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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 127 records · Page 7Linked to original sources
bioRxiv2017

Production of Multilayer Cell Mass from Olfactory Stem Cell and Its Utilization Potential in Nervous Tissue Regeneration

BackgroundExperimental studies performed with human olfactory nerve stem cells haveshown that these cells can ameliorate nerve cell regeneration. Developing a method of repairing nerve damage solely using stem cells without the need of any supporting material is important.\n\nMethodsA multilayer cell mass was obtained from olfactory tissue-derived mesenchymal stem cells with high viability and proliferation capability using a protocol devoid of scaffolds or any other artificial supporting material. First, human olfactory tissue-derived mesenchymal stem cells were isolated, cultured, and characterized. Next, consecutive passages were conducted to obtain multilayer cell growth. The resulting cell mass could be suitable for tissue engineering models as well as nerve cell or tissue regeneration studies in the future.\n\nResultsViability and adhesive properties of the resulting cell mass were examined and found to be suitable for use in nerve tissue regeneration.\n\nConclusionIt is suggested that an in vitro-produced olfactory stem cell mass can be applied to a very small damaged region and could have a high potential for microenvironment formation.

bioengineering↗
bioRxiv2017

Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform

Cell-free transcription-translation platforms have been widely utilized to express soluble proteins in basic synthetic biological circuit prototyping. From a synthetic biology point of view, it is critical to express membrane proteins in cell-free transcription-translation systems, and use them directly in biocircuits, considering the fact that histidine kinases, G-protein coupled receptors (GPCRs) and other important biosensors are all membrane proteins. Previous studies have expressed membrane proteins in cell-free systems with the help of detergents, liposomes or nanodiscs, but have not demonstrated the ability to prototype circuit behavior for the purpose of testing more complex circuit functions involving membrane-bound proteins. Built on previous efforts, in this work we demonstrated that we could co-translationally express solubilized and active membrane proteins in our cell-free TX-TL platform with membrane-like materials. We first tested the expression of several constructs with {beta}1 and {beta}2 adrenergic receptors in TX-TL and observed significant insoluble membrane protein production. The addition of nanodiscs to the cell free expression system enabled solubilization of membrane proteins. Nanodisc is lipoprotein-based membrane-like material. The activity of {beta}2 adrenergic receptor was tested with both fluorescence and Surface Plasmon Resonance (SPR) binding assays by monitoring the specific binding response of small-molecule binders, carazolol and norepinephrine. Our results suggest that it is promising to use cell-free expression systems to prototype synthetic biocircuits involving single chain membrane proteins without extra procedures. This data made us one step closer to testing complex membrane protein circuits in cell-free environment.

bioengineering↗
bioRxiv2017

Advanced Protection Against Marine Biofouling Using Solar Light

We undertook prolonged testing of a new xerogel coating (AquaSun) to protect from marine biofouling a surface probe immersed in the seawater of Abu Dhabi. Electron microscopy, Raman mapping, and photoluminescence experiments coupled to extensive analyses of marine water before and after 122 days of testing showed excellent action against biofouling when irradiating the probe with visible light. Considering the high chemical and mechanical stability and the low cost of the sol-gel coating, the technology has significant potential en route to replacing conventional antifouling and foul release coatings with a single product of broad applicability.

bioengineering↗
bioRxiv2017

On the relationship between indenation hardness and modulus, and the damage resistance of biological materials.

The remarkable mechanical performance of biological materials is based on intricate structure-function relationships. Nanoindentation has become the primary tool for characterising biological materials, as it allows to relate structural changes to variations in mechanical properties on small scales. However, the respective theoretical background and associated interpretation of the parameters measured via indentation derives largely from research on traditional engineering materials such as metals or ceramics. Here, we discuss the functional relevance of indentation hardness in biological materials by presenting a meta-analysis of its relationship with indentation modulus. Across seven orders of magnitude, indentation hardness was directly proportional to indentation modulus, illustrating that hardness is not an independent material property. Using a lumped parameter model to deconvolute indentation hardness into components arising from reversible and irreversible deformation, we establish criteria which allow to interpret differences in indentation hardness across or within biological materials. The ratio between hardness and modulus arises as a key parameter, which is a proxy for the ratio between irreversible and reversible deformation during indentation, and the materials yield strength. Indentation hardness generally increases upon material dehydration, however to a larger extend than expected from accompanying changes in indentation modulus, indicating that water acts as a plasticiser. A detailed discussion of the role of indentation hardness, modulus and toughness in damage control during sharp or blunt indentation yields comprehensive guidelines for a performance-based ranking of biological materials, and suggests that quasi-plastic deformation is a frequent yet poorly understood damage mode, highlighting an important area of future research.

bioengineering↗
bioRxiv2017

High-Throughput Controlled Mechanical Stimulation and Functional Imaging In Vivo

Understanding mechanosensation and other sensory behavior in genetic model systems such as C. elegans is relevant to many human diseases. These studies conventionally require immobilization by glue and manual delivery of stimuli, leading to low experimental throughput and high variability. Here we present a microfluidic platform that delivers precise mechanical stimuli robustly. The system can be easily used in conjunction with functional imaging and optical interrogation techniques, as well as other capabilities such as sorting or more sophisticated fluid delivery schemes. The platform is fully automated, thereby greatly enhancing the throughput and robustness of experiments. We show that behavior of the well-known gentle and harsh touch neurons and their receptive fields can be recapitulated in our system. Using calcium dynamics as a readout, we demonstrate the ability to perform a drug screen in vivo. Furthermore, using an integrated chip platform that can deliver both mechanical and chemical stimuli, we examine sensory integration in interneurons in response to multimodal sensory inputs. We envision that this system will be able to greatly accelerate the discovery of genes and molecules involved in mechanosensation and multimodal sensory behavior, as well as the discovery of therapeutics for related diseases.

bioengineering↗
bioRxiv2017

Sorting of molecular shuttles by designing electrical and mechanical properties of microtubules

Kinesin-driven microtubules have been a focus to serve as molecular shuttles to replace multiple on-chip functions in micro total analysis systems ({micro}TAS). Although transport, concentration, and detection of target molecules have been demonstrated, controllability of transport directions is still a major challenge. To define multiple moving directions for selective molecular transport, we integrated the bottom-up molecular design of microtubules and the top-down design of a microfluidic device. The surface charge density and stiffness of microtubules were controlled, allowing us to create three different types of microtubules with different gliding directions corresponding to their electrical and mechanical properties. The measured curvature of gliding microtubules enabled us to optimize the size and design of the device for molecular sorting in a top-down approach. The integrated bottom-up and top-down design achieved separation of stiff microtubules from negatively-charged soft microtubules with approximately 80% efficiency under an electric field. Our method is the first to sort multiple microtubules by integrating molecular control and microfluidic device design, and is applicable to multiplexed molecular sorters.

bioengineering↗
bioRxiv2017

Denitrification of water in a microbial fuel cell (MFC) using seawater bacteria

The sea contains various microbes which have an ability to reduce and oxidize substances like iron, sulphur, and nitrate. Most of these processes happen in the seawater, but can also be applied for puri[fi]cation of wastewater. In the present work, a consortium of seawater bacteria has been used for the first time in a microbial fuel cell to reduce nitrate in synthetic water samples and produce electricity by oxidizing organic matter. The concentrations of [Formula] and [Formula] were reduced to well below their permissible limits. Moreover, the growth of the bacterial consortium at cathode causes an increased electricity production in the cell because of the increased bacterial activity. The performance of the cell with a bicarbonate buffered solution (BBS) at the cathode was superior to that obtained with the commonly used phosphate buffered solution (PBS). As BBS is the natural buffering agent found in the sea, the use of BBS is eco-friendly. The same seawater bacterial consortium could be used at both the anode and the cathode, confirming their adaptability to different environments. Unfortunately, denitrification was accompanied by the generation of high concentrations of [Formula] at the anode and the cathode, probably because of the use of N2 gas for sparging the anolyte. This aspect merits further investigation.

bioengineering↗
bioRxiv2017

Open Design 3D-Printable Adjustable Micropipette that meets ISO Standard for Accuracy

Scientific communities are drawn to the open source model as an increasingly utilitarian method to produce and share work. Initially used as a means to develop freely available software, open source projects have been applied to hardware including scientific tools. Increasing convenience of 3D printing has fueled the proliferation of open labware projects aiming to develop and share designs for scientific tools that can be produced in-house as cheap alternatives to commercial products. We present our design of a micropipette that is assembled from 3D-printable parts and some hardware that works by actuating a disposable syringe to a user adjustable limit. Graduations on the syringe are used to accurately adjust the set point to the desired volume. Our open design printed micropipette is assessed in comparison to a commercial pipette and meets ISO 8655 standards.

bioengineering↗
bioRxiv2017

Controlling cell shape on hydrogels using lift-off patterning

Polyacrylamide gels functionalized with extracellular matrix proteins are commonly used as cell culture platforms to evaluate the combined effects of extracellular matrix composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a high-fidelity photoresist lift-off patterning method to pattern ECM proteins on polyacrylamide hydrogels ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing method. Lift-off patterning achieves higher protein transfer efficiency, increases pattern accuracy, increases pattern yield, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape and enable long-term imaging of actin intracellular structure and lamellipodia dynamics when we culture epithelial cells on these substrates.

bioengineering↗
bioRxiv2017

Micropatterned Substrates To Promote And Dissect Reprogramming Of Human Somatic Cells

Reprogramming of human somatic cells to induce pluripotent stem cells (iPSCs) generates valuable precursors for disease modeling and regenerative medicine. However, the reprogramming process can be inefficient and noisy, creating many partially reprogrammed cells in addition to fully reprogrammed iPSCs. To address these shortcomings, we developed a micropatterned substrate that allows for dynamic live-cell microscopy of thousands of cell subpopulations undergoing reprogramming. Micropatterning facilitated a change in shape, size and clustering of nuclei to promote somatic identity erasure. Increased proliferation, cell density and decreased intercellular YAP signaling accompanied these nuclear changes. A combination of eight nuclear characteristics could be used to track reprogramming progression and distinguish partially reprogrammed cells from those that were fully reprogrammed.\n\nMicropatterned substrates constitute a new tool for facile iPSC production and can be used in high-throughput to probe and understand the subcellular changes that accompany human cell fate transitions.

bioengineering↗
bioRxiv2017

Multiplexed confocal and super-resolution fluorescence imaging of cytoskeletal and neuronal synapse proteins

Neuronal synapses contain dozens of protein species whose expression levels and localizations are key determinants of synaptic transmission and plasticity. The spectral properties of fluorophores used in conventional microscopy limit the number of measured proteins to four species within a given sample. The ability to perform high-throughput confocal or super-resolution imaging of many proteins simultaneously without limitation in target number imposed by this spectral limit would enable large-scale characterization of synaptic protein networks in situ. Here, we introduce PRISM: Probe-based Imaging for Sequential Multiplexing, a method that sequentially utilizes either high affinity Locked Nucleic Acid (LNA) or low affinity DNA probes to enable diffraction-limited confocal and PAINT-based super-resolution imaging. High-affinity LNA probes offer high-throughput, confocal-based imaging compared with PAINT, which uses low affinity probes to realize localization-based super-resolution imaging. Simultaneous immunostaining of all targets is performed prior to imaging, followed by sequential LNA/DNA probe exchange that requires only minutes under mild wash conditions. We apply PRISM to quantify the co-expression levels and nanometer-scale organization of one dozen cytoskeletal and synaptic proteins within individual neuronal synapses. Our approach is scalable to dozens of target proteins and is compatible with high-content screening platforms commonly used to interrogate phenotypic changes associated with genetic and drug perturbations in a variety of cell types.

bioengineering↗
bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗
bioRxiv2017

Micro-Scale Transport Processes for Advanced Healthcare and Point of Care Diagnostics

Recent outbreaks like Zika and Ebola highlights the challenges associated with pathogen diagnostics in the developing world. With the outbreak in Africa, and isolated cases on other continents, the need for an affordable, rapid and portable diagnostic solution has been repeatedly stressed and is one of the most critical issues confronting global health. Unfortunately, the current conventional PCR instrumentation needed to perform gold standard DNA-based diagnostic tests is bulky, slow, and expensive, making it unsuitable for resource-limited settings in developing countries where dedicated laboratory facilities are not available. Advances in micro-fluidics and smartphone based technology has paved way for novel implementations of traditional molecular diagnostic techniques. We present a series of advances in molecular diagnostic techniques by harnessing convective flow to actuate biochemical reactions.

bioengineering↗
bioRxiv2017

Scaling up DNA data storage and randomaccess retrieval

Current storage technologies can no longer keep pace with exponentially growing amounts of data. 1 Synthetic DNA offers an attractive alternative due to its potential information density of ~ 1018 B/mm3, 107 times denser than magnetic tape, and potential durability of thousands of years.2 Recent advances in DNA data storage have highlighted technical challenges, in particular, coding and random access, but have stored only modest amounts of data in synthetic DNA. 3,4,5 This paper demonstrates an end-to-end approach toward the viability of DNA data storage with large-scale random access. We encoded and stored 35 distinct files, totaling 200MB of data, in more than 13 million DNA oligonucleotides (about 2 billion nucleotides in total) and fully recovered the data with no bit errors, representing an advance of almost an order of magnitude compared to prior work. 6 Our data curation focused on technologically advanced data types and historical relevance, including the Universal Declaration of Human Rights in over 100 languages,7 a high-definition music video of the band OK Go,8 and a CropTrust database of the seeds stored in the Svalbard Global Seed Vault.9 We developed a random access methodology based on selective amplification, for which we designed and validated a large library of primers, and successfully retrieved arbitrarily chosen items from a subset of our pool containing 10.3 million DNA sequences. Moreover, we developed a novel coding scheme that dramatically reduces the physical redundancy (sequencing read coverage) required for error-free decoding to a median of 5x, while maintaining levels of logical redundancy comparable to the best prior codes. We further stress-tested our coding approach by successfully decoding a file using the more error-prone nanopore-based sequencing. We provide a detailed analysis of errors in the process of writing, storing, and reading data from synthetic DNA at a large scale, which helps characterize DNA as a storage medium and justify our coding approach. Thus, we have demonstrated a significant improvement in data volume, random access, and encoding/decoding schemes that contribute to a whole-system vision for DNA data storage.

bioengineering↗
bioRxiv2017

Protein Fingerprinting With A Binary Alphabet

Abstract.If protein sequences are recoded with a binary alphabet derived from a division of the 20 amino acids into two subsets, a protein can be identified from its subsequences by searching through a recoded sequence database. A binary-coded primary sequence can be obtained for an unbroken protein molecule from current blockades in a nanopore. Only two (instead of 20) blockade levels need to be recognized to identify a residues subset; a hard or soft detector can do this with two current thresholds. Computations were done on the complete proteome of Helicobacter pylori (http://www.uniprot.org; database id UP000000210, 1553 sequences) using a binary alphabet based on published data for residue volumes in the range [~]0.06 nm3 to [~]0.225 nm3. Assuming normally distributed volumes, more than 93% of binary subsequences of length 20 from the primary sequences of H. pylori are correct with a confidence level of 90-95%; they can uniquely identify over 98% of the proteins. Recently published work shows that a 0.7 nm diameter nanopore can measure residue volume with a resolution of [~]0.07 nm3; this makes the procedure described here both feasible and practical. This is a non-destructive single-molecule method without the vagaries of proteolysis.

bioengineering↗
bioRxiv2017

A Microfluidic Module For Real-Time Generation Of Complex Multi-Molecule Temporal Concentration Profiles

We designed a microfluidic module that generates complex, dynamic concentration profiles of multiple molecules over a large concentration range using pulse-width modulation (PWM). Our PWM device can arbitrarily combine up to 6 different inputs and select between three downstream mixing channels as required by the application. The module can produce arbitrary concentrations with a dynamic range of up to 3-5 decades. We created complex concentration profiles of 2 molecules, with each concentration independently controllable, and show that the PWM module can execute rapid concentration changes as well as long-timescale pharmacokinetic profiles. Concentration profiles were generated for molecules with molecular weights ranging from 560 Da to 150 kDa. Our PWM module produces robust and precise concentration profiles under a variety of operating conditions, making it ideal for integration with existing microfluidic devices for advanced cell and pharmacokinetic studies.

bioengineering↗
bioRxiv2017

Optimising 3D scaffold for otic neural progenitor differentiation

Hearing loss is a condition highly prevalent worldwide. It affects people of a broad age range since the causes and risk factors are varied. At present, some types of hearing impairments have a palliative treatment whereas some, especially for those where otic neurons are damaged, cannot be properly treated. Recent findings had shown it possible to use human embryonic stem cell-derived otic neural progenitors (ONPs) as a new mode of treating hearing loss caused by damage to the spiral ganglion neurons (SGNs). To improve the efficiency and overcome some limitations of this potential treatment, we have applied principles of tissue engineering which involves an interaction between cells and an extracellular matrix -mimicking scaffold. Here, we describe the influence of poly(l-lactic acid)(PLLA) aligned fibres on ONP cell morphology, proliferation, neuronal differentiation and establishment of neural polarity under both progenitor and neuralising conditions. The results show that most of ONPs on aligned fibres exhibited bipolar morphology and extended their neurites along the major fibre axis. Their proliferation was lower than those in 2D culture but the differentiation of ONPs on aligned fibres was significantly enhanced in both progenitor and neuralising conditions as indicated by the fluorescence intensity and number of cells that were positive for neuronal markers ({beta}-tubulin III and NF200) and the expression pattern of spiral ganglion molecular markers (MMP13, NPR2 and NTNG1). Moreover, axonal and dendritic markers (TAU and MAP2 respectively) were also induced after 14 days in culture.

bioengineering↗