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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]

Precisely pinpointing the positions of emitters within the diffraction limit is crucial for quantitative analysis or molecular mechanism investigation in biomedical research but has remained challenging unless exploiting single molecule localization microscopy (SMLM). Via integrating experimental spot dataset with deep learning, we develop a new approach, Digital-SMLM, to accurately predict emitter numbers and positions for sub-diffraction-limit spots with an accuracy of up to 98 % and a root mean square error as low as 14 nm. Digital-SMLM can accurately resolve two emitters at a close distance, e.g. 30 nm. Digital-SMLM outperforms Deep-STORM in predicting emitter numbers and positions for sub-diffraction-limited spots and recovering the ground truth distribution of molecules of interest. We have validated the generalization capability of Digital-SMLM using independent experimental data. Furthermore, Digital-SMLM complements SMLM by providing more accurate event number and precise emitter positions, enabling SMLM to closely approximate the natural state of high-density cellular structures. [ABSTRACT FROM AUTHOR]

Nanoelectromechanical systems (NEMS)‐based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real‐time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single‐molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E. coli GroEL molecules (801 kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40 μm diameter with a maximum flux of 25 molecules/second. The mass spectrum obtained with NEMS‐MS shows good agreement with the known mass of GroEL. [ABSTRACT FROM AUTHOR]

MicroRNA is regarded as a significant biomarker for cancer diagnosis, disease process evaluation and therapeutic guidance, and dual-parameter measurement may contribute to a more accurate and realistic assessment. To meet the urgent need for simultaneous detection of multiple biomarkers, we combined three-dimensional DNAzyme motors with single molecule imaging technique to construct a convenient, intuitive, and sensitive approach for the simultaneous detection of dual miRNAs in the free state or in extracellular vesicles. Quantification of target miRNAs can be realized through the detection of amplified fluorescence signals generated by the target miRNA-initiated cleavage of fluorescent substrate strands by the DNAzyme motors. The practicability was systematically validated with microRNA-21-5p and microRNA-10b-5p as targets, acquiring a satisfactory sensitivity sufficient to detect low abundance targets at 0.5 or 1 pM to 100 pM. Besides, the extracellular vesicular miRNAs can be conveniently detected without extraction. The clinical applicability was verified with a series of extracellular vesicles from clinical samples, which exhibited good distinguishability between colorectal cancer patients and healthy donors. In addition to the advantages of good specificity and high sensitivity, the system has potential to be easily adapted by minor alteration of the DNA sequences and fluorophore sets for detection of multiple miRNAs and even other types of biomarkers such as proteins. Therefore, it shows promise to be widely applied in various fields such as early diagnosis of cancer and its prognostic assessment. [ABSTRACT FROM AUTHOR]

Vinculin binds to specific sites of mechanically unfolded talin rod domains to reinforce the coupling of the cell's exterior to its force generation machinery. Force-dependent vinculin–talin complexation and dissociation was previously observed as contraction or extension of the unfolded talin domains respectively using magnetic tweezers. However, the structural mechanism underlying vinculin recognition of unfolded vinculin binding sites (VBSs) in talin remains unknown. Using molecular dynamics simulations, we demonstrate that a VBS dynamically refolds under force, and that vinculin can recognize and bind to partially unfolded VBS states. Vinculin binding enables refolding of the mechanically strained VBS and stabilizes its folded α-helical conformation, providing resistance against mechanical stress. Together, these results provide an understanding of a recognition mechanism of proteins unfolded by force and insight into the initial moments of how vinculin binds unfolded talin rod domains during the assembly of this mechanosensing meshwork. Author summary: Cells attach to and interact with their surroundings, and sense mechanical signals from the microenvironment. Vinculin and talin are classical examples of mechanosensitive proteins that complex in a force-dependent manner. Talin domains unfold under mechanical loads exposing buried vinculin binding sites, namely single α-helices. Such mechanical stretching enables recognition by vinculin, however, the structural states of vinculin's binding site when under force and the complexation mechanisms are poorly understood. We used molecular dynamics simulations to study the behavior of a single vinculin binding helix under mechanical load and during the initial moments of the vinculin–talin binding process. Our results reveal the highly dynamic nature of a vinculin binding helix under mechanical load. Depending on the force magnitude, the helix frequently reforms to a partially folded conformation on the nanosecond to microsecond timescale. Crucially, vinculin recognizes such partially folded helices in talin and promotes their refolding against force. Our findings are beyond the resolution limits imposed by experimental single molecule methods, suggesting a model which may be useful as a general mechanism for protein-protein binding under mechanical load. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), crucial mediators of cell‐to‐cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV‐mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan‐vesicular affinity ligands for both EVs and their non‐canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV‐associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin‐lacking Red Blood Cell‐derived EVs, overcoming limitations associated with antibody‐based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first‐ever peptide‐based application in SiMoA technology. [ABSTRACT FROM AUTHOR]

In order to reveal the migration and evolution of gas molecules in the actual oil–paper insulation composite system of transformer from the molecular level, the diffusion behavior of seven gas molecules (H2, CO, CO2, CH4, C2H2, C2H4, C2H6) generated during the operation and aging of oil-immersed transformers in the oil–paper composite insulation system is studied by molecular dynamics. Firstly, based on the molecular dynamics software, the model of the oil–paper composite insulation system and the gas molecule model is constructed. In order to compare and analyze the diffusion properties of gas molecules in a single medium, a single model of insulating oil and cellulose is also constructed. Then, the diffusion coefficients of gas molecules in the insulating oil, cellulose, and oil–paper insulating composite system are simulated and calculated. And the differences in the diffusion properties of gas molecules in the three insulating mediums are discussed. Finally, the microscopic mechanism of diffusion of different gas molecules in the three mediums is analyzed. The simulation results show that among the three mediums, the diffusion coefficient of H2 is the largest, while the diffusion coefficients of the other gas molecules are not very different. The diffusion coefficients of the seven gas molecules are the smallest in the oil-immersed paper composite insulation system, followed by cellulose, while the diffusion coefficients are the largest in mineral oil. It indicates that the diffusion of gas molecules is inhibited in oil–paper insulation systems where the insulating paper is completely immersed in oil. This is mainly due to the fact that the insulating oil completely penetrates into the paper, filling the pores and voids between the fibers, resulting in a reduction in the transition vacancies of the intermediate gas molecules, which hinders the diffusion of the gas molecules. [ABSTRACT FROM AUTHOR]

Carvacrol and thymol are broad-spectrum natural antimicrobial agents. To reduce their volatility and improve their antimicrobial performance, synergistic systems were prepared loading the active molecules in zinc-modified clays. Montmorillonite (MMT) and zeolite (ZEO) were modified with zinc ions (ZnMMT and ZnZEO), with well-known antimicrobial properties, and then with carvacrol or thymol, reaching the 26 ± 3% and 33 ± 2% w/w of loading, respectively. The resulting hybrid materials were characterized by FT-IR, XPS, XRD, TGA, and GC-MS to evaluate carvacrol/thymol release in simulating food matrices. Antimicrobial assays carried out using spoiler and pathogenic bacterial strains showed that the antimicrobial activity of both thymol and carvacrol was largely preserved once they were loaded into Zn-modified clays. However, MMT hybrids showed an antibacterial activity significantly higher than ZEO hybrids at 50 mg/mL of thymol and carvacrol. For this reason, deeper antimicrobial evaluations were carried out only for ZnMMT composites. ZnMMT loaded with thymol or carvacrol produced inhibition zones against most of the target strains, also at 3.12 mg/mL, while the positive controls represented by the single molecule thymol or carvacrol were not active. The hybrid materials can be useful for applications in which the antimicrobial activity of natural molecules need to be displayed over time as requested for the control of microbial pathogens and spoilage bacteria in different applications, such as active packaging, biomaterials, and medical devices. [ABSTRACT FROM AUTHOR]

A total of 43 novel HLA alleles detected in haematopoietic cell donors using single molecule real‐time DNA sequencing. [ABSTRACT FROM AUTHOR]

Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3′-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs. [ABSTRACT FROM AUTHOR]

Heterocyclic chemistry is one of the emerging branches of organic chemistry due to its growing attention in pharmacology. Piperazine is a dinitrogen-containing heterocycle that spans many applications, including anticancer, anticonvulsant, antidepressant, antimicrobial, antimalarial, antiviral, antitubercular, anti-inflammatory, activities etc. In a similar fashion, the second moiety, i.e., acetanilide, an N-phenylacetamide, also executed a large number of bioactivities, which included anticancer, antileishmanial, antipyretic, analgesic, antidiabetic, antiviral potential, etc. Both moieties have large-scale potential to show bioactivities in different aspects. When both moieties combine in a single molecule, it forms a potent biological compound. A number of precursors have been used for the synthesis of piperazine-based acetanilides. In this review, we have addressed different synthetic strategies that have been reported in the past. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to compare the clinical outcomes of deep sclerectomy and trabeculectomy surgeries, both utilizing 5-fluorouracil. Methods: In this study, 11 eyes of 10 patients (14.3%) in the deep sclerectomy group and 66 eyes of 61 patients (85.7%) in the trabeculectomy group were retrospectively examined. Success in intraocular pressure (IOP) was defined as being below 21 mmHg without medication, partially successful if achieved below 21 mmHg with a single molecule, considered less successful with two to four molecules for maintaining IOP below 21 mmHg, and deemed unsuccessful if IOP exceeded 21 mmHg. Results: The deep sclerectomy group showed a success rate of 81.8% on post-operative day 1, with 18.2% considered unsuccessful; at post-operative 6 months, the success rate was 12.5%, with 50% partially successful and 37.5% less successful. In the trabeculectomy group, the success rate was 97% on post-operative day 1, with 3% considered unsuccessful; at post-operative 6 months, the success rate was 60.8%, with 15.7% partially successful, 13.7% less successful, and 9.8% unsuccessful. Trabeculectomy patients experienced complications such as choroidal detachment (6.1%), shallow anterior chamber (4.5%), tight suturing (3%), internal ostium occlusion (3%), posterior synechia (3%), bleb failure (1.5%), cyclodialysis/iridodialysis (1.5%), and hyphema (1.5%). In the deep sclerectomy group, tight suturing (36.3%), and dellen (9.09%) were observed, with no other complications reported. Conclusion: Trabeculectomy surgery has been observed to be superior to deep sclerectomy in follow-ups up to 48 months postoperatively. However, it is noted that there is a higher risk of post-operative complications. [ABSTRACT FROM AUTHOR]

Hindered phenol antioxidants and benzophenone UV absorbers are common polymer additives and often used in combination applications to enhance the anti-aging performance of polymer materials. This study primarily aims to incorporate hindered phenol and benzophenone structures into a single molecule to develop a multifunctional polymer additive with good anti-aging performance. Thus, a novel potential polymer anti-aging agent, namely 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid 3-(4-benzoyl-3-hydroxyphenoxy)propyl ester (3C), was synthesized using 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid, 3-bromo-1-propanol, and 2,4-dihydroxybenzophenone as raw materials by two-step procedure. The structure of compound 3C was characterized by nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), Fourier-transform infrared (FT-IR) spectroscopy, and X-ray single crystal diffraction. Its thermal stability and UV resistance were assessed using thermogravimetric analysis (TGA) and UV absorption spectroscopy (UV). The compound 3C as an additive was incorporated into the preparation of polyolefin elastomer (POE) films. The anti-aging performance of POE films was evaluated by measuring parameters such as oxidation induction time, melt flow index, transmittance, and infrared spectra of the artificially aged POE films. The results indicate that the compound 3C exhibits a promising anti-aging performance in both thermo-oxidative aging and ultraviolet aging tests of POE films and is a potential polymer anti-aging agent. [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]

Complex materials exhibit fascinating features especially in situations far from equilibrium. Thus, methods for investigating structural dynamics with sub‐second time resolution are becoming a question of interest at varying spatial scales. With novel microscopy techniques steadily improving, the temporal and spatial limits of multiple imaging methods are investigated with an emphasis on the important role of correlative imaging and cryo‐fixation. A deep‐dive is taken into cryo‐correlative light and electron microscopy (CLEM) as a starting point for multimodal investigations of ultrastructural dynamics at high spatiotemporal resolution. The focus is on highlighting the different microscopy methods that capture the following key aspects: 1) samples are as close to native state as possible 2) dynamic process information is captured, 3) high structural resolution is enabled. Additionally, the size of samples that can be imaged under these conditions is looked at and approaches not only focusing on single molecules, but larger structures are highlighted. [ABSTRACT FROM AUTHOR]

Surface enhanced Raman scattering (SERS) provides a label free method of analyzing molecules from diverse and complex signals, potentially with single molecule sensitivity. The chemical specificity inherent in the SERS spectrum can identify molecules; however signal variability arising from the diversity of plasmonic environments can limit quantification, particularly at low concentrations. Here we show that digitizing, or counting SERS events, can decrease the limit of detection in flowing solutions enabling quantification of single molecules. By using multivariate curve resolution and establishing a score threshold, each individual spectrum can be classified as containing an event or not. This binary "yes/no" can then be quantified, and a linear region can be established. This method was shown to lower the limit of detection to the lowest physical limit, and lowered the limit of detection by an order of magnitude from the traditional, intensity based LOD calculations. [ABSTRACT FROM AUTHOR]

In biological membranes, lipids often interact with membrane proteins (MPs), regulating the localization and activity of MPs in cells. Although elucidating lipid–MP interactions is critical to comprehend the physiological roles of lipids, a systematic and comprehensive identification of lipid-binding proteins has not been adequately established. Therefore, we report the development of lipid-immobilized beads where lipid molecules were covalently immobilized. Owing to the detergent tolerance, these beads enable screening of water-soluble proteins and MPs, the latter of which typically necessitate surfactants for solubilization. Herein, two sphingolipid species—ceramide and sphingomyelin—which are major constituents of lipid rafts, were immobilized on the beads. We first showed that the density of immobilized lipid molecules on the beads was as high as that of biological lipid membranes. Subsequently, we confirmed that these beads enabled the selective pulldown of known sphingomyelin- or ceramide-binding proteins (lysenin, p24, and CERT) from protein mixtures, including cell lysates. In contrast, commercial sphingomyelin beads, on which lipid molecules are sparsely immobilized through biotin–streptavidin linkage, failed to capture lysenin, a well-known protein that recognizes clustered sphingomyelin molecules. This clearly demonstrates the applicability of our beads for obtaining proteins that recognize not only a single lipid molecule but also lipid clusters or lipid membranes. Finally, we demonstrated the screening of lipid-binding proteins from Neuro2a cell lysates using these beads. This method is expected to significantly contribute to the understanding of interactions between lipids and proteins and to unravel the complexities of lipid diversity. [ABSTRACT FROM AUTHOR]

Electrochemiluminescence (ECL) is rapidly evolving from an analytical method into an optical microscopy. The orthogonality of the electrochemical trigger and the optical readout distinguishes it from classic microscopy and electrochemical techniques, owing to its near‐zero background, remarkable sensitivity, and absence of photobleaching and phototoxicity. In this minireview, we summarize the recent advances in ECL imaging technology, emphasizing original configurations which enable the imaging of biological entities and the improvement of the analytical properties by increasing the complexity and multiplexing of bioassays. Additionally, mapping the (electro)chemical reactivity in space provides valuable information on nanomaterials and facilitates deciphering ECL mechanisms for improving their performances in diagnostics and (electro)catalysis. Finally, we highlight the recent achievements in imaging at the ultimate limits of single molecules, single photons or single chemical reactions, and the current challenges to translate the ECL imaging advances to other fields such as material science, catalysis and biology. [ABSTRACT FROM AUTHOR]

Pancreatic cancer, ranking as the third factor in cancer‐related deaths, necessitates enhanced diagnostic measures through early detection. In response, SiMoT‐Single‐molecule with a large Transistor multiplexing array, achieving a Technology Readiness Level of 5, is proposed for a timely identification of pancreatic cancer precursor cysts and is benchmarked against the commercially available chemiluminescent immunoassay SIMOA (Single molecule array) SP‐X System. A cohort of 39 samples, comprising 33 cyst fluids and 6 blood plasma specimens, undergoes detailed examination with both technologies. The SiMoT array targets oncoproteins MUC1 and CD55, and oncogene KRAS, while the SIMOA SP‐X planar technology exclusively focuses on MUC1 and CD55. Employing Principal Component Analysis (PCA) for multivariate data processing, the SiMoT array demonstrates effective discrimination of malignant/pre‐invasive high‐grade or potentially malignant low‐grade pancreatic cysts from benign non‐mucinous cysts. Conversely, PCA analysis applied to SIMOA assay reveals less effective differentiation ability among the three cyst classes. Notably, SiMoT unique capability of concurrently analyzing protein and genetic markers with the threshold of one single molecule in 0.1 mL positions it as a comprehensive and reliable diagnostic tool. The electronic response generated by the SiMoT array facilitates direct digital data communication, suggesting potential applications in the development of field‐deployable liquid biopsy. [ABSTRACT FROM AUTHOR]

Ultra-sensitive sensors based on the resonant properties of whispering gallery modes (WGMs) can detect fractional changes in nanoscale environments down to the length and time scales of single molecules. However, it is challenging to isolate single-molecule signals from competing noise sources in experiments, such as thermal and mechanical sources of noise, and—at the most fundamental level—the shot noise limit of classical light. Additionally, in contrast to traditional bulk refractive index measurements, analyzing single-molecule signals is complicated by the localized nature of their interactions with nanoscale field gradients. This perspective discusses multifaceted solutions to these challenges, including the use of quantum light sources to boost the signal-to-noise ratio in experiments and leveraging the power of supercomputers to predict the electronic response of molecules to WGM optoplasmonic fields. We further discuss the role of machine learning in WGM sensing, including several advanced models that can predict molecular polarizability and solvent effects. These advancements in WGM spectroscopy and computational modeling can help to decipher the molecular mechanics of enzymes, enable studies of catalysis on the nanoscale, and probe the quantum nature of molecules. [ABSTRACT FROM AUTHOR]

The complement is a conserved cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP). Human C4BP is a macromolecular glycoprotein composed of two distinct subunits, C4BPα and C4BPβ. These associate with vitamin K-dependent protein S (ProS) forming an ensemble of co-occurring higher-order structures. Here, we characterize these C4BP assemblies. We resolve and quantify isoforms of purified human serum C4BP using distinct single-particle detection techniques: charge detection mass spectrometry, and mass photometry accompanied by high-speed atomic force microscopy. Combining cross-linking mass spectrometry, glycoproteomics, and structural modeling, we report comprehensive glycoproteoform profiles and full-length structural models of the endogenous C4BP assemblies, expanding knowledge of this key complement inhibitor's structure and composition. Finally, we reveal that an increased C4BPα to C4BPβ ratio coincides with elevated C-reactive protein levels in patient plasma samples. This observation highlights C4BP isoform variation and affirms a distinct role of co-occurring C4BP assemblies upon acute phase inflammation. Synopsis: C4b-binding protein complement inhibitor forms co-occurring assemblies in human serum. Here, by using an integrative structural biology approach, structural and compositional details of C4BP are exposed, resulting in structural models of full-length glycosylated C4BP assemblies. Integrating high-speed atomic force microscopy and cross-linking mass spectrometry we present full-length structural models of C4BP, a spider-like assembly of C4BPα and C4BPβ, non-covalently interacting with vitamin K-dependent protein S. Dominant C4BP variants in healthy human serum are C4BP(β+) captured and analyzed by single molecule mass photometry and charge detection mass spectrometry. Quantitative serum proteomics reveals that C4BP variant composition changes during acute phase inflammation, with an increase in α7 variant abundance. Structual analysis of complement inhibitor C4BP uncovers full-length spider-like assemblies of C4BPα and C4BPβ subunits and composition changes during acute phase inflammation. [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]

Thanks to their giant, yet tunable, Q-factor resonances, all-dielectric metasurfaces supporting the quasi-bound states in the continuum (q-BIC) resonances are well-suited to provide a promising platform for quantum-coherent light–matter interactions. Yet, the strong coupling regime, characterized by the hybrid light–matter states – polaritons, has not yet been fully explored in the mid-infrared regime. This paper investigates the parameter space of vibrational strong coupling (VSC) between material and metasurface cavities supporting q-BIC resonances in the mid-infrared spectral range. We outline the effects of transition dipole strength, damping rate, and the number of molecules coupled to a single cavity, as well as the cavity damping rates, to understand their respective impacts on VSC. By tuning the Q-factor of the metasurface and material parameters, a new transition light–matter coupling zone is introduced, bridging the gap between weak and strong coupling, where polaritons form but their linewidths prohibit their spectral identification. The study further identifies the effects of cavity linewidth on polariton peak separability in strongly coupled systems, highlighting that the cavities with smaller nonradiative losses and narrower linewidths facilitate better polariton separability. Moreover, we found that matching cavity and material loss, satisfying the critical strong coupling condition, enhances the coupling strength between cavity and material. Overall, these findings can guide the design of photonic cavities suited for VSC experiments, contributing to the burgeoning fields of polaritonic chemistry, light-mediated modulation of chemical reactivity, and highly sensitive molecular spectroscopy. [ABSTRACT FROM AUTHOR]

Fluorescence spectroscopy serves as an ultrasensitive sophisticated tool where background noises which serve as a major impediment to the detection of the desired signals can be safely avoided for detections down to the single-molecule levels. One such way of bypassing background noise is plasmon-enhanced fluorescence (PEF), where the interactions of fluorophores at the surface of metals or plasmonic nanoparticles are probed. The underlying condition is a significant spectral overlap between the localized surface plasmon resonance (LSPR) of the nanoparticle and the absorption or emission spectra of the fluorophore. The rationale being the coupling of the excited state of the fluorophore with the localized surface plasmon leads to an augmented emission, owing to local field enhancement. It is manifested in enhanced quantum yields concurrent with a decrease in fluorescence lifetimes, owing to an increase in radiative rate constants. This improvement in detection provided by PEF allows a significant scope of expansion in the domain of weakly emitting fluorophores which otherwise would have remained unperceivable. The concept of coupling of weak emitters with plasmons can bypass the problems of photobleaching, opening up avenues of imaging with significantly higher sensitivity and improved resolution. Furthermore, amplification of the emission signal by the coupling of free electrons of the metal nanoparticles with the electrons of the fluorophore provides ample opportunities for achieving lower detection limits that are involved in biological imaging and molecular sensing. One avenue that has attracted significant attraction in the last few years is the fast, label-free detection of bio-analytes under physiological conditions using plasmonic nanoparticles for point-of-care analysis. This review focusses on the applications of plasmonic nanomaterials in the field of biosensing, imaging with a brief introduction on the different aspects of LSPR and fabrication techniques. [ABSTRACT FROM AUTHOR]

Self-assembly of M(ClO4)2 (M2+ = Ni2+, Cu2+, and Zn2+) with (1S,1′S,1′′S,2R,2′R,2′′R)-(benzenetricarbonyltris(azanediyl))tris(2,3-dihydro-1H-indene-2,1-diyl) trinicotinate (s,r-L) and the corresponding enantiomer (r,s-L) as a pair of chiral tridentate donors gives rise to the chiral cage pairs [M3(s,r- and r,s-L)2](ClO4)6. For the two pairs of [(Me2CO)(H2O)@M3(r,-s and s,r-L)2](ClO4)6 (M2+ = Ni2+ and Zn2+), the inner cavity is occupied by both an acetone and a single water molecule, whereas for the copper(II) pair of [Me2CO@Cu3(r,s- and s,r-L)2](ClO4)6 under the same conditions, the cavity is filled by only one acetone molecule. Thus, the encapsulation of guest molecules into the cages during self-assembly shows significant metal(II) ion effects. These chiral cages are effective for the enantio-recognition of chiral (S)-2-butanol and (R)-2-butanol via the shifts of the electrochemical oxidation potentials obtained by the linear sweep voltammetry (LSV) technique, density functional theory (DFT) calculations, and the chiral 2-butanol adsorption in the single-crystal-to-single-crystal (SCSC) mode. [ABSTRACT FROM AUTHOR]

Incorporating transition metal atoms into metal–molecule–metal junctions presents opportunities for exploring the electronic properties of coordination complexes, organometallics and metal–organic materials on the single molecule level. Recent single molecule conductance studies have shown that in situ incorporation of electrode metal atoms into coordination chains formed in the junction can occur with deprotonated, negatively charged organic ligands, such as the imidazolate (Im−) anion. However, the mechanism and chemical principles, such as the role of the charge state of the ligand, for the construction of such coordination chains are still debated. Here, we probe the role of the ligand charge state and electronic structure in single-molecule conductance and formation of metal–molecule coordination chains. We perform break junction measurements with triazole isomers, which can bridge junctions both in their neutral and charged forms, and find that prior deprotonation of the ligands is not required for coordination complex assembly, but can affect the molecular conductance and junction formation probability. Our results indicate that coordination chains can form with neutral ligands, as long as the electron density in the frontier MOs is concentrated at the binding sites and along the direction of pulling, promoting ligand binding and incorporation of gold atoms into the junction during elongation. Our findings may provide insight into design principles for in situ assembled molecular wires with transition metal atoms and open the door to electronic and spintronic studies of such materials. [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]

Background: Biotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16. Results: This work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain. Conclusions: This investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation. [ABSTRACT FROM AUTHOR]

The practical application of new single molecule protein sequencing (SMPS) technologies requires accurate estimates of their associated sequencing error rates. Here, we describe the development and application of two distinct parameter estimation methods for analyzing SMPS reads produced by fluorosequencing. A Hidden Markov Model (HMM) based approach, extends whatprot, where we previously used HMMs for SMPS peptide-read matching. This extension offers a principled approach for estimating key parameters for fluorosequencing experiments, including missed amino acid cleavages, dye loss, and peptide detachment. Specifically, we adapted the Baum-Welch algorithm, a standard technique to estimate transition probabilities for an HMM using expectation maximization, but modified here to estimate a small number of parameter values directly rather than estimating every transition probability independently. We demonstrate a high degree of accuracy on simulated data, but on experimental datasets, we observed that the model needed to be augmented with an additional error type, N-terminal blocking. This, in combination with data pre-processing, results in reasonable parameterizations of experimental datasets that agree with controlled experimental perturbations. A second independent implementation using a hybrid of DIRECT and Powell's method to reduce the root mean squared error (RMSE) between simulations and the real dataset was also developed. We compare these methods on both simulated and real data, finding that our Baum-Welch based approach outperforms DIRECT and Powell's method by most, but not all, criteria. Although some discrepancies between the results exist, we also find that both approaches provide similar error rate estimates from experimental single molecule fluorosequencing datasets. Author summary: Diverse new technologies are being developed for single-molecule protein sequencing, capable of identifying and quantifying mixtures of proteins at the level of individual molecules. There are many biochemical challenges intrinsic to high-throughput studies of proteins at such high sensitivity arising from their heterogeneous chemistries, sizes, and abundances. Beyond these challenges, the technologies themselves involve complex multi-step analytical processes. Thus, in developing and optimizing these technologies, it is important to consider the accuracy of each step and to have reliable approaches for estimating these accuracies. We focus on one particular single-molecule sequencing technology known as flourosequencing. We report and validate two methods for simultaneously determining the error-rates of each of the various steps of the fluorosequencing process. These new error estimation techniques will help researchers to better interpret the effects of changes to the chemistry and sample preparation used in fluorosequencing so that these steps can be improved. Further, more accurate determination of error rates will aid in the creation of better tools for the interpretation of this data. [ABSTRACT FROM AUTHOR]

The success of a nanopore experiment relies not only on the quality of the experimental design but also on the performance of the analysis program utilized to decipher the ionic perturbations necessary for understanding the fundamental molecular intricacies. An event extraction framework is developed that leverages parallel computing, efficient memory management, and vectorization, yielding significant performance enhancement. The newly developed abf‐ultra‐simple function extracts key parameters from the header critical for the operation of open‐seek‐read‐close data loading architecture running on multiple cores. This underpins the swift analysis of large files where an ≈ × 18 improvement is found for a 100 min‐long file (≈4.5 GB) compared to the more traditional single (cell) array data loading method. The application is benchmarked against five other analysis platforms showcasing significant performance enhancement (>2 ×–1120 ×). The integrated provisions for batch analysis enable concurrently analyzing multiple files (vital for high‐bandwidth experiments). Furthermore, the application is equipped with multi‐level data fitting based on abrupt changes in the event waveform. The application condenses the extracted events to a single binary file improving data portability (e.g., 16 GB file with 28 182 events reduces to 47.9 MB–343 × size reduction) and enables a multitude of post‐analysis extractions to be done efficiently. [ABSTRACT FROM AUTHOR]

Background: The global management of type 2 diabetes mellitus (T2D) presents ongoing challenges, including the need for strategies that reduce cardiovascular risk and manage metabolic comorbidities. Incretin hormones, specifically glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are integral to metabolic regulation, yet individuals with T2D exhibit diminished incretin responses. Tirzepatide, a novel GIP/GLP-1 receptor agonist, has emerged as a promising therapeutic option, combining the advantages of both incretin hormones in a single molecule to manage T2D. Objective: This systematic review aims to evaluate the efficacy of tirzepatide as a twincretin therapy for T2D and its associated adverse effects reported in previous studies. Methods: Utilizing PRISMA guidelines, a comprehensive literature search was conducted across four key databases (PubMed, SCOPUS, Web of Science, and Science Direct) for English-language publications on tirzepatide and its impact on T2D management. Inclusion and exclusion criteria guided the selection process, while data extraction focused on participant characteristics, clinical outcomes, and risk of bias assessment. Results: The analysis included various studies highlighting tirzepatide's effects. It consistently demonstrated significant reductions in HbA1c levels, with studies reporting mean reductions ranging from 0.47% to 2.58%, and substantial average weight loss of 12.4 kg or more. Notably, tirzepatide surpassed traditional therapies such as semaglutide and insulin glargine, displaying superior glycemic control with a comparable safety profile--primarily mild gastrointestinal side effects. Conclusion: Tirzepatide represents a transformative advancement in T2D management, offering robust improvements in glycemic control and weight reduction alongside beneficial metabolic effects. Its favorable safety profile positions it as a substantial first-line therapy option. Future research should focus on long-term effects and practical application across various patient populations to optimize treatment strategies and improve patient outcomes significantly. [ABSTRACT FROM AUTHOR]

Chaetonotidae is the most diversified family of the entire phylum Gastrotricha; it comprises ~430 species distributed across 16 genera. The current classification, established mainly on morphological traits, has been challenged in recent years by phylogenetic studies, indicating that the cuticular ornamentations used to discriminate among species may be misleading when used to identify groupings, which has been the practice until now. Therefore, a consensus is developing toward implementing novel approaches to better define species identity and affiliation at a higher taxonomic ranking. Using an integrative morphological and molecular approach, including annotation of the mitogenome, we report on some freshwater gastrotrichs characterised by a mixture of two types of cuticular scales diagnostic of the genera Aspidiophorus and Heterolepidoderma. Our specimens' overall anatomical characteristics find no correspondence in the taxa of these two genera, calling for their affiliation to a new species. Phylogenetic analyses based on the sequence of the ribosomal RNA genes of 96 taxa consistently found the new species unrelated to Aspidiophorus or Heterolepidoderma but allied with Chaetonotus aff. subtilis, as a subset of a larger clade, including mostly planktonic species. Morphological uniqueness and position along the non-monophyletic Chaetonotidae branch advocate erecting a new genus to accommodate the current specimens; consequently, the name Litigonotus ghinii gen. nov., sp. nov. is proposed. The complete mitochondrial genome of the new taxon resulted in a single circular molecule 14,384 bp long, including 13 protein-coding genes, 17 tRNA genes and 2 rRNAs genes, showing a perfect synteny and collinearity with the only other gastrotrich mitogenome available, a possible hint of a high level of conservation in the mitochondria of Chaetonotidae. ZooBank: Gastrotricha (hairy-bellied worms) is a phylum of microscopic aquatic invertebrates whose traditional morphology-based classification has been challenged in recent years. We report a new genus and species found using an integrative morphological and molecular approach, which is unrelated to morphologically similar species in the genera Aspidiophorus and Heterolepidoderma. The mitochondrial genome of the new taxon has the same number of genes and gene order as the only other gastrotrich mitogenome available, suggesting the conservation of mitochondria at the family level. (Image credit: M. Antonio Todaro.) [ABSTRACT FROM AUTHOR]

The [ZnL(ONO2)2] 1 and [ZnL(NCS)2] 2 complexes were synthesized using self-assembly of the s-triazine tridentate ligand (L) with Zn(NO3)2·6H2O and Zn(ClO4)2·6H2O/NH4SCN, respectively. The Zn(II) is further coordinated by two nitrate and two isothiocyanate groups as monodentate ligands in 1 and 2, respectively. Both complexes have distorted square pyramidal coordination environments where the extent of distortion is found to be greater in 2 (τ5 = 0.41) than in 1 (τ5 = 0.28). Hirshfeld calculations explored the significant C···O, C···C, N···H, and O···H contacts in the molecular packing of both complexes. The energy framework analysis gave the total interaction energies of −317.8 and −353.5 kJ/mol for a single molecule in a 3.8 Å cluster of 1 and 2, respectively. The total energy diagrams exhibited a strong resemblance to the dispersion energy frameworks in both complexes. NBO charge analysis predicted the charges of the Zn(II) in complexes 1 and 2 to be 1.217 and 1.145 e, respectively. The electronic configuration of Zn1 is predicted to be [core] 4S0.32 3d9.98 4p0.45 4d0.02 5p0.01 for 1 and [core] 4S0.34 3d9.97 4p0.53 4d0.02 for 2. The increased occupancy of the valence orbitals is attributed to the donor→acceptor interactions from the ligand groups to Zn(II). The Zn(II) complexes were examined for their cytotoxic and antimicrobial activities. Both 1 and 2 have good cytotoxic efficiency towards HCT-116 and A-549 cancerous cell lines. We found that 1 is more active (IC50 = 29.53 ± 1.24 and 35.55 ± 1.69 µg/mL) than 2 (IC50 = 41.25 ± 2.91 and 55.05 ± 2.87 µg/mL) against both cell lines. Also, the selectivity indices for the Zn(II) complexes are higher than one, indicating their suitability for use as anticancer agents. In addition, both complexes have broad-spectrum antimicrobial activity (IC50 = 78–625 μg/mL) where the best result is found for 2 against P. vulgaris (IC50 = 78 μg/mL). Its antibacterial activity is found to be good compared to gentamycin (5 μg/mL) as a positive control against this microbe. [ABSTRACT FROM AUTHOR]

Recent advances in chromatin conformation capture technologies, such as SPRITE and Pore-C, have enabled the detection of simultaneous contacts among multiple chromatin loci. This has made it possible to investigate the cooperative transcriptional regulation involving multiple genes and regulatory elements at the resolution of a single molecule. However, these technologies are unavoidably subject to the random polymer looping effect and technical biases, making it challenging to distinguish genuine regulatory relationships directly from random polymer interactions. Here, we present HyperloopFinder, a method for identifying regulatory multi-way chromatin contacts (hyperloops) by jointly modeling the random polymer looping effect and technical biases to estimate the statistical significance of multi-way contacts. The results show that our model can accurately estimate the expected interaction frequency of multi-way contacts based on the distance distribution of pairwise contacts, revealing that most multi-way contacts can be formed by randomly linking the pairwise contacts adjacent to each other. Moreover, we observed the spatial colocalization of the interaction sites of hyperloops from image-based data. Our results also revealed that hyperloops can function as scaffolds for the cooperation among multiple genes and regulatory elements. In summary, our work contributes novel insights into higher-order chromatin structures and functions and has the potential to enhance our understanding of transcriptional regulation and other cellular processes. [ABSTRACT FROM AUTHOR]

Gaseous signaling molecules (GSMs) including nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) have presented excellent therapeutic efficacy such as anti‐inflammatory, anti‐microbial and anti‐cancer effects and multiple biomedical applications in recent years. As the three most vital signaling molecules in human physiology, these three GSMs show so intertwined and orchestrated interactions that the synergy of multiple gases may demonstrate a more complex therapeutic potential than single gas delivery. Consequently, researchers have been devoted to developing codelivery systems of GSMs by synthesizing a single molecule as a dual donor to maximize the gaseous therapeutic efficacy. In this minireview, we summarize the recent developments of molecules or materials enabling codelivery of GSMs for biomedical applications. It appears that compared with the abundant cases of codelivery of NO and H2S, research on codelivery of CO and the other two GSMs separately remains to be explored. [ABSTRACT FROM AUTHOR]

In modern cancer therapy, blockage of more than one target is a standard approach, and there are already many dual‐target drugs that can achieve multiple inhibition through a single molecule. Herein, we designed and synthesized a series of novel derivatives with signal transducer and activator of transcription 3 (STAT3) and histone deacetylase (HDAC) inhibitory activity through strategy of combining pharmacophore based on the STAT3 inhibitor E28 and HDAC inhibitor MS‐275. Among them, compound 24 (IC50 = 8.22 ± 0.27 μM) showed better anti‐tumor activity than the clinical Class I HDAC inhibitor MS‐275 (IC50 = 14.65 ± 0.24 μM) in MCF‐7 breast cancer cells. Furthermore, the dual inhibition to HDAC and STAT3 of compound 24 was validated by western blot analysis. The study provides new tool compounds for further exploration of STAT3–HDAC pathway inhibitor achieved with a single molecule. [ABSTRACT FROM AUTHOR]

Increased levels of glial fibrillary acidic protein (GFAP) in blood have been identified as a valuable biomarker for some neurological disorders, such as Alzheimer's disease and multiple sclerosis. However, most blood GFAP quantifications so far were performed using the same bead-based assay, and to date a routine clinical application is lacking. In this study, we validated a novel second-generation (2nd gen) Ella assay to quantify serum GFAP. Furthermore, we compared its performance with a bead-based single molecule array (Simoa) and a homemade GFAP assay in a clinical cohort of neurological diseases, including 210 patients. Validation experiments resulted in an intra-assay variation of 10 %, an inter-assay of 12 %, a limit of detection of 0.9 pg/mL, a lower limit of quantification of 2.8 pg/mL, and less than 20 % variation in serum samples exposed to up to five freeze-thaw cycles, 120 h at 4 °C and room temperature. Measurement of the clinical cohort using all assays revealed the same pattern of GFAP distribution in the different diagnostic groups. Moreover, we observed a strong correlation between the 2nd gen Ella and Simoa (r=0.91 (95 % CI: 0.88–0.93), p<0.0001) and the homemade immunoassay (r=0.77 (95 % CI: 0.70–0.82), p<0.0001). Our results demonstrate a high reliability, precision and reproducibility of the 2nd gen Ella assay. Although a higher assay sensitivity for Simoa was observed, the new microfluidic assay might have the potential to be used for GFAP analysis in daily clinical workups due to its robustness and ease of use. [ABSTRACT FROM AUTHOR]

[Display omitted] • Plasma glycoproteomics provides broad and reproducible coverage for clinical applications. • Chemometric analyses demonstrates great potential of glycoproteomics for biomarker applications. • Site-specific glycosylation changes delineate glycobiology processes underlying disease. • Bioinformatic analyses of disease samples enable integral interpretation of glycoproteomes. • Glycosylation changes can be site-specific on single protein molecules depending on disease. The human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease. Here, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics. Comparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data. Glycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors. Our work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease. [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. [ABSTRACT FROM AUTHOR]

Among the many methods to image molecular structure, laser-induced electron diffraction (LIED) can image a single gas-phase molecule by locating all of a molecule's atoms in space and time. The method is based on attosecond electron recollision driven by a laser field and can reach attosecond temporal resolution. Implementation with a mid-IR laser and cold-target recoil ion-momentum spectroscopy, single molecules are measured with picometer resolution due to the keV electron impact energy without ensemble averaging or the need for molecular orientation. Nowadays, the method has evolved to detect single complex and chiral molecular structures in 3D. The review will touch on the various methods to discuss the implementations of LIED toward single-molecule imaging and complement the discussions with noteworthy experimental findings in the field. [ABSTRACT FROM AUTHOR]

The dynamic characteristics of single DNA molecules translocating within micro/nano-channels are fundamental for a wide range of applications such as stretching, separating, mapping, and even sequencing of DNA molecules. In this study, a type of tapered microchannel chip with uniform height for all configurations was fabricated, with the major tapered structure having a length of 13 μm and a width that tapers from 5 μm to 20 μm. The dynamic characteristics such as the trajectories and velocities of λ-DNA molecules translocating from different positions driven by an external DC electric field force were systematically investigated by single-molecule fluorescence imaging technology. Some dynamic characteristics of DNA molecules translocation were found. Considering simply the effects of electrophoretic force and electro-osmotic force on the DNA molecules, the dynamic characteristics of DNA molecules are well understood. For example, the velocity of the DNA molecule is inversely proportional to the diameter of the tapered channel and the turning phenomena of the trajectory of the DNA molecules translocating through microchannels. This study is helpful and proposes new ideas for the design and development of microfluidic chips for the quantitative manipulation of DNA molecules. [ABSTRACT FROM AUTHOR]

Efficient distribution of oxygen (O2) to the tissues in mammals depends on the evolved ability of red blood cell (RBC) hemoglobin (Hb) to sense not only O2 levels, but metabolic cues such as pH, PCO2, and organic phosphates, and then dispense or take up oxygen accordingly. O2 delivery is the product of not only oxygen release fromRBCs, but also blood flow,which itself is also governed by vasoactivemolecular mediators exported by RBCs. These vascular signals, including ATP and S-nitrosothiols (SNOs) are produced and exported as a function of the oxygen and metabolic milieu, and then fine-tune peripheral metabolism through context-sensitive vasoregulation. Emerging and repurposed RBC-oriented therapeutics can modulate either or both of these allosteric and vasoregulatory activities, with a single molecule or other intervention influencing both arms of O2 transport in some cases. For example, organic phosphate repletion of stored RBCs boosts the negative allosteric effector 2,3 biphosphoglycerate (BPG) as well as the anti-adhesive molecule ATP. In sickle cell disease, aromatic aldehydes such as voxelotor can disfavor sickling by increasing O2 affinity, and in newer generations, these molecules have been coupled to vasoactive nitric oxide (NO)-releasing adducts. Activation of RBC pyruvate kinase also promotes a left shift in oxygen binding by consuming and lowering BPG, while increasing the ATP available for cell health and export on demand. Further translational and clinical investigation of these novel allosteric and/or vasoregulatory approaches to modulating O2 transport are expected to yield new insights and improve the ability to correct or compensate for anemia and other O2 delivery deficits. [ABSTRACT FROM AUTHOR]

Lanthanide luminescence is dominated by quenching by high-energy oscillators in the chemical environment. The rate of non-radiative energy transfer to a single H2O molecule coordinated to a Eu3+ ion exceeds the usual rates of emission by an order of magnitude. We know these rates, but the details of these energy transfer processes are yet to be established. In this work, we study the quenching rates of [Eu(D2O)9]3+ and [Eu(DOTA)(D2O)]− in H2O/D2O mixtures by sequentially exchanging the deuterons with protons. Flash freezing the solutions allows us to identify species with various D/H contents in solution and thus to quantify the energy transfer processes to individual OH-oscillators. This is not possible in solution due to fast exchange in the ensembles present at room temperature. We conclude that the energy transfer processes are accurately described, predicted, and simulated using a step-wise addition of the rates of quenching by each O–H oscillator. This documents the sequential increase in the rate of the energy transfer processes in the quenching of lanthanide luminescence, and further provides a methodology to identify isotopic impurities in deuterated lanthanide systems in solution. [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]

Background: LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson's disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. Methods: Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. Results: pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil degranulation, antigenic responses, and suppressed platelet activation. Conclusions: The extracellular serum ratio of pT73-Rab10 to total Rab10 is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics that mitigate associated deleterious immunological responses. [ABSTRACT FROM AUTHOR]

Nonlinear photochromic reactions that work with weak incoherent light are important for molecular operations with high spatial resolution and multiple photofunctions based on single molecules. However, nonlinear photochromic compounds generally require complex molecular design, restricting accessibility in various fields. Herein, we report nonlinear photochromic properties in a perylene‐substituted rhodamine spirolactam derivative (Rh−Pe), which is synthesized from rhodamine B in facile procedures. Direct excitation of Rh−Pe produces the triplet excited state via the charge‐transfer (CT) state. The triplet excited state causes triplet–triplet annihilation to bring the generation of the intensely colored ring‐open form with nonlinear behavior. Furthermore, green‐ and red‐light‐induced photochromism was achieved in Rh−Pe using triplet sensitizers, although Rh−Pe can be directly excited only by ultraviolet and blue light. Our findings are expected to contribute to the development of photofunctional materials showing nonlinear behavior and low‐energy light responsivity. [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]