Imaging therapeutic peptide transport across intestinal barriers.

Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides, due to the biological barriers associated with the gastrointestinal tract. In this review, we start by summarizing the pharmacological performance of several clinically relevant orally administrated therapeutic peptides, highlighting their low bioavailabilities. Thus, there is a strong need to increase the transport of peptide drugs across the intestinal barrier to realize future treatment needs and further development in the field. Currently, progress is hampered by a lack of understanding of transport mechanisms that govern intestinal absorption and transport of peptide drugs, including the effects of the permeability enhancers commonly used to mediate uptake. We describe how, for the past decades, mechanistic insights have predominantly been gained using functional assays with end-point read-out capabilities, which only allow indirect study of peptide transport mechanisms. We then focus on fluorescence imaging that, on the other hand, provides opportunities to directly visualize and thus follow peptide transport at high spatiotemporal resolution. Consequently, it may provide new and detailed mechanistic understanding of the interplay between the physicochemical properties of peptides and cellular processes; an interplay that determines the efficiency of transport. We review current methodology and state of the art in the field of fluorescence imaging to study intestinal barrier transport of peptides, and provide a comprehensive overview of the imaging-compatible in vitro, ex vivo, and in vivo platforms that currently are being developed to accelerate this emerging field of research.

Single-molecule DNA-mapping and whole-genome sequencing of individual cells.

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.

Contributions of the glycocalyx, endothelium, and extravascular compartment to the blood-brain barrier.

The endothelial cells that form the blood-brain barrier (BBB) are coated with glycocalyx, on the luminal side, and with the basement membrane and astrocyte endfeet, on the abluminal side. However, it is unclear how exactly the glycocalyx and extravascular structures contribute to BBB properties. We used two-photon microscopy in anesthetized mice to record passive transport of four different-sized molecules-sodium fluorescein (376 Da), Alexa Fluor (643 Da), 40-kDa dextran, and 150-kDa dextran-from blood to brain, at the level of single cortical capillaries. Both fluorescein and Alexa penetrated nearly the entire glycocalyx volume, but the dextrans penetrated less than 60% of the volume. This suggested that the glycocalyx was a barrier for large but not small molecules. The estimated permeability of the endothelium was the same for fluorescein and Alexa but several-fold lower for the larger dextrans. In the extravascular compartment, co-localized with astrocyte endfeet, diffusion coefficients of the dyes were an order of magnitude lower than in the brain parenchyma. This suggested that the astrocyte endfeet and basement membrane also contributed to BBB properties. In conclusion, the passive transport of small and large hydrophilic molecules through the BBB was determined by three separate barriers: the glycocalyx, the endothelium, and the extravascular compartment. All three barriers must be taken into account in drug delivery studies and when considering BBB dysfunction in disease states.

How To Characterize Individual Nanosize Liposomes with Simple Self-Calibrating Fluorescence Microscopy.

Nanosize lipid vesicles are used extensively at the interface between nanotechnology and biology, e.g., as containers for chemical reactions at minute concentrations and vehicles for targeted delivery of pharmaceuticals. Typically, vesicle samples are heterogeneous as regards vesicle size and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles-of their lipid bilayers and lumens, separately. A vesicle then images as two spots, one in each color channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles. They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Nonideal vesicles in the same images were characterized by how their four data violate the calibrated relationship established for ideal vesicles. In this way, our method yields size, shape, lamellarity, and encapsulation efficiency of each imaged vesicle. Applying this procedure to extruded samples of vesicles, we found that, contrary to common assumptions, only a fraction of vesicles are ideal.

Single-particle trajectories reveal two-state diffusion-kinetics of hOGG1 proteins on DNA.

We reanalyze trajectories of hOGG1 repair proteins diffusing on DNA. A previous analysis of these trajectories with the popular mean-squared-displacement approach revealed only simple diffusion. Here, a new optimal estimator of diffusion coefficients reveals two-state kinetics of the protein. A simple, solvable model, in which the protein randomly switches between a loosely bound, highly mobile state and a tightly bound, less mobile state is the simplest possible dynamic model consistent with the data. It yields accurate estimates of hOGG1's (i) diffusivity in each state, uncorrupted by experimental errors arising from shot noise, motion blur and thermal fluctuations of the DNA; (ii) rates of switching between states and (iii) rate of detachment from the DNA. The protein spends roughly equal time in each state. It detaches only from the loosely bound state, with a rate that depends on pH and the salt concentration in solution, while its rates for switching between states are insensitive to both. The diffusivity in the loosely bound state depends primarily on pH and is three to ten times higher than in the tightly bound state. We propose and discuss some new experiments that take full advantage of the new tools of analysis presented here.

Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing.

Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

Erratum: "Calibration-on-the-spot": How to calibrate an EMCCD camera from its images.

This corrects the article DOI: 10.1038/srep28680.

Classification of DNA nucleotides with transverse tunneling currents.

It has been theoretically suggested and experimentally demonstrated that fast and low-cost sequencing of DNA, RNA, and peptide molecules might be achieved by passing such molecules between electrodes embedded in a nanochannel. The experimental realization of this scheme faces major challenges, however. In realistic liquid environments, typical currents in tunneling devices are of the order of picoamps. This corresponds to only six electrons per microsecond, and this number affects the integration time required to do current measurements in real experiments. This limits the speed of sequencing, though current fluctuations due to Brownian motion of the molecule average out during the required integration time. Moreover, data acquisition equipment introduces noise, and electronic filters create correlations in time-series data. We discuss how these effects must be included in the analysis of, e.g., the assignment of specific nucleobases to current signals. As the signals from different molecules overlap, unambiguous classification is impossible with a single measurement. We argue that the assignment of molecules to a signal is a standard pattern classification problem and calculation of the error rates is straightforward. The ideas presented here can be extended to other sequencing approaches of current interest.

"Calibration-on-the-spot": How to calibrate an EMCCD camera from its images.

In order to count photons with a camera, the camera must be calibrated. Photon counting is necessary, e.g., to determine the precision of localization-based super-resolution microscopy. Here we present a protocol that calibrates an EMCCD camera from information contained in isolated, diffraction-limited spots in any image taken by the camera, thus making dedicated calibration procedures redundant by enabling calibration post festum, from images filed without calibration information.

How to determine local stretching and tension in a flow-stretched DNA molecule.

We determine the nonuniform stretching of and tension in a mega base pairs-long fragment of deoxyribonucleic acid (DNA) that is flow stretched in a nanofluidic chip. We use no markers, do not know the contour length of the DNA, and do not have the full DNA molecule inside our field of view. Instead, we analyze the transverse thermal motion of the DNA. Tension at the center of the DNA adds up to 16 pN, giving almost fully stretched DNA. This method was devised for optical mapping of DNA, specifically, DNA denaturation patterns. It may be useful also for other studies, e.g., DNA-protein interactions, specifically, their tension dependence. Generally, wherever long strands of DNA-e.g., native DNA extracted from human cells or bacteria-must be stretched with ease for inspection, this method applies.

Transition state theory demonstrated at the micron scale with out-of-equilibrium transport in a confined environment.

Transition state theory (TST) provides a simple interpretation of many thermally activated processes. It applies successfully on timescales and length scales that differ several orders of magnitude: to chemical reactions, breaking of chemical bonds, unfolding of proteins and RNA structures and polymers crossing entropic barriers. Here we apply TST to out-of-equilibrium transport through confined environments: the thermally activated translocation of single DNA molecules over an entropic barrier helped by an external force field. Reaction pathways are effectively one dimensional and so long that they are observable in a microscope. Reaction rates are so slow that transitions are recorded on video. We find sharp transition states that are independent of the applied force, similar to chemical bond rupture, as well as transition states that change location on the reaction pathway with the strength of the applied force. The states of equilibrium and transition are separated by micrometres as compared with angstroms/nanometres for chemical bonds.

How to connect time-lapse recorded trajectories of motile microorganisms with dynamical models in continuous time.

We provide a tool for data-driven modeling of motility, data being time-lapse recorded trajectories. Several mathematical properties of a model to be found can be gleaned from appropriate model-independent experimental statistics, if one understands how such statistics are distorted by the finite sampling frequency of time-lapse recording, by experimental errors on recorded positions, and by conditional averaging. We give exact analytical expressions for these effects in the simplest possible model for persistent random motion, the Ornstein-Uhlenbeck process. Then we describe those aspects of these effects that are valid for any reasonable model for persistent random motion. Our findings are illustrated with experimental data and Monte Carlo simulations.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly.

Configurational Statistics of Magnetic Bead Detection with Magnetoresistive Sensors.

Magnetic biosensors detect magnetic beads that, mediated by a target, have bound to a functionalized area. This area is often larger than the area of the sensor. Both the sign and magnitude of the average magnetic field experienced by the sensor from a magnetic bead depends on the location of the bead relative to the sensor. Consequently, the signal from multiple beads also depends on their locations. Thus, a given coverage of the functionalized area with magnetic beads does not result in a given detector response, except on the average, over many realizations of the same coverage. We present a systematic theoretical analysis of how this location-dependence affects the sensor response. The analysis is done for beads magnetized by a homogeneous in-plane magnetic field. We determine the expected value and standard deviation of the sensor response for a given coverage, as well as the accuracy and precision with which the coverage can be determined from a single sensor measurement. We show that statistical fluctuations between samples may reduce the sensitivity and dynamic range of a sensor significantly when the functionalized area is larger than the sensor area. Hence, the statistics of sampling is essential to sensor design. For illustration, we analyze three important published cases for which statistical fluctuations are dominant, significant, and insignificant, respectively.

Harmonic force spectroscopy measures load-dependent kinetics of individual human ß-cardiac myosin molecules.

Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using 'harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human ß-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.

Error filtering, pair assembly and error correction for next-generation sequencing reads.

MOTIVATION: Next-generation sequencing produces vast amounts of data with errors that are difficult to distinguish from true biological variation when coverage is low. RESULTS: We demonstrate large reductions in error frequencies, especially for high-error-rate reads, by three independent means: (i) filtering reads according to their expected number of errors, (ii) assembling overlapping read pairs and (iii) for amplicon reads, by exploiting unique sequence abundances to perform error correction. We also show that most published paired read assemblers calculate incorrect posterior quality scores. AVAILABILITY AND IMPLEMENTATION: These methods are implemented in the USEARCH package. Binaries are freely available at http://drive5.com/usearch. CONTACT: robert@drive5.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Concentrating genomic length DNA in a microfabricated array.

We demonstrate that a microfabricated bump array can concentrate genomic-length DNA molecules efficiently at continuous, high flow velocities, up to 40 µm/s, if the single-molecule DNA globule has a sufficiently large shear modulus. Increase in the shear modulus is accomplished by compacting the DNA molecules to minimal coil size using polyethylene glycol (PEG) derived depletion forces. We map out the sweet spot, where concentration occurs, as a function of PEG concentration and flow speed using a combination of theoretical analysis and experiment. Purification of DNA from enzymatic reactions for next-generation DNA-sequencing libraries will be an important application of this development.

Optimal estimation of diffusion coefficients from single-particle trajectories.

How does one optimally determine the diffusion coefficient of a diffusing particle from a single-time-lapse recorded trajectory of the particle? We answer this question with an explicit, unbiased, and practically optimal covariance-based estimator (CVE). This estimator is regression-free and is far superior to commonly used methods based on measured mean squared displacements. In experimentally relevant parameter ranges, it also outperforms the analytically intractable and computationally more demanding maximum likelihood estimator (MLE). For the case of diffusion on a flexible and fluctuating substrate, the CVE is biased by substrate motion. However, given some long time series and a substrate under some tension, an extended MLE can separate particle diffusion on the substrate from substrate motion in the laboratory frame. This provides benchmarks that allow removal of bias caused by substrate fluctuations in CVE. The resulting unbiased CVE is optimal also for short time series on a fluctuating substrate. We have applied our estimators to human 8-oxoguanine DNA glycolase proteins diffusing on flow-stretched DNA, a fluctuating substrate, and found that diffusion coefficients are severely overestimated if substrate fluctuations are not accounted for.