Search

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]

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]

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]

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]

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]

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]

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]

Single-molecule localization microscopy (SMLM) surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states, precisely pinpointing the resulted isolated emission patterns, thereby reconstructing the super-resolution images based on the accumulated locations of thousands to millions of single molecules. This technique achieves a ten-fold improvement in resolution, unveiling the intricate details of molecular activities and structures in cells and tissues. Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes, providing insights into structural intricacies and spatial relationships among different targets. This review explores recent advancements in multicolor SMLM, evaluates the strengths and limitations of each variant, and discusses the future prospects. [ABSTRACT FROM AUTHOR]

Dengue virus, an arbovirus of genus Flavivirus, is an infectious disease causing organisms in the tropical environment leading to numerous deaths every year. No therapeutic is available against the virus till date with only symptomatic relief available. Here, we have tried to design therapeutic compounds from scratch by fragment based method followed by pharmacophore based modelling to find suitable similar structure molecules and validated the same by MD simulation, followed by binding energy calculations and ADMET analysis. The receptor binding region of the dengue envelope protein was considered as the target for prevention of viral host cell entry and thus infection. This resulted in the final selection of kanamycin as a stable binding molecule against the Dengue virus envelope protein receptor binding domain. This study results in selection of a single molecule having high binding energy and prominent stable interactions as determined by post simulation analyses. This study aims to provide a direction for development of small molecule therapeutics against the dengue virus in order to control infection. This study may open a new avenue in the arena of structure based and fragment based therapeutic design to obtain novel molecules with therapeutic potential. [ABSTRACT FROM AUTHOR]

The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family. [ABSTRACT FROM AUTHOR]

We refresh the design of zero-mode waveguides (ZMWs) by introducing metamaterials that makes the zeroth order resonant mode existence. Of particular importance, the resulting electromagnetic field exhibits nearly constant distribution but not a trivial solution of Maxwell's equation, showing great advantage to equalize the excitation rate of molecules throughout the waveguides. A closed form expression for the wave impedance is derived which is verified by the finite-difference time-domain simulations. Benefitted from the cavity Purcell effect which is lacking in existing ZMWs, fluorescence amplification and lifetime reduction are simultaneously enhanced. A practical design where the excitation volume reduced down to sub-zeptoliter and the fluorescence lifetime shortened to picosecond scale is illustrated. This result makes single molecule real time (SMRT) sensing of biochemical reactions at biophysically relevant concentrations (~ μM) possible, combining off-the-shelf ultrafast lasers. [ABSTRACT FROM AUTHOR]

Imaging-based spatial transcriptomics technologies such as Multiplexed error-robust fluorescence in situ hybridization (MERFISH) can capture cellular processes in unparalleled detail. However, rigorous and robust analytical tools are needed to unlock their full potential for discovering subcellular biological patterns. We present Intracellular Spatial Transcriptomic Analysis Toolkit (InSTAnT), a computational toolkit for extracting molecular relationships from spatial transcriptomics data at single molecule resolution. InSTAnT employs specialized statistical tests and algorithms to detect gene pairs and modules exhibiting intriguing patterns of co-localization, both within individual cells and across the cellular landscape. We showcase the toolkit on five different datasets representing two different cell lines, two brain structures, two species, and three different technologies. We perform rigorous statistical assessment of discovered co-localization patterns, find supporting evidence from databases and RNA interactions, and identify associated subcellular domains. We uncover several cell type and region-specific gene co-localizations within the brain. Intra-cellular spatial patterns discovered by InSTAnT mirror diverse molecular relationships, including RNA interactions and shared sub-cellular localization or function, providing a rich compendium of testable hypotheses regarding molecular functions. Here, the authors introduce the Intracellular Spatial Transcriptomic Analysis Toolkit (InSTAnT), which allows researchers to study the information encoded in the physical proximity of individual transcripts by detecting persistent subcellular patterns. [ABSTRACT FROM AUTHOR]

Inferring the 3D structure and conformation of disordered biomolecules, e.g., single stranded nucleic acids (ssNAs), remains challenging due to their conformational heterogeneity in solution. Here, we use escape-time electrometry (ETe) to measure with sub elementary-charge precision the effective electrical charge in solution of short to medium chain length ssNAs in the range of 5–60 bases. We compare measurements of molecular effective charge with theoretically calculated values for simulated molecular conformations obtained from Molecular Dynamics simulations using a variety of forcefield descriptions. We demonstrate that the measured effective charge captures subtle differences in molecular structure in various nucleic acid homopolymers of identical length, and also that the experimental measurements can find agreement with computed values derived from coarse-grained molecular structure descriptions such as oxDNA, as well next generation ssNA force fields. We further show that comparing the measured effective charge with calculations for a rigid, charged rod—the simplest model of a nucleic acid—yields estimates of molecular structural dimensions such as linear charge spacings that capture molecular structural trends observed using high resolution structural analysis methods such as X-ray scattering. By sensitively probing the effective charge of a molecule, electrometry provides a powerful dimension supporting inferences of molecular structural and conformational properties, as well as the validation of biomolecular structural models. The overall approach holds promise for a high throughput, microscopy-based biomolecular analytical approach offering rapid screening and inference of molecular 3D conformation, and operating at the single molecule level in solution. [ABSTRACT FROM AUTHOR]

Previously [A.O. Savostianov, I.Yu. Eremchev, T. Plakhotnik, and A.V. Naumov, Phys. Rev. B 110, 045430 (2024)], we have shown that the zero-phonon line broadening of single tetra-tert-butylterrylene molecules embedded in the matrix of amorphous polyisobutylene is a result of coupling with resonant vibrational modes arising due to the impurity effect on the host normal modes. However, the question remained whether this model would be compatible with the extensive single-molecule spectroscopy and photon echo data previously obtained for the same host/guest system. In the present work we demonstrate the high predictive power of the resonant mode model treated in the framework of the non-perturbative theory of electron–phonon coupling, and good agreement with all experimental data. Furthermore, for single-molecule data we find an unexpectedly large dispersion of the force constants for impurity molecules. To explain this dispersion, we propose a simple microscopic model that assumes fluctuations in the distance between the impurity molecule and its nearest surroundings. [ABSTRACT FROM AUTHOR]

Non-equilibrium Green's function (NEGF) and density functional theory (DFT) calculations are used to explore the impact of doping on the electron transport properties in a single tetracene molecule linked to gold electrodes using isocyanide anchoring groups. Boron (B) and Nitrogen (N) atoms are used for doping and co-doping (BN) of the carbon atoms placed at the edge of the tetracene molecule. It was found that the chemical doping of tetracene molecules mainly impacts the rectification trends compared to non-doped molecules. Our findings indicate that B doping significantly improves the rectification ratio compared to other dopants because of a greater difference between the current values under positive and negative biases as a result of asymmetric I-V characteristics. These inferences have also been assessed in terms of MPSH and transmission spectra. In addition, novel characteristic of negative differential resistance (NDR) is attained in single dopant molecular junctions. [ABSTRACT FROM AUTHOR]

The Asian green mussel, Perna viridis is an important aquaculture species in the family Mytilidae contributing substantially to molluscan aquaculture. We generated a high-quality chromosome level assembly of this species by combining PacBio single molecule sequencing technique (SMRT), Illumina paired-end sequencing, high-throughput chromosome conformation capture technique (Hi-C) and Bionano mapping. The final assembly resulted in a genome of 723.49 Mb in size with a scaffold N50 of 49.74 Mb with 99% anchored into 15 chromosomes. A total of 49654 protein-coding genes were predicted from the genome. The presence of 634 genes associated with the cancer pathway and 408 genes associated with viral carcinogenesis indicates the potential of this species to be used as a model for cancer studies. The chromosome-level assembly of this species is also a valuable resource for further genomic selection and selective breeding for improving economically important aquaculture traits and augmenting aquaculture productivity. [ABSTRACT FROM AUTHOR]

Lujo virus (LUJV) is a human pathogen that was the cause of a deadly hemorrhagic fever outbreak in Africa. LUJV is a divergent member of the Arenaviridae with some similarities to both the "Old World" and "New World" serogroups, but it uses a cell-entry receptor, neuropilin-2 (NRP2), that is distinct from the receptors of OW and NW viruses. Though the receptor binding domain of LUJV has been characterized structurally, the overall organization of the trimeric spike complex and how NRP2 is recognized in this context were unknown. Here, we present the structure of the membrane-embedded LUJV spike complex determined by cryo-electron microscopy. Analysis of the structure suggested that a single NRP2 molecule is bound at the apex of the trimeric spike and that multiple subunits of the trimer contact the receptor. The binding of NRP2 involves an intriguing arginine-methionine interaction, which we analyzed using quantum mechanical modeling methods. We compare the LUJV spike structure with the only other available structure of a complete arenaviral spike, which is the Lassa virus. The similarities and differences between them shed light on Arenavirus evolution, inform vaccine design, and provide information that will be useful in combating future Arenavirus outbreaks. The Lujo virus is a deadly human pathogen. Here, Eilon-Ashkenazy et al. determine the structure of the viral spike complex and elucidate key aspects of its function. [ABSTRACT FROM AUTHOR]

A regular method has been proposed to quantitatively describe the fluorescence of molecules randomly moving in a nanowell. Correlation functions of the fluorescence of single molecules depending on the geometry of the nanowell and the penetration depth of an exciting field into it have been determined. The obtained results can be used to quantitatively analyze the properties of molecules and to extract information on the parameters of the nanowell. [ABSTRACT FROM AUTHOR]

Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under conditions which recapitulate the neutral pH and ionic strength of the cytosol. The formation of protein aggregates is a hallmark of Parkinson's disease, with small oligomeric species implicated as a major source of toxicity. In this work, Xu et al. determine their mechanism of formation and role in aggregation. [ABSTRACT FROM AUTHOR]

The capability to excite, probe, and manipulate vibrational modes is essential for understanding and controlling chemical reactions at the molecular level. Recent advancements in tip-enhanced Raman spectroscopies have enabled the probing of vibrational fingerprints in a single molecule with Ångström-scale spatial resolution. However, achieving controllable excitation of specific vibrational modes in individual molecules remains challenging. Here, we demonstrate the selective excitation and probing of vibrational modes in single deprotonated phthalocyanine molecules utilizing resonance Raman spectroscopy in a scanning tunneling microscope. Selective excitation is achieved by finely tuning the excitation wavelength of the laser to be resonant with the vibronic transitions between the molecular ground electronic state and the vibrational levels in the excited electronic state, resulting in the state-selective enhancement of the resonance Raman signal. Our approach contributes to setting the stage for steering chemical transformations in molecules on surfaces by selective excitation of molecular vibrations. The authors demonstrate selective excitation of atomic vibrations in a single molecule utilizing tip-enhanced resonance Raman spectroscopy, by precisely tuning the wavelength of the exciting laser to be resonant with the molecular vibronic transitions. [ABSTRACT FROM AUTHOR]

Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs. [ABSTRACT FROM AUTHOR]

Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies. New optical methods for investigating the activity of small molecules in cells may facilitate development of anticancer therapeutics. Here, the authors use a super-resolution optical platform and single molecule tracking to gain insight into WRN regulation in cancer. [ABSTRACT FROM AUTHOR]

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing. Cas9 remains bound to DNA after cleavage and its removal is required for DNA double-strand break repair. Here, the authors show that the HLTF translocase disrupts the Cas9- DNA post-cleavage complexes in a process that requires the HLTF HIRAN domain and ATPase activity. [ABSTRACT FROM AUTHOR]

Hydroxyurea (HU; hydroxycarbamide) is a chemotherapy medication used to treat various types of cancer and other diseases such as sickle cell anemia. HU inhibits DNA synthesis by targeting ribonucleotide reductase (RNR). Recent studies have suggested that HU also causes oxidative stress in living systems. In the present study, we investigated if HU could directly affect the activity and/or conformation of DNA. We measured in vitro gene expression in the presence of HU by adapting a cell-free luciferase assay. HU exhibited a bimodal effect on gene expression, where promotion or inhibition were observed at lower or higher concentrations (mM range), respectively. Using atomic force microscopy (AFM), the higher-order structure of DNA was revealed to be partially-thick with kinked-branching structures after HU was added. An elongated coil conformation was observed by AFM in the absence of HU. Single DNA molecules in bulk aqueous solution under fluctuating Brownian motion were imaged by fluorescence microscopy (FM). Both spring and damping constants, mechanical properties of DNA, increased when HU was added. These experimental investigations indicate that HU directly interacts with DNA and provide new insights into how HU acts as a chemotherapeutic agent and targets other diseases. [ABSTRACT FROM AUTHOR]

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging. [ABSTRACT FROM AUTHOR]

Electrokinetic force has been the major choice for driving the translocation of molecules through a nanopore. However, the use of this approach is limited by an uncontrollable translocation speed, resulting in non-uniform conductance signals with low conformational sensitivity, which hinders the accurate discrimination of the molecules. Here, we show the use of inertial-kinetic translocation induced by spinning an in-tube micro-pyramidal silicon nanopore fabricated using photovoltaic electrochemical etch-stop technique for biomolecular sensing. By adjusting the kinetic properties of a funnel-shaped centrifugal force field while maintaining a counter-balanced state of electrophoretic and electroosmotic effect in the nanopore, we achieved regulated translocation of proteins and obtained stable signals of long and adjustable dwell times and high conformational sensitivity. Moreover, we demonstrated instantaneous sensing and discrimination of molecular conformations and longitudinal monitoring of molecular reactions and conformation changes by wirelessly measuring characteristic features in current blockade readouts using the in-tube nanopore device. The authors report a strategy to achieve high S/N ratio signal readout in single molecule sensing by incorporating the inertial forces as a new channel for independently controlling the translocation parameters with high precision. [ABSTRACT FROM AUTHOR]

The advent of civilian spaceflight challenges scientists to precisely describe the effects of spaceflight on human physiology, particularly at the molecular and cellular level. Newer, nanopore-based sequencing technologies can quantitatively map changes in chemical structure and expression at single molecule resolution across entire isoforms. We perform long-read, direct RNA nanopore sequencing, as well as Ultima high-coverage RNA-sequencing, of whole blood sampled longitudinally from four SpaceX Inspiration4 astronauts at seven timepoints, spanning pre-flight, day of return, and post-flight recovery. We report key genetic pathways, including changes in erythrocyte regulation, stress induction, and immune changes affected by spaceflight. We also present the first m6A methylation profiles for a human space mission, suggesting a significant spike in m6A levels immediately post-flight. These data and results represent the first longitudinal long-read RNA profiles and RNA modification maps for each gene for astronauts, improving our understanding of the human transcriptome's dynamic response to spaceflight. Here the authors explore the role of chemical modifications within RNA molecules in spaceflight response, observing increased m6A mRNA modifications immediately post-spaceflight in gene markers associated with stress response. [ABSTRACT FROM AUTHOR]

Background: Blood-based biomarkers for Alzheimer's disease (AD) are promising to be used in clinical settings. The liver is an important degradation organ of the body. Whether liver function affects the levels of AD biomarkers needs to be studied. Objective: To investigate the associations between liver function and the plasma levels of AD biomarkers. Methods: We conducted an ADNI cohort-based cross-sectional study. Thirteen liver function markers commonly used in clinical settings were analyzed: total protein (TP), albumin (AL), globulin (GL), AL/GL ratio (A/G), total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), AST/ALT ratio, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (GGT). Liquid chromatography-tandem mass spectrometry was used to detect the plasma Aβ42 and Aβ40 concentrations. Single Molecule array technique was used to measure the plasma p-tau181 and NfL concentrations. We used linear regression models to analyze the associations between liver function markers and the levels of AD plasma biomarkers. Results: ALP was positively associated with the levels of plasma Aβ42 (β = 0.16, P = 0.018) and Aβ40 (β = 0.21, P = 0.004). LDH was positively associated with the levels of plasma p-tau181 (β = 0.09, P = 0.022). While NfL was correlated with multiple liver function markers, including AL, A/G, ALT, AST/ALT, and LDH. Conclusion: Liver function was associated with the plasma levels of AD biomarkers. It needs to consider the potential influence of liver function on the reference ranges and the interpretation of results for AD biomarkers before clinical use. [ABSTRACT FROM AUTHOR]

Metasurfaces are currently among the most attractive research fields, in no small measure due to their usability as refractometric sensors, offering unparalleled sensitivity up to sensing single atoms or molecules. This is rooted in their ability to localize electromagnetic fields in volumes orders of magnitude below the diffraction limit. Numerous materials, both conductive (metals, transparent conductive oxides, graphene, MXenes...) and all-dielectric (oxides, low-loss semiconductors) can be used to build them, thus tailoring metasurfaces, imparting multifunctionalities and enhancing design freedom. However, the fundamental sensing mechanism is practically identical across refractometric platforms – spectral dispersion changes of scattering parameters due to a difference between the real parts of refractive index of the analyte and the environment. Here we describe the use of exceptional capabilities of metasurfaces in transforming optical space to recognize analytes with identical real parts of refractive index but different imaginary parts. We consider a metasurface biosensor with cruciform openings array in an ultrathin silicon layer on silica. We simulate variations of the imaginary part of the effective refractive index in the openings while keeping the real part constant. The circular power flow that increases the optical path, field localization and intrinsically low losses in the optical range jointly cause that adding even the minuscule amounts of analyte with slightly increased optical absorption significantly reduces transmission through the structure, despite its exceptionally low thickness. The described principle can be used to distinguish bio-analytes with identical real part of refractive index like RNA, DNA, proteins, lipids, but also non-biological analytes. [ABSTRACT FROM AUTHOR]

Controllable photofluorochromic systems with high contrast and multicolor in both solutions and solid states are ideal candidates for the development of dynamic artificial intelligence. However, it is still challenging to realize multiple photochromism within one single molecule, not to mention good controllability. Herein, we report an aggregation-induced emission luminogen TPE-2MO2NT that undergoes oxidation cleavage upon light irradiation and is accompanied by tunable multicolor emission from orange to blue with time-dependence. The photocleavage mechanism revealed that the self-generation of reactive oxidants driving the catalyst-free oxidative cleavage process. A comprehensive analysis of TPE-2MO2NT and other comparative molecules demonstrates that the TPE-2MO2NT molecular scaffold can be easily modified and extended. Further, the multicolor microenvironmental controllability of TPE-2MO2NT photoreaction within polymer matrices enables the fabrication of dynamic fluorescence images and 4D information codes, providing strategies for advanced controllable information encryption. Photochromic systems with high contrast and multiple colours are useful for responsive materials, but it is challenging to achieve multiple colours with one molecule. Here, the authors report a photochromic system controlled by oxidative cleavage on light irradiation. [ABSTRACT FROM AUTHOR]

The rotational dynamics of a molecule is sensitive to neighboring atoms or molecules, which can be used to probe the intermolecular interactions in the gas phase. Here, we real-time track the laser-driven rotational dynamics of a single N2 molecule affected by neighboring Ar atoms using coincident Coulomb explosion imaging. We find that the alignment trace of N-N axis decays fast and only persists for a few picoseconds when an Ar atom is nearby. We show that the decay rate depends on the rotational geometry of whether the Ar atom stays in or out of the rotational plane of the N2 molecule. Additionally, the vibration of the van der Waals bond is found to be excited through coupling with the rotational N-N axis. The observations are well reproduced by solving the time-dependent Schrödinger equation after taking the interaction potential between the N2 and Ar into consideration. Our results demonstrate that environmental effects on a molecular level can be probed by directly visualizing the rotational dynamics. The authors demonstrate, using coincident Coulomb explosion imaging, that the rotational dynamics of single nitrogen molecules can be used as a probe to sense the interactions with surrounding Ar atoms in gas-phase clusters. [ABSTRACT FROM AUTHOR]

This study employs ab initio calculations based on density functional theory (DFT) to investigate the structural properties, 1H-NMR spectra, and vibrational spectra of methane sulfonic acid (MSA) at low degree of hydration. The findings reveal that energetically stable structures are formed by small clusters consisting of one or two MSA molecules (m = 1 and 2) and one or two water molecules in (MSA)m·(H2O)n (m = 1–2 and n = 1–5).These stable structures arise from the formation of strong cyclic hydrogen bonds between the proton of the hydroxyl (OH) group in MSA and the water molecules. However, clusters containing three or more water molecules (n > 2) exhibit proton transfer from MSA to water, resulting in the formation of ion-pairs composed of CH3SO3− and H3O+species. The measured 1H-NMR spectra demonstrate the presence of hydrogen-bonded interactions between MSA and water, with a single MSA molecule interacting with water molecules. This interaction model accurately represents the hydrogen bonding network, as supported by the agreement between the experimental and calculated NMR chemical shift results. [ABSTRACT FROM AUTHOR]

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments. This study combines lattice light sheet microscopy and single molecule imaging to study protein dynamics and chromatin structure in live cells. The authors describe how nucleosomes and proteins move and are organised in relation to chromatin density. [ABSTRACT FROM AUTHOR]

Single particle imaging at atomic resolution is perhaps one of the most desired goals for ultrafast X-ray science with X-ray free-electron lasers. Such a capability would create great opportunity within the biological sciences, as high-resolution structural information of biosamples that may not crystallize is essential for many research areas therein. In this paper, we report on a comprehensive computational study of diffraction image formation during single particle imaging of a macromolecule, containing over one hundred thousand non-hydrogen atoms. For this study, we use a dedicated simulation framework, SIMEX, available at the European XFEL facility. Our results demonstrate the full feasibility of computational single-particle imaging studies for biological samples of realistic size. This finding is important as it shows that the SIMEX platform can be used for simulations to inform relevant single-particle-imaging experiments and help to establish optimal parameters for these experiments. This will enable more focused and more efficient single-particle-imaging experiments at XFEL facilities, making the best use of the resource-intensive XFEL operation. [ABSTRACT FROM AUTHOR]

Translation initiation in bacteria is frequently regulated by various structures in the 5′ untranslated region (5′UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aim to explore how G4 formation in mRNA (RG4) at 5′UTR impacts translation using a T7-based in vitro translation system and in E. coli. We show that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We find that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We propose a physical barrier model in which bulky structures in 5′UTR biases ribosome movement toward the downstream start codon, thereby increasing the translation output. This study provides biophysical insights into the regulatory role of 5′UTR structures in in vitro and bacterial translation, highlighting their potential applications in tuning gene expression. In eukaryotes, G-quadruplex in mRNA (RG4) 5′ UTR inhibit translation initiation. Here the authors employ single molecule assay to show that RG4 in E. coli reporter increases translation efficiency by preventing ribosome dislodging. [ABSTRACT FROM AUTHOR]

Protein diffusion is a critical factor governing the functioning and organization of a cell's cytoplasm. In this study, we investigate the influence of (poly)ribosome distribution, cell aging, protein aggregation, and biomolecular condensate formation on protein mobility within the E. coli cytoplasm. We employ nanoscale single-molecule displacement mapping (SMdM) to determine the spatial distribution of the proteins and to meticulously track their diffusion. We show that the distribution of polysomes does not impact the lateral diffusion coefficients of proteins. However, the degradation of mRNA induced by rifampicin treatment leads to an increase in protein mobility within the cytoplasm. Additionally, we establish a significant correlation between cell aging, the asymmetric localization of protein aggregates and reduced diffusion coefficients at the cell poles. Notably, we observe variations in the hindrance of diffusion at the poles and the central nucleoid region for small and large proteins, and we reveal differences between the old and new pole of the cell. Collectively, our research highlights cellular processes and mechanisms responsible for spatially organizing the bacterial cytoplasm into domains with different structural features and apparent viscosity. Single molecule diffusion study to determine the impact of (poly)ribosome distribution, cell aging, protein aggregation, and biomolecular condensate formation on the spatial organization and mobility of proteins in bacteria. [ABSTRACT FROM AUTHOR]

Direct RNA sequencing offers the possibility to simultaneously identify canonical bases and epi-transcriptomic modifications in each single RNA molecule. Thus far, the development of computational methods has been hampered by the lack of biologically realistic training data that carries modification labels at molecular resolution. Here, we report on the synthesis of such samples and the development of a bespoke algorithm, mAFiA (m6A Finding Algorithm), that accurately detects single m6A nucleotides in both synthetic RNAs and natural mRNA on single read level. Our approach uncovers distinct modification patterns in single molecules that would appear identical at the ensemble level. Compared to existing methods, mAFiA also demonstrates improved accuracy in measuring site-level m6A stoichiometry in biological samples. Direct RNA-seq offers the possibility to identify RNA modifications on single molecules. Here, the authors report on the synthesis of biologically realistic training data and the development of mAFiA that accurately detects m6A on single read level. [ABSTRACT FROM AUTHOR]

Single molecule fluorescence in situ hybridisation (smFISH) has become a valuable tool to investigate the mRNA expression of single cells. However, it requires a considerable amount of programming expertise to use currently available open-source analytical software packages to extract and analyse quantitative data about transcript expression. Here, we present FISHtoFigure, a new software tool developed specifically for the analysis of mRNA abundance and co-expression in QuPath-quantified, multi-labelled smFISH data. FISHtoFigure facilitates the automated spatial analysis of transcripts of interest, allowing users to analyse populations of cells positive for specific combinations of mRNA targets without the need for computational image analysis expertise. As a proof of concept and to demonstrate the capabilities of this new research tool, we have validated FISHtoFigure in multiple biological systems. We used FISHtoFigure to identify an upregulation in the expression of Cd4 by T-cells in the spleens of mice infected with influenza A virus, before analysing more complex data showing crosstalk between microglia and regulatory B-cells in the brains of mice infected with Trypanosoma brucei brucei. These analyses demonstrate the ease of analysing cell expression profiles using FISHtoFigure and the value of this new tool in the field of smFISH data analysis. [ABSTRACT FROM AUTHOR]

Biopolymer topology is critical for determining interactions inside cell environments, exemplified by DNA where its response to mechanical perturbation is as important as biochemical properties to its cellular roles. The dynamic structures of chiral biopolymers exhibit complex dependence with extension and torsion, however the physical mechanisms underpinning the emergence of structural motifs upon physiological twisting and stretching are poorly understood due to technological limitations in correlating force, torque and spatial localization information. We present COMBI-Tweez (Combined Optical and Magnetic BIomolecule TWEEZers), a transformative tool that overcomes these challenges by integrating optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy, demonstrated on single DNA molecules, that can controllably form and visualise higher order structural motifs including plectonemes. This technology combined with cutting-edge MD simulations provides quantitative insight into complex dynamic structures relevant to DNA cellular processes and can be adapted to study a range of filamentous biopolymers. It is hard to correlate force, torque and localization information. The authors report Combined Optical and Magnetic BIomolecule TWEEZers, COMBI-Tweez, that integrates optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy: they demonstrate visualisation of higher order structural motifs in DNA. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) could modulate expression of immune checkpoints (ICPs) by cooperating with immunity genes in tumor immunization. However, precise functions in immunity and potential for predicting ICP inhibitors (ICI) response have been described for only a few lncRNAs. Here we present an integrated framework that leverages network-based analyses and Bayesian network inference to identify the regulated relationships including lncRNA, ICP and immunity genes as ICP-related LncRNAs mediated Core Regulatory Circuitry Triplets (ICP-LncCRCTs) that can make robust predictions. Hub ICP-related lncRNAs such as MIR155HG and ADAMTS9-AS2 were highlighted to play central roles in immune regulation. Specific ICP-related lncRNAs could distinguish cancer subtypes. Moreover, the ICP-related lncRNAs are likely to significantly correlated with immune cell infiltration, MHC, CYT. Some ICP-LncCRCTs such as CXCL10-MIR155HG-ICOS could better predict one-, three- and five-year prognosis compared to single molecule in melanoma. We also validated that some ICP-LncCRCTs could effectively predict ICI-response using three kinds of machine learning algorithms follow five independent datasets. Specially, combining ICP-LncCRCTs with the tumor mutation burden (TMB) improves the prediction of ICI-treated melanoma patients. Altogether, this study will improve our grasp of lncRNA functions and accelerating discovery of lncRNA-based biomarkers in ICI treatment. An integrated framework to identify immune checkpoint-related lncRNA core regulatory circuitry triplets has the potential to improve our understanding of lncRNA functions and accelerate the discovery of lncRNA-based biomarkers for immune checkpoint inhibitor treatment. [ABSTRACT FROM AUTHOR]

Interactions between monatomic ions and water molecules are fundamental to understanding the hydration of complex polyatomic ions and ionic process. Among the simplest and well-established ion-related reactions is dissolution of salt in water, which is an endothermic process requiring an increase in entropy. Extensive efforts have been made to date; however, most studies at single-ion level have been limited to theoretical approaches. Here, we demonstrate the salt dissolution process by manipulating a single water molecule at an under-coordinated site of a sodium chloride film. Manipulation of molecule in a controlled manner enables us to understand ion–water interaction as well as dynamics of water molecules at NaCl interfaces, which are responsible for the selective dissolution of anions. The water dipole polarizes the anion in the NaCl ionic crystal, resulting in strong anion–water interaction and weakening of the ionic bonds. Our results provide insights into a simple but important elementary step of the single-ion chemistry, which may be useful in ion-related sciences and technologies. The strong ionic bond in salt is broken by electrostatic interactions with water, but direct observation at the level of a single ion is challenging. Here, the authors have visualized the preferential dissolution of an anion by manipulating a single water molecule. [ABSTRACT FROM AUTHOR]

Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need of complex sample pretreatment, direct and rapid analysis of fruits in a hand-held device has never been previously reported. Nanopore, a versatile single molecule sensor, can be specially engineered to perform this task. A hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore modified with a sole phenylboronic acid (PBA) adapter is prepared. This engineered MspA accurately recognizes 1,2-diphenols, alditols, α-hydroxy acids and saccharides in prune, grape, lemon, different varieties of kiwifruits and commercial juice products. Assisted with a custom machine learning program, an accuracy of 99.3% is reported and the sample pretreatment is significantly simplified. Enantiomers such as DL-malic acids can also be directly identified, enabling sensing of synthetic food additives. Though demonstrated with fruits, these results suggest wide applications of nanopore in food and drug administration uses. Fruits contain a large variety of cis-diols but their rapid and direct analysis without complex sample pretreatment was not achieved. Here, the authors report a Mycobacterium smegmatis porin A nanopore modified with a phenylboronic acid adapter for recognition of 1,2-diphenols, alditols, α-hydroxy acids and saccharides in fruits. [ABSTRACT FROM AUTHOR]

This article discusses the development of a new model of immune cells called Langerhans-like cells (LCs) for studying tick-borne encephalitis virus (TBEV) infection. LCs play a crucial role in the early-stage infection by transporting viral antigens from the skin to the lymph nodes. The researchers derived primary LCs from murine bone marrow and confirmed their suitability for TBEV infection experiments. The LCs were found to be susceptible to TBEV infection, with high cell viability and viral replication. This new model provides a valuable tool for studying TBEV infection and the dissemination of the virus. [Extracted from the article]

There has been a long fundamental pursuit to enhance and levitate the Raman scattering signal intensity of molecule by a huge number of ~ 14–15 orders of magnitude, to the level comparable with the molecule fluorescence intensity and truly entering the regime of single-molecule Raman spectroscopy. In this work we report unambiguous observation of single-molecule Raman spectroscopy via synergic action of electromagnetic and chemical enhancement for rhodamine B (RhB) molecule absorbed within the plasmonic nanogap formed by gold nanoparticle sitting on the two-dimensional (2D) monolayer WS2 and 2 nm SiO2 coated gold thin film. Raman spectroscopy down to an extremely dilute value of 10–18 mol/L can still be clearly visible, and the statistical enhancement factor could reach 16 orders of magnitude compared with the reference detection sample of silicon plate. The electromagnetic enhancement comes from local surface plasmon resonance induced at the nanogap, which could reach ~ 10–11 orders of magnitude, while the chemical enhancement comes from monolayer WS2 2D material, which could reach 4–5 orders of magnitudes. This synergic route of Raman enhancement devices could open up a new frontier of single molecule science, allowing detection, identification, and monitor of single molecules and their spatial–temporal evolution under various internal and external stimuli. [ABSTRACT FROM AUTHOR]

Introduction: Alzheimer's Disease (AD) is complex and novel approaches are urgently needed to aid in diagnosis. Blood is frequently used as a source for biomarkers; however, its complexity prevents proper detection. The analytical power of metabolomics, coupled with statistical tools, can assist in reducing this complexity. Objectives: Thus, we sought to validate a previously proposed panel of metabolic blood-based biomarkers for AD and expand our understanding of the pathological mechanisms involved in AD that are reflected in the blood. Methods: In the validation cohort serum and plasma were collected from 25 AD patients and 25 healthy controls. Serum was analysed for metabolites using nuclear magnetic resonance (NMR) spectroscopy, while plasma was tested for markers of neuronal damage and AD hallmark proteins using single molecule array (SIMOA). Results: The diagnostic performance of the metabolite biomarker panel was confirmed using sparse-partial least squares discriminant analysis (sPLS-DA) with an area under the curve (AUC) of 0.73 (95% confidence interval: 0.59–0.87). Pyruvic acid and valine were consistently reduced in the discovery and validation cohorts. Pathway analysis of significantly altered metabolites in the validation set revealed that they are involved in branched-chain amino acids (BCAAs) and energy metabolism (glycolysis and gluconeogenesis). Additionally, strong positive correlations were observed for valine and isoleucine between cerebrospinal fluid p-tau and t-tau. Conclusions: Our proposed panel of metabolites was successfully validated using a combined approach of NMR and sPLS-DA. It was discovered that cognitive-impairment-related metabolites belong to BCAAs and are involved in energy metabolism. [ABSTRACT FROM AUTHOR]

The effect of the changes in buffer concentrations or any additives like surfactants in the protein samples during the analysis on the single-biomolecule diffusion is one of the hidden points in the single-molecule time-resolved measurements. In the current work, phosphoglycerate kinase (PGK) labeled with Atto-647 has been investigated on the single molecule level while it diffuses in Na2CO3 buffer at concentrations that vary from 10 mg/l up to 50 mg/l. The fluorescence lifetime of PGK labeled with Atto-647 in 50 mg/ml Na2CO3 has been measured, and it was found to be 2.7 ns. The fluorescence cross correlation of the diffused protein has also been measured, which confirms that the used samples are at a single molecule level. Time decay fluorescence anisotropy has been performed for PGK labeled with Atto-647 in different concentrations of Na2CO3, and the results confirmed that there is a clear impact on the molecular translational and rotational diffusion even with slight changes in the buffer concentration. [ABSTRACT FROM AUTHOR]

Break junction (BJ) measurements provide insights into the electrical properties of diverse molecules, enabling the direct assessment of single-molecule conductances. The BJ method displays potential for use in determining the dynamics of individual molecules, single-molecule chemical reactions, and biomolecules, such as deoxyribonucleic acid and ribonucleic acid. However, conductance data obtained via single-molecule measurements may be susceptible to fluctuations due to minute structural changes within the junctions. Consequently, clearly identifying the conduction states of these molecules is challenging. This study aims to develop a method of precisely identifying conduction state traces. We propose a novel single-molecule analysis approach that employs total variation denoising (TVD) in signal processing, focusing on the integration of information technology with measured single-molecule data. We successfully applied this method to simulated conductance traces, effectively denoise the data, and elucidate multiple conduction states. The proposed method facilitates the identification of well-defined plateau lengths and supervised machine learning with enhanced accuracies. The introduced TVD-based analytical method is effective in elucidating the states within the measured single-molecule data. This approach exhibits the potential to offer novel perspectives regarding the formation of molecular junctions, conformational changes, and cleavage. [ABSTRACT FROM AUTHOR]

Hypericum is a large genus that includes more than 500 species of pharmacological, ecological and conservation value. Although latest advances in sequencing technologies were extremely exploited for generating and assembling genomes of many living organisms, annotated whole genome sequence data is not publicly available for any of the Hypericum species so far. Bioavailability of secondary metabolites varies for different tissues and the data derived from different cultures will be a valuable tool for comparative studies. Here, we report the single molecule real-time sequencing (SMRT) data sets of Hypericum perforatum L. plantlets and cell suspension cultures for the first time. Sequencing data from cell suspension cultures yielded more than 33,000 high-quality transcripts from 20 Gb of raw data, while more than 55,000 high-quality transcripts were obtained from 35 Gb of raw data from plantlets. This dataset is a valuable tool for comparative transcriptomic analysis and will help to understand the unknown biosynthetic pathways of high medicinal value in the Hypericum genus. [ABSTRACT FROM AUTHOR]

High resolution force measurements of molecules on surfaces, in non-contact atomic force microscopy, are often only performed at cryogenic temperatures, due to needing a highly stable system, and a passivated probe tip (typically via CO-functionalisation). Here we show a reliable protocol for acquiring three-dimensional force map data over both single organic molecules and assembled islands of molecules, at room temperature. Isolated cobalt phthalocyanine and islands of C60 are characterised with submolecular resolution, on a passivated silicon substrate (B:Si(111)- ( 3 × 3 ) R 3 0 ∘ ). Geometries of cobalt phthalocyanine are determined to a ~ 10 pm accuracy. For the C60, the protocol is sufficiently robust that areas spanning 10 nm × 10 nm are mapped, despite the difficulties of room temperature operation. These results provide a proof-of-concept for gathering high-resolution three-dimensional force maps of networks of complex, non-planar molecules on surfaces, in conditions more analogous to real-world application. High resolution force measurements of molecules on surfaces using non-contact atomic force microscopy are typically performed at cryogenic temperatures. Here, the authors outline a reliable protocol for acquiring three-dimensional force map data at room temperature, demonstrating such capabilities on isolated cobalt phthalocyanine molecules and islands of C60 molecules. [ABSTRACT FROM AUTHOR]