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DNA‐PAINT enabled super‐resolution imaging through the transient binding of fluorescently‐labelled single‐stranded DNA (ssDNA) imagers to target ssDNA. However, its performance is constrained by imager background fluorescence, resulting in relatively long image acquisition and potential artifacts. We designed a molecular beacon (MB) as the PAINT imager. Unbound MB in solution reduces the background fluorescence due to its natively quenched state. They are fluorogenic upon binding to target DNA to create individual fluorescence events. We demonstrate that MB‐PAINT provides localization precision similar to traditional linear imager DNA‐PAINT. We also show that MB‐PAINT is ideally suited for fast super‐resolution imaging of molecular tension probes in living cells, eliminating the potential of artifacts from free‐diffusing imagers in traditional DNA‐PAINT at the cell‐substrate interface. [ABSTRACT FROM AUTHOR]

DNA‐PAINT enabled super‐resolution imaging through the transient binding of fluorescently‐labelled single‐stranded DNA (ssDNA) imagers to target ssDNA. However, its performance is constrained by imager background fluorescence, resulting in relatively long image acquisition and potential artifacts. We designed a molecular beacon (MB) as the PAINT imager. Unbound MB in solution reduces the background fluorescence due to its natively quenched state. They are fluorogenic upon binding to target DNA to create individual fluorescence events. We demonstrate that MB‐PAINT provides localization precision similar to traditional linear imager DNA‐PAINT. We also show that MB‐PAINT is ideally suited for fast super‐resolution imaging of molecular tension probes in living cells, eliminating the potential of artifacts from free‐diffusing imagers in traditional DNA‐PAINT at the cell‐substrate interface. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are highly promising as drug delivery vehicles due to their nanoscale size, stability and biocompatibility. EVs possess natural targeting abilities and are known to traverse long distances to reach their target cells. This long-range organotropism and the ability to penetrate hard-to-reach tissues, including the brain, have sparked interest in using EVs for the targeted delivery of pharmaceuticals. In addition, EVs can be readily harvested from an individual's biofluids, making them especially suitable for personalized medicine applications. However, the targeting abilities of unmodified EVs have proven to be insufficient for clinical applications. Multiple attempts have been made to bioengineer EVs to fine-tune their on-target binding. Here, we summarize the current state of knowledge on the natural targeting abilities of native EVs. We also critically discuss the strategies to functionalize EV surfaces for superior long-distance targeting of specific tissues and cells. Finally, we review the challenges in achieving specific on-target binding of EV nanocarriers. [ABSTRACT FROM AUTHOR]

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (~4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation. [ABSTRACT FROM AUTHOR]

Hydrophobic interactions guide protein folding, multidomain protein assembly, receptor–ligand binding, membrane formation, and cellular transportation. On the macroscale, hydrophobic interactions consist of the aggregation of “oil-like” objects in water by minimizing the interfacial energy. However, studies of the hydration behavior of small hydrophobic molecules have shown that the microscopic (∼1 nm) hydration mechanism differs fundamentally from its macroscopic counterpart. Theoretical studies over the last two decades have pointed to an intricate dependence of molecular hydration mechanisms on the length scale. The microscopic-to-macroscopic crossover length scale is critically important to hydrophobic interactions in polymers, proteins, and other macromolecules. Accurate experimental determination of hydration mechanisms and interaction strengths directly influence our understanding of protein folding. [ABSTRACT FROM AUTHOR]

We describe a bacterial reporter system, FRep, for rapid and facile detection of protein-DNA recognition. The bioprobe reporter comprises genes of two fluorescent proteins (FPs) separated by a potential DNA target. If a coexpressed transcription factor binds the DNA target, transcription of the second FP is impeded, resulting in loss of FRET partner. Using ratiometric FRET, we show that evaluation of protein-DNA recognition can be reliably made on bZIP and bHLHZ transcription factors and their DNA targets. FRep displays similar thresholds of detection regarding protein-DNA binding affinities, as compared to well-established electrophoretic and yeast assays, although we observed variations in the intensity of fluorescence signals and detection thresholds that may depend on differences between DNA-binding protein production levels and/or stability in the cell, or the expressed bioprobe linker between the two FPs. FRep can potentially be applied to high-throughput searches of both protein and DNA libraries; in a mock library screen, binding and nonbinding complexes can even be distinguished by visual inspection of colonies on plates. FRep presents notable advantages over existing technologies when applied to assessing protein-DNA interactions in vivo, and this approach has the potential for applications in assaying protein-protein interactions and screening molecules that influence specific macromolecular interactions. [ABSTRACT FROM AUTHOR]

Hydrophobicity underpins self-assembly in many natural and synthetic molecular and nanoscale systems. A signature of hydrophobicity is its temperature dependence. The first experimental evaluation of the temperature and size dependence of hydration free energy in a single hydrophobic polymer is reported, which tests key assumptions in models of hydrophobic interactions in protein folding. Herein, the hydration free energy required to extend three hydrophobic polymers with differently sized aromatic side chains was directly measured by single molecule force spectroscopy. The results are threefold. First, the hydration free energy per monomer is found to be strongly dependent on temperature and does not follow interfacial thermodynamics. Second, the temperature dependence profiles are distinct among the three hydrophobic polymers as a result of a hydrophobic size effect at the subnanometer scale. Third, the hydration free energy of a monomer on a macromolecule is different from a free monomer; corrections for the reduced hydration free energy due to hydrophobic interaction from neighboring units are required. [ABSTRACT FROM AUTHOR]

The effect of temperature during solvent annealing on the microdomain orientation of block copolymer ultrathin films was investigated using symmetric polystyrene-block-poly(methyl methacrylate) diblock copolymers. When acetone solvent annealing was performed at low temperatures, 2 °C, 38% of samples exhibited a pattern of cylinders perpendicular to the substrate, while 62% of the samples exhibited a mixture of cylinders orientated parallel and perpendicular to the substrate. In the case of acetone solvent annealing at room temperature, only 6% of samples exhibited a pattern with cylinders perpendicular to the substrate, while 73% of the samples exhibited a mixture of cylinders orientated parallel and perpendicular to the substrate. We discuss how the morphology is affected by temperature during solvent annealing. The low-temperature method was used to pattern cylinders perpendicular to the substrate using PS-b-PMMA copolymers with molecular weight ranging from 52 000 g/mol per block to 160 000 g/mol per block. [ABSTRACT FROM AUTHOR]

The hydrophobic interaction is significantly responsible for driving protein folding and self-assembly. To understand it, the thermodynamics, the role of water structure, the dewetting process surrounding hydrophobes, and related aspects have undergone extensive investigations. Here, we examine the hypothesis that polymer-solvent interfacial free energy is adequate to describe the energetics of the collapse of a hydrophobic homopolymer chain at fixed temperature, which serves as a much simplified model for studying the hydrophobic collapse of a protein. This implies that changes in polymer-solvent interfacial free energy should be directly proportional to the force to extend a collapsed polymer into a bad solvent. To test this hypothesis, we undertook single-molecule force spectroscopy on a collapsed, single, polystyrene chain in water-ethanol and water-salt mixtures where we measured the monomer solvation free energy from an ensemble average conformations. Different proportions within the binary mixture were used to create solvents with different interfacial free energies with polystyrene. In these mixed solvents, we observed a linear correlation between the interfacial free energy and the force required to extend the chain into solution, which is a direct measure of the solvation free energy per monomer on a single chain at room temperature. A simple analytical model compares favorably with the experimental results. This knowledge supports a common assumption that explicit water solvent may not be necessary for cases whose primary concerns are hydrophobic interactions and hydrophobic hydration. [ABSTRACT FROM AUTHOR]

Lake Winnipeg in Manitoba, Canada is heavily impacted by harmful algal blooms that contain non-protein amino acids (NPAAs) produced by cyanobacteria: N-(2-aminoethyl)glycine (AEG), β-aminomethyl-L-alanine (BAMA), β-N-methylamino-L-alanine (BMAA), and 2,4-diaminobutyric acid (DAB). Our objective was to investigate the impact of microbial diversity on NPAA production by cyanobacteria using semi-purified crude cyanobacterial cultures established from field samples collected by the Lake Winnipeg Research Consortium between 2016 and 2021. NPAAs were detected and quantified by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) using validated analytical methods, while Shannon and Simpson alpha diversity scores were determined from 16S rRNA metagenomic sequences. Alpha diversity in isolate cultures was significantly decreased compared to crude cyanobacterial cultures (p < 0.001), indicating successful semi-purification. BMAA and AEG concentrations were higher in crude compared to isolate cultures (p < 0.0001), and AEG concentrations were correlated to the alpha diversity in cultures (r = 0.554; p < 0.0001). BAMA concentrations were increased in isolate cultures (p < 0.05), while DAB concentrations were similar in crude and isolate cultures. These results demonstrate that microbial community complexity impacts NPAA production by cyanobacteria and related organisms. [ABSTRACT FROM AUTHOR]

Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell's surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level. [ABSTRACT FROM AUTHOR]

The article discusses Sleep Paper Reader, a fictional device that allows the user to read scientific research publications while sleeping.

Objective: Type 2 diabetes mellitus (T2D) is the result of a dysregulation of insulin production and signalling, leading to an increase in both glucose concentration and pro-inflammatory cytokines such as interleukin (IL)-6 and tumour necrosis factor (TNF)-α. Previous work showed that T2D patients exhibited immune dysfunction associated with increased adhesion molecule expression on endothelial cell surfaces, accompanied by decreased neutrophil rolling velocity on the endothelial cell surface. Changes in cell rolling adhesion have direct vascular and immune complications such as atherosclerosis and reduced healing time in T2D patients. While previous studies focused primarily on how endothelial cells affect neutrophil rolling under T2D conditions, little is known about changes to neutrophils that affect their rolling. In this study, we aim to show how the rolling behaviour of neutrophils is affected by T2D conditions on a controlled substrate. Results: We found that neutrophils cultured in T2D-serum mimicking media increased cell rolling velocity compared to neutrophils under normal conditions. Specifically, glucose alone is responsible for higher rolling velocity. While cytokines further increase the rolling velocity, they also reduce the cell size. Both glucose and cytokines likely reduce the function of P-selectin Glycoprotein Ligand-1 (PSGL-1) on neutrophils. [ABSTRACT FROM AUTHOR]

It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford--Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard--Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system. [ABSTRACT FROM AUTHOR]

To eliminate the precise microchannel alignment requirement for parallel surface acoustic wave (pSSAW) fabrication, a tilted standing surface acoustic wave (tSSAW) is incorporated in many studies recently. In tSSAW, the microchannel is tilted at a specific angle (θ) instead of being parallel to interdigital transducer fingers. This causes the pressure node lines to be formed in the tilted direction to the fluid flow, which leads to better controllability in the separation or manipulation of the particles and higher efficiency. In this study, we developed a three‐dimensional (3D) finite element analysis (FEA) simulation to study the effect of the tSSAW device parameters to maximize its performance in particle separation. The outcomes of the 3D simulation of tSSAW devices indicate that the optimum values of the microchannel tilt angle (θ) and the aperture length must be between 5° ≤ θ ≤ 15° and the same as the microchannel length, respectively. The optimum values are considered in the fabrication of the tSSAW device, where it is tested with 20 μm, 15 μm, and 600 nm polystyrene particles. [ABSTRACT FROM AUTHOR]

Melatonin is a human neurotransmitter and plant signalling metabolite that perceives and directs plant metabolism. The mechanisms of melatonin action in plants remain undefined. We hypothesized that roots have a melatonin-specific receptor and/or transporter that can respond to melatonin-mediating pharmaceuticals. To test this hypothesis Arabidopsis seedlings were grown with melatonin pharmaceutical receptor agonists: ramelteon and tasimelteon, and/or antagonists: luzindole and 4-P-PDOT. Ramelteon was found both to mimic and competitively inhibit melatonin metabolism in plants. Due to the higher selectivity of ramelteon for the MT1 receptor type in humans, a sequence homology search for MT1 in Arabidopsis identified the rhomboid-like protein 7 (RBL7). In physiological studies, Arabidopsis rbl7 mutants were less responsive to ramelteon and melatonin. Quantum dot visualizations of the effects of ramelteon on melatonin binding to root cell membranes revealed a potential mechanism. We propose that RBL7 is a melatonin-interacting protein that directs root architecture and growth in a mechanism that is responsive to environmental factors. [ABSTRACT FROM AUTHOR]

Amyloid fibril diseases are characterized by the abnormal production of aggregated proteins and are associated with many types of neuro- and physically degenerative diseases. X-ray diffraction techniques, solid-state magic-angle spinning NMR spectroscopy, circular dichroism (CD) spectroscopy, and transmission electron microscopy studies have been utilized to detect and examine the chemical, electronic, material, and structural properties of amyloid fibrils at up to angstrom spatial resolution. However, X-ray diffraction studies require crystals of the fibril to be analyzed, while other techniques can only probe the bulk solution or solid samples. In the work reported here, apertureless near-field scanning infrared microscopy (ANSIM) was used to probe the secondary structure of individual amyloid fibrils made from an in vitro solution. Simultaneous topographic and infrared images of individual amyloid fibrils synthesized from the #21-31 peptide fragment of β2-microglobulin were acquired. Using this technique, IR spectra of the amyloid fibrils were obtained with a spatial resolution of less than 30 nm. It is observed that the experimental scattered field spectrum correlates strongly with that calculated using the far-field absorption spectrum. The near-field images of the amyloid fibrils exhibit much lower scattering of the IR radiation at approximately 1630 cm-1. In addition, the near-field images also indicate that composition and/or structural variations among individual amyloid fibrils were present. [ABSTRACT FROM AUTHOR]

The mechanical properties of DNA have enabled it to be a structural and sensory element in many nanotechnology applications. While specific base-pairing interactions and secondary structure formation have been the most widely utilized mechanism in designing DNA nanodevices and biosensors, the intrinsic mechanical rigidity and flexibility are often overlooked. In this article, we will discuss the biochemical and biophysical origin of double-stranded DNA rigidity and how environmental and intrinsic factors such as salt, temperature, sequence, and small molecules influence it. We will then take a critical look at three areas of applications of DNA bending rigidity. First, we will discuss how DNA's bending rigidity has been utilized to create molecular springs that regulate the activities of biomolecules and cellular processes. Second, we will discuss how the nanomechanical response induced by DNA rigidity has been used to create conformational changes as sensors for molecular force, pH, metal ions, small molecules, and protein interactions. Lastly, we will discuss how DNA's rigidity enabled its application in creating DNA-based nanostructures from DNA origami to nanomachines. [ABSTRACT FROM AUTHOR]

Melatonin and serotonin are important phytochemicals enabling plants to redirect growth in response to environmental stresses. Despite much research on their biosynthetic routes, localization of their biosynthetic enzymes and recent identification of a phytomelatonin receptor, localization of the molecules themselves has to date not been possible. Elucidation of their locations in living tissues can provide an effective tool to facilitate indolamine research across systems including both plants and animals. In this study, we employed a novel technique, quantum dot nanoparticles, to directly visualize melatonin and serotonin in axenic roots. Melatonin was absorbed through epidermal cells, travelled laterally, and accumulated in endodermal and rapidly dividing pericycle cells. Serotonin was absorbed by cells proximal to the crown with rapid polar movement toward the root tip. Thermal stress disrupted localization and dispersed melatonin and serotonin across cells. These data demonstrate the natural movement of melatonin and serotonin in roots directing cell growth and suggest that plants have a mechanism to disperse the indolamines throughout tissues as antioxidants in response to environmental stresses. [ABSTRACT FROM AUTHOR]

Nuclear speckles are self-assembled organelles composed of RNAs and proteins. They are proposed to act as structural domains that control distinct steps in gene expression, including transcription, splicing and mRNA export. Earlier studies identified differential localization of a few components within the speckles. It was speculated that the spatial organization of speckle components might contribute directly to the order of operations that coordinate distinct processes. Here, by performing multi-color structured illumination microscopy, we characterized the multilayer organization of speckles at a higher resolution. We found that SON and SC35 (also known asSRSF2) localize to the central region of the speckle, whereas MALAT1 and small nuclear (sn)RNAs are enriched at the speckle periphery. Coarse-grained simulations indicate that the non-random organization arises due to the interplay between favorable sequence-encoded intermolecular interactions of speckle-resident proteins and RNAs. Finally, we observe positive correlation between the total amount of RNA present within a speckle and the speckle size. These results imply that speckle size may be regulated to accommodate RNA accumulation and processing. Accumulation of RNA from various actively transcribed speckle-associated genes could contribute to the observed speckle size variations within a single cell. [ABSTRACT FROM AUTHOR]