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Therefore, it is very likely that the no occurrence of the far-red band in the aggregated core antenna complexes is directly related to the absence of lutein1 in their structures. The absence of the far-red band also suggests the possibility that aggregation-induced conformational change of the core antenna complexes does not yield a chlorophyll-carotenoid interaction associated energy dissipation channel.A mainstay of personal protective equipment during the coronavirus disease 2019 pandemic is the N95 filtering facepiece respirator. selleckchem N95 respirators are commonly used to protect healthcare workers from respiratory pathogens, including the novel coronavirus severe acute respiratory syndrome coronavirus 2, and are increasingly employed by other frontline workers and the general public. Under routine circumstances, these masks are disposable, single-use items, but extended use and reuse practices have been broadly enacted to alleviate critical supply shortages during the coronavirus disease 2019 pandemic. Although extended-time single use presents a low risk of pathogen transfer, repeated donning and doffing of potentially contaminated masks presents increased risk of pathogen transfer. Therefore, efficient and safe decontamination methods for N95 masks are needed to reduce the risk of reuse and mitigate local supply shortages. Here, we review the available literature concerning use of germicidal ultraviolet-C (UV-C) light to decontaminate N95 masks. We propose a practical method for repeated point-of-use decontamination using commercially available UV-C cross-linker boxes from molecular biology laboratories to expose each side of the mask to 800-1200 mJ/cm2 of UV-C. We measure the dose that penetrated to the interior of the respirators and model the potential germicidal action on coronaviruses. Our experimental results, in combination with modeled data, suggest that such a UV-C treatment cycle should induce a >3-log-order reduction in viral bioburden on the surface of the respirators and a 2-log-order reduction throughout the interior. We find that a dose 50-fold greater does not impair filtration or fit of 3M 8210 N95 masks, indicating that decontamination can be performed repeatedly. link2 As such, UV-C germicidal irradiation is a practical strategy for small-scale point-of-use decontamination of N95s.Agonists are evaluated by a concentration-response curve (CRC), with a midpoint (EC50) that indicates potency, a high-concentration asymptote that indicates efficacy, and a low-concentration asymptote that indicates constitutive activity. A third agonist attribute, efficiency (η), is the fraction of binding energy that is applied to the conformational change that activates the receptor. We show that η can be calculated from EC50 and the asymptotes of a CRC derived from either single-channel or whole-cell responses. For 20 agonists of skeletal muscle nicotinic receptors, the distribution of η-values is bimodal with population means at 51% (including acetylcholine, nornicotine, and dimethylphenylpiperazinium) and 40% (including epibatidine, varenicline, and cytisine). The value of η is related inversely to the size of the agonist's headgroup, with high- versus low-efficiency ligands having an average volume of 70 vs. 102 Å3. Most binding site mutations have only a small effect on acetylcholine efficiency, except for αY190A (35%), αW149A (60%), and those at αG153 (42%). If η is known, the EC50 and high-concentration asymptote can be calculated from each other. Hence, an entire CRC can be estimated from the response to a single agonist concentration, and efficacy can be estimated from EC50 of a CRC that has been normalized to 1. Given η, the level of constitutive activity can be estimated from a single CRC.Single-molecule fluorescence detection of protein and other biomolecules requires a polyethylene glycol (PEG)-passivated surface. Individual channels on a PEG-passivated slide are typically used only a few times, limiting the number of experiments per slide. Here, we report several strategies for regenerating PEG surfaces for multiple rounds of experiments. First, we show regeneration of DNA- or RNA-tethered surfaces by washing out the bound protein by 0.1% sodium dodecyl sulfate, which is significantly more effective than 6 M urea, 6 M GdmCl, or 100 μM proteinase K. Strikingly, 10 consecutive experiments in five different systems produced indistinguishable results both in molecule count and protein activity. Second, duplexed DNA unwound by helicase or denatured by 50 mM NaOH was reannealed with a complementary strand to regenerate the duplexed substrate with an exceptionally high recovery rate. Third, the biotin-PEG layer was regenerated by using 7 M NaOH to strip off NeutrAvidin, which can be reapplied for additional experiments. We demonstrate five cycles of regenerating antibody immobilized surface by which three different protein activity was measured. Altogether, our methods represent reliable and reproducible yet simple and rapid strategies that will enhance the efficiency of single-molecule experiments.During clathrin-mediated endocytosis, a patch of flat plasma membrane is deformed into a vesicle. In walled cells, such as plants and fungi, the turgor pressure is high and pushes the membrane against the cell wall, thus hindering membrane internalization. selleckchem In this work, we study how a patch of membrane is deformed against turgor pressure by force and by curvature-generating proteins. We show that a large amount of force is needed to merely start deforming the membrane and an even larger force is needed to pull a membrane tube. link2 selleckchem The magnitude of these forces strongly depends on how the base of the membrane is constrained and how the membrane is coated with curvature-generating proteins. In particular, these forces can be reduced by partially, but not fully, coating the membrane patch with curvature-generating proteins. Our theoretical results show excellent agreement with experimental data.Beyond the critical role of cell nuclei in gene expression and DNA replication, they also have a significant influence on cell mechanosensation and migration. Nuclear stiffness can impact force transmission and, furthermore, act as a physical barrier to translocation across tight spaces. link3 As such, it is of wide interest to accurately characterize nucleus mechanical behavior. In this study, we present a computational investigation of the in situ deformation of a heterogeneous chondrocyte nucleus. A methodology is developed to accurately reconstruct a three-dimensional finite-element model of a cell nucleus from confocal microscopy. By incorporating the reconstructed nucleus into a chondrocyte model embedded in pericellular and extracellular matrix, we explore the relationship between spatially heterogeneous nuclear DNA content, shear stiffness, and resultant shear strain. We simulate an externally applied extracellular matrix shear deformation and compute intranuclear strain distributions, which are directly coleus becoming more ellipsoidal. link2 Our findings may have broad implications for current understanding of how local DNA concentrations and associated strain amplification can impact cell mechanotransduction and drive cell behavior in development, migration, and tumorigenesis.Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A2A receptor (A2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A2AR C-terminus from 412 to 316 amino acids (A2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A2AR levels. As cAMP production is downstream of the first activation event-coupling of G protein to its receptor-investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gαs proteins, we characterize the association of A2AR and A2AΔ316R to Gαs with and without GDP or GTPγs using surface plasmon resonance (SPR). Gαs affinity for A2AR was greatest for apo-Gαs, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A2AR C-terminus (A2AΔ316R) decreased the affinity of the unliganded receptor for Gαs by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.Signal transduction within crowded cellular compartments is essential for the physiological function of cells. Although the accuracy with which receptors can probe the concentration of ligands has been thoroughly investigated in dilute systems, the effect of macromolecular crowding on the inference of concentration remains unclear. In this work, we develop an algorithm to simulate reversible reactions between reacting Brownian particles. Our algorithm facilitates the calculation of reaction rates and correlation times for ligand-receptor systems in the presence of macromolecular crowding. Using this method, we show that it is possible for crowding to increase the accuracy of estimated ligand concentration based on receptor occupancy. In particular, we find that crowding can enhance the effective association rates between small ligands and receptors to a degree sufficient to overcome the increased chance of rebinding due to caging by crowding molecules. For larger ligands, crowding decreases the accuracy of the receptor's estimate primarily by decreasing the microscopic association and dissociation rates.All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. link3 Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressthway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. link3 Two glycan chains are conserved in the Fc region in IgG; however, their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. After deglycosylation using peptideN-glycosidase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16-0.27 S. This change was attributed to the reduction in mass after glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. Although the radius of gyration (RG) was unchanged, the cross-sectional radius of gyration (RXS-1) increased by 0.
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