 3D-printed CCs by a combination of modelling and experimentation. The functionality and reliability of 3D-printed trigger valves-an essential fluidic element that stops one liquid until flow is triggered by a second liquid-was tested for different geometries and different solutions. Trigger valves with geometries up to 80-fold larger than cleanroom-fabricated ones were found to function reliably. We designed 3D-printed retention burst valves that encode sequential liquid drainage and delivery using capillary pressure differences encoded by varying valve height and width. Using an electrical circuit analogue of the CC, we established circuit design rules for ensuring strictly sequential liquid delivery. We realized a 3D-printed CC with reservoir volumes 60 times larger than cleanroom-fabricated circuits and autonomously delivered eight liquids in a pre-determined sequence in < 7 min, exceeding the number of sequentially-encoded, self-regulated fluidic delivery events apreviously reported. Taken together, our results demonstrate that 3D-printing enables rapid prototyping of reliable CCs with improved functionality and potential applications in diagnostics, research and education.

Bioengineering↗

bioRxiv2016

Differential stimulation of the retina with glutamate toward a neurotransmitter-based retinal prosthesis

Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no retinal stimulation study exploiting the inner retinal pathways exists. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses.

Bioengineering↗

bioRxiv2016

Active delivery to the brain by chemotaxis

In recent years, scientists have created artificial microscopic and nanoscopic self-propelling particles, often referred to as nano- or micro-swimmers, capable of mimicking biological locomotion and taxis. This active diffusion enables the engineering of complex operations that so far have not been possible at the micro- and nanoscale. One of the most promising task is the ability to engineer nanocarriers that can autonomously navigate within tissues and organs, accessing nearly every site of the human body guided by endogenous chemical gradients. Here we report a fully synthetic, organic, nanoscopic system that exhibits attractive chemotaxis driven by enzymatic conversion of glucose. We achieve this by encapsulating glucose oxidase -- alone or in combination with catalase -- into nanoscopic and biocompatible asymmetric polymer vesicles (known as polymersomes). We show that these vesicles self-propel in response to an external gradient of glucose by inducing a slip velocity on their surface, which makes them move in an extremely sensitive way towards higher concentration regions. We finally demonstrate that the chemotactic behaviour of these nanoswimmers enables a four-fold increase in penetration to the brain compared to non-chemotactic systems.

Bioengineering↗

bioRxiv2016

Live Cell Partial Wave Spectroscopic microscopy: Label-free Imaging of the Native, Living Cellular Nanoarchitecture

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10nm) to the chromosomal (>200nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using Partial Wave Spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20-200nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nano-architecture. Therefore, we developed a live cell PWS technique which allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real-time. In this work, we employ live cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live cell DNA binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Since biological function is tightly paired with structure, live cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.\n\nSignificance StatementChromatin is one of the most critical structures within the cell because it houses most genetic information. Its structure is well understood at the nucleosomal (<20nm) and chromosomal (>200nm) levels, however, due to the lack of quantitative imaging modalities to study this organization, little is known about the higher-order structure between these length scales in live cells. We present a label-free technique, live cell Partial Wave Spectroscopic (PWS) microscopy with sensitivity to structures between 20-200nm that can quantify the nano-architecture in live cells. With this technique, we can detect DNA fragmentation and expand on the link between metabolic function and higher-order chromatin structure. Live cell PWS allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in live cells.

Bioengineering↗

bioRxiv2016

Genome Editing With Targeted Deaminases

Precise genetic modifications are essential for biomedical research and gene therapy. Yet, traditional homology-directed genome editing is limited by the requirements for DNA cleavage, donor DNA template and the endogenous DNA break-repair machinery. Here we present programmable cytidine deaminases that enable site-specific cytidine to thymidine (C-to-T) genomic edits without the need for DNA cleavage. Our targeted deaminases are efficient and specific in Escherichia coli, converting a genomic C-to-T with 13% efficiency and 95% accuracy. Edited cells do not harbor unintended genomic abnormalities. These novel enzymes also function in human cells, leading to a site-specific C-to-T transition in 2.5% of cells with reduced toxicity compared with zinc-finger nucleases. Targeted deaminases therefore represent a platform for safer and effective genome editing in prokaryotes and eukaryotes, especially in systems where DSBs are toxic, such as human stem cells and repetitive elements targeting.

Bioengineering↗