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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]

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]

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]

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]

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]

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]

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]

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.

Cadherin-mediated tension at adherens junctions (AJs) is fundamental for cell-cell adhesion and maintaining epithelial integrity. Despite the importance of manipulating AJs to dissect cell-cell interactions, existing three-dimensional (3D) multicellular models have not adequately addressed the precise manipulation of these junctions. To fill this gap, we introduce E-cadherin-modified tension gauge tethers (TGTs) at the junctions within spheroids. The system enables both quantification and modulation of junctional tension with specific DNA triggers. Using rupture-induced fluorescence, we successfully measure mechanical forces in 3D spheroids. Furthermore, mechanically strong TGTs can maintain normal E-cadherin-mediated adhesion. Employing toehold-mediated strand displacement allowed us to disrupt E-cadherin-specific cell-cell adhesion, consequently altering intracellular tension within the spheroids. Our methodology offers a robust and precise way to manipulate cell-cell adhesion and intracellular mechanics in spheroid models.

Just as a single polypeptide strand can self-fold into a complex 3D structure, a single strand of DNA can self-fold into DNA origami. Most DNA origami structures (i.e., the scaffold-staple and DNA tiling systems) utilize hundreds of short single-stranded DNA. As such, these structures come with challenges inherent to intermolecular construction. Many assembly challenges involving intermolecular interactions can be resolved if the origami structure is constructed from one DNA strand, where folding is not concentration dependent, the folded structure is more resistant to nuclease degradation, and the synthesis can be achieved at an industrial scale at a thousandth of the cost. This review discusses the design principles and considerations employed in single-stranded DNA origami and its potential benefits and drawbacks., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yang, Bakker, Raghu and Li.)

The advancement in microfluidics has provided an excellent opportunity for shifting from conventional sub-micron-sized isolation and purification methods to more robust and cost-effective lab-on-chip platforms. The acoustic-driven separation approach applies differential forces acting on target particles, guiding them towards different paths in a label-free and biocompatible manner. The main challenges in designing the acoustofluidic-based isolation platforms are minimizing the reflected radio frequency signal power to achieve the highest acoustic radiation force acting on micro/nano-sized particles and tuning the bandwidth of the acoustic resonator in an acceptable range for efficient size-based binning of particles. Due to the complexity of the physics involved in acoustic-based separations, the current existing lack in performance predictive understanding makes designing these miniature systems iterative and resource-intensive. This study introduces a unique approach for design automation of acoustofluidic devices by integrating the machine learning and multi-objective heuristic optimization approaches. First, a neural network-based prediction platform was developed to predict the resonator's frequency response according to different geometrical configurations of interdigitated transducers In the next step, the multi-objective optimization approach was executed for extracting the optimum design features for maximum possible device performance according to decision-maker criteria. The results show that the proposed methodology can significantly improve the fine-tuned IDT designs with minimum power loss and maximum working frequency range. The examination of the power loss and bandwidth on the alternation and distribution of the acoustic pressure inside the microfluidic channel was carried out by conducting a 3D finite element-based simulation. The proposed methodology improves the performance of the acoustic transducer by overcoming the constraints related to bandwidth operation, the magnitude of acoustic radiation force on particles, and the distribution of pressure acoustic inside the microchannel., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Talebjedi, Heydari, Taatizadeh, Tasnim, Li and Hoorfar.)

Hyaluronic acid is a native extra-cellular matrix derivative that promises unique properties, such as anti-inflammatory response and cell-signaling with tissue-specific applications under its bioactive properties. Here, we investigate the importance of the duration of synthesis to obtain photocrosslinkable methacrylated hyaluronic acid (MeHA) with high degree of substitution. MeHA with high degree of substitution can result in rapid photocrosslinking and can be used as a bioink for stereolithographic (SLA) three dimensional 3D bioprinting. Increased degree of substitution results Our findings show that a ten-day synthesis results in an 88% degree of methacrylation (DM), whereas three-day and five-day syntheses result in 32% and 42% DM, respectively. The rheological characterization revealed an increased rate of photopolymerization with increasing DM. Further, we developed a hybrid bioink to overcome the non-cell-adhesive nature of MeHA by combining it with gelatin methacryloyl (GelMA) to fabricate 3D cell-laden hydrogel scaffolds. The hybrid bioink exhibited a 55% enhancement in stiffness compared to MeHA only and enabled cell-adhesion while maintaining high cell viability. Investigations also revealed that the hybrid bioink was a more suitable candidate for stereolithography (SLA) 3D bioprinting than MeHA because of its mechanical strength, printability, and cell-adhesive nature. This research lays out a firm foundation for the development of a stable hybrid bioink with MeHA and GelMA for first-ever use with SLA 3D bioprinting., (© 2021 IOP Publishing Ltd.)

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

Traditional cell/particle isolation methods are time-consuming and expensive and can lead to morphology disruptions due to high induced shear stress. To address these problems, novel lab-on-a-chip-based purification methods have been employed. Among various methods introduced for the separation and purification of cells and synthetics particles, acoustofluidics has been one of the most effective methods. Unlike traditional separation techniques carried out in clinical laboratories based on chemical properties, the acoustofluidic process relies on the physical properties of the sample. Using acoustofluidics, manipulating cells and particles can be achieved in a label-free, contact-free, and highly biocompatible manner. To optimize the functionality of the platform, the numerical study should be taken into account before conducting experimental tests to save time and reduce fabrication expenses. Most current numerical studies have only considered one-dimensional harmonic standing waves to simulate the acoustic pressure distribution. However, one-dimensional simulations cannot calculate the actual acoustic pressure distribution inside the microchannel due to its limitation in considering longitudinal waves. To address this limitation, a two-dimensional numerical simulation was conducted in this study. Our numerical simulation investigates the effects of the platform geometrical and operational conditions on the separation efficiency. Next, the optimal values are tested in an experimental setting to validate these optimal parameters and conditions. This work provides a guideline for future acoustofluidic chip designs with a high degree of reproducibility and efficiency., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Leukocyte rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic process in which cells roll along the endothelial surface of blood vessel walls to reach the site of infection. The most common approach to investigate cell-substrate adhesion is to analyze the cell rolling velocity in response to shear stress changes. It is assumed that changes in rolling velocity indicate changes in adhesion strength. In general, cell rolling velocity is studied at the population level as an average velocity corresponding to given shear stress. However, no statistical investigation has been performed on the instantaneous velocity distribution. In this study, we first developed a method to remove systematic noise and revealed the true velocity distribution to exhibit a log-normal profile. We then demonstrated that the log-normal distribution describes the instantaneous velocity at both the population and single-cell levels across the physiological flow rates. The log-normal parameters capture the cell motion more accurately than the mean and median velocities, which are prone to systematic error. Lastly, we connected the velocity distribution to the molecular adhesion force distribution and showed that the slip-bond regime of the catch-slip behavior of the P-selectin/PSGL-1 interaction is responsible for the variation of cell velocity., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Molecular force sensors (MFSs) have grown to become an important tool to study the mechanobiology of cells and tissues. They provide a minimally invasive means to optically report mechanical interactions at the molecular level. One of the challenges in molecular force sensor studies is the interpretation of the fluorescence readout. In this review, we divide existing MFSs into three classes based on the force-sensing mechanism (reversibility) and the signal output (analog/digital). From single-molecule force spectroscopy (SMFS) perspectives, we provided a critical discussion on how the sensors respond to force and how the different sensor designs affect the interpretation of their fluorescence readout. Lastly, the review focuses on the limitations and attention one must pay in designing MFSs and biological experiments using them; in terms of their tunability, signal-to-noise ratio (SNR), and perturbation of the biological system under investigation.

To understand the mechanical forces involved in cell adhesion, molecular force sensors have been developed to study tension through adhesion proteins. Recently, a class of molecular force sensors called tension gauge tethers (TGTs) have been developed that rely on irreversible force-dependent dissociation of a DNA duplex to study cell adhesion forces. Although the TGT offers a high signal-to-noise ratio and is ideal for studying fast/single-molecular adhesion processes, quantitative interpretation of experimental results has been challenging. Here, we use a computational approach to investigate how TGT fluorescence readout can be quantitatively interpreted. In particular, we studied force sensors made of a single TGT, multiplexed single TGTs, and two TGTs connected in series. Our results showed that fluorescence readout using a single TGT can result from drastically different combinations of force history and adhesion event density that span orders of magnitude. In addition, the apparent behavior of the TGT is influenced by the tethered receptor-ligand, making it necessary to calibrate the TGT with every new receptor-ligand. To solve this problem, we proposed a system of two serially connected TGTs. Our result shows that not only is the ratiometric readout of serial TGT independent of the choice of receptor-ligand, it is able to reconstruct force history with sub-pN force resolution. This is also not possible by simply multiplexing different types of TGTs together. Last, we systematically investigated how the sequence composition of the two serially connected TGTs can be tuned to achieve different dynamic range. This computational study demonstrated how serially connected irreversible molecular dissociation processes can accurately quantify molecular force and laid the foundation for subsequent experimental studies., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Exosomes are small (∼30-140 nm) lipid bilayer-enclosed particles of endosomal origin. They are a subset of extracellular vesicles (EVs) that are secreted by most cell types. There has been growing interest in exosome research in the last decade due to their emerging role as intercellular messengers and their potential in disease diagnosis. Indeed, exosomes contain proteins, lipids, and RNAs that are specific to their cell origin and could deliver cargo to both nearby and distant cells. As a result, investigation of exosome cargo contents could offer opportunities for disease detection and treatment. Moreover, exosomes have been explored as natural drug delivery vehicles since they can travel safely in extracellular fluids and deliver cargo to destined cells with high specificity and efficiency. Despite significant efforts made in this relatively new field of research, progress has been held back by challenges such as inefficient separation methods, difficulties in characterization, and lack of specific biomarkers. In this review, we summarize the current knowledge in exosome biogenesis, their roles in disease progression, and therapeutic applications and opportunities in bioengineering. Furthermore, we highlight the established and emerging technological developments in exosome isolation and characterization. We aim to consider critical challenges in exosome research and provide directions for future studies.

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 as SRSF2) 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., Competing Interests: Competing interestsS.M.F. is an employee of Ionis Pharmaceuticals and receives salary from the company., (© 2017. Published by The Company of Biologists Ltd.)

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 β(2)-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., (© 2011 American Chemical Society)

A caspase-7 biosensor (vDEVDc) based on FRET (fluorescence resonance energy transfer) was used to study the proteolytic properties of caspase-7, an executioner protease in cellular apoptosis. An active isoform of caspase-7 with the 56 N-terminal residues truncated (57casp7) cleaved vDEVDc at the recognition sequence, resulting in a FRET efficiency decrease of 61%. In contrast, an isoform with the 23 N-terminal residues truncated (24casp7) bound to vDEVDc but did not cleave the substrate, resulting in a FRET increase of 15%. Kinetic results showed an exponential substrate cleavage and binding curve for the 57casp7 and 24casp7 isoforms, respectively. FRET changes of the vDEVDc biosensor were also monitored in cos-7 cells upon STS-induced apoptosis. Finally, we modeled caspase-7 binding to vDEVDc and estimated a FRET emission ratio increase of 31.7%, which agrees with the 15% experimental result. We showed that two differently truncated isoforms of caspase-7 exhibit different enzymatic properties, namely binding by 24casp7 and hydrolysis by 57casp7.

Background: To infer homology and subsequently gene function, the Smith-Waterman (SW) algorithm is used to find the optimal local alignment between two sequences. When searching sequence databases that may contain hundreds of millions of sequences, this algorithm becomes computationally expensive., Results: In this paper, we focused on accelerating the Smith-Waterman algorithm by using FPGA-based hardware that implemented a module for computing the score of a single cell of the SW matrix. Then using a grid of this module, the entire SW matrix was computed at the speed of field propagation through the FPGA circuit. These modifications dramatically accelerated the algorithm's computation time by up to 160 folds compared to a pure software implementation running on the same FPGA with an Altera Nios II softprocessor., Conclusion: This design of FPGA accelerated hardware offers a new promising direction to seeking computation improvement of genomic database searching.

Calmodulin (CaM) is a highly versatile Ca(2+) signaling transducer known to regulate over a hundred proteins. In this paper, we further demonstrate the versatility of CaM binding by showing that it binds to a synthetic peptide (revCKKp) made by reversing the amino acid sequence of the CaM-binding peptide (CKKp) from CaM-dependent protein kinase kinase (CaMKK) (residues 438-463). Sequence comparison between revCKKp and other CaM-binding peptides (CBPs) from the CaM target databank showed that revCKKp does not resemble any existing classes of CBPs, except CKKp [M. Zhang, T. Yuan, Molecular mechanisms of calmodulin's functional versatility, Biochem. Cell Biol. 76 (1998) 313-323; S.W. Vetter, E. Leclerc, Novel aspects of calmodulin target recognition and activation, Eur. J. Biochem. 270 (2003) 404-414]. Furthermore, computational modeling showed that revCKKp could bind CaM in a similar manner to CKKp. Lastly, we experimentally showed that our synthetic revCKKp binds to CaM in a reversible Ca(2+)-dependent manner.

In the past two decades, protein engineering has advanced significantly with the emergence of new chemical and genetic approaches. Modification and recombination of existing proteins not only produced novel enzymes used commercially and in research laboratories, but furthermore, they revealed the mechanisms of protein function. In this chapter, we will describe the applications and significance of current protein engineering approaches.