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Automated segmentation of skin strata in reflectance confocal microscopy depth stacks

Reflectance confocal microscopy (RCM) is a powerful tool for in-vivo examination of a variety of skin diseases. However, current use of RCM depends on qualitative examination by a human expert to look for specific features in the different strata of the skin. Developing approaches to quantify features in RCM imagery requires an automated understanding of what anatomical strata is present in a given en-face section. This work presents an automated approach using a bag of features approach to represent en-face sections and a logistic regression classifier to classify sections into one of four classes (stratum corneum, viable epidermis, dermal-epidermal junction and papillary dermis). This approach was developed and tested using a dataset of 308 depth stacks from 54 volunteers in two age groups (20-30 and 50-70 years of age). The classification accuracy on the test set was 85.6%. The mean absolute error in determining the interface depth for each of the stratum corneum/viable epidermis, viable epidermis/dermal-epidermal junction and dermal-epidermal junction/papillary dermis interfaces were 3.1 m, 6.0 m and 5.5 m respectively. The probabilities predicted by the classifier in the test set showed that the classifier learned an effective model of the anatomy of human skin.

Bioengineering

Graphene oxide chemically decorated with hybrid Ag-Ru/chitosan nanoparticles: fabrication and properties

Hybridization of distinct materials into a single nanoplatform is relevant to advance materials properties for functional application such as biosensor platform. We report the synthesis and characterization of nanosheets of graphene oxide decorated with hybrid nanoparticles of silver-ruthenium bipyridine complex (Ag@[Ru(bpy)3]2+) core and chitosan shell. Hybrid nanoparticles were first obtained through a sequential wet-chemical approach using in situ reduction, electrostatic and coordination reaction. Oxygenated functional groups of graphene oxide and abundant amine groups of chitosan layer on the surface of hybrid nanoparticles allowed the functionalization reaction. Changes in intrinsic optical, chemical and structural properties of graphene oxide due to hybrid nanoparticles were studied in depth using spectroscopic techniques and an electron microscope. Electrodes modified with nanosheets of graphene oxide-hybrid nanoparticles retain the biocompatibility and displayed an amplified redox property suitable for a broad range of sensing studies.

Bioengineering

Adaptive Evolution for Increased Biomass and Magnetic Nanoparticle Productivity of Magnetotactic Bacteria

Large magnetic nanoparticles (over 25 nm diameter) are a valuable commodity but remain difficult to synthesize using traditional chemical synthesis methods. Magnetotactic bacteria (MTBs) have evolved mechanisms to produce monodisperse, protected magnetic nanostructures within organelles (magnetosomes). Genomic diversity of MTB species result in unique particle properties that vary in shape and size ranging from 30 nm to 150 nm based on the genetic background. Culturing and engineering MTBs for the production of magnetic nanoparticles carries tremendous potential but is underdeveloped. This primarily because MTBs are difficult to culture and genetically manipulate, limitations that could be alleviated with adaptive evolution. We propose the magnetotrophic reactor, a novel bioreactor system for adaptively evolving MTBs for better growth and magnetosome production. This platform is projected to be superior to the traditional evolution methods since robust growth phenotypes can be selected for while maintaining selective pressure for magnetotaxis. We provide, herein, a quantitative basis for our platform including considerations of continuous evolution, sizing, and magnetic field pulsing. Our proposed fermentation process anticipates scalable production of 1 g/L per day of monodisperse magnetic nanoparticles to enable industrial applications.

Bioengineering

Automatic Synthesis of Anthropomorphic Pulmonary CT Phantoms

The great density and structural complexity of pulmonary vessels and airways impose limitations on the generation of accurate reference standards, which are critical in training and in the validation of image processing methods for features such as pulmonary vessel segmentation or artery-vein (AV) separations. The design of synthetic computed tomography (CT) images of the lung could overcome these difficulties by providing a database of pseudorealistic cases in a constrained and controlled scenario where each part of the image is differentiated unequivocally. This work demonstrates a complete framework to generate computational anthropomorphic CT phantoms of the human lung automatically. Starting from biological and image-based knowledge about the topology and relationships between structures, the system is able to generate synthetic pulmonary arteries, veins, and airways using iterative growth methods that can be merged into a final simulated lung with realistic features. Visual examination and quantitative measurements of intensity distributions, dispersion of structures and relationships between pulmonary air and blood flow systems show good correspondence between real and synthetic lungs.

Bioengineering

A spatio-temporal model for spontaneous thrombus formation in cerebral aneurysms

We propose a new numerical model to describe thrombus formation in cerebral aneurysms. This model combines CFD simulations with a set of bio-mechanical processes identified as being the most important to describe the phenomena at a large space and time scales. The hypotheses of the model are based on in vitro experiments and clinical observations. We document that we can reproduce very well the shape and volume of patient specific thrombus segmented in giant aneurysms.

Bioengineering

A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo.

The Cas9/CRISPR system is a powerful tool for studying gene function. Here we describe a method that allows temporal control of Cas9/CRISPER activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-CAS9) enables conditional Cas9 expression in vitro in the presence of an FKBP12 synthetic ligand and temporal control of gene-editing. Further, we show that this strategy can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest, without the latter being co-modulated. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic identification of essential genes and the interrogation of genes functional interactions.

Bioengineering

OptoDyCE: Automated System for High-Throughput All-Optical Dynamic Cardiac Electrophysiology

The improvement of preclinical cardiotoxicity testing, the discovery of new ion-channel-targeted drugs, and the phenotyping and use of stem-cell-derived cardiomyocytes and other biologics all necessitate high-throughput (HT), cellular-level electrophysiological interrogation tools. Optical techniques for actuation and sensing provide instant parallelism, enabling contactless dynamic HT testing of cells and small-tissue constructs, not affordable by other means. Here, we consider, computationally and experimentally, the limits of all-optical electrophysiology when applied to drug testing, then implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We validate optical actuation by virally introducing optogenetic drivers in (rat and human) cardiomyocytes or through the modular use of dedicated light-sensitive somatic \"spark\" cells. We show that this automated all-optical approach provides high-throughput means of cellular interrogation, i.e. allows for dynamic testing of >600 multicellular samples or compounds per hour, and yields high-content information about the action of a drug over time, space and doses.

Bioengineering

A circuit theory of protein structure

Protein secondary and tertiary structure is modeled as a linear passive analog lumped electrical circuit. Modeling is based on the structural similarity between helix, sheet, turn/loop, and helix pair in proteins and inductor, capacitor, resistor, and transformer in electrical circuits; it includes methods from circuit analysis and synthesis. A protein circuit is a one-port with a restrictive circuit topology (for example, the circuit for a secondary structure cannot be a Foster II ladder or a Wheatstone-like bridge). It has a rational positive real impedance function whose pole-zero distribution serves as a compact descriptor of secondary and tertiary structure, which is reminiscent of the Ramachandran plot. Standard circuit analysis methods such as node/loop equations and pole-zero maps may be used to study differences at the secondary and tertiary levels within and across proteins. Pairs of interacting proteins can be modeled as two-ports and studied via transfer functions. Similarly circuit synthesis methods can be used to construct protein circuits whose real counterparts may or may not exist. An analysis example shows how a protein circuit is constructed for thioredoxin and its pole-zero map obtained. A synthesis example shows how an electrical circuit with a single Brune section is obtained from a specified set of poles and zeros and then mapped to an artificial protein with a helix pair (corresponding to the transformer in the Brune section). Possible applications to folding, drug design, and visualization are indicated.

Bioengineering

A digital approach to protein identification and quantity estimation using tandem nanopores, peptidases, and database search

A digital approach to protein identification and quantity estimation using electrical measurements and database search is proposed. It is based on an electrolytic cell with two (three) nanopores and one (two) peptidase(s) covalently attached to the trans side of a pore. An unknown protein is digested by a reagent or peptidase into peptides ending in a known amino acid; the peptides enter the cell, pass through the first pore, and are fragmented by a high-specificity endopeptidase. The second enzyme, if present, is an exopeptidase that cleaves the fragments into single residues after the second pore. Level transitions in an ionic blockade or transverse current pulse due to residues in a fragment or individual pulses due to single residues are counted. This yields the positions of the endopeptidases target in the peptide, and, together with the peptides terminal residue, a partial sequence. Search through the Uniprot database for such sequences identifies over 90% of the proteins in the human proteome. The percentage can be increased by repeating the procedure with other reagents and cells specific to other residues, close to 100% may be possible. Sample purification to homogeneity is not required as the method applies to an arbitrary mixture of proteins; the quantity of a protein in the sample is estimated from the number of identifying peptides sensed over a long run. A Fokker-Planck model gives minimum enzyme turnover intervals required for ordered sensing of peptide fragments. With thick (80-100 nm) pores, required pulse resolution times are within the capability of CMOS detectors. The method can be implemented with existing technology; several related issues are discussed.

Bioengineering

Targeted insertion in well-characterized Drosophila cell lines using φC31 integrase

We describe an adaptation of {varphi}C31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with and without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrated the technology by integrating a cassette containing a Cu++-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays - a major emphasis of cell-based studies.

Bioengineering

Integrating Gene Synthesis and Microfluidic Protein Analysis for Rapid Protein Engineering

The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology, and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification, and characterization (mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 440 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF - DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

Bioengineering

Growing neuronal islands on multi-electrode arrays using an Accurate Positioning-μCP device

BackgroundMulti-electrode arrays (MEAs) allow non-invasive multiunit recording in-vitro from cultured neuronal networks. For sufficient neuronal growth and adhesion on such MEAs, substrate preparation is required. Plating of dissociated neurons on a uniformly prepared MEAs surface results in the formation of spatially extended random networks with substantial inter-sample variability. Such cultures are not optimally suited to study the relationship between defined structure and dynamics in neuronal networks. To overcome these shortcomings, neurons can be cultured with pre-defined topology by spatially structured surface modification. Spatially structuring a MEA surface accurately and reproducibly with the equipment of a typical cell-culture laboratory is challenging.\n\nNew MethodIn this paper, we present a novel approach utilizing microcontact printing (CP) combined with a custom-made device to accurately position patterns on MEAs with high precision. We call this technique AP-CP (accurate positioning micro-contact printing).\n\nComparison with existing MethodsOther approaches presented in the literature using CP for patterning either relied on facilities or techniques not readily available in a standard cell culture laboratory, or they did not specify means of precise pattern positioning.\n\nConclusionHere we present a relatively simple device for reproducible and precise patterning in a standard cell-culture laboratory setting. The patterned neuronal islands on MEAs provide a basis for high throughput electrophysiology to study the dynamics of single neurons and neuronal networks.

Bioengineering

An introduction to antibiotic-free techniques to eliminate Staphylococcus aureus from blood

Here, we describe the implementation of three techniques for capturing and killing Staphylococcus aureus in blood in vitro inside a medical tube. The first technique involves capturing and removing pathogens using antibodies that are coated, via a simple chemical process, on the inner walls of a modified medical tube (tube capturing technique). In the second technique, a photosensitizer-antibody conjugate adheres to the pathogens while in circulation. When blood flows through the same kind of tube, the conjugate is activated by near-infrared (NIR) light to kill pathogens (photodynamic therapy technique). For the third technique, pathogens are exposed to light in the ultraviolet (UV) range while circulating through a similar tube (UV technique), which kills the pathogens. We spiked blood with S. aureus, starting with about 107 CFU/mL and ending at 108 CFU/mL after 5 hours. While the spiked bacteria rapidly grew in nutrition-rich whole blood, each of the three techniques were able to independently remove between 61% and 84% more S. aureus in the experimental blood sample compared to the controls groups. When combined, these techniques demonstrated a removal rate between 87% and 92%.

Bioengineering

On the optimal use of isotherm models for the characterization of biosorption of lead onto algae

For the first time we apply a new method based on the mathematical derivation of some known isotherm from the Burr function which describes many birth-death (sorption-desorption) phenomena in ecology and economy. Therefore, in this study the experimental isotherm data of biosorption of Pb(II) onto algae was modeled to Langmuir, Hill-Sips, Brouers-Sotolongo, Brouers-Gaspard, and Redlich-Peterson isotherm models. The parameters of each model were determined by non linear fitting algorithms using Mathematica program. The maximum Pb(II) removal rate increased with the increase of temperature and reached the maximum value (98%) at the temperature of 40 {degrees}C. The results showed that the Hill-Sips and the Brouers-Sotolongo isotherms were definitely the most suitable models to satisfactorily describe biosorption of Pb(II) on the algal biomass. In addition, as these two models gave very close results, the use of an intermediate one the Brouers-Gaspard isotherm model could also describe the sorption in most cases. High coefficient of determination values were obtained by using non linear methods and these findings are contrary to most works in this field that use linearization methods. Further, this study showed that a complete set of data is necessary to have a good representation of the isotherm and using only coefficient of determination is not always an adequate tool to compare the goodness of the non linear fit of an isotherm models.

Bioengineering

Frequency Transformations and Spectral Gaps in Cochlear Implant Processing

In previous work[1] we created a mathematical model and identified a major source of distortion in Cochlear Implant(CI) processing which manifests itself in three forms, all of which are due to the nonlinear envelope processing algorithms which are widely used in some form or another in many current models. The first are spectral gaps or dead zones within the claimed frequency coverage range. This means that there exist regions of the spectrum for which there is no possible input that can produce an output at those frequencies. The second are frequency transformations which convert input tones of one frequency to tones of another frequency. Because this is a many-to-one transformation, it renders following a melody impossible, as the fundamental frequency of two different notes may be mapped to the same output frequency. This makes them impossible to distinguish, (although there may be differences in higher order harmonics that we will discuss). The third type of distortion are intermodulation products between input tones which yield additional output tones that were not present in either input. In the case of multiple talkers, these will compound the comprehension difficulty, as not only are the original spectral components of each speaker transformed, but additional nonexistent components have been added into the mix. This accounts for the great difficulty of CI users in noise.\n\nIn this work, we extend our earlier work in three ways. First, we clarify our description of spectral gaps which a number of readers pointed out was unclear, in that it implied that certain input tones will produce no response at all. In fact, all input tones will produce a response, but in most cases, the output will be frequency-transformed to a different frequency which the CI is capable of producing. Second, we graphically illustrate the input/output frequency transformation, so that the reader can clearly see at a glance how each frequency is altered. The form of this transformation is a staircase over most of the usable range, meaning that for single, pure tones all frequencies in the passband of a particular channel are mapped to a single frequency--the center frequency of that channel. As frequency continues to increase, all frequencies in the passband of the next channel become mapped to the center frequency of that channel, and so on. The exception is in the low frequencies, for reasons that we discuss. Third, in our earlier work we analyzed the simple case of only two pure tones within a single channel. Here we extend to the more realistic case of mixtures of complex tones, such as musical notes or the vowels of speech which may each have multiple harmonics extending throughout much of the audible frequency range. We find that, as expected, the output components of a source within a single channel often clash (are dissonant) with each other, and with those output components of that source (higher harmonics) which fall within other channels. So that instead of there being a harmonic or integral relationship among the output spectral components of each source, these components are no longer related to each other harmonically as they were at the input, thus producing a dissonant and grating percept. Furthermore, in the case two or more complex tones, additional intermodulation components are produced that further distort the sound. All these assertions are derived from theoretical considerations, and also noted from the authors own listening experience, and further confirmed from correspondence with other CI users.

Bioengineering

Open Hardware: Towards a Fully-Wireless Sub-Cranial Neuro-Implant for Measuring Electrocorticography Signals

Implantable neuronal interfaces to the brain are an important keystone for future medical applications. However, entering this field of research is difficult since such an implant requires components from many different areas of technology. Since the complete avoidance of wires is important due to the risk of infections and other long-term problems, means for wireless transmitting data and energy are a necessity which adds to the requirements. In recent literature many high-tech components for such implants are presented with remarkable properties. However, these components are typically not freely available for your system. Every group needs to re-develop their own solution. This raises the question if it is possible to create a reusable design for an implant and its external base-station, such that it allows other groups to use it as a starting point. In this article we try to answer this question by presenting a design based exclusively on commercial off-the-shelf components and studying the properties of the resulting system. Following this idea, we present a fully wireless neuronal implant for simultaneously measuring electrocorticography signals at 128 locations from the surface of the brain. All design files are available as open source.

Bioengineering

A Genetically Encoded Reporter for Diffusion Weighted Magnetic Resonance Imaging

The ability to monitor gene expression in intact, optically opaque animals is important for a multitude of applications including longitudinal imaging of transgene expression and long term tracking of cell based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatial and temporal resolution. However, existing MRI reporter genes, based primarily on metal-binding proteins or chemical exchange saturation transfer probes, are limited by their reliance on metal ions or relatively low sensitivity. In this work, we introduce a new class of genetically encoded reporters for MRI that work by altering water diffusivity. We show that overexpression of the human water channel aquaporin 1 (AQP1) produces robust contrast in diffusion weighted MRI by increasing effective water diffusivity in tissues by over 100% without affecting cell viability or morphology. Low levels of AQP1 expression ({small tilde}1 M), or mixed populations comprising as few as 10% AQP1-expressing cells, produce sufficient contrast to be observed by MRI. We demonstrate the utility of AQP1 in vivo by imaging gene expression in intracranial tumor xenografts. Overall, our results establish AQP1 as a new, metal-free, nontoxic and sensitive genetically encoded reporter for diffusion weighted MRI.

Bioengineering

DNA sequencing with stacked nanopores and exonuclease: a simulation-based analysis

Experiments (Clarke et al., Nat. Nanotech., 2009, 4, 265-270) have shown that DNA could be sequenced using a nanopore-based electrolytic cell in which an exonuclease enzyme in the cis chamber cleaves the leading base of a strand of DNA. The base is identified (with a reported accuracy that exceeds 99%) by the level of the current blockade it causes in the pore; a biological adapter inside slows down the base to lower the detection bandwidth required. This approach, which has been mathematically modeled, analyzed, and simulated (Reiner et al., J. Chem. Phys., 2012, 137, 214903; Brady and Reiner, ibid., 2015, 143, 074904), is error-prone because bases may be lost to diffusion or enter the pore out of order. Here a modified cell with three stacked nanopores (UNP, MNP, and DNP) and the enzyme attached to the trans side of UNP is proposed. Translocation of a base is simulated with the random walk of a dimensionless particle; the results show that bases translocate through MNP and DNP in sequence order without loss. If this holds in practice then with a suitably designed adapter and compatible enzyme turnover rates base calling accuracy would be limited only by the accuracy of base discrimination. Potential implementation issues are discussed.

Bioengineering