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The transport of particles through channels holds immense significance in physics, chemistry, and biological sciences. For instance, the motion of solutes through biological membranes is facilitated by specialized proteins that create water-filled channels. Valuable insights can be obtained by studying the transition paths of particles through a channel and gathering information on their lifetimes inside the channel as well as their exit probabilities. In a similar vein, we consider a one-dimensional model of channel-facilitated transport where a diffusive particle is subject to attractive interactions with the walls of the channel. We study the statistics of conditional and unconditional escape times in the presence of resetting—an intermittent dynamics that brings the particle back to its initial coordinate stochastically. We determine analytically the physical conditions under which such a resetting mechanism becomes beneficial for the faster escape of the particles from the channel, thus enhancing transport. Our theory has been verified with the aid of Brownian dynamics simulations for various interaction strengths and extents. The overall results presented herein highlight the scope of resetting-based strategies to be universally promising for complex transport processes of single or long molecules through biological membranes. [ABSTRACT FROM AUTHOR]

A recent ground-breaking experimental study [Lyons et al., Phys. Rev. X 14(1), 011017 (2024)] reports on measuring the temporal duration and the spatial extent of failed attempts to cross an activation barrier (i.e., "loops") for a folding transition in a single molecule and for a Brownian particle trapped within a bistable potential. Within the model of diffusive dynamics, however, both of these quantities are, on average, exactly zero because of the recrossings of the barrier region boundary. That is, an observer endowed with infinite spatial and temporal resolution would find that finite loops do not exist (or, more precisely, form a set of measure zero). Here we develop a description of the experiment that takes the "fuzziness" of the boundaries caused by finite experimental resolution into account and show how the experimental uncertainty of localizing the point, in time and space, where the barrier is crossed leads to observable distributions of loop times and sizes. Although these distributions generally depend on the experimental resolution, this dependence, in certain cases, may amount to a simple resolution-dependent factor and, therefore, the experiments do probe inherent properties of barrier crossing dynamics. [ABSTRACT FROM AUTHOR]

Both sugars and lipids are important biomolecular building blocks with exceptional conformational flexibility and adaptability to their environment. Glycolipids bring together these two molecular components in the same assembly and combine the complexity of their conformational landscapes. In the present study, we have used selective double resonance vibrational spectroscopy, in combination with a computational approach, to explore the conformational preferences of two glycolipid models (3-0-acyl catechol and guaiacol α-D-glucopyranosides), either fully isolated in the gas phase or controlled interaction with a single water molecule. We could identify the preferred conformation and structures of the isolated and micro-hydrated species and evidence of the presence of a strong water pocket, which may influence the conformational flexibility of such systems, even in less controlled environments. [ABSTRACT FROM AUTHOR]

Hydrogen clathrate hydrates are ice-like crystalline substances in which hydrogen molecules are trapped inside polyhedral cages formed by the water molecules. Small cages can host only a single H2 molecule, while each large cage can be occupied by up to four H2 molecules. Here, we present a neutron scattering study on the structure of the sII hydrogen clathrate hydrate and on the low-temperature dynamics of the hydrogen molecules trapped in its large cages, as a function of the gas content in the samples. We observe spectral features at low energy transfer (between 1 and 3 meV), and we show that they can be successfully assigned to the rattling motion of a single hydrogen molecule occupying a large water cage. These inelastic bands remarkably lose their intensity with increasing the hydrogen filling, consistently with the fact that the probability of single occupation (as opposed to multiple occupation) increases as the hydrogen content in the sample gets lower. The spectral intensity of the H2 rattling bands is studied as a function of the momentum transfer for partially emptied samples and compared with three distinct quantum models for a single H2 molecule in a large cage: (i) the exact solution of the Schrödinger equation for a well-assessed semiempirical force field, (ii) a particle trapped in a rigid sphere, and (iii) an isotropic three-dimensional harmonic oscillator. The first model provides good agreement between calculations and experimental data, while the last two only reproduce their qualitative trend. Finally, the radial wavefunctions of the three aforementioned models, as well as their potential surfaces, are presented and discussed. [ABSTRACT FROM AUTHOR]

The observation that materials can change their properties when placed inside or near an optical resonator has sparked a fervid interest in understanding the effects of strong light–matter coupling on molecular dynamics, and several approaches have been proposed to extend the methods of computational chemistry into this regime. Whereas the majority of these approaches have focused on modeling a single molecule coupled to a single cavity mode, changes to chemistry have so far only been observed experimentally when very many molecules are coupled collectively to multiple modes with short lifetimes. While atomistic simulations of many molecules coupled to multiple cavity modes have been performed with semi-classical molecular dynamics, an explicit description of cavity losses has so far been restricted to simulations in which only a very few molecular degrees of freedom were considered. Here, we have implemented an effective non-Hermitian Hamiltonian to explicitly treat cavity losses in large-scale semi-classical molecular dynamics simulations of organic polaritons and used it to perform both mean-field and surface hopping simulations of polariton relaxation, propagation, and energy transfer. [ABSTRACT FROM AUTHOR]

The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.

The interplay of molecules gives rise to collective phenomena absent in a single molecule. Many examples of collective phenomena have been reported as their knowledge is essential for understanding the behavior of matter. Here, we consider molecules sufficiently separated from each other to not form chemical bonds. If these molecules are excited, e.g., by a weak laser, can they concertedly relax by emitting a single high-energy photon possessing the total energy of all the relaxing molecules? We show that this concerted emission process is indeed possible. We estimate its probability and analyze its dependence on molecular properties, intermolecular distances, and relative orientations of the molecules. A numerical example on two pyridine molecules is given. The concerted emission found is a fundamental process expected to be operative in gas phase and clusters. Its true relevance lies in its intimate relationship to concerted emission of virtual photons and thus to collective energy transfer ionizing neighboring systems. The estimated rates and examples discussed of this collective intermolecular Coulombic decay shed much light on recent puzzling experiments. [ABSTRACT FROM AUTHOR]

Strong light–matter interactions significantly modify the optical properties of molecules in the vicinity of plasmonic metal nanoparticles. Since the dimension of the plasmonic cavity approaches that of the molecules, it is critical to explicitly describe the nanoparticle junctions. In this work, we use the discrete interaction model/quantum mechanical (DIM/QM) method to model the coupling between the plasmonic near-field and molecular excited states. DIM/QM is a combined electrodynamics/quantum mechanical model that uses an atomistic description of the nanoparticle. We extend the DIM/QM method to include the local field effects in the sum-over-state formalism of time-dependent density functional theory. As a test of the method, we study the interactions between small organic chromophores and metal nanoparticles. In particular, we examine how the inclusion of multiple electronic transitions and intermolecular interactions modify the coupling between molecules and nanoparticles. Using the sum-over-state formalism of DIM/QM, we show that two-state models break down when the plasmon excitation is detuned from the molecular excitations. To gain further insight, we compare the simple coupled-dipole model (CDM) with the DIM/QM model. We find that CDM works well for simple systems but fails when going beyond the single molecule or single nanoparticle cases. We also find that the coupling depends strongly on the site of the nanoparticle in which the chromophore couples to. Our work suggests the importance of explicitly describing the cavity to capture the atomistic level local field environment in which the molecule strongly couples to. [ABSTRACT FROM AUTHOR]

With growing propensity towards personalised medication, the application and impact of molecular theragnostic (or, theranostic) is expected to increase dramatically. Molecular theragnostic is the combination of diagnostics and therapeutics in single targeted molecule. If successful, molecular theragnostic will determine the treatment strategy and therapeutic outcome apart from favourably influencing the time of treatment and comfort of the patient. Recent developments such as [64Cu]Cu‐ATSM for imaging tumor‐hypoxia, 99Y silicate for arthritis, 1⁷⁷Lu‐PSMA‐617 for prostate cancer etc has been discussed in detail in the literature.[1,2] This sectioned review deals in particular with the development of nuclear molecular theragnostic agents. The genesis and design strategies are presented after compiling recent reports. [ABSTRACT FROM AUTHOR]

Compared with aggregate spin behavior, single‐molecule spin behavior can be accurately understood, controlled, and applied at the level of basic building blocks. The potential of single‐molecule electronic and nuclear spins for monitoring and control represents a beacon of promise for the advancement of molecular spin devices, which are fabricated by connecting a single molecule between two electrodes. Metal complexes, celebrated for their superior magnetic attributes, are widely used in the devices to explore spin effects. Moreover, single‐molecule electrical techniques with high signal‐to‐noise ratio, temporal resolution, and reliability help to understand the spin characteristics. In this review, the focus is on the devices with metal complexes, especially single‐molecule magnets, and systematically present experimental and theoretical state of the art of this field at the single‐molecule level, including the fundamental concepts of the electronic and nuclear spin and their basic spin effects. Then, several experimental methods developed to regulate the spin characteristics of metal complexes at single‐molecule level are introduced, as well as the corresponding intrinsic mechanisms. A brief discussion is provided on the comprehensive applications and the considerable challenges of single‐molecule spin devices in detail, along with a prospect on the potential future directions of this field. [ABSTRACT FROM AUTHOR]

Infrared (IR) spectroscopy stands as a workhorse for exploring bond vibrations, offering a wealth of chemical insights across diverse frontiers. With increasing focus on the regime of single molecules, obtaining IR‐sensitive information from individual molecules at room temperature would provide essential information about unknown molecular properties. Here, we leverage bond‐selective fluorescence microscopy, facilitated by narrowband picosecond mid‐IR and near‐IR double‐resonance excitation, for high‐throughput mid‐IR structural probing of single molecules. We robustly capture single‐molecule images and analyze the combined polarization dependence, vibrational peaks, linewidths, and lifetimes of probe molecules with representative scaffolds. From bulk to single molecules, we find that vibrational lifetimes remain consistent, while linewidths are narrowed by approximately twofold and anisotropy becomes more pronounced. Additionally, unexpected peak shifts from single molecules were observed, attributed to the generation of new modes due to previously unexplored dimerization, supported by quantum chemistry calculations. These findings underscore the importance of infrared analysis on individual single molecules in ambient environments, offering molecular information crucial for functional imaging and the investigation of the fundamental properties and utilities of luminescent molecules. [ABSTRACT FROM AUTHOR]

β-carotene (BCR) is the most abundant carotenoid, a colorant, antioxidant, and provitamin A. The extreme hydrophobicity of this hydrocarbon requires special mechanisms for distribution in aqueous media, including water-soluble carotenoproteins. However, all known carotenoproteins prefer oxygenated carotenoids and bind BCR inefficiently. Here, we present the crystal structure of the BCR-binding protein (BBP) from gregarious male locusts, which is responsible for their vivid yellow body coloration, in complex with its natural ligand, BCR. BBP forms an antiparallel tubular homodimer with α/β-wrap folded monomers, each forming a hydrophobic 47 Å long, coaxial tunnel that opens outward and is occupied by one s-cisC6-C7, all-trans BCR molecule. In the BCR absence, BBP accepts a range of xanthophylls, with reduced efficiency depending on the position and number of oxygen atoms, but rejects lycopene. The structure captures a pigment complex with a Takeout 1 protein and inspires potential applications of BBP as a BCR solubilizer. [Display omitted] • Crystal structure of a soluble protein in complex with β-carotene (BBP) determined • Extended BBP dimer has tubular subunits each binding a single β-carotene molecule • BBP is selective for β-carotene, rejects lycopene, and tolerates some xanthophylls • BBP apoform shows efficient ligand-assisted refolding when dissolved in 8 M urea Among soluble carotenoproteins, those binding carotenes are rare. Egorkin et al. determined the crystal structure of genuine β-carotene-binding protein (BBP) from gregarious locusts responsible for their yellow body coloration. Reconstitution studies revealed a selectivity for β-carotene, inability to bind lycopene, and unusual blue spectral shifts for carotenoids upon binding to BBP. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron entry involves spike (S) glycoprotein-mediated fusion of viral and late endosomal membranes. Here, using single-molecule Förster resonance energy transfer (sm-FRET) imaging and biochemical measurements, we directly visualized conformational changes of individual spike trimers on the surface of SARS-CoV-2 Omicron pseudovirions during fusion activation. We observed that the S2 domain of the Omicron spike is a dynamic fusion machine. S2 reversibly interchanges between the pre-fusion conformation and two previously undescribed intermediate conformations. Acidic pH shifts the conformational equilibrium of S2 toward an intermediate conformation and promotes the membrane hemi-fusion reaction. Moreover, we captured conformational reversibility in the S2 domain, which suggests that spike can protect itself from pre-triggering. Furthermore, we determined that Ca2+ directly promotes the S2 conformational change from an intermediate conformation to post-fusion conformation. In the presence of a target membrane, low pH and Ca2+ stimulate the irreversible transition to S2 post-fusion state and promote membrane fusion. [Display omitted] • Omicron spike fusion domain S2 shows dynamic plasticity • S2 spontaneously samples two functional intermediate conformations to fusion pathway • Low pH promotes formation of fusion competent intermediate conformations • Ca2+ catalyzes an S2 conformational change to a stable post fusion conformation at low pH Dey et al. used single molecule FRET for a real-time visualization of SARS-CoV-2 spike conformational dynamics on the virion surface during fusion triggered by low pH and Ca2+. The intermediates identified during dynamic S protein mediated fusion provide mechanistic insights for Omicron entry that may guide CoV inhibitor design. [ABSTRACT FROM AUTHOR]

High‐mobility emissive n‐type polymer semiconductors (HMEN‐CPs) are essential for organic optoelectronic devices. However, due to the lack of highly electron‐deficient building blocks with excellent physicochemical properties and the molecular design trade‐offs, the development of HMEN‐CPs has significantly lagged behind that of p‐type counterparts. Here, a new electron‐deficient acceptor, TFBVDPP is developed, by integrating the efficient carrier‐transporting diketopyrrolopyrrole and intense light‐emitting phenylene‐vinylene into a single molecule system. A series of all‐acceptor‐type polymers are synthesized by Pd‐catalyzed Suzuki polycondensation. The introduction of a vinyl bridge induces intra‐/intermolecular non‐covalent interactions, significantly narrowing the optical bandgap while enhancing coplanarity and crystallinity of the conjugated backbone, thus one of the polymers P1 exhibits ideal ambipolar transports with hole and electron mobilities reaching 4.6 and 3.7 cm2 V−1 s−1, respectively. Additionally, by directly homopolymerizing TFBVDPP, the non‐radiative transition is effectively suppressed, maintaining high electron transport while enhancing emission. This system shows excellent unipolar electron mobility (µe) and high quantum yield (Φ), achieving a record Φ · µe value exceeding 10−2 cm2 V−1 s−1, which is at least three orders of magnitude higher than the reported values. This study provides a new approach for developing HMEN‐CPs, and demonstrates their great potential in advancing organic optoelectronic technologies. [ABSTRACT FROM AUTHOR]

The mammalian plasma membrane is known to contain domains with varying lipid composition and biophysical properties. However, studying these membrane lipid domains presents challenges due to their predicted morphological similarity to the bulk membrane and their scale being below the classical resolution limit of optical microscopy. To address this, we combine the solvatochromic probe di-4-ANEPPDHQ, which reports on its biophysical environment through changes in its fluorescence emission, with spectrally resolved single-molecule localisation microscopy. The resulting data comprises nanometre-precision localisation coordinates and a generalised polarisation value related to the probe's environment – a marked point pattern. We introduce quantification algorithms based on topological data analysis (PLASMA) to detect and map nano-domains in this marked data, demonstrating their effectiveness in both artificial membranes and live cells. By leveraging environmentally sensitive fluorophores, multi-modal single molecule localisation microscopy, and advanced analysis methods, we achieve nanometre scale mapping of membrane properties and assess changes in response to external perturbation with methyl-β-cyclodextrin. This integrated methodology represents an integrated toolset for investigating marked point pattern data at nanometre spatial scales. Studying lipid nano-domains within the mammalian cell plasma membrane remains a very challenging task. Here, the authors use the solvatochromic probe di-4-ANEPPDHQ, which is sensitive to its biophysical environment, to map membrane nano-environments via single-molecule localisation microscopy. [ABSTRACT FROM AUTHOR]

After the recent double revolutions in structural biology, which include the use of direct detectors for cryo‐electron microscopy resulting in a significant improvement in the expected resolution of large macromolecule structures, and the advent of AlphaFold which allows for near‐accurate prediction of any protein structures, the field of structural biology is now pursuing more ambitious targets, including several MDa assemblies. But complex target systems cannot be tackled using a single biophysical technique. The field of integrative structural biology has emerged as a global solution. The aim is to integrate data from multiple complementary techniques to produce a final three‐dimensional model that cannot be obtained from any single technique. The absence of atomic force microscopy data from integrative structural biology platforms is not necessarily due to its nm resolution, as opposed to Å resolution for x‐ray crystallography, nuclear magnetic resonance, or electron microscopy. Rather a significant issue was that the AFM topographic data lacked interpretability. Fortunately, with the introduction of the AFM‐Assembly pipeline and other similar tools, it is now possible to integrate AFM topographic data into integrative modeling platforms. The advantages of single molecule techniques, such as AFM, include the ability to confirm experimentally any assembled molecular models or to produce alternative conformations that mimic the inherent flexibility of large proteins or complexes. The review begins with a brief overview of the historical developments of AFM data in structural biology, followed by an examination of the strengths and limitations of AFM imaging, which have hindered its integration into modern modeling platforms. This review discusses the correction and improvement of AFM topographic images, as well as the principles behind the AFM‐Assembly pipeline. It also presents and discusses a series of challenges that need to be addressed in order to improve the incorporation of AFM data into integrative modeling platform. [ABSTRACT FROM AUTHOR]

In single‐molecule microscopy, a big question is how precisely we can estimate the location of a single molecule. Our research shows that by using iterative localisation microscopy and factoring in the prior information, we can boost precision and reduce the number of photons needed. Leveraging the Van Trees inequality aids in determining the optimal precision achievable. Our approach holds promise for wider application in discerning the optimal precision across diverse imaging scenarios, encompassing various illumination strategies, point spread functions and overarching control methodologies. [ABSTRACT FROM AUTHOR]

Correlative super‐resolution microscopy has the potential to accurately visualize and validate new biological structures past the diffraction limit. However, combining different super‐resolution modalities, such as deterministic stimulated emission depletion (STED) and stochastic single‐molecule localization microscopy (SMLM), is a challenging endeavour. For correlative STED and SMLM, the following poses a significant challenge: (1) the photobleaching of the fluorophores in STED; (2) the subsequent reactivation of the fluorophores for SMLM and (3) finding the right fluorochrome and imaging buffer for both imaging modalities. Here, we highlight how the deep ultraviolet (DBUE) wavelengths of the Mercury (Hg) arc lamp can help recover STED bleaching and allow for the reactivation of single molecules for SMLM imaging. We also show that Alexa Fluor 594 and the commercially available Prolong Diamond to be excellent fluorophores and imaging media for correlative STED and SMLM. [ABSTRACT FROM AUTHOR]

Human parainfluenza viruses (hPIVs) are causative agents of upper and lower respiratory tract infections and they have four serotypes. The virion surface displays hemagglutinin‐neuraminidase (HN), having hemagglutinating (HA) and neuraminidase (NA) activities in a single molecule. The HA activity binds the virion to sialic acid on the viral receptor on host cells and the NA releases the progeny viruses from the cell surface. There are several methods for assaying viral NA activity, such as the thiobarbituric acid assay, 4‐methylumbelliferyl‐N‐acetyl‐α‐d‐neuraminic acid assay, NA‐Star assay, and enzyme‐linked lectin assay (ELLA). However, these are mainly used for influenza viruses and not for hPIVs. A fluorescent‐based cytochemical NA assay using BTP3‐Neu5Ac as the substrate was recently developed and used for orthomyxo‐ and paramyxoviruses, including types 1 and 3 hPIVs. In this study, we used the ELLA, and BTP‐Neu5Ac assay for 14 field isolate strains of hPIVs including all four serotypes. The reaction in ELLA at pH 6.5 using peanut agglutinin (PNA) as a lectin was very low for all tested viruses except a type 3 virus strain with the maximum reaction at pH 6.5 and the acidic conditions did not enhance the reaction. ELLA with another lectin, Erythrina cristagalli agglutinin exhibited significant and stronger reactions than with PNA in some strains of types 1 and 3 viruses. The BTP3‐Neu5Ac assay showed a fluorescent signal on cells infected with all the viruses except the hPIV1/Sendai/713/2018 strain in LLC‐MK2 and/or MNT‐1. The signal was detected in cell‐free virus, as well, in all the viruses except the hPIV4a/Sendai/3935/2003 strain. The strength of the signal varied among viral strains but it was stronger in the reaction at pH 4.0 than pH 7.0 and strongest in type 2 hPIVs. [ABSTRACT FROM AUTHOR]

Long-term single-molecule fluorescence measurements are widely used powerful tools to study the conformational dynamics of biomolecules in real time to further elucidate their conformational dynamics. Typically, thousands or even more single-molecule traces are analyzed to provide statistically meaningful information, which is labor-intensive and can introduce user bias. Recently, several deep-learning models have been developed to automatically classify single-molecule traces. In this study, we introduce DEBRIS (Deep lEarning Based fRagmentatIon approach for Single-molecule fluorescence event identification), a deep-learning model focusing on classifying local features and capable of automatically identifying steady fluorescence signals and dynamically emerging signals of different patterns. DEBRIS efficiently and accurately identifies both one-color and two-color single-molecule events, including their start and end points. By adjusting user-defined criteria, DEBRIS becomes the pioneer in using a deep learning model to accurately classify four different types of single-molecule fluorescence events using the same trained model, demonstrating its universality and ability to enrich the current toolbox. DEBRIS is a deep learning-based model that can classify local features and identify steady events and dynamically emerging events within two-color single-molecule traces of varying lengths based on user-defined criteria. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) pathogenesis involves dysregulation in diverse biochemical processes. Nevertheless, plasma tau phosphorylated at threonine 181 (P‐tau181), a recognised AD biomarker, has been described to reflect early‐stage cortical amyloid‐β (Aβ) deposition in cognitively normal (CN) adults. Therefore, identifying changes in plasma metabolites associated with plasma P‐tau181 at the pre‐clinical stage may provide insights into underlying biochemical mechanisms to better understand initial AD pathogenesis. In the current study, plasma P‐tau181, quantified via single molecule array (Simoa) technology, and plasma metabolites, quantified via targeted‐mass spectrometry, were investigated for associations in CN older adults and upon stratification by positron emission tomography (PET)‐Aβ load. In addition, the P‐tau181‐linked metabolites were evaluated for cognitive performance and neuroimaging markers of AD and the potential to distinguish between CN Aβ− and CN Aβ+ individuals. Significant positive associations of medium‐ and long‐chain acylcarnitines (ACs) were observed with P‐tau181 in the entire cohort, CN Aβ− and CN Aβ+, suggesting a link between initial Aβ pathology and fatty acid oxidation‐mediated energy metabolism pathways. However, in CN Aβ−, additional linear associations of P‐tau181 were observed with muscle metabolism and nitric oxide homeostasis‐associated metabolites. Upon investigating the P‐tau181‐linked metabolites for cognitive performance, significant inverse correlations of the verbal and visual episodic memory and the global composite score were noted in CN Aβ+ with medium‐ and long‐chain ACs, suggesting prognostic value of ACs accompanying weaker cognitive performance. While investigating neuroimaging markers, ACs had positive associations with PET‐Aβ load and inverse associations with hippocampal volume in CN Aβ+, indicating connections of ACs with initial AD pathogenesis. Furthermore, based on receiver operating characteristics analysis, the associated ACs potentially classified PET‐Aβ status in older adults. Therefore, plasma P‐tau181‐linked circulating ACs may serve as potential prognostic markers for initial AD pathogenesis in CN older adults. However, further cross‐sectional and longitudinal research in highly characterised AD cohorts is needed to validate current findings. [ABSTRACT FROM AUTHOR]

The giant freshwater prawn (Macrobrachium rosenbergii) has many advantages in aquaculture, such as fast growth rate, short breeding cycle and good nutrition, which makes it a freshwater shrimp with high economic value. Herein, high-quality chromosome-level genome of both female and male prawns were obtained by combining Illumina paired-end sequencing, PacBio single molecule sequencing technique and High-through chromosome conformation capture (Hi-C) technologies. In ZZ male prawn, a final contig assembly of 3118.58 Mb with a N50 length of 956,237 bp was obtained. In WW female prawn, a final contig assembly of 3333.31 Mb with a N50 length of 1,143,555 bp was obtained. The assembled genome sequences from prawns were anchored to 59 chromosomes. Moreover, the sex chromosomes including W chromosome and Z chromosome were generated in prawn with the length of 36.23 Mb and 27.33 Mb, respectively. The sequence similarity of Z chromosome and W chromosome reached to 74.90%. The high-quality genome resource will be useful for further molecular breeding and functional genomic research of giant freshwater prawns. [ABSTRACT FROM AUTHOR]

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylcytosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we study TDG in the presence and absence of 5-formylcytosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of base modifications. TDG active site variants and truncated N-terminus, reveals these variants alter base modification search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG is found to either bypass, colocalize with, or encounter but not bypass the nucleosome. Truncating the N-terminus reduces the number of interactions with the nucleosome. Our findings provide mechanistic insights into how TDG searches for modified DNA bases in chromatin. Thymine DNA glycosylase is an essential DNA repair enzyme involved in oxidative demethylation of 5- methylC. In this study the authors have tracked specific and non-specific DNA binding of TDG at the single molecule level. TDG is found to hop and slide on DNA and found to interact with nucleosomes. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms underlying amyloid‐β (Aβ) aggregation is pivotal in the context of Alzheimer's disease. This study aims to elucidate the secondary nucleation process of Aβ42 peptides by combining experimental and computational methods. Using a newly developed nanopipette‐based amyloid seeding and translocation assay, confocal fluorescence spectroscopy, and molecular dynamics simulations, the influence of the seed properties on Aβ aggregation is investigated. Both fragmented and unfragmented seeds played distinct roles in the formation of oligomers, with fragmented seeds facilitating the formation of larger aggregates early in the incubation phase. The results show that secondary nucleation leads to the formation of oligomers of various sizes and structures as well as larger fibrils structured in β‐sheets. From these findings a mechanism of secondary nucleation involving two types of aggregate populations, one released and one growing on the mother fiber is proposed. [ABSTRACT FROM AUTHOR]

Double phosphorescence components from a single organic molecule is rarely reported before because most organic molecules must follow Kasha's rule and merely emit phosphorescence from T1 state. We discover a unique phenomenon of conformation‐controlled expression of double phosphorescence components. The green phosphorescence component and the red phosphorescence component are assigned to the dinaphthylamine moiety and the pyrene unit of N,N‐di(naphthalen‐2‐yl)pyren‐1‐amine (NaPy), respectively. NaPy shows time‐dependent and excitation‐dependent afterglow color change in copolymerized MMA film at room temperature due to simultaneous expression of the double phosphorescence components. However, in PMMA film and copolymerized MA film, NaPy just expresses the red phosphorescence component, suggesting that matrix rigidity greatly affects expression of the double phosphorescence components via altering conformation of NaPy. In low‐rigidity matrix, the NaPy conformation is dyanmic due to free rotation and triplet excitons relax to T1 state via inner conversion or vibration relaxation, leading to the sole expression of the red phosphorescence component. In high‐rigidity matrix, the highly twisted conformation of NaPy is stabilized due to limitation of rotation and triplet excitons at T1 state and T2 state return to S0 state separately, resulting in simultaneous expression of the double phosphorescence components. Thus, NaPy follows Kasha's rule in low‐rigidity matrix but disobeys Kasha's rule in high‐rigidity matrix. In high‐rigidity matrix, the dinaphthylamine moiety and the pyrene unit can function relatively independently, resulting in time‐dependent and excitation‐dependent wide‐range afterglow color change. [ABSTRACT FROM AUTHOR]

Organic photovoltaics (OPVs) have made significant strides with efficiencies now exceeding 20%, positioning them as potential competitors to inorganic solar technologies. One of the most critical challenges toward this goal is the severe open‐circuit voltage (Voc) loss caused by the nonradiative charge recombination (NRCR). Herein, this review comprehensively summarizes the NRCR mechanisms and suppression techniques of OPVs across various scales from molecule to device. Specifically, the origins of NRCR in a single molecule are first summarized, and molecular design principles toward high photoluminescence quantum yield are reviewed following the Marcus theory. Next, the effect of aggregation on NRCR is reviewed, as well as the molecular and processing strategies to modulate the film packing for NRCR suppression. Furthermore, the progresses in the avoidance of nonradiative loss pathways mediated by charge transfer states and triplet states in donor:acceptor bulk heterojunctions are tracked. Besides, the interfacial optimization and device structure design to maximize the electroluminescent quantum efficiency are presented. Finally, several potential pathways toward curtailing NRCR for high‐performance OPVs are outlined. Therefore, this review shows an insightful perspective to understand and mitigate the NRCR at multi‐scales, and is poised to provide a clear roadmap for the next breakthrough of OPVs. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) single molecule localization microscopy (SMLM) plays an important role in biomedical applications, but its data processing is very complicated. Deep learning is a potential tool to solve this problem. As the state of art 3D super-resolution localization algorithm based on deep learning, FD-DeepLoc algorithm reported recently still has a gap with the expected goal of online image processing, even though it has greatly improved the data processing throughput. In this paper, a new algorithm Lite-FD-DeepLoc is developed on the basis of FD-DeepLoc algorithm to meet the online image processing requirements of 3D SMLM. This new algorithm uses the feature compression method to reduce the parameters of the model, and combines it with pipeline programming to accelerate the inference process of the deep learning model. The simulated data processing results show that the image processing speed of Lite-FD-DeepLoc is about twice as fast as that of FD-DeepLoc with a slight decrease in localization accuracy, which can realize real-time processing of 256×256 pixels size images. The results of biological experimental data processing imply that Lite-FD-DeepLoc can successfully analyze the data based on astigmatism and saddle point engineering, and the global resolution of the reconstructed image is equivalent to or even better than FD-DeepLoc algorithm. [ABSTRACT FROM AUTHOR]

The single-molecule tracking of transmembrane receptors in living cells has provided significant insights into signaling mechanisms, such as mobility and clustering upon their activation/inactivation, making it a potential screening method for drug discovery. Here we show that single-molecule tracking-based screening can be used to explore compounds both detectable and undetectable by conventional methods for disease-related receptors. Using an automated system for a fast large-scale single-molecule analysis, we screen for epidermal growth factor receptor (EGFR) from 1134 of FDA approved drugs. The 18 hit compounds include all EGFR-targeted tyrosine kinase inhibitors (TKIs) in the library that suppress any phosphorylation-dependent mobility shift of EGFR, proving the concept of this approach. The remaining hit compounds are not reported as EGFR-targeted drugs and do not inhibit EGF-induced EGFR phosphorylation. These non-TKI compounds affect the mobility and/or clustering of EGFR without EGF and induce EGFR internalization, to impede EGFR-dependent cell growth. Thus, single-molecule tracking provides an alternative modality for discovering therapeutics on various receptor functions with previously untargeted mechanisms. EGFR is a primary target molecule in drug exploration for cancer therapeutics. Here, the authors show a demonstration of single-molecule tracking-based drug screening for EGFR, proving the selectivity for tyrosine kinase inhibitors and potential to find drugs with previously untargeted mechanisms. [ABSTRACT FROM AUTHOR]

The reversible photo-induced conformation transition of a single molecule with a [5]helicene backbone has garnered considerable interest in recent studies. Based on such a switching process, one can build molecular photo-driven switches for potential applications of nanoelectronics. But the achievement of high-performance reversible single-molecule photoswitches is still rare. Here, we theoretically propose a 13,14-dimethylcethrene switch whose photoisomerization between the ring-closed and ring-open forms can be triggered by ultraviolet (UV) and visible light irradiation. The electronic structure transitions and charge transport characteristics, concurrent with the photo-driven electrocyclization of the molecule, are calculated by the non-equilibrium Green's function (NEGF) in combination with density functional theory (DFT). The electrical conductivity bears great diversity between the closed and open configurations, certifying the switching behavior and leading to a maximum on–off ratio of up to 103, which is considerable in organic junctions. Further analysis confirms the evident switching behaviors affected by the molecule–electrode interfaces in molecular junctions. Our findings are helpful for the rational design of organic photoswitches at the single-molecule level based on cethrene and analogous organic molecules. [ABSTRACT FROM AUTHOR]

Generative models, such as diffusion models, have made significant advancements in recent years, enabling the synthesis of high‐quality realistic data across various domains. Here, the adaptation and training of a diffusion model on super‐resolution microscopy images are explored. It is shown that the generated images resemble experimental images, and that the generation process does not exhibit a large degree of memorization from existing images in the training set. To demonstrate the usefulness of the generative model for data augmentation, the performance of a deep learning‐based single‐image super‐resolution (SISR) method trained using generated high‐resolution data is compared against training using experimental images alone, or images generated by mathematical modeling. Using a few experimental images, the reconstruction quality and the spatial resolution of the reconstructed images are improved, showcasing the potential of diffusion model image generation for overcoming the limitations accompanying the collection and annotation of microscopy images. Finally, the pipeline is made publicly available, runnable online, and user‐friendly to enable researchers to generate their own synthetic microscopy data. This work demonstrates the potential contribution of generative diffusion models for microscopy tasks and paves the way for their future application in this field. [ABSTRACT FROM AUTHOR]

Stable open‐shell luminescent radicals have recently attracted much attention due to their unique luminescence properties. However, a radical molecule with both Kasha and anti‐Kasha doublet emission properties has not been reported. Herein, we have successfully synthesized a stable chlorine‐substituted Chichibabin's hydrocarbon, TTM‐TTM, along with its mono‐radical counterpart, TTM‐HTTM. The emission of TTM‐TTM follows Kasha's rule in the near infrared region. However, TTM‐HTTM shows dual channel doublet emissions of Kasha and anti‐Kasha. Remarkably, these two types of emission compete dynamically in both solution and condensed states. Our findings provide valuable insights into the rational design and discovery of stable radicals that possess distinctive luminescent properties, thus broadening the horizons of luminescent materials research. [ABSTRACT FROM AUTHOR]

Based on the same bis(4-piperidine) phenyl derivative donor, isophorone derivative bridge, and phenyl trifluoromethyl tricyclofuran (3F-TCF) acceptor, two different isolating groups, tert butyldimethylsilyl (TBDMS) and pentafluorophenyl ring (5F-Ph), were introduced into the donor and bridge to synthesize two chromophores XY 1-2. Eight 5F-Ph groups are respectively attached to the donor and bridge segments of the chromophore, which can prevent the molecules from tightly packing and thus improve polarization efficiency. This article systematically investigates the UV absorption, density functional theory (DFT) calculations, thermal stability, and electro-optic activity of these chromophores. The chromophores XY 1-2 exhibit good thermal stability at decomposition temperatures (Td) greater than 250 °C. Most importantly, rigid steric hindrance groups can effectively reduce dipole–dipole interactions between molecules and improve the conversion efficiency of the first-order hyperpolarization (β) of chromophores into large electro-optic (EO) coefficients (r33). And the poling efficiency of a single XY2 molecule at 1310 nm (0.69 ± 0.01 nm2 V−2/(1020 molecules per cm3)) is much higher than that of a single XY1 molecule when polarizing the polymer with chromophore XY 1-2. [ABSTRACT FROM AUTHOR]

One of the still-debated topics regarding the olfactory function concerns the presence or absence of sex-related differences in individuals. In this study, we checked for a relationship between the olfactory function of females and males and their ability to perceive single molecules, and researched how this can influence the intensity with which the complex odor formed by a set of single molecules is perceived. First, females and males were classified as normosmic or hyposmic based on the TDI olfactory score obtained using the Sniffin' Sticks test. Subsequently, the headspace of roasted coffee beans, as a complex olfactory stimulus, was broken down into single molecules by means of a chromatographic column; these were simultaneously conveyed to a mass spectrometer (for their subsequent classification) and to the human nose, which acts as a chemical sensor by means of an olfactometer port. The results obtained with this gas chromatography–olfactometry approach show both qualitative and quantitative differences between females and males, with females performing better than males. In addition, the odor intensity reported by females when sniffing pen #10, containing coffee aroma, is significantly higher than that reported by males. In conclusion, these data highlight that the human ability to perceive both single compounds and complex odors is strongly conditioned, not only by the olfactory function of individuals, but also by their sex. [ABSTRACT FROM AUTHOR]

Recent advances in light microscopy have enabled single molecules to be imaged and tracked within living cells and this approach is impacting our understanding of cell biology. Computer modeling and simulation are important adjuncts to the experimental cycle since they aid interpretation of experimental results and help refine, test and generate hypotheses. Object-oriented computer modeling is particularly well-suited for simulating random, thermal, movements of individual molecules as they interact with other molecules and subcellular structures, but current models are often limited to idealized systems consisting of unit volumes or planar surfaces. Here, a simulation tool is described that combines a 3-dimensional, voxelated, representation of the cell consisting of subcellular structures (e.g. nucleus, endoplasmic reticulum, cytoskeleton, vesicles, and filopodia) combined with numerical floating-point precision simulation of thousands of individual molecules moving and interacting within the 3-dimensional space. Simulations produce realistic time-series video sequences comprising single fluorophore intensities and realistic background noise which can be directly compared to experimental fluorescence video microscopy data sets. [ABSTRACT FROM AUTHOR]

Background: Blood-based biomarkers are gaining grounds for the detection of Alzheimer's disease (AD) and related disorders (ADRDs). However, two key obstacles remain: the lack of methods for multi-analyte assessments and the need for biomarkers for related pathophysiological processes like neuroinflammation, vascular, and synaptic dysfunction. A novel proteomic method for pre-selected analytes, based on proximity extension technology, was recently introduced. Referred to as the NULISAseq CNS disease panel, the assay simultaneously measures ~ 120 analytes related to neurodegenerative diseases, including those linked to both core (i.e., tau and amyloid-beta (Aβ)) and non-core AD processes. This study aimed to evaluate the technical and clinical performance of this novel targeted proteomic panel. Methods: The NULISAseq CNS disease panel was applied to 176 plasma samples from 113 individuals in the MYHAT-NI cohort of predominantly cognitively normal participants from an economically underserved region in southwestern Pennsylvania, USA. Classical AD biomarkers, including p-tau181, p-tau217, p-tau231, GFAP, NEFL, Aβ40, and Aβ42, were independently measured using Single Molecule Array (Simoa) and correlations and diagnostic performances compared. Aβ pathology, tau pathology, and neurodegeneration (AT(N) statuses) were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and an MRI-based AD-signature composite cortical thickness index, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA and neuroimaging-determined AT(N) biomarkers. Results: NULISA concurrently measured 116 plasma biomarkers with good technical performance (97.2 ± 13.9% targets gave signals above assay limits of detection), and significant correlation with Simoa assays for the classical biomarkers. Cross-sectionally, p-tau217 was the top hit to identify Aβ pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878–0.983). Fourteen markers were significantly decreased in Aβ-PET + participants, including TIMP3, BDNF, MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aβ PET-dependent yearly increases in Aβ-PET + participants. Novel plasma biomarkers with tau PET-dependent longitudinal changes included proteins associated with neuroinflammation, synaptic function, and cerebrovascular integrity, such as CHIT1, CHI3L1, NPTX1, PGF, PDGFRB, and VEGFA; all previously linked to AD but only reliable when measured in cerebrospinal fluid. The autophagosome cargo protein SQSTM1 exhibited significant association with neurodegeneration after adjusting age, sex, and APOE ε4 genotype. Conclusions: Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes, consistent with the recently revised biological and diagnostic framework. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD. [ABSTRACT FROM AUTHOR]

Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how human U1 snRNP recognizes particular 5′ splice sites (5′SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5′SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5′SS modulation. We have determined a sequential binding mechanism for modulation of −1A bulged 5′SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged with a −1A bulged 5′SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5′SS complexes. Branaplam targets a ribonucleoprotein, not only an RNA duplex, and its action depends on fundamental properties of 5′SS recognition. Splice sites must be correctly identified for introns to be removed from pre-mRNAs and for expression of the correct mRNA isoforms. Here, the authors elucidate how the human U1 snRNP splicing factor recognizes 5′ splice sites and how a splicing modulating drug can enhance splice site recognition. [ABSTRACT FROM AUTHOR]

Photoactivatable fluorescent probes are valuable tools in bioimaging for tracking cells down to single molecules and for single molecule localization microscopy. For the latter application, green emitting dyes are in demand. We herein developed an efficient green‐emitting photoactivatable furanyl‐BODIPY (PFB) and we established a new mechanism of photoactivation called Directed Photooxidation Induced Activation (DPIA) where the furan is photo‐oxidized in a directed manner by the singlet oxygen produced by the probe. The efficient photoconverter (93‐fold fluorescence enhancement at 510 nm, 49 % yield conversion) is functionalizable and allowed targeting of several subcellular structures and organelles, which were photoactivated in live cells. Finally, we demonstrated the potential of PFB in super‐resolution imaging by performing PhotoActivated Localization Microscopy (PALM) in live cells. [ABSTRACT FROM AUTHOR]

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis. The mechanisms controlling the formation of cellular adhesions are not fully understood. Here, the authors use single molecule tracking and super-resolution imaging to show that the actin cytoskeleton regulates integrin diffusion in the developing Drosophila embryo. [ABSTRACT FROM AUTHOR]

The advent of third‐generation sequencing (TGS) represents a significant shift in the field of genetic sequencing, enabling single‐molecule sequencing to overcome limitations of short‐read NGS platforms. Several studies have assessed the utilisation of TGS in HLA genotyping, though many of these studies have described the high error rate as an obstacle to achieving a robust and highly accurate HLA typing assay. In 2021, Oxford Nanopore Technologies (ONT) released the high‐accuracy sequencing Kit 14 and the MinION flow cell model R10.4.1, which were reported to achieve sequencing accuracies up to 99%. The aim of this study was to validate this novel high‐accuracy sequencing kit for HLA genotyping coupled with a full‐gene HLA PCR assay. Comparison with historical data obtained using legacy flow cell models such as R9.4, R10.3 and R10.4 was also done to assess progressive improvement in sequencing performance with each sequential release. The workflow was validated based on data throughput, sequence quality and accuracy, and HLA genotyping resolution. An initial validation was performed using an internal reference panel of 42 samples representing common HLA allele groups, followed by an analysis of data obtained from 111 sequencing batch runs since the implementation, to assess assay performance and define quality control metrics to assess inter‐run variability and monitor quality. Furthermore, challenges arising from MinION flow cell stability and use, and assessment of barcode contamination are discussed. The findings of this study highlight advantages of ONT sequencing kit 14/R10.4.1 for HLA genotyping and the implementation considerations for the routine diagnostic HLA laboratory. [ABSTRACT FROM AUTHOR]

This study showed that the predictor in logistic regression can be applied to estimating the Gibbs free energy of tRNAs' recognition of and binding to their aminoacyl-tRNA synthetases. Then, 24 linear logistic regression models predicting different classes of tRNAs loaded with a corresponding amino acid were trained in a machine learning classification method, reducing the misclassification error to zero. The models were based on minimal subsets of Boolean explanatory variables describing the favorite presence of nucleotides or nucleosides localized in the different parts of the tRNA. In 90% of cases, they agree with the components of the consensus strand in a class of tRNAs loaded by a given amino acid. According to the proposed theoretical model, the values of the free energy for the entry of the recognition state in the process of tRNA charging were obtained, and the inputs from identity nucleotides and the tRNA strand backbone were distinguished. Almost all the resulting models indicated leading anticodon tandems defining the first and second positions of the anticodon (positions 35 and 36 of the tRNA strand) and the small sets (up to six positions) of the other nucleotides as the natural identity nucleotides most influential in the free energy balance. The magnitude of their input to this energy depends on the position in the strand, favoring positions −1, 35, and 36. The role of position 34 is relatively smaller. These identity attributes may not always be fully arranged in a real single adaptor molecule but were comprehensively present in a given tRNA class. A detailed analysis of the resulting models showed that the absolute value of the energy of binding the tandem 35–36 decreases with the number of identity positions, as well as with the decreasing number of possible hydrogen bonds. On the other hand, in these conditions, the absolute value of the energy of binding of other identity nucleotides increases. All the models indicate that the nucleotide-independent energy of the repulsion tRNA backbone decreases with the number of identity nucleotides. It was also shown that the total free energy change in entering the recognition state increases with the amino acid mass, making this process less spontaneous, which may have an evolutionary reference. [ABSTRACT FROM AUTHOR]

Simple Summary: Scientists have developed new techniques that allow them to study the heart at an incredibly detailed level, focusing on individual cells rather than looking at groups of cells together. This is important because the heart is made up of many different types of cells, each with its own role, and understanding how they interact is key to discovering what happens in both healthy hearts and in heart disease. By using methods that look at the genes, proteins, and other molecules inside single cells, researchers can uncover how cells communicate and change over time. These discoveries are helping scientists learn more about how the heart develops, how it stays healthy, and what goes wrong in heart diseases. This knowledge could lead to better treatments and new ways to diagnose heart conditions, helping people live longer, healthier lives. By directly measuring multiple molecular features in hundreds to millions of single cells, single-cell techniques allow for comprehensive characterization of the diversity of cells in the heart. These single-cell transcriptome and multi-omic studies are transforming our understanding of heart development and disease. Compared with single-dimensional inspections, the combination of transcriptomes with spatial dimensions and other omics can provide a comprehensive understanding of single-cell functions, microenvironment, dynamic processes, and their interrelationships. In this review, we will introduce the latest advances in cardiac health and disease at single-cell resolution; single-cell detection methods that can be used for transcriptome, genome, epigenome, and proteome analysis; single-cell multi-omics; as well as their future application prospects. [ABSTRACT FROM AUTHOR]

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H+) act to directly and indirectly (via decreased Ca2+ activation) inhibit myosin's force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases. [ABSTRACT FROM AUTHOR]

Experimental methods of single-molecule enzymology allow scientists to determine physicochemical properties of distinct single molecules of various enzymes and to perform direct monitoring of functioning of enzymes at different steps of their catalytic cycle. The approach based on the use of solid-state nanopores is a promising tool for studying the functioning of single-enzyme molecules. Herein, this approach is employed for monitoring the functioning of cytochrome P450 BM3, which represents a very convenient model of cytochrome P450-containing monooxygenase systems. A nanopore of ~5 nm in diameter has been formed in a 40 nm-thick silicon nitride chip by electron beam drilling (EBD), and a single molecule of the BM3 enzyme has been entrapped in the pore. The functioning of the enzyme molecule has been monitored by recording the time dependence of the ion current through the nanopore during the reaction of laurate hydroxylation. In our experiments, the enzyme molecule has been found to be active for 1500 s. The results of our research can be further used in the development of highly sensitive detectors for single-molecule studies in enzymology. [ABSTRACT FROM AUTHOR]

Limit of detection (LoD) is a term that is used to characterize the sensitivity of an analytical method. The existing limitation of the sensitivity of analysis using modern mass spectrometry methods has been experimentally shown to be a limiting factor in the application of proteomic technologies in medicine. This article proposes a concept of a new technology that will set a new vector of development in the development of systems for solving problems of medical diagnostics and deals with theoretical and practical aspects of creating a new technology for the detection of single biomacromolecules (in particular, proteins) in biological samples. Such technology should be based on the principle of signal registration similar to that used in a Geiger counter (also known as a Geiger–Müller counter or G-M counter), a device that automatically counts the number of ionizing particles that hit it. This counter is free from probabilistic components; it registers a signal if there is at least one target molecule in the analysis chamber. Predictive medical diagnostics require technology based on methods where sensitivity allows for the detection of single marker molecules in a biological sample volume of 1–10 µL, the smallest volume of biomaterial used in laboratory diagnostics. Creation of a detector with a sensitivity of 10−18 M would allow for the detection of one molecule in 1 µL of the sample, which fundamentally makes this approach analogous to a G-M counter for solutions. To date, bioanalytical methods are limited to a sensitivity of 10−12 M (which is approximately 1 million molecules per 1 μL), which is insufficient to capture the early stages of pathological processes. [ABSTRACT FROM AUTHOR]

Huntington disease (HD) is a genetic neurodegenerative disease caused by cytosine, adenine, guanine (CAG) expansion in the Huntingtin (HTT) gene, translating to an expanded polyglutamine tract in the HTT protein. Age at disease onset correlates to CAG repeat length but varies by decades between individuals with identical repeat lengths. Genome-wide association studies link HD modification to DNA repair and mitochondrial health pathways. Clinical studies show elevated DNA damage in HD, even at the premanifest stage. A major DNA repair node influencing neurodegenerative disease is the PARP pathway. Accumulation of poly adenosine diphosphate (ADP)-ribose (PAR) has been implicated in Alzheimer and Parkinson diseases, as well as cerebellar ataxia. We report that HD mutation carriers have lower cerebrospinal fluid PAR levels than healthy controls, starting at the premanifest stage. Human HD induced pluripotent stem cell-derived neurons and patient-derived fibroblasts have diminished PAR response in the context of elevated DNA damage. We have defined a PAR-binding motif in HTT, detected HTT complexed with PARylated proteins in human cells during stress, and localized HTT to mitotic chromosomes upon inhibition of PAR degradation. Direct HTT PAR binding was measured by fluorescence polarization and visualized by atomic force microscopy at the single molecule level. While wild-type and mutant HTT did not differ in their PAR binding ability, purified wild-type HTT protein increased in vitro PARP1 activity while mutant HTT did not. These results provide insight into an early molecular mechanism of HD, suggesting possible targets for the design of early preventive therapies. [ABSTRACT FROM AUTHOR]

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. Here, we present two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb its function. We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry, compatible with a transcyclooctene coupled fluorescent dye. Additionally, by inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we can incorporate a fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population. We identified T468 as a rapid labeling site (~5 min) based on functional and biochemical assays in HEK293T/17 cells. Through adapting linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, we generated a fully functional channel construct, TRPV1exCellHalo, with intact wild-type gating properties. We used TRPV1exCellHalo in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The application of these extracellular label TRPV1 (exCellTRPV1) constructs to track surface localization of the channel will shed significant light on the mechanisms regulating its expression and provide a general scheme to introduce similar modifications to other cell surface receptors. [ABSTRACT FROM AUTHOR]

Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro -infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription. [Display omitted] • We uncovered a heterogeneous transcriptional termination of HBV transcripts in cellular models and in patients with CHB. • HBV transcription termination is functionally linked to HBV replication. • RNA helicases DDX5 and DDX17 promote HBV transcriptional read-through and viral RNA destabilisation. • DDX5 and DDX17 are host HBV restriction factors and are downregulated in patients with CHB. [ABSTRACT FROM AUTHOR]

Cadherins are calcium dependent adhesion proteins that establish and maintain the intercellular mechanical contact by bridging the gap between adjacent cells. Desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) are tissue specific cadherin isoforms of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2 -gene and in the DSC2 -gene are related to arrhythmogenic right ventricular cardiomyopathy (ARVC) a rare but severe heart muscle disease. Here, several possible homophilic and heterophilic binding interactions of wild-type Dsg2, wild-type Dsc2, as well as one Dsg2- and two Dsc2-variants, each associated with ARVC, are investigated. Using single molecule force spectroscopy (SMFS) with atomic force microscopy (AFM) and applying Jarzynski's equality the kinetics and thermodynamics of Dsg2/Dsc2 interaction can be determined. The free energy landscape of Dsg2/Dsc2 dimerization exposes a high activation energy barrier, which is in line with the proposed strand-swapping binding motif. Although the binding motif is not affected by any of the mutations, the binding kinetics of the interactions differ significantly from the wild-type. While wild-type cadherins exhibit an average complex lifetime of approx. 0.3 s interactions involving a variant consistently show - lifetimes that are substantially larger. The lifetimes of the wild-type interactions give rise to the picture of a dynamic adhesion interface consisting of continuously dissociating and (re)associating molecular bonds, while the delayed binding kinetics of interactions involving an ARVC-associated variant might be part of the pathogenesis. Our data provide a comprehensive and consistent thermodynamic and kinetic description of cardiac cadherin binding, allowing detailed insight into the molecular mechanisms of cell adhesion. [Display omitted] • Integrity of heart muscle tissue relies on weak molecular bonds between cadherins. • Single molecule force spectroscopy reveals cadherin binding kinetics. • Wild-type cadherin bonds exhibit characteristic fast (re-) binding kinetics. • Specific cardiomyopathy associated cadherin variants slow binding kinetics. • Impaired molecular dynamics could be a cardiomyopathy-specific pathomechanism. [ABSTRACT FROM AUTHOR]

Cancer remains a significant global health challenge, prompting exploration into alternative treatments such as those derived from natural compounds found in traditional medicine. Recent research has underscored the role of proteins like Focal Adhesion Kinase (FAK), Vascular Endothelial Growth Factor (VEGF), and Metastasis-Associated Protein 1 (MTA1) in driving cancer cell proliferation and survival. Here, we investigated the potential of a single molecule to modulate these key proteins involved in metastasis, offering a promising avenue for cancer therapy. Terminalia elliptica, commonly known as Asna, possesses a diverse range of medicinal properties, including antimicrobial, anti-inflammatory, anticancer, antidiabetic, antiaging, hepatoprotective, antioxidant, and neuroprotective activities. Our study aimed to explore the anticancer potential of Terminalia elliptica by identifying bioactive compounds capable of targeting FAK, VEGF, and MTA1 to impede cancer metastasis. Through in silico analysis, we conducted network analysis using Cytoscape to assess the significance of these bioactive compounds in the inhibition of signaling pathways driving metastasis. The utilization of these bioactives as potential candidates for targeted therapy of VEGF, FAK, and MTA1 regulated pathways was preliminarily assessed by Molecular Docking and MD Simulation. Our findings revealed that phytobioactives namely, Chebulinic Acid of Terminalia elliptica, exhibited notable binding affinity and interaction with FAK, and Chebulagic Acid with VEGF, and MTA1. This discovery holds promise as a novel therapeutic approach for combating cancer, offering potential benefits in cancer treatment and management. [ABSTRACT FROM AUTHOR]

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter‐ and intra‐molecular interactions. Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes. Furthermore, the principle of protein assembly gives guidelines for new biomimetic materials with potential applications in medicine, energy, and nanotechnology. Atomic force microscopy (AFM) is a powerful tool for investigating protein assembly and interactions across spatial scales (single molecules to cells) and temporal scales (milliseconds to days). It has significantly contributed to understanding nanoscale architectures, inter‐ and intra‐molecular interactions, and regulatory elements that determine protein structures, assemblies, and functions. This review describes recent advancements in elucidating protein assemblies with in situ AFM. We discuss the structures, diffusions, interactions, and assembly dynamics of proteins captured by conventional and high‐speed AFM in near‐native environments and recent AFM developments in the multimodal high‐resolution imaging, bimodal imaging, live cell imaging, and machine‐learning‐enhanced data analysis. These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research. [ABSTRACT FROM AUTHOR]