Super-Resolution Tension PAINT Imaging with a Molecular Beacon.

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.

Development and Results of the First Certification Examination in the American Board of Medical Specialties Neurocritical Care Subspecialty.

This article reviews the development of the American Board of Medical Specialties subspecialty in neurocritical care (NCC) and describes the requirements for certification and the results of the first certification examination administered in October 2021. The American Board of Psychiatry and Neurology (ABPN) is the administrative board, and the sponsoring boards are the American Board of Anesthesiology (ABA), American Board of Emergency Medicine (ABEM), American Board of Internal Medicine (ABIM), and American Board of Neurological Surgery. The American Board of Medical Specialties approved the subspecialty in 2018, and the Accreditation Council for Graduate Medical Education developed and approved the training requirements in 2021. The fellowship programs are either 12 or 24 months in length and may become available in Academic Year 2022-2023. The first NCC examination was developed by a multispecialty group of subject matter experts following established test development procedures and was successfully administered to 1,011 candidates in October 2021. There were 406 (40.2%) ABIM candidates, 356 (35.2%) ABPN candidates, 208 (20.6%) ABA candidates, and 41 (4.1%) ABEM candidates. The end-of-test survey indicated that most examinees were satisfied with their test taking experience, and the .92 reliability index indicated that the test scores were reliable. An established process was also followed to set the criterion-referenced passing standard, and the resulting pass rate of 72.7% was judged to be reasonable. In summary, the combined efforts of representatives from the ABPN, ABA, ABEM, ABIM, and American Board of Neurological Surgery yielded a quality assessment instrument to identify physicians who possess the expertise required to be certified in NCC. The test development committee will continue to expand and improve the pool of test questions for the next examination, which is scheduled for October 2022.

Quantitative interpretation of cell rolling velocity distribution.

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.

Antireflux Surgery at National Surgical Quality Improvement Program-Pediatric Hospitals.

PURPOSE: This study aims to examine contemporary practice patterns and compare short-term outcomes for vesicoureteral reflux procedures (ureteral reimplant/endoscopic injection) using National Surgical Quality Improvement Program-Pediatric data. MATERIALS AND METHODS: Procedure-specific variables for antireflux surgery were developed to capture data not typically collected in National Surgical Quality Improvement Program-Pediatric (eg vesicoureteral reflux grade, urine cultures, 31-60-day followup). Descriptive statistics were performed, and logistic regression assessed associations between patient/procedural factors and outcomes (urinary tract infection, readmissions, unplanned procedures). RESULTS: In total, 2,842 patients (median age 4 years; 76% female; 68% open reimplant, 6% minimally invasive reimplant, 25% endoscopic injection) had procedure-specific variables collected from July 2016 through June 2018. Among 88 hospitals, a median of 24.5 procedures/study period were performed (range 1-148); 95% performed ≥1 open reimplant, 30% ≥1 minimally invasive reimplant, and 70% ≥1 endoscopic injection, with variability by hospital. Two-thirds of patients had urine cultures sent preoperatively, and 76% were discharged on antibiotics. Outcomes at 30 days included emergency department visits (10%), readmissions (4%), urinary tract infections (3%), and unplanned procedures (2%). Over half of patients (55%) had optional 31-60-day followup, with additional outcomes (particularly urinary tract infections) noted. Patients undergoing reimplant were younger, had higher reflux grades, and more postoperative occurrences than patients undergoing endoscopic injections. CONCLUSIONS: Contemporary data indicate that open reimplant is still the most common antireflux procedure, but procedure distribution varies by hospital. Emergency department visits are common, but unplanned procedures are rare, particularly for endoscopic injection. These data provide basis for comparing short-term complications and developing standardized perioperative pathways for antireflux surgery.

Mechanical Flexibility of DNA: A Quintessential Tool for DNA Nanotechnology.

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.

Quantifying Molecular Forces with Serially Connected Force Sensors.

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.

Quantitative analysis of multilayer organization of proteins and RNA in nuclear speckles at super resolution.

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.

Quantifying molecular tension-classifications, interpretations and limitations of force sensors.

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.

Direct visualization of location and uptake of applied melatonin and serotonin in living tissues and their redistribution in plants in response to thermal stress.

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.

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory.

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.

Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo.

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.

Synthesis, self-assembly and photophysical properties of oligo(2,5-dihexyloxy-1,4-phenylene vinylene)-block-poly(ethylene glycol).

We describe the synthesis and characterization of a family of diblock copolymers with 5 units of a dihexyloxy-phenylenevinylene block (OHPV) connected to a series of poly(ethylene glycol) (PEG) chains of different average lengths (12, 45 and 115 PEG units: OHPV5-b-PEG12, OHPV5-b-PEG45, OHPV5-b-PEG115). All three polymers underwent self-assembly in ethanol, a good solvent for the PEG units, but poor for the OHPV segment. The nature of the structures formed depends sensitively on the length of the PEG block. OHPV5-b-PEG115 formed long fiber-like micelles of uniform width, whereas OHPV5-b-PEG45 formed fragile broad ribbons. We also obtained thin ribbons with OHPV5-b-PEG12 but they tend to fold and twist upon themselves. The structures obtained were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM), as well as by wide-angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC). In addition, their photophysical properties were examined by UV-vis, steady state fluorescence and fluorescence decay measurements. The results of these experiments indicate that the OHPV groups pack differently in the fiber-like micelles of OHPV5-b-PEG115 than in the lamellar structures formed by OHPV5-b-PEG45.

Signature of hydrophobic hydration in a single polymer.

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.

Altering Cell Junctional Tension in Spheroids through E-Cadherin Engagement Modulation.

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.

Microbial Diversity Impacts Non-Protein Amino Acid Production in Cyanobacterial Bloom Cultures Collected from Lake Winnipeg.

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), ß-aminomethyl-L-alanine (BAMA), ß-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.

Single polymer studies of hydrophobic hydration.

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. In this Account, we discuss our recent measurements of the hydration energies of single hydrophobic homopolymers as they unfold. We describe in detail our single molecule force spectroscopy technique, the interpretation of the single polymer force curve, and how it relates to the hydration free energy of a hydrophobic polymer. Specifically, we show how temperature, side-chain sizes and solvent conditions, affect the driving force of hydrophobic collapse. The experiments reveal that the size of the nonpolar polymer side-chains changes the thermal signatures of hydration. The sizes of the polymer side-chains bridge the length scale where theories had predicted a transition between entropically driven microscopic hydration and enthalpically driven macroscopic hydrophobic hydration. Our experimental results revealed a crossover length scale of approximately 1 nm, similar to the results from recent theoretical studies. Experiments that probe solvent dependency show that the microscopic polymer hydration is correlated with macroscopic interfacial tension. Consistent with theoretical predictions, the solvent conditions affect the microscopic and macroscopic hydrophobic strengths in similar ways. Although the extended polymers and proteins span hundreds of nanometers, the experiments show that their hydration behavior is determined by the size of a single hydrophobic monomer. As the hydrophobic particle size decreases from the macroscopic to the microscopic regime, the scaling relationship changes from a dependence on interfacial area to a dependence on volume. Therefore, under these conditions, the driving force for the aggregation of hydrophobic molecules is reduced, which has significant implications for the strength of hydrophobic interactions in molecular systems, particularly in protein folding.

A single strand: A simplified approach to DNA origami.

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.

Targeting Capabilities of Native and Bioengineered Extracellular Vesicles for Drug Delivery.

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.

The effect of type-2 diabetes conditions on neutrophil rolling adhesion.

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.