Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.

The Z-disk or Z-line is located at the lateral borders of sarcomere, the fundamental unit of striated muscle. They provide mechanical stability and can boost contractility of cardiac myocytes. In this paper, we propose to generate a 3D model of Z-disks within single adult cardiac cells from an automated segmentation of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The proposed fully automated segmentation scheme is comprised of three main modules including “pre-processing”, “segmentation” and “refinement”. We represent a timely-efficient, simple, yet effective model to perform segmentation and refinement steps. Contrast stretching, and Gaussian kernels are used to pre- process the dataset, and well-known “Sobel operators” are used in the segmentation module. We have validated our model by comparing segmentation results with ground-truth annotated Z-disks in terms of pixel-wise accuracy. The results show that our model correctly detects Z-disks with 90.56% accuracy. Finally, the underlying network of Z-disks are rendered in 3D using ImageJ and IMARIS.

We report the preparation of degradable capsules via layer-by-layer assembly using polyelectrolyte (PE) polyrotaxanes (PRXs). The PRX capsules were prepared by the sequential deposition of PRXs onto silica particles followed by the dissolution of the silica cores. The colloidal stability of the PRX capsules that are formed depends on the salt/buffer solution used in the assembly process. Various salt/buffer combinations were examined to avoid aggregation of the core-shell particles during PRX assembly and core dissolution. Using appropriate assembly conditions, we prepared colloidally stable, robust capsules. PRX capsules consisting of eight layers of PE PRXs had a wall thickness of ~15 nm. The degradation of the PRX capsules was demonstrated through the disassembly of the PE PRXs using glutathione, which cleaves the disulfide bonds linking the end-capping groups of the PE PRXs. Given the supramolecular noncovalent structure of PRXs and their adjustable properties, it is expected that PRXs will be used as building blocks for assembling advanced capsules with unique and tailored properties.

The dynamic collision of emulsified water drops in the presence of non-ionic surfactants plays a crucial role in many practical applications. Interaction force between water drops coated with non-ionic food grade surfactants is expected to exhibit rich dynamic behavior that is not yet explored. The collision forces between immobilized water drops in canola oil in the presence of a well-known food grade surfactant polyglycerol polyricinoleate (PGPR) are measured at concentrations well below typically used to form stable emulsions. An extension or kink, attributed to a short-range attractive interaction due to PGPR bridging between the drops, was observed in the retract portion of the force curves at higher applied forces or slower collision velocities. The Stokes-Reynolds-Young-Laplace (SRYL) model was used to calculate theoretical force curves. For higher collisions velocities, the agreement between the calculated and experiment data was acceptable, but the SRYL model failed to describe the extension or kink feature observed at slower velocities below. Both the AFM data and the comparison to the model calculation indicated the presence of a short-range attractive force, not of a hydrodynamic origin, attributed to the bridging and extension of PGPR molecules on the surface of water drops below saturation of the interface.

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

AIMS: Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. METHODS: hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. RESULTS: The presence of hMSCs-either hAMSCs or hBMSCs-increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. CONCLUSIONS: This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

Multilayered graphene-based membranes are promising for a variety of applications related to ion or molecule transport, such as energy storage and water treatment. However, the complex three-dimensional cascading nanoslit-like structure embedded in the membrane makes it difficult to interpret and rationalize experimental results, quantitatively compare with the traditional membrane systems, and quantitatively design new membrane structures. In this paper, systematic numerical simulations were performed to establish an equivalent one-dimensional (1D) nanochannel model to represent the structure of multilayered graphene membranes. We have established a quantitative relationship between effective diffusion length and cross-section area of the 1D model and our recently developed two dimensional (2D) representative microstructure for graphene membranes. We find that only in the cases of a relatively large lateral size (> ~100 nm) and a small slit size (< 2 nm), the effective diffusion length and can be calculated by an over-simplified but often used model. Otherwise, they show complex dependence on all three structural parameters of the 2D structural model. Our equivalent 1D nano-channel model can reproduce experimental results very well except for h < 0.5 nm. The discrepancy could be attributed to the anomalous behaviour of molecules under nano-confinement that is not considered in our simulations. This model can also be extended to multilayered membranes assembled by other 2D materials.

Plants have a complex passive fluid transport system capable of internalizing small molecules from the environment, and this system offers an ideal route for augmenting plants with functional nanomaterials. Current plant augmentation techniques use pre-formed nanomaterials and permeabilizing agents or plant cuttings. A so far unexplored concept is the formation of the functional material, in situ, from precursors small enough to be passively internalized through the roots without harming the plants. Metal-organic frameworks are ideal for in situ synthesis as they are composed of metal ions coordinated with organic ligands and have recently been mineralized around single-celled organisms in mild aqueous conditions. Herein, the synthesis of two types of metal-organic frameworks, zinc(2-methylimidazole)2 and lanthanide2 (terephthalate)3 , are reported inside a variety of plants. In situ synchrotron experiments help elucidate the formation kinetics and crystal phases of the nano-biohybrid plants. Plants augmented with luminescent metal-organic frameworks are utilized for small molecule sensing, although other applications, such as pathogen sensing, proton conductive plants, improved CO2 capture, bacteria-free nitrogen fixation, drought and fungi-resistance, and enhanced photosynthesis and photocatalysis, are foreseeable. Overall, the generation of functional materials inside of fully intact plants could lead to more complex nano-biohybrid sensors and organisms augmented with superior performance characteristics.

Ion transport in nanoconfinement differs from that in bulk and has been extensively researched across scientific and engineering disciplines1-4. For many energy and water applications of nanoporous materials, concentration-driven ion diffusion is simultaneously subjected to a local electric field arising from surface charge or an externally applied potential. Due to the uniquely crowded intermolecular forces under severe nanoconfinement (<2 nm), the transport behaviours of ions can be influenced by the interfacial electrical double layer (EDL) induced by a surface potential, with complex implications, engendering unusual ion dynamics5-7. However, it remains an experimental challenge to investigate how such a surface potential and its coupling with nanoconfinement manipulate ion diffusion. Here, we exploit the tunable nanoconfinement in layered graphene-based nanoporous membranes to show that sub-2 nm confined ion diffusion can be strongly modulated by the surface potential-induced EDL. Depending on the potential sign, the combination and concentration of ion pairs, diffusion rates can be reversibly modulated and anomalously enhanced by 4~7 times within 0.5 volts, across a salt concentration gradient up to seawater salinity. Modelling suggests that this anomalously enhanced diffusion is related to the strong ion-ion correlations under severe nanoconfinement, and cannot be explained by conventional theoretical predictions.