Search bioRxiv⌕ Search

SEARCH · Search bioRxiv

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
bioRxiv2018

Supercharging enables organized assembly of synthetic biomolecules

There are few methods for the assembly of defined protein oligomers and higher order structures that could serve as novel biomaterials. Using fluorescent proteins as a model system, we have engineered novel oligomerization states by combining oppositely supercharged variants. A well-defined, highly symmetrical 16-mer (two stacked, circular octamers) can be formed from alternating charged proteins; higher order structures then form in a hierarchical fashion from this discrete protomer. During SUpercharged PRotein Assembly (SuPrA), electrostatic attraction between oppositely charged variants drives interaction, while shape and patchy physicochemical interactions lead to spatial organization along specific interfaces, ultimately resulting in protein assemblies never before seen in nature.

bioengineering↗
bioRxiv2018

High Temporal-Resolution Dynamic PET Image Reconstruction Using A New Spatiotemporal Kernel Method

Current clinical dynamic PET has an effective temporal resolution of 5-10 seconds, which can be adequate for traditional compartmental modeling but is inadequate for exploiting the benefit of more advanced tracer kinetic modeling. There is a need to improve dynamic PET to allow fine temporal sampling of 1-2 seconds. However, reconstruction of these shorttime frames from tomographic data is extremely challenging as the count level of each frame is very low and high noise presents in both spatial and temporal domains. Previously the kernel framework has been developed and demonstrated as a statistically efficient approach to utilizing image prior for low-count PET image reconstruction. Nevertheless, the existing kernel methods mainly explore spatial correlations in the data and only have a limited ability in suppressing temporal noise. In this paper, we propose a new kernel method which extends the previous spatial kernel method to the general spatiotemporal domain. The new kernelized model encodes both spatial and temporal correlations obtained from image prior information and is incorporated into the PET forward projection model to improve the maximum likelihood (ML) image reconstruction. Computer simulations and an application to real patient scan have shown that the proposed approach can achieve effective noise reduction in both spatial and temporal domains and outperform the spatial kernel method and conventional ML reconstruction method for improving high temporal-resolution dynamic PET imaging.

bioengineering↗
bioRxiv2018

Achilles tendon structure in distance runners does not change following a competitive season

Achilles tendon structure differs between trained distance runners and healthy controls, but the progression of tendon remodeling over the course of a competitive season is poorly understood. Therefore, the purpose of this study was to quantify Achilles tendon structure at the beginning and completion of a cross country season. We hypothesized that athletes who did not develop tendinopathy would not present with changes in tendon structure. Ultrasound assessments of the right Achilles tendon mid-substance were performed to quantify tendon organization, thickness, and echogenicity. Subjective structural measures and reported outcomes were also collected to determine if tendinopathy was present in any of the subjects. None of the subjects developed symptomatic tendinopathy over the course of the competitive season, but one runner did show signs of mild neovascularization. Tendon organization and echogenicity did not change over the course of the season. Tendon thickness increased by 7% (P < 0.001) but the effect size was small (d = 0.36). Runners who do not develop symptomatic tendinopathy have habituated tendon structure that may serve as a protective mechanism against the rigors of distance running. Monitoring tendon structure may serve as a means of detecting signs of structural indicators of tendinopathy prior to the presentation of symptoms.

bioengineering↗
bioRxiv2018

S-MiRAGE: A quantitative, secreted RNA-based reporter of gene expression and cell persistence

Non-destructive measurements of cell persistence and gene expression are crucial for longitudinal research studies and for prognostic assessment of cell therapies. Here we describe S-MiRAGE, a platform that utilizes small secreted RNA molecules as sensitive and quantitatively accurate reporters of cellular processes. We demonstrate the utility of S-MiRAGE by monitoring the differentiation status of human embryonic stem cells in vitro, and tumor growth in a mouse model in vivo.

bioengineering↗
bioRxiv2018

Optimization of Tenocyte Lineage-related Factors from Tonsil-derived Mesenchymal Stem Cells using Response Surface Methodology

Researchers should consider various potential factors that affect tenogenic differentiation of mesenchymal stem cells (MSCs); however, this requires numerous experimental settings, which are associated with high cost and time. We aimed to assess the differential effects of transforming growth factor beta 3 (TGF-{beta}3) on the tenogenesis of tonsil-derived MSCs (T-MSCs) and bone marrow-derived MSCs (BM-MSCs) using design of experiments (DoE). Bone marrow and tonsillar tissue was collected from four patients; mononuclear cells were separated and treated with 5 and 10 ng/mL of TGF-{beta}3 with vehicle control. A full-factorial experimental design with a categorical factor of 0 was employed to study the effect of tension based on T-MSCs. Eighty-four trials were utilized, fitted with RSM, and then used to obtain mathematical prediction models. Exposure of T-MSCs and BM-MSCs to TGF-{beta}3 increased the expression of scleraxis (SCX), tenomodulin (TNMD), decorin, collagen I, and tenascin C. Expression of most of these factors reached a maxima after 2-3 days of treatment. Considering all of the tenocyte lineage-related factors that were assessed, the predicted value of the factors from T-MSCs was significantly induced at 2.7 ng/mL of TGF-{beta}3 during 2.5-day culture, whereas the predicted value of the factors from BM-MSCs was significantly induced during 2.3-day culture, regardless of TGF-{beta}3 concentration. This study demonstrated that tenogenic differentiation of T-MSCs and BM-MSCs under TGF-{beta}3 stimulation showed a similar culture time for peak expression of tenocyte-related mRNAs using RSM. This study suggests the potential of using the DoE approach for optimization of the culture protocol for tenogenesis of MSCs.

bioengineering↗
bioRxiv2018

Effect of eco-remediation and microbial community using multilayer solar planted floating island (MS-PFI) in the drainage channel

A multilayer solar planted floating island (MS-PFI) planted with Eichhornia crassipes are potential alternatives to traditional PFI. The highest removal rates of suspended solids, total nitrogen, total phosphorus, ammonia nitrogen and chemical oxygen demand was 86%, 75%, 80%, 95% and 84%, respectively. Proteobacteria (average 43.4% of total sequences) and Actinobacteria (19.9%) were the dominant phyla. Numerous genus had obvious differences between influent and effluent water, for instance, 13, 12 and 7 % in effluent water were assigned to the hgcl_clade, Norank_c_Cyanobacteria, and Rhizorhapis, while their relative abundances were decreased to 5, 3 and 0 %. In contrast, a distinct increase among Flavobacterium (10%), Limnohabitans (7%), Alpinimonas (4%), norank_p_Saccharibacteria (4%), Erwinia (3%) after MS-PFI treatment. MS-PFI brings various bacteria involved in contaminant degradation and nutrient removal in biological wastewater treatment systems. An amount of {yen} 1,843 was totally inputted to construct floating bed, which was rarely needed operation and maintenance costs.\n\nImportanceIn-situ micro-polluted water ecological remediation, microorganisms and plants are effective to improve environmental quality and provide essential ecosystem services. Recently, we invent a new multilayer solar with an excellent pollutant removal efficiency. Microbes can decompose or mineralize organic matter effectively, also provide food for aquatic animals and increase nutrients or substances for plants, it is an important part of biogeochemical cycles and energy flows in aquatic ecological systems. However, few study explain the bacteria diversity and its responses between influent and effluent water in a planted floating island. The significance of our study is in identifying-in greater detail-the responses of bacteria in the new MS-PFI. This will greatly enhance our knowledge of bacteria communities, and can be widely used in micro-polluted water remediation.

bioengineering↗
bioRxiv2018

Practical bioinstrumentation developments for AC magnetic field-mediated magnetic nanoparticle heating applications.

Heat dissipation during magnetization reversal processes in magnetic nanoparticles (MNP), upon exposure to alternating magnetic fields (AMF), has been extensively studied in relation to applications in magnetic fluid hyperthermia (MFH). This current paper demonstrates the design, fabrication, and evaluation of an efficient instrument, operating on this principle, for use as (i) a non-contact, in vitro, real-time temperature monitor; (ii) a drug release analysis system (DRAS); (iii) a high flux density module for AMF-mediated MNP studies; and (iv) an in vivo coil setup for real-time, whole body thermal imaging. The proposed DRAS is demonstrated by an AMF-mediated drug release proof-of-principle experiment. Also, the technique described facilitates non-contact temperature measurements of specific absorption rate (SAR) as accurately as temperature measurements using a probe in contact with the sample. Numerical calculations estimating the absolute and root mean squared flux densities, and other MNP - AMF studies suggest that the proposed stacked planar coil module could be employed for calorimetry. Even though the proposed in vivo coil setup could be used for real-time, whole body thermal imaging (within the limitations due to issues of penetration depth), further design effort is required in order to enhance the energy transfer efficiency.

bioengineering↗
bioRxiv2018

Tendon slack length is the primary determinant of plantarflexor muscle-tendon function in computational simulations of gait

1Background: Locomotion is partly dictated by plantarflexor function and structure. Computational simulations are powerful tools capable of testing the isolated effects of muscle-tendon structure on gait function. Research Question: The purpose of this study was to characterize the sensitivity of plantarflexor muscle function based on muscle-tendon unit (MTU) parameters. We hypothesized that plantarflexor metabolics and shortening dynamics would be sensitive to MTU parameters. Methods: Stance phase of gait was simulated using a musculoskeletal model and computed muscle control algorithm. Optimal muscle fiber length, tendon slack length, and tendon stiffness parameters were systematically changed to test the effects on plantarflexor metabolics and shortening dynamics. Results and Significance: Plantarflexor metabolic demands were 8 and 28 times more sensitive to muscle fiber and tendon slack lengths, respectively, compared to the effect of tendon stiffness. Shortened tendon slack lengths induced a large passive plantarflexion moment during early stance, which required non-physiologic dorsiflexor contractions. Conversely, longer muscle fiber and tendon slack lengths increased the shortening demands of the plantarflexors to account for the added length of the MTU. These findings highlight the importance of carefully selecting MTU parameters when modeling gait with musculoskeletal models, especially in pathologic or high-performance athlete populations.

bioengineering↗
bioRxiv2018

Biophysically Realistic Neuron Models for Simulation of Cortical Stimulation

1.ObjectiveWe implemented computational models of human and rat cortical neurons for simulating the neural response to cortical stimulation with electromagnetic fields.\n\nApproachWe adapted model neurons from the library of Blue Brain models to reflect biophysical and geometric properties of both adult rat and human cortical neurons and coupled the model neurons to exogenous electric fields (E-fields). The models included 3D reconstructed axonal and dendritic arbors, experimentally-validated electrophysiological behaviors, and multiple, morphological variants within cell types. Using these models, we characterized the single-cell responses to intracortical microstimulation (ICMS) and uniform E-field with dc as well as pulsed currents.\n\nMain resultsThe strength-duration and current-distance characteristics of the model neurons to ICMS agreed with published experimental results, as did the subthreshold polarization of cell bodies and axon terminals by uniform dc E-fields. For all forms of stimulation, the lowest threshold elements were terminals of the axon collaterals, and the dependence of threshold and polarization on spatial and temporal stimulation parameters was strongly affected by morphological features of the axonal arbor, including myelination, diameter, and branching.\n\nSignificanceThese results provide key insights into the mechanisms of cortical stimulation. The presented models can be used to study various cortical stimulation modalities while incorporating detailed spatial and temporal features of the applied E-field.

bioengineering↗
bioRxiv2018

Pre-existing Technological Core and Roots for the CRISPR Breakthrough

This paper applies objective methods to explore the technological origins of the widely acclaimed CRISPR breakthrough in the technological domain of genome engineering. Previously developed patent search techniques are first used to recover a set of patents that well-represent the genome editing domain before CRISPR. Main paths are then determined from the citation network associated with this patent set allowing identification of the three major knowledge trajectories. The most significant of these trajectories for CRISPR involves the core of genome editing with less significant trajectories involving cloning and endonuclease specific developments. The major patents on the core trajectory are consistent with qualitative expert knowledge of the topical area. A second set of patents that we call the CRISPR roots are obtained by finding the patents directly cited by the recent CRISPR patents along with patents cited by that set of patents. We find that the CRISPR roots contain 8 key patents from the genome engineering main path associated with restriction endonucleases and the expected strong connection of CRISPR to prior genome editing technology such as Zn finger nucleases. Nonetheless, analysis of the full CRISPR roots shows that a very wide array of technological knowledge beyond genome engineering has contributed to achieving the CRISPR breakthrough. Such breadth in origins is not surprising since \"spillover\" is generally perceived as important and previous qualitative studies of CRISPR have shown not only technological breadth in origins but scientific breadth as well. In addition, we find that the estimated rate of functional performance improvement of the CRISPR roots set is about 9% per year compared to the genome engineering set ({small tilde}4 % per year). These estimates indicate below average rates of improvement and may indicate that CRISPR (and perhaps yet undiscovered) genome engineering developments could evolve in effectiveness over an upcoming long rather than short time period.

bioengineering↗
bioRxiv2018

Automated High-Throughput Light-Sheet Fluorescence Microscopy of Larval Zebrafish

AO_SCPCAPBSTRACTC_SCPCAPLight sheet fluorescence microscopy enables fast, minimally phototoxic, three-dimensional imaging of live specimens, but is currently limited by low throughput and tedious sample preparation. Here, we describe an automated high-throughput light sheet fluorescence microscope in which specimens are positioned by and imaged within a fluidic system integrated with the sheet excitation and detection optics. We demonstrate the ability of the instrument to rapidly examine live specimens with minimal manual intervention by imaging fluorescent neutrophils over a nearly 0.3 mm3 volume in dozens of larval zebrafish. In addition to revealing considerable inter-individual variability in neutrophil number, known previously from labor-intensive methods, three-dimensional imaging allows assessment of the correlation between the bulk measure of total cellular fluorescence and the spatially resolved measure of actual neutrophil number per animal. We suggest that our simple experimental design should considerably expand the scope and impact of light sheet imaging in the life sciences.

bioengineering↗
bioRxiv2018

Transparent Titanium Dioxide Nanotubes: Processing, Characterization, and Application in Establishing Cellular Response Mechanisms

The therapeutic applications of titanium dioxide nanotubes (TiO2 NTs) as osteogenic surface treatments for titanium (Ti)-based implants are largely due to the finely tunable physical characteristics of these nanostructures. As these characteristics change, so does the cellular response, yet the exact mechanisms for this relationship remains largely undefined. We present a novel TiO2 NT imaging platform that is suitable for use with live-cell imaging techniques, thereby enabling, for the first time, dynamic investigation of those mechanisms. In this work, fabrication methods for producing transparent TiO2 NTs with diameters of 56 {+/-} 6 nm, 75 {+/-} 7 nm, 92 {+/-} 9 nm, and 116 {+/-} 10 nm are described. To demonstrate the diagnostic potential of these TiO2 NT imaging platforms, the focal adhesion protein vinculin and actin cytoskeletal filaments were fluorescently tagged in osteoblasts and real-time, high-resolution fluorescent microscopy of live-cell interactions with TiO2 NT substrates were observed. The scope of such a platform is expected to extend far beyond the current proof-of-concept, with great potential for addressing the dynamic response of cells interacting with nanostructured substrates.

bioengineering↗
bioRxiv2018

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

bioengineering↗
bioRxiv2018

Linking single-cell measurements of mass, growth rate, and gene expression

We introduce a microfluidic platform that enables single-cell mass and growth rate measurements upstream of single-cell RNA-sequencing (scRNA-seq) to generate paired single-cell biophysical and transcriptional data sets. Biophysical measurements are collected with a serial suspended microchannel resonator platform (sSMR) that utilizes automated fluidic state switching to load individual cells at fixed intervals, achieving a throughput of 120 cells per hour. Each single-cell is subsequently captured downstream for linked molecular analysis using an automated collection system. From linked measurements of a murine leukemia (L1210) and pro-B cell line (FL5.12), we identify gene expression signatures that correlate significantly with cell mass and growth rate. In particular, we find that both cell lines display a cell-cycle signature that correlates with cell mass, with early and late cell-cycle signatures significantly enriched amongst genes with negative and positive correlations with mass, respectively. FL5.12 cells also show a significant correlation between single-cell growth efficiency and a G1-S transition signature, providing additional transcriptional evidence for a phenomenon previously observed through biophysical measurements alone. Importantly, the throughput and speed of our platform allows for the characterization of phenotypes in dynamic cellular systems. As a proof-of-principle, we apply our system to characterize activated murine CD8+ T cells and uncover two unique features of CD8+ T cells as they become proliferative in response to activation: i) the level of coordination between cell cycle gene expression and cell mass increases, and ii) translation-related gene expression increases and shows a correlation with single-cell growth efficiency. Overall, our approach provides a new means of characterizing the transcriptional mechanisms of normal and dysfunctional cellular mass and growth rate regulation across a range of biological contexts.

bioengineering↗
bioRxiv2018

Molecular inactivation of exopolysaccharide biosynthesis in Paenibacillus polymyxa DSM 365 for enhanced 2,3-butanediol production

Formation of Exopolysaccharides (EPS) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa decreases 2,3-BD yield, increases medium viscosity and impacts 2,3-BD downstream processing. Therefore, additional purification steps are required to rid the fermentation broth of EPS prior to 2,3-BD purification, which adds to the production cost. To eliminate EPS production during 2,3-BD fermentation, we explored a metabolic engineering strategy to disable the EPS production pathway of P. polymyxa, thereby increasing 2,3-BD yield and productivity. The levansucrase gene which encodes levansucrase, the enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The resulting P. polymyxa levansucrase null mutant showed 34% and 54% increases in growth with 6.4- and 2.4-folds decrease in EPS formation in sucrose and glucose cultures, respectively. The observed decrease in EPS formation by the levansucrase null mutant may account for the 27% and 4% increase in 2,3-BD yield, and 4% and 128% increases in 2,3-BD productivity when grown on sucrose and glucose media, respectively. Genetic stability of the levansucrase null mutant was further evaluated. Interestingly, the levansucrase null mutant remained genetically stable over fifty generations with no observable decrease in growth and 2,3- BD formation with or without antibiotic supplementations. Collectively, our results show that P. polymyxa levansucrase null mutant has potential for improving 2,3-BD yield, and ultimately, the economics of large-scale microbial 2,3-BD production.

bioengineering↗
bioRxiv2018

Flux Balance Analysis Identifies Distinct NADPH Production Strategies Across NCI 60 Cancer Cell Lines

Flux Balance Analysis is a linear mathematical procedure which determines the set of reaction fluxes to produce a maximum flux of a reaction of interest. In this study, a core cancer model developed by Zielinski et al. 2017 is constrained by a set of 59 cancer cell type specific uptake and secretion rates. Optimizing for cell type specific biomass objective reactions and examining serine flux distributions reveals variability in production of NADPH. In many cell lines, production of NADPH is correlated to biosynthetic demand, however, outliers exist that produce excess NADPH beyond that of biomass demand. These outliers are first characterized by their NADPH production strategy (pentose phosphate pathway or a combination of One Folate Cycle and Malic Enzyme) and then the factors responsible for the different NADPH production strategies are identified. Results indicate that pentose phosphate pathway (PPP) producing NADPH cell lines had reprogrammed tricarboxylic acid cycle metabolism to meet the demand for decreased flux through glycolytic enzymes, while one folate cycle and malic enzyme (OFC + ME) producing NADPH cell lines had higher threonine, tyrosine and serine uptake.

bioengineering↗
bioRxiv2018

Exploiting Index Cross-Talk to Modify Variant Calls

Modern next-generation DNA sequencers support multiplex sequencing to improve throughput and decrease costs. This is done by pooling and sequencing samples together in parallel, which are later demultiplexed according to their unique indexes1, 2. When reads are assigned to the wrong index, called index cross-talk, information is leaked between samples3-6. This creates a physical information side-channel, a well known class of vulnerabilities in information security7-10, that may be used to modify downstream results. Here we demonstrate the feasibility of such an attack through the use of a separately indexed library that causes a wild-type human exome to be misclassified as heterozygous at the sickle-cell locus. Simple methods can be used to minimize or detect attempts to modify genetic variants using this side-channel, such as filtering by read quality or finding outliers in read coverage. To further minimize this risk we recommend the use of new library preparation methods that reduce index cross-talk, like unique dual indexes11, 12, whenever samples are sequenced together in important applications. Biotechnology that interfaces molecular and digital information, like DNA sequencers, may have security risks typically associated with information systems, including the side-channel vulnerability described in this study. We encourage the community to consider the security of genomics-information pipelines before they reach mass adoption.

bioengineering↗
bioRxiv2018

Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

Intervertebral disc degeneration is a complex, cell-mediated process originating in the nucleus pulposus (NP) and is associated with extracellular matrix catabolism leading to disc height loss and impaired spine kinematics. Previously, we developed an acellular bovine NP (ABNP) for NP replacement that emulated human NP matrix composition and supported cell seeding; however, its mechanical properties were lower than those reported for human NP. To address this, we investigated ethanol-mediated compaction and crosslinking to enhance the ABNPs dynamic mechanical properties and degradation resistance while maintaining its cytocompatibility. First, volumetric and mechanical effects of compaction only were confirmed by evaluating scaffolds after various immersion times in buffered 28% ethanol. It was found that compaction reached equilibrium at [~]30% compaction after 45 min, and dynamic mechanical properties significantly increased 2-6x after 120 min of submersion. This was incorporated into a crosslinking treatment, through which scaffolds were subjected to 120 min pre-compaction in buffered 28% ethanol prior to carbodiimide crosslinking. Their dynamic mechanical properties were evaluated before and after accelerated degradation by ADAMTS-5 or MMP-13. Cytocompatibility was determined by seeding stem cells onto scaffolds and evaluating viability through metabolic activity and fluorescent staining. Compacted and crosslinked scaffolds showed significant increases in DMA properties without detrimentally altering their cytocompatibility, and these mechanical gains were maintained following enzymatic exposure.

bioengineering↗