A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.

Cell-free nucleic acids (cfNAs) such as short non-coding microRNA (miRNA) and circulating tumor DNA (ctDNA) that reside in bodily fluids have emerged as potential cancer biomarkers. Methods for the rapid, highly specific, and sensitive monitoring of cfNAs in biofluids have, therefore, become increasingly attractive as clinical diagnosis tools. As a next generation technology, we provide a practical guide for an amplification-free, single molecule Förster resonance energy transfer (smFRET)-based kinetic fingerprinting approach termed intramolecular single molecule recognition through equilibrium Poisson sampling, or iSiMREPS, for the rapid detection and counting of miRNA and mutant ctDNA with virtually unlimited specificity and single molecule sensitivity. iSiMREPS utilizes a pair of fluorescent detection probes, wherein one probe immobilizes the target molecules on the surface, and the other probe transiently and reversibly binds to the target to generate characteristic time-resolved fingerprints as smFRET signal that are detected in a total internal reflection fluorescence microscope. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and nonspecific background binding. By accelerating kinetic fingerprinting using the denaturant formamide and reducing background signals by removing target-less probes from the surface via toehold-mediated strand displacement, iSiMREPS has been demonstrated to count miR-141 and EGFR exon 19 deletion ctDNA molecules with a limit of detection (LOD) of ~1 and 3 fM, respectively, as well as mutant allele fractions as low as 0.0001%, during a standard acquisition time of only ~10 s per field of view. In this review, we provide a detailed roadmap for implementing iSiMREPS more broadly in research and clinical diagnostics, combining rapid analysis, high specificity, and high sensitivity.

Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor.

Biofluid-derived cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that also increase the risk of sample loss and cross-contamination. To achieve the direct, rapid, and amplification-free detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein designed a single-molecule kinetic fingerprinting assay, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS). iSiMREPS exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures a target molecule onto the surface, while the other transiently interrogates the target to generate kinetic fingerprints by intramolecular single-molecule Förster resonance energy transfer (smFRET) that are recorded by single-molecule fluorescence microscopy and identify the target after kinetic filtering and data analysis. We optimize the sensor design, use formamide to further accelerate the fingerprinting kinetics, and maximize sensitivity by removing non-target-bound probes using toehold-mediated strand displacement to reduce background. We show that iSiMREPS can detect, in as little as 10 s, two distinct, promising cancer biomarkers-miR-141 and a common EGFR exon 19 deletion-reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.

Persistent increases of PKMζ in memory-activated neurons trace LTP maintenance during spatial long-term memory storage.

PKMζ is an autonomously active PKC isoform crucial for the maintenance of synaptic long-term potentiation (LTP) and long-term memory. Unlike other kinases that are transiently stimulated by second messengers, PKMζ is persistently activated through sustained increases in protein expression of the kinase. Therefore, visualizing increases in PKMζ expression during long-term memory storage might reveal the sites of its persistent action and thus the location of memory-associated LTP maintenance in the brain. Using quantitative immunohistochemistry validated by the lack of staining in PKMζ-null mice, we examined the amount and distribution of PKMζ in subregions of the hippocampal formation of wild-type mice during LTP maintenance and spatial long-term memory storage. During LTP maintenance in hippocampal slices, PKMζ increases in the pyramidal cell body and stimulated dendritic layers of CA1 for at least 2 hr. During spatial memory storage, PKMζ increases in CA1 pyramidal cells for at least 1 month, paralleling the persistence of the memory. During the initial expression of the memory, we tagged principal cells with immediate-early gene Arc promoter-driven transcription of fluorescent proteins. The subset of memory-tagged CA1 cells selectively increases expression of PKMζ during memory storage, and the increase persists in dendritic compartments within stratum radiatum for 1 month, indicating long-term storage of information in the CA3-to-CA1 pathway. We conclude that persistent increases in PKMζ trace the molecular mechanism of LTP maintenance and thus the sites of information storage within brain circuitry during long-term memory.

Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers.

Methods for detecting and quantifying disease biomarkers in biofluids with high specificity and sensitivity play a pivotal role in enabling clinical diagnostics, including point-of-care tests. The most widely used molecular biomarkers include proteins, nucleic acids, hormones, metabolites, and other small molecules. While numerous methods have been developed for analyzing biomarkers, most techniques are challenging to implement for clinical use due to insufficient analytical performance, high cost, and/or other practical shortcomings. For instance, the detection of cell-free nucleic acid (cfNA) biomarkers by digital PCR and next-generation sequencing (NGS) requires time-consuming nucleic acid extraction steps, often introduces enzymatic amplification bias, and can be costly when high specificity is required. While several amplification-free methods for detecting cfNAs have been reported, these techniques generally suffer from low specificity and sensitivity. Meanwhile, the quantification of protein biomarkers is generally performed using immunoassays such as enzyme-linked immunosorbent assay (ELISA); the analytical performance of these methods is often limited by the availability of antibodies with high affinity and specificity as well as the significant nonspecific binding of antibodies to assay surfaces. To address the drawbacks of existing biomarker detection methods and establish a universal diagnostics platform capable of detecting different types of analytes, we have developed an amplification-free approach, named single-molecule recognition through equilibrium Poisson sampling (SiMREPS), for the detection of diverse biomarkers with arbitrarily high specificity and single-molecule sensitivity. SiMREPS utilizes the transient, reversible binding of fluorescent detection probes to immobilized target molecules to generate kinetic fingerprints that are detected by single-molecule fluorescence microscopy. The analysis of these kinetic fingerprints enables nearly perfect discrimination between specific binding to target molecules and any nonspecific binding. Early proof-of-concept studies demonstrated the in vitro detection of miRNAs with a limit of detection (LOD) of approximately 1 fM and >500-fold selectivity for single-nucleotide polymorphisms. The SiMREPS approach was subsequently expanded to the detection of rare mutant DNA alleles from biofluids at mutant allele fractions of as low as 1 in 1 million, corresponding to a specificity of >99.99999%. Recently, SiMREPS was generalized to protein quantification using dynamically binding antibody probes, permitting LODs in the low-femtomolar to attomolar range. Finally, SiMREPS has been demonstrated to be suitable for the in situ detection of miRNAs in cultured cells, the quantification of small-molecule toxins and drugs, and the monitoring of telomerase activity at the single-molecule level. In this Account, we discuss the principles of SiMREPS for the highly specific and sensitive detection of molecular analytes, including considerations for assay design. We discuss the generality of SiMREPS for the detection of very disparate analytes and provide an overview of data processing methods, including the expansion of the dynamic range using super-resolution analysis and the improvement of performance using deep learning algorithms. Finally, we describe current challenges, opportunities, and future directions for the SiMREPS approach.

Ultraspecific analyte detection by direct kinetic fingerprinting of single molecules.

The detection and quantification of biomarkers have numerous applications in biological research and medicine. The most widely used methods to detect nucleic acids require amplification via the polymerase chain reaction (PCR). However, errors arising from the imperfect copying fidelity of DNA polymerases, limited specificity of primers, and heat-induced damage reduce the specificity of PCR-based methods, particularly for single-nucleotide variants. Furthermore, not all analytes can be amplified efficiently. While amplification-free methods avoid these pitfalls, the specificity of most such methods is strictly constrained by probe binding thermodynamics, which for example hampers detection of rare somatic mutations. In contrast, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) provides ultraspecific detection with single-molecule and single-nucleotide sensitivity by monitoring the repetitive interactions of a fluorescent probe with surface-immobilized targets. In this review, we discuss SiMREPS in comparison with other analytical approaches, and describe its utility in quantifying a range of nucleic acids and other analytes.

Development of bone screw using novel biodegradable composite orthopedic biomaterial: from material design to in vitro biomechanical and in vivo biocompatibility evaluation.

A novel composite biomaterial for bone-soft tissue fixation applications was developed. MgO-Silk-PCL, Silk-PCL and MgO-PCL composites were prepared with variable filler concentrations (0, 10, 20 and 30% w/w of MgO nanoparticles and 0%, 5%, 10%, 20% and 30% of degummed silk fiber) in PCL polymer. The highest mechanical properties were obtained with 10% MgO and 20% Silk composite (MSP) wih 1.7× better tensile strength and 7.5× tensile modulus over PCL. It exhibited good cell viability, adhesion and hemocompatibility, increased cell proliferation and differentiation. MgO filler contributed more in increasing tensile strength, whereas silk fiber towards modulus, imparting a synergistic effect on mechanical performance. Prototype bone screws were molded using the MSP composite in a custom-designed mold. It showed significantly increased degradation (2.7 fold after 60 days) in PBS attributable to binary filler phase as compared to PCL. In vivo biosafety studies of MgO-silk-PCL composite screw in SD rats by subcutaneous implantation showed moderate inflammation at 2 weeks which subsided after 4th week. No toxic effect was seen in histopathology of vital organs and in blood parameters. Composite screw showed 2× pull-out strength of PCL in synthetic bone, therefore a potential candidate for bone-soft fixation applications like resorbable orthopedic screws for ACL reconstruction.

Oral dosing of pentoxifylline, a pan-phosphodiesterase inhibitor restores bone mass and quality in osteopenic rabbits by an osteogenic mechanism: A comparative study with human parathyroid hormone.

The non-selective phosphodiesterase inhibitor pentoxifylline (PTX) is used for the treatment of intermittent claudication due to artery occlusion. Previous studies in rodents have reported salutary effects of the intraperitoneal administration of PTX in segmental bone defect and fracture healing, as well as stimulation of bone formation. We determined the effect of orally dosed PTX in skeletally mature ovariectomized (OVX) rabbits with osteopenia. The half-maximal effective concentration (EC50) of PTX in rabbit bone marrow stromal cells was 3.07 ±â€¯1.37 nM. The plasma PTX level was 2.05 ±â€¯0.522 nM after a single oral dose of 12.5mg/kg, which was one-sixth of the adult human dose of PTX. Four months of daily oral dosing of PTX at 12.5 mg/kg to osteopenic rabbits completely restored bone mineral density, bone mineral content (BMC), microarchitecture and bone strength to the level of the sham-operated (ovary intact) group. The bone strength to BMC relationship between PTX and sham was similar. The bone restorative effect of PTX was observed in both axial and appendicular bones. In osteopenic rabbits, PTX increased serum amino-terminal propeptide, mineralized nodule formation by stromal cells and osteogenic gene expression in bone. PTX reversed decreased calcium weight percentage and poor crystal packing found in osteopenic rabbits. Furthermore, similar to parathyroid hormone (PTH), PTX had no effect on bone resorption. Taken together, our data show that PTX completely restored bone mass, bone strength and bone mineral properties by an anabolic mechanism. PTX has the potential to become an oral osteogenic drug for the treatment of post-menopausal osteoporosis.

Magnesium oxide nanoparticle-loaded polycaprolactone composite electrospun fiber scaffolds for bone-soft tissue engineering applications: in-vitro and in-vivo evaluation.

The objective of the present investigation was to assess the potential of magnesium oxide nanoparticle (MgO NP)-loaded electrospun polycaprolactone (PCL) polymer composites as a bone-soft tissue engineering scaffold. MgO NPs were synthesized using a hydroxide precipitation sol-gel method and characterized using field emission gun-scanning electron microscopy/energy-dispersive x-ray spectroscopy (FEG-SEM/EDS), field emission gun-transmission electron microscopy (FEG-TEM), and x-ray diffraction (XRD) analysis. PCL and MgO-PCL nanocomposite fibers were fabricated using electrospinning with trifluoroethanol as solvent at 19 kV applied voltage and 1.9 ml h-1 flow rate as optimized process parameters, and were characterized by FEG-TEM, FEG-SEM/EDS, XRD, and differential scanning calorimetry analyses. Characterization studies of as-synthesized nanoparticles revealed diffraction peaks indexed to various crystalline planes peculiar to MgO particles with hexagonal and cubical shape, and 40-60 nm size range. Significant improvement in mechanical properties (tensile strength and elastic modulus) of nanocomposites was observed as compared to neat polymer specimens (fourfold and threefold, respectively), due to uniform dispersion of nanofillers along the polymer fiber length. There was a remarkable bioactivity shown by nanocomposite scaffolds in immersion test, as indicated by formation of surface hydroxyapatite layer by the third day of incubation. MgO-loaded electrospun PCL mats showed enhanced in-vitro biological performance with osteoblast-like MG-63 cells in terms of adhesion, proliferation, and marked differentiation marker activity owing to greater surface roughness, nanotopography, and hydrophilicity facilitating higher protein adsorption. In-vivo subcutaneous implantation study in Sprague Dawley rats revealed initial moderate inflammatory tissue response near implant site at the second week timepoint that subsided later (eighth week) with no adverse effect on vital organ functionalities as seen in histopathological analysis supported by serum biochemical and hematological parameters which did not deviate significantly from normal physiological range, indicating good biocompatibility in-vivo. Thus, MgO-PCL nanocomposite electrospun fibers have potential as an efficient scaffold material for bone-soft tissue engineering applications.

Bioconductive 3D nano-composite constructs with tunable elasticity to initiate stem cell growth and induce bone mineralization.

Bioactive 3D composites play an important role in advanced biomaterial design to provide molecular coupling and improve integrity with the cellular environment of the native bone. In the present study, a hybrid lyophilized polymer composite blend of anionic charged sodium salt of carboxymethyl chitin and gelatin (CMChNa-GEL) reinforced with nano-rod agglomerated hydroxyapatite (nHA) has been developed with enhanced biocompatibility and tunable elasticity. The scaffolds have an open, uniform and interconnected porous structure with an average pore diameter of 157±30µm and 89.47+0.03% with four dimensional X-ray. The aspect ratio of ellipsoidal pores decrease from 4.4 to 1.2 with increase in gelatin concentration; and from 2.14 to 1.93 with decrease in gelling temperature. The samples were resilient with elastic stain at 1.2MPa of stress also decreased from 0.33 to 0.23 with increase in gelatin concentration. The crosslinker HMDI (hexamethylene diisocyanate) yielded more resilient samples at 1.2MPa in comparison to glutaraldehyde. Increased crosslinking time from 2 to 4h in continuous compression cycle show no improvement in maximum elastic stain of 1.2MPa stress. This surface elasticity of the scaffold enables the capacity of these materials for adherent self renewal and cultivation of the NTERA-2 cL.D1 (NT2/D1), pluripotent embryonal carcinoma cell with biomechanical surface, as is shown here. Proliferation with MG-63, ALP activity and Alizarin red mineralization assay on optimized scaffold demonstrated ***p<0.001 between different time points thus showing its potential for bone healing. In pre-clinical study histological bone response of the scaffold construct displayed improved activity of bone regeneration in comparison to self healing of control groups (sham) up to week 07 after implantation in rabbit tibia critical-size defect. Therefore, this nHA-CMChNa-GEL scaffold composite exhibits inherent and efficient physicochemical, mechanical and biological characteristics based on gel concentrations, gelatin mixing and gelling temperature thus points to creating bioactive 3D scaffolds with tunable elasticity for orthopedic applications.

A Pilot Study Assessing the Impact of 3-D Printed Models of Aortic Aneurysms on Management Decisions in EVAR Planning.

INTRODUCTION: Endovascular repair of aortic aneurysms with difficult anatomy is challenging. There is no consensus for planning such procedures. METHODS: Six cases of aortic aneurysms with challenging anatomical features, such as short, angulated, and conical necks and tortuous iliacs were harvested. The computed tomography (CT) scans were anonymized. Lifesize 3-dimensional (3-D) printed models were created of the lumen. Endovascular operators were asked to review the CT angiography (CTA), make a management plan, and give an indication of their confidence. They were then presented with the equivalent model and asked to review their decision. Their attitudes to such models were briefly surveyed. RESULTS: A total of 28 endovascular operators reviewed 144 cases. After review of the physical model, the management plan changed in 29 (20.1%) of 144 cases. Initial plan after CTA review was endovascular 73.6%, open repair 22.9%, and second opinion 3.5%. After model review, this became endovascular 67.4%, open repair 19.4%, and second opinion 4.8%. Although the general trend was toward more open procedures, off-label techniques reduced from 19.4% to 15.2% following model review. When the management plan did not change, level of confidence did increase in 37 (43.5%) of 85 cases. The majority of operators stated that they would find models useful for planning in some procedures. For 1 case, the change in the percentage of participants being sure in the management plan was statistically significant (P = .031). CONCLUSION: The 3-D printed models may be potentially useful in planning cases with EVAR. It is a paradigm that warrants further investigation.

An expedient stereoselective and chemoselective synthesis of bicyclic oxazolidinones from quinols and isocyanates.

A mild and efficient synthesis of bicyclic oxazolidinones from quinols and isocyanates, under DBU-mediated conditions at room temperature, is described. The aza-Michael addition to substituted cyclohexadienones is stereoselective and chemoselective.

Multivalent niacin nanoconjugates for delivery to cytoplasmic lipid droplets.

We report here the design, synthesis, and properties, of multifunctional niacin nanoconjugates based on dendritic, miktoarm and linear backbone nanocarriers, using "click" chemistry. The conjugates were in this instance used to deliver the therapeutic agent niacin to lipid droplets. The desired combination of niacin, a lipophilic fluorescent dye (BODIPY), and polyethylene glycol (PEG), was achieved by covalently linking the desired agents to the selected carrier. The nanocarriers containing niacin and BODIPY were found almost exclusively within cytoplasmic lipid droplets in the cells used in this study (living hepatocytes and microglia), whereas the trifunctional carrier containing niacin, BODIPY and PEG was partially localized within these organelles but also elsewhere in the cytoplasmic compartment. Spectrofluorometric analyses, confocal microscopy and fluorescence cell sorting revealed different rates and extent of multifunctional conjugate(s) internalization in the two cell types. Even micromolar concentrations of the internalized multifunctional conjugates did not cause significant cell death or mitochondrial functional impairment, suggesting that they are suitable candidate nanostructures for lipid droplet imaging and for targeting drugs to these cellular organelles. These studies provide an efficient and easy way to synthesize multifunctional nanocarriers by click chemistry, applicable to the synthesis of related multifunctional nanostructures and to their use in the targeting of cellular organelles, including lipid droplets.

Bilayer edge and curvature effects on partitioning of lipids by tail length: atomistic simulations.

The partitioning of lipids among different microenvironments in a bilayer is of considerable relevance to characterization of composition variations in biomembranes. Atomistic simulation has been ill-suited to model equilibrated lipid mixtures because the time required for diffusive exchange of lipids among microenvironments exceeds typical submicrosecond molecular dynamics trajectories. A method to facilitate local composition fluctuations, using Monte Carlo mutations to change lipid structures within the semigrand-canonical ensemble (at a fixed difference in component chemical potentials, Deltamu), was recently implemented to address this challenge. This technique was applied here to mixtures of dimyristoylphosphatidylcholine and a shorter-tail lipid, either symmetric (didecanoylphosphatidylcholine (DDPC)) or asymmetric (hexanoyl-myristoylphosphatidylcholine), arranged in two types of structure: bilayer ribbons and buckled bilayers. In ribbons, the shorter-tail component showed a clear enrichment at the highly curved rim, more so for hexanoyl-myristoylphosphatidylcholine than for DDPC. Results on buckled bilayers were variable. Overall, the DDPC content of buckled bilayers tended to exceed by several percent the DDPC content of flat ones simulated at the same Deltamu, but only for mixtures with low overall DDPC content. Within the buckled bilayer structure, no correlation could be resolved between the sign or magnitude of the local curvature of a leaflet and the mean local lipid composition. Results are discussed in terms of packing constraints, surface area/volume ratios, and curvature elasticity.

Complementarity and clustering in a simple model mixed bilayer.

A bilayer of uniform thickness containing a mixture of long and short lipids is simulated using a parallel hard-rod model to illustrate the effect of transbilayer repulsions between the tails of the long component. Monte Carlo simulations show considerable entropy-driven clustering within each layer. Demixing reaches a maximum at the highest packing fraction of the liquid state and decreases as the system orders. The formation of complementary clusters of long and short rods on opposite sides of the bilayer increases translational freedom within each cluster by reducing constraints imposed by the opposing leaflet, an effect that becomes less important as rods lock into facing hexagonally ordered arrays.