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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Mechanosensing of Integrin α6 and EGFR Converges atCalpain 2

Introduction Introduction Materials and Methods Results Discussion Conclusions Author Contributions Conflict of Interest References Carcinoma progression is associated with deposition of ECM that stiffens the local microenvironment [1, 2]. This tissue stiffening results in deposition of additional matrix proteins, initiating a positive feedback loop between cells and the evolving stroma [3]. Cells sense and respond to the stiffness of their environment via RhoA GTPase activation, which feeds back to increase cell contractility via activation of myosin light chain kinase [4]. These ECM-driven changes in cytoskeletal tension regulate motility in a cell-type specific manner [5]. In vi ...

bioengineering↗

bioRxiv2017

Optimization of Alginante- poly-L-lysine Microencapsules Strength by Box-Behnken Model for CHO Culture in Stirred Tank Bioreactor

Summary statementBox-Behnken model is an efficient method to optimized process conditions of microencapsules strength for long-term cell culture, promotes CHO cells viability and protein production in a stirred tank bioreactor.\n\nABSTRACTCell microencapsulation technology has been proved to be a valuable technology in the fields of large-scale cell culture. It is important to accurately construct the microcapsule membranes with desired properties including a certain thickness suitable for cell growth, and maximum strength for the stability of microencapsules. As single factor experiments are time-consuming to obtain the desired membrane preparation conditions, Box-Behnken model was used to investigate the interactions among reaction conditions, predict the optimized reaction conditions for given purpose, that was membrane with maximum strength and desired thickness for microencapsulated cell culture. Significant values of R2 in this study indicated the model theoretical values are very close to the measured values. Based on the desired membrane thickness, the process of maximum strength was optimized, and the prediction agreement of measured value and model theoretical value was 91.11%. The optimized microencapsules with maximum strength and 15 m membrane thickness promote CHO cells viability and protein production in stirred tank bioreactor. The result shows that Box-Behnken model is an efficient method to optimized process conditions of microencapsules strength for long-term cell culture.

bioengineering↗

bioRxiv2017

Time-gated detection of protein-protein interactions with transcriptional readout

Transcriptional assays such as yeast two hybrid, split ubiquitin, and Tango that convert transient protein-protein interactions (PPIs) in cells into stable expression of transgenes are powerful tools for PPI discovery, high-throughput screens, and analysis of large cell populations. However, these assays frequently suffer from high background and they lose all information about PPI dynamics. To address these limitations, we developed a light-gated transcriptional assay for PPI detection called PPI-FLARE (PPI-Fast Light- and Activity-Regulated Expression). PPI-FLARE requires both a PPI to deliver TEV protease proximal to its cleavage peptide, and externally-applied blue light to uncage the cleavage peptide, in order to release a membrane-tethered transcription factor (TF) for translocation to the nucleus. We used PPI-FLARE to detect the ligand-induced association of 12 different PPIs in living mammalian cells, with a temporal resolution of 5 minutes and a {+/-}ligand signal ratio up to 37. By systematically shifting the light irradiation window, we could reconstruct PPI time-courses, distinguishing between GPCRs that engage in transient versus sustained interactions with the cytosolic effector arrestin. When combined with FACS, PPI-FLARE enabled >100-fold enrichment of cells experiencing a specific GPCR-arrestin PPI during a short 10-minute light window over cells missing that PPI during the same time window. Due to its high specificity, sensitivity, and generality, PPI-FLARE should be a broadly useful tool for PPI analysis and discovery.

bioengineering↗

bioRxiv2017

Printed Droplet Microfluidics for on demand dispensing of picoliter droplets and cells

Although the elementary unit of biology is the cell, high throughput methods for the microscale manipulation of cells and reagents are limited. The existing options are either slow, lack single cell specificity, or utilize fluid volumes out of scale with those of cells. Here, we present Printed Droplet Microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single cell printer, enabling high throughput generation of intricate arrays of droplets, cells, and microparticles. Printed Droplet Microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

bioengineering↗

bioRxiv2017

A Biomaterial Screening Approach to Reveal Microenvironmental Mechanisms of Drug Resistance

TOC FigureDrug response screening, gene expression, and kinome signaling were combined across biomaterial platforms to combat adaptive resistance to sorafenib.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC=\"FIGDIR/small/168039_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (60K):\norg.highwire.dtl.DTLVardef@a7af0eorg.highwire.dtl.DTLVardef@d5f2bborg.highwire.dtl.DTLVardef@330bd6org.highwire.dtl.DTLVardef@14e1f1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Insight BoxWe combined biomaterial platforms, drug screening, and systems biology to identify mechanisms of extracellular matrix-mediated adaptive resistance to RTK-targeted cancer therapies. Drug response was significantly varied across biomaterials with altered stiffness, dimensionality, and cell-cell contacts, and kinome reprogramming was responsible for these differences in drug sensitivity. Screening across many platforms and applying a systems biology analysis were necessary to identify MEK phosphorylation as the key factor associated with variation in drug response. This method uncovered the combination therapy of sorafenib with a MEK inhibitor, which decreased viability on and within biomaterials in vitro, but was not captured by screening on tissue culture plastic alone. This combination therapy also reduced tumor burden in vivo, and revealed a promising approach for combating adaptive drug resistance.\n\nAbstractTraditional drug screening methods lack features of the tumor microenvironment that contribute to resistance. Most studies examine cell response in a single biomaterial platform in depth, leaving a gap in understanding how extracellular signals such as stiffness, dimensionality, and cell-cell contacts act independently or are integrated within a cell to affect either drug sensitivity or resistance. This is critically important, as adaptive resistance is mediated, at least in part, by the extracellular matrix (ECM) of the tumor microenvironment. We developed an approach to screen drug responses in cells cultured on 2D and in 3D biomaterial environments to explore how key features of ECM mediate drug response. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments.

bioengineering↗

bioRxiv2017

Imaging action potential in single mammalian neurons by tracking the accompanying sub-nanometer mechanical motion

Action potentials in neurons have been studied traditionally by the patch clamp and more recently by the fluorescence detection methods. Here we describe a label-free optical imaging method that can measure mechanical motion in single cells with sub-nanometer detection limit and sub-millisecond temporal resolution. Using the method, we have observed sub-nanometer mechanical motion accompanying the action potential in single mammalian neurons. The shape and width of the transient displacement are similar to those of the electrically recorded action potential, but the amplitude varies from neuron to neuron, and from one region of a neuron to another, ranging from 0.2 - 0.4 nm. The work indicates that action potentials may be studied non-invasively in single mammalian neurons by label-free imaging of the accompanying subnanometer mechanical motion.

bioengineering↗

bioRxiv2017

Synthetic single domain antibodies for the conformational trapping of membrane proteins

Single domain antibodies called nanobodies are excellent affinity reagents for membrane proteins. However, their generation relies on immunizations, which is only amenable to robust proteins and impedes selections in the presence of non-covalent or toxic ligands. To overcome these key limitations, we developed a novel in vitro selection platform, which builds on synthetic nanobodies called sybodies. Inspired by the shape diversity of natural nanobodies, three sybody libraries exhibiting different randomized surface shapes were engineered for high thermal stability. Using ribosome display, exceptionally large libraries were pre-enriched against membrane protein targets and subsequently funneled into a robust phage display process, thereby reducing selection bias. We successfully generated conformation-selective, high affinity sybodies against the human glycine transporter GlyT1, the human equilibrative nucleotide transporter ENT1 and a bacterial ABC transporter. Our platform builds exclusively on commercially available reagents and enables non-specialized labs to generate conformation-specific binders against previously intractable protein targets.

bioengineering↗

bioRxiv2017

Biphasic, Switch-Like Isothermal DNA Amplification

We report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.

bioengineering↗

bioRxiv2017

RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems

Autofocus methods used in biomicroscopy are either based on the search of an optimal focus position - which requires suspending data collection during the optimization process - or on the continuous monitoring of the position of a fiducial plane - which may not coincide with the sample itself. Here, we introduce RAPID (Rapid Autofocus via Pupil-split Image phase Detection), a method for real-time image-based focus stabilization, applicable in all wide-field microscopy systems. We demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to light-sheet microscopy. RAPID provides a universal autofocus solution for automated microscopy, and enables quantitative assays otherwise impossible in a standard microscope, such as 3D tracking of fast-moving organisms.

bioengineering↗

bioRxiv2017

OptoGap: an optogenetics-enabled assay for quantification of cell-cell coupling in multicellular cardiac tissue

Intercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and nonexcitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g. cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells, e.g. cardiomyocytes, that are light-insensitive. Cell-cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. Its potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modeling and proper calibration.\n\nIntercellular coupling is a fundamental form of communication between cells, essential for the synchronization of physiological processes in different organs. Pathologically altered coupling or the emergence of de novo coupling between native and donor cells are problems of interest in many cardiac applications, e.g. during cell delivery and cell integration for cardiac repair therapy1,2. In particular, interactions between cardiomyocytes and fibroblasts are of interest, especially the pro-arrhythmic increase in coupling as the latter transition to myofibroblasts3-6.\n\nElectrical coupling in cardiac tissue is mediated primarily by low-resistance paths formed by gap-junctional proteins (connexins), that can link cardiomyocytes (CMs) to each other and to non-cardiomyocytes (nCMs), such as fibroblasts. Qualitative and quantitative methods, e.g. immunofluorescence, messenger RNA and Western blots, are often used to assay connexin expression levels as a surrogate measure of coupling, but they do not provide functional information. A method for direct quantification of cell-cell coupling within the multicellular tissue context is highly desirable.

bioengineering↗

bioRxiv2017

System-level studies of a cell-free transcription-translation platform for metabolic engineering

Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development.

bioengineering↗

bioRxiv2017

Quantifying the thermostability landscape separating plant sesquiterpene synthase orthologs using Maximum Entropy

As global temperatures rise due to climate change, crops are becoming increasingly vulnerable to failure. We tested the accessibility of thermodynamic phenotypic diversity within limited positional changes using 29=512 mutants of a terpene synthase (TPS), Tobacco 5-epi-Aristolchene synthase. First, we measured the thermal unfolding curves of each mutant and found that mutations shifted the Tms both higher and lower, including a cohort of mutants that failed to fold. The low correlation coefficient between the Tms of these mutants and the proportions of each terpene product output by the enzymes revealed that thermostability and product output are independent traits. Maximum Noise Entropy analyses were used to analyze the impact of the 9 mutational positions on thermostability, revealing that three of these positions were mainly responsible for the trait of increased Tm. These positions form a functional network as measured by the nonlinearity of their combined effects on thermostability. Unexpectedly, the strongly destabilizing positions combine nonlinearly to ameliorate each others deleterious effects resulting in a synergistic dampening. Taken together, our study shows the high potential for specialized metabolic enzyme engineering but also reveals a complex interconnected system of amino acids that will continue to evade perfect predictability.

bioengineering↗

bioRxiv2017

An Open-Source, Programmable Pneumatic Setup for Operation and Automated Control of Single- and Multi-Layer Microfluidic Devices

Microfluidic technologies have been used across diverse disciplines (e.g. high-throughput biological measurement, fluid physics, laboratory fluid manipulation) but widespread adoption has been limited due to the lack of openly disseminated resources that enable non-specialist labs to make and operate their own devices. Here, we report the open-source build of a pneumatic setup capable of operating both single and multilayer (Quake-style) microfluidic devices with programmable scripting automation. This setup can operate both simple and complex devices with 48 device valve control inputs and 18 sample inputs, with modular design for easy expansion, at a fraction of the cost of similar commercial solutions. We present a detailed step-by-step guide to building the pneumatic instrumentation, as well as instructions for custom device operation using our software, Geppetto, through an easy-to-use GUI for live on-chip valve actuation and a scripting system for experiment automation. We show robust valve actuation with near real-time software feedback and demonstrate use of the setup for high-throughput biochemical measurements on-chip. This open-source setup will enable specialists and novices alike to run microfluidic devices easily in their own laboratories.\n\nO_TBL View this table:\norg.highwire.dtl.DTLVardef@d25ba2org.highwire.dtl.DTLVardef@7dfd9eorg.highwire.dtl.DTLVardef@1959573org.highwire.dtl.DTLVardef@186dceaorg.highwire.dtl.DTLVardef@111af70_HPS_FORMAT_FIGEXP M_TBL O_TABLECAPTIONSpecifications table\n\nC_TABLECAPTION C_TBL

bioengineering↗

bioRxiv2017

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites.\n\nSignificance StatementGenome editing has been revolutionized by the engineering of molecular scissors that cut DNA at a predetermined location on the chromosome. When these molecular scissors are expressed within cells, these scissors cut the genomic DNA at the designated target site, and the cells respond by repairing the cut sites. This repair process frequently introduces mutations at the target cut site. The more efficient the molecular scissors, the more number of cells in a treated culture dish exhibit these mutations at the cut site. Investigators therefore design several molecular scissors targeting the same region on the chromosome to identify the best ones. We describe a new recipe to measure scissors efficiency in target site cutting.

bioengineering↗

bioRxiv2017

Dynamic, non-contact 3D sample rotation for microscopy

In vivo imaging of growing and developing samples requires a dynamic adaptation of the sample orientation to continuously achieve optimal performance. Here, we present how, after the injection of magnetic beads, a sample can be freely positioned by applying a magnetic field. We demonstrate its performance for zebrafish on an epi-fluorescence microscope and on a light sheet system for superior multi-view acquisition.

bioengineering↗

bioRxiv2017

Targeting individual cells by barcode in pooled sequence libraries

There is rising interest in applying single-cell transcriptome analysis and other single-cell sequencing methods to resolve differences between cells. Pooled processing of thousands of single cells is now routinely practiced by introducing cell-specific DNA barcodes early in cell processing protocols1-5. However, researchers must sequence a large number of cells to sample rare subpopulations6-8, even when fluorescence-activated cell sorting (FACS) is used to pre-enrich rare cell populations. Here, a new molecular enrichment method is used in conjunction with FACS enrichment to enable efficient sampling of rare dendritic cell (DC) populations, including the recently identified AXL+SIGLEC6+ (AS DCs) subset7, within a 10X Genomics single-cell RNA-Seq library. DC populations collectively represent 1-2% of total peripheral blood mononuclear cells (PBMC), with AS DC representing only 1-3% of human blood DCs and 0.01-0.06% of total PBMCs.

bioengineering↗

bioRxiv2017

Measuring Biotherapeutic Viscosity and Degradation On-Chip with Particle Diffusometry

In absence of efficient ways to test drug stability and efficacy, pharmaceuticals that have been stored outside of set temperature conditions are destroyed, often at great cost. This is especially problematic for biotherapeutics, which are highly sensitive to temperature fluctuations. Current platforms for assessing the stability of protein-based biotherapeutics in high throughput and in low volumes are unavailable outside of research and development laboratories and are not efficient for use in production, quality control, distribution, or clinical settings. In these alternative environments, microanalysis platforms could provide significant advantages for the characterization of biotherapeutic degradation. Here we present particle diffusometry (PD), a new technique to study degradation of biotherapeutic solutions. PD uses a simple microfluidic chip and microscope setup to calculate the Brownian motion of particles in a quiescent solution using a variation of particle image velocimetry (PIV) fundamentals. We show that PD can be used to measure the viscosity of protein solutions to discriminate intact protein from degraded samples as well as to determine the change in viscosity as a function of therapeutic concentration. PD viscosity analysis is applied to two particularly important biotherapeutic preparations: insulin, a commonly used protein for diabetic patients, and monoclonal antibodies which are an emerging class of biotherapeutics used to treat a variety of diseases such as autoimmune disorders and cancer. PD-based characterization of solution viscosity is a new tool for biotherapeutic analysis, and owing to its easy setup could readily be implemented at key points of the pharmaceutical delivery chain and in clinical settings.

bioengineering↗

bioRxiv2017

cytoNet: Network Analysis of Cell Communities

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNets capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin 4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains. Availability and ImplementationQutubLab.org/how | cytoNet contact: cytoNetProject@gmail.com Brain Initiative Alliance Toolmaker cytoNet site: https://www.braininitiative.org/toolmakers/resources/cytonet/ Author / Lay SummarycytoNet provides an online tool to rapidly characterize relationships between objects within images and video frames. To study complex tissue, cell and subcellular topologies, cytoNet integrates vision science with the mathematical technique of graph theory. This allows the method to simultaneously identify environmental effects on single cells and on network topology. cytoNet has versatile use across neuroscience, stem cell biology and regenerative medicine. cytoNet applications described in this study include: (1) characterizing how sensing pain alters neural circuit activity, (2) quantifying how vascular cells respond to neurotrophic stimuli overexpressed in the brain after injury or exercise, (3) delineating features of fat tissue that may confer resistance to obesity and (4) uncovering structure-function relationships of human stem cells as they transform into neurons.

bioengineering↗