Hyperspectral Mapping of Human Primary and Stem Cells at Cell-Matrix Interfaces.

Extracellular matrices interface with cells to promote cell growth and tissue development. Given this critical role, matrix mimetics are introduced to enable biomedical materials ranging from tissue engineering scaffolds and tumor models to organoids for drug screening and implant surface coatings. Traditional microscopy methods are used to evaluate such materials in their ability to support exploitable cell responses, which are expressed in changes in cell proliferation rates and morphology. However, the physical imaging methods do not capture the chemistry of cells at cell-matrix interfaces. Herein, we report hyperspectral imaging to map the chemistry of human primary and embryonic stem cells grown on matrix materials, both native and artificial. We provide the statistical analysis of changes in lipid and protein content of the cells obtained from infrared spectral maps to conclude matrix morphologies as a major determinant of biochemical cell responses. The study demonstrates an effective methodology for evaluating bespoke matrix materials directly at cell-matrix interfaces.

Folding-Mediated DNA Delivery by α-Helical Amphipathic Peptides.

The renaissance gene therapy experiences these days requires specialist biomaterials and a systemic understanding of major factors influencing their ability to deliver genetic material. Peptide transfection systems represent a major class of such biomaterials. Several peptidic reagents have been commercialized to date. However, a comparative assessment of peptide sequences alone without auxiliary support or excipients against a common determinant for their ability to complex and deliver DNA has been lacking. This study cross-compares commercial and experimental transfection reagents from the same family of helical amphiphiles. Factors defining the efficacy of DNA delivery including cell uptake and gene expression are assessed along with cytotoxicity and DNA complexation. The results show that despite differences in sequence composition, length, and origin, peptide reagents of the same structural family exhibit similar characteristics and limitations with common variability trends. The cross-comparison revealed that functional DNA delivery is independent of the peptide sequence used but is mediated by the ability of the reagents to co-fold with DNA. Peptide folding proved to be the common determinant for DNA complexation and delivery by peptidic transfection reagents.

Peptide Nanoparticles for Gene Packaging and Intracellular Delivery.

Efficient gene transfer is necessary for advanced biotechnologies ranging from gene therapy to synthetic biology. Peptide nanoparticles provide suitable packaging systems promoting targeted gene expression or silencing. Though these systems have yet to match the transfection efficacy of viruses, they are typically devoid of drawbacks characteristic of virus-based vectors, including insertional mutagenesis, low packaging capacities, and strong immune responses. Given the promise nanoparticle formulations hold for gene delivery, methods of their preparation and accurate analysis of their physicochemical and biological properties become indispensable for progress toward systems that seek to outperform viral vectors. Herein, we report a comprehensive protocol for the preparation and characterization of archetypal peptide nanoparticles resulting from nonspecific and noncovalent complexation with RNA and DNA.

Cholesterol Anchors Enable Efficient Binding and Intracellular Uptake of DNA Nanostructures.

DNA nanostructures constitute a rapidly advancing tool-set for exploring cell-membrane functions and intracellular sensing or advancing delivery of biomolecular cargo  into cells. Chemical conjugation with lipid anchors can mediate binding of DNA nanostructures to synthetic lipid bilayers, yet how such structures interact with biological membranes and internalize cells has not been shown. Here, an archetypal 6-duplex nanobundle is used to investigate how lipid conjugation influences DNA cell binding and internalization kinetics. Cellular interactions of DNA nanobundles modified with one and three cholesterol anchors were assessed using flow cytometry and confocal microscopy. Nuclease digestion was used to distinguish surface-bound DNA, which is nuclease accessible, from internalized DNA. Three cholesterol anchors were found to enhance cellular association by up to 10-fold when compared with unmodified DNA. The bundles were endocytosed efficiently within 24 h. The results can help design controlled DNA binding and trafficking into cells.

DNA Origami Inside-Out Viruses.

A synthetic topology for everted viruses is reported. The topology is a single-stranded virion DNA assembled into a hollow cube with exterior decorated with HIV-Tat transduction domains. The cube incorporates a pH-responsive lid allowing for the controlled encapsulation of functional proteins and their transfer and release into live cells. Unlike viruses, which are protein shells with a [3,5]-fold rotational symmetry that encase nucleic acids, these cubes are [3, 4]-fold DNA boxes encapsulating proteins. Like viruses, such everted DNA-built viruses are monodisperse nanoscale assemblies that infect human cells with a specialist cargo. The design offers a bespoke bottom-up platform for engineering nonpolyhedral, nonprotein synthetic viruses.

Antimicrobial peptide capsids of de novo design.

The spread of bacterial resistance to antibiotics poses the need for antimicrobial discovery. With traditional search paradigms being exhausted, approaches that are altogether different from antibiotics may offer promising and creative solutions. Here, we introduce a de novo peptide topology that-by emulating the virus architecture-assembles into discrete antimicrobial capsids. Using the combination of high-resolution and real-time imaging, we demonstrate that these artificial capsids assemble as 20-nm hollow shells that attack bacterial membranes and upon landing on phospholipid bilayers instantaneously (seconds) convert into rapidly expanding pores causing membrane lysis (minutes). The designed capsids show broad antimicrobial activities, thus executing one primary function-they destroy bacteria on contact.

Autonomously folded α-helical lockers promote RNAi.

RNAi is an indispensable research tool with a substantial therapeutic potential. However, the complete transition of the approach to an applied capability remains hampered due to poorly understood relationships between siRNA delivery and gene suppression. Here we propose that interfacial tertiary contacts between α-helices can regulate siRNA cytoplasmic delivery and RNAi. We introduce a rationale of helical amphipathic lockers that differentiates autonomously folded helices, which promote gene silencing, from helices folded with siRNA, which do not. Each of the helical designs can deliver siRNA into cells via energy-dependent endocytosis, while only autonomously folded helices with pre-locked hydrophobic interfaces were able to promote statistically appreciable gene silencing. We propose that it is the amphipathic locking of interfacing helices prior to binding to siRNA that enables RNAi. The rationale offers structurally balanced amphipathic scaffolds to advance the exploitation of functional RNAi.

A De Novo Virus-Like Topology for Synthetic Virions.

A de novo topology of virus-like assembly is reported. The design is a trifaceted coiled-coil peptide helix, which self-assembles into ultrasmall, monodisperse, anionic virus-like shells that encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, these shells share the same physical characteristics of viruses being anionic, nonaggregating, abundant, hollow, and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virus-like structures reported to date, both synthetic and native, with the ability to adapt and transfer small and large nucleic acids. The design thus offers a promising solution for engineering bespoke artificial viruses with desired functions.

Structurally plastic peptide capsules for synthetic antimicrobial viruses.

A conceptual design for artificial antimicrobial viruses is described. The design emulates viral assembly and function to create self-assembling peptide capsules that promote efficient gene delivery and silencing in mammalian cells. Unlike viruses, however, the capsules are antimicrobial, which allows them to exhibit a dual biological function: gene transport and antimicrobial activity. Unlike other antimicrobials, the capsules act as pre-concentrated antimicrobial agents that elicit rapid and localised membrane-disrupting responses by converting into individual pores at their precise landing positions on membranes. The concept holds promise for engineering virus-like scaffolds with biologically tuneable properties.

Evaluation of standardization capability of current cardiac troponin I assays by a correlation study: results of an IFCC pilot project.

BACKGROUND: As a part of an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) project to prepare a commutable reference material for cardiac troponin I (cTnI), a pilot study evaluated current cTnI assays for measurement equivalence and their standardization capability. METHODS: cTnI-positive samples collected from 90 patients with suspected acute myocardial infarction were assessed for method comparison by 16 cTnI commercial assays according to predefined testing protocols. Seven serum pools prepared from these samples were also assessed. RESULTS: Each assay was assessed against median cTnI concentrations measured by 16 cTnI assays using Passing-Bablok regression analysis of 79 patient samples with values above each assay's declared detection limit. We observed a 10-fold difference in cTnI concentrations for lowest to highest measurement results. After mathematical recalibration of assays, the between-assay variation for patient samples reduced on average from 40% to 22% at low cTnI concentration, 37%-20% at medium concentration, and 29%-14% at high concentration. The average reduction for pools was larger at 16%, 13% and 7% for low, medium and high cTnI concentrations, respectively. Overall, assays demonstrated negligible bias after recalibration (y-intercept: -1.4 to 0.3 ng/L); however, a few samples showed substantial positive and/or negative differences for individual cTnI assays. CONCLUSIONS: All of the 16 commercial cTnI assays evaluated in the study demonstrated a significantly higher degree of measurement equivalence after mathematical recalibration, indicating that measurement harmonization or standardization would be effective at reducing inter-assay bias. Pooled sera behaved similarly to individual samples in most assays.

Exploitable length correlations in peptide nanofibres.

Sequence-prescribed biomolecular assemblies find increasing use in the development of novel nanostructured materials. Critical requirements for emerging designs remain in matching form with function. Peptide assembly diversifies form and supports function, but lacks control over both. Herein we exploit length correlations in peptide nanoscale fibres (form) using a model helical template. We establish that different assembly patterns result from a synergistic interplay between peptide length, net charge and folding and supra-molecular cooperativity, while correlating with increases in cell proliferation (function) as a function of peptide length. The revealed correlations offer an efficient rationale for the programming of longitudinally finite and biologically active nanoscale fibres.

Quantification of protein concentration using UV absorbance and Coomassie dyes.

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used.

Bayesian analysis of an international ELISA comparability study.

BACKGROUND: Immunoassays are biochemical tests applied to measure even very small amounts of substance using the highly specific binding between an antibody and its antigen. They have a wide range of applications. The measurement however, might be associated with substantial uncertainty; this can have significant consequences for any diagnosis, or clinical decision. An international comparability study was thus performed to assess the sources of uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. METHODS: In contrast to the original publication for this international comparability study, we reanalyse the data using Bayesian inference. This provides a statistically coherent approach to estimate ELISA concentrations and their associated uncertainties. RESULTS: The Bayesian uncertainties of individual ELISAs and laboratory estimates are considerably larger than previously reported uncertainties. The average concentrations estimated here differ from the ones estimated by each study participant. In general, this leads to different conclusions about the study. In particular, the inter- and intra-laboratory consistency is increased, and repeatability problems occur for fewer laboratories. CONCLUSIONS: Decisions which are based on plausible ranges of measurements (such as credible intervals), are generally superior to those solely based on point estimates (such as the mean). Reliable uncertainties are thus vital, and not only in metrology. In this paper, a general method is developed to derive concentration estimates and valid uncertainties for ELISAs. Guidance on applying this Bayesian method is provided and the importance of reliable uncertainties associated with ELISAs is underlined. The applicability and virtues of the presented method are demonstrated in the context of an international comparability study.

Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation.

In this study, the first steps in the development of a secondary reference measurement procedure (RMP) 'higher metrological order measurement procedure' to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 µg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies.

Polymer- and colloid-mediated bioassays, sensors and diagnostics.

Synthetic polymers and colloids are increasingly being exploited in bioassays to help measure gene expression, sequence genomes, monitor metabolic disorders and detect the presence of disease. This can be attributed to their potential to reduce reaction scales, improve throughput, lower costs and improve the sensitivity, selectivity, stability and reproducibility of assays. This review highlights the newest application areas, including some of the strategies employed, as well as major technical challenges and future opportunities. The move away from conventional assay approaches is being driven by a desire to improve our basic understanding of human biology, to diagnose diseases earlier, and to manage healthcare resources more efficiently. These endeavors are important owing to a rising world population and an increasing average life span.

Standardisation of cardiac troponin I measurement: past and present.

The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut-off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between-method variation was approximately 2- to 5-fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum-based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long-term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG-TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.

Quantitation of protein.

The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Methods are described to provide information on how to analyze protein concentration using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chemical mechanisms that are prone to interference from chemicals prevalent in many biological buffer preparations. A discussion of which assays are prone to interference and the selection of alternative methods is included.

An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.

BACKGROUND: Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. METHODS: The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories. RESULTS: Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54+/-0.22 mg/L, with the spread of data ranging around +/-35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty. CONCLUSIONS: Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation.

Fluorescent peptide probes for high-throughput measurement of protein phosphatases.

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.