metaProbiotics: a tool for mining probiotic from metagenomic binning data based on a language model.

Beneficial bacteria remain largely unexplored. Lacking systematic methods, understanding probiotic community traits becomes challenging, leading to various conclusions about their probiotic effects among different publications. We developed language model-based metaProbiotics to rapidly detect probiotic bins from metagenomes, demonstrating superior performance in simulated benchmark datasets. Testing on gut metagenomes from probiotic-treated individuals, it revealed the probioticity of intervention strains-derived bins and other probiotic-associated bins beyond the training data, such as a plasmid-like bin. Analyses of these bins revealed various probiotic mechanisms and bai operon as probiotic Ruminococcaceae's potential marker. In different health-disease cohorts, these bins were more common in healthy individuals, signifying their probiotic role, but relevant health predictions based on the abundance profiles of these bins faced cross-disease challenges. To better understand the heterogeneous nature of probiotics, we used metaProbiotics to construct a comprehensive probiotic genome set from global gut metagenomic data. Module analysis of this set shows that diseased individuals often lack certain probiotic gene modules, with significant variation of the missing modules across different diseases. Additionally, different gene modules on the same probiotic have heterogeneous effects on various diseases. We thus believe that gene function integrity of the probiotic community is more crucial in maintaining gut homeostasis than merely increasing specific gene abundance, and adding probiotics indiscriminately might not boost health. We expect that the innovative language model-based metaProbiotics tool will promote novel probiotic discovery using large-scale metagenomic data and facilitate systematic research on bacterial probiotic effects. The metaProbiotics program can be freely downloaded at https://github.com/zhenchengfang/metaProbiotics.

Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Identification and Structural Characterization of Twisted Atomically Thin Bilayer Materials by Deep Learning.

Two-dimensional materials are expected to play an important role in next-generation electronics and optoelectronic devices. Recently, twisted bilayer graphene and transition metal dichalcogenides have attracted significant attention due to their unique physical properties and potential applications. In this study, we describe the use of optical microscopy to collect the color space of chemical vapor deposition (CVD) of molybdenum disulfide (MoS2) and the application of a semantic segmentation convolutional neural network (CNN) to accurately and rapidly identify thicknesses of MoS2 flakes. A second CNN model is trained to provide precise predictions on the twist angle of CVD-grown bilayer flakes. This model harnessed a data set comprising over 10,000 synthetic images, encompassing geometries spanning from hexagonal to triangular shapes. Subsequent validation of the deep learning predictions on twist angles was executed through the second harmonic generation and Raman spectroscopy. Our results introduce a scalable methodology for automated inspection of twisted atomically thin CVD-grown bilayers.

Xanthomonas chitinilytica sp. nov., a novel chitinolytic bacterium isolated from a microbial fermentation bed material.

A Gram-stain-negative bacterium, H13-6T, was isolated from a microbial fermentation bed material collected from a pig farm located in Yan'an, Shaanxi, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain H13-6T was affiliated with the genus Xanthomonas and showed highest similarity to strain Xanthomonas maliensis M97T (98.38%), Xanthomonas prunicola CFBP 8353T (98.26%) and Xanthomonas oryzae ATCC 35933T (98.11%). The pairwise ortho Average Nucleotide Identity values and the digital DNA-DNA hybridization values between strain H13-6T and the other Xanthomonas species were all below their respective cut-offs. Two genes encoding for chitinase were found and the strain showed a strong chitin-degrading activity. The major fatty acids were Iso-C15:0 (55.9%), Antesio-C15:0 (7.4%) and Iso-C11:0 (5.5%) and the major polar lipids were diphosphatidylglycerol, phosphatidyglycerol and phosphatidylethanolamine. Based on the phenotypic properties and phylogenetic distinctiveness, Xanthomonas chitinilytica was proposed as a novel species of the genus Xanthomonas, with strain H13-6T (= CGMCC 1.61317T = NBRC 115641T) as type strain.

Effects of resveratrol on DLD and NDUFB9 decrease in frozen semen of Mongolian sheep.

Mongolian sheep are a breed of sheep in China known for their excellent cold and drought resistance. Sperm from Mongolian sheep are often cryopreserved to improve breeding outcomes. However, cryopreservation of sperm often results in issues such as reduced vitality and altered morphology. Therefore, the objective of this study was to investigate the impact of the cryoprotectant resveratrol on frozen sperm from Mongolian sheep, specifically examining its effects on key proteins during cryopreservation. In this study, sperm samples were obtained from three adult Mongolian rams and processed through semen centrifugation. The sperm motility parameters of Fresh Sperm Group (FR), Resveratrol added before freezing group (FF-Res), Resveratrol-free frozen sperm group (FT), and Resveratrol added after freeze-thawing group (FA-Res) were determined. The tandem mass tags (TMT) peptide labeling combined with LC-MS/MS was used for proteomic analysis of the total proteins in FR and FT groups. A total of 2651 proteins were identified, among which 41 proteins were upregulated and 48 proteins were downregulated after freezing. In-depth bioinformatics analysis of differentially abundant proteins (DAPs) revealed their close association with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathway. The energy-related protein dihydrolipoamide dehydrogenase (DLD) and the reactive oxygen species (ROS)-related protein NADH dehydrogenase 1 beta subcomplex subunit 9 (NDUFB9) exhibited significant decreases, indicating their potential role as key proteins contributing to reduced sperm vitality. The study demonstrated that the addition of resveratrol (RES) to semen could elevate the expression levels of DLD and NDUFB9 proteins. This study represents the pioneering proteomic analysis of Mongolian ram sperm before and after cryopreservation, establishing the significance of DLD and NDUFB9 as key proteins influencing the decline in vitality following cryopreservation of Mongolian ram sperm. These findings clarify that resveratrol can enhance the levels of DLD and NDUFB9 proteins in cryopreserved Mongolian ram sperm, consequently enhancing their vitality.

Kaistella yananensis sp. nov., a Novel Indoleacetic Acid-Producing Bacterium Isolated from the Root Nodules of Sophora davidii (Franch.) Skeels.

A novel endophytic bacterium, designated strain BT6-1-3T, was isolated from the root nodules of a leguminous shrub named Sophora davidii (Franch.) Skeels, found growing wild in Yan'an, Shaanxi Province, China. Cells were Gram-staining-negative, non-motile, catalase-positive, oxidase-positive, and did not produce H2S. Strain BT6-1-3T grew at 15-40 °C (optimum 30 °C), at pH 6.0-10.0 (optimum pH 9.0), and with 0-1% (w/v) NaCl (optimum 0.5%). The quinone system was menaquinone 6. The major fatty acids present in BT6-1-3T were iso-C11:0, iso-C15:0, and C16:0. The G+C content of genomic DNA was 39.4 mol% by whole genome sequencing. According to the analysis of 16S rRNA gene sequence, the closest relative was Kaistella montana WG4 (nucleotide identity was 97.6%). The genome of strain BT6-1-3T was sequenced, and the genome similarity was calculated using average nucleotide identity and genome-to-genome distance analysis with the genomes of other strains of Kaistella. Both strongly supported that the strain BT6-1-3T belonged to the genus Kaistella as a representative of a new species. Based on phylogenetic analysis, chemotaxonomic data, and physiological and biochemical characteristics, strain BT6-1-3T represents a new species of the genus Kaistella and is named as Kaistella yananensis sp. nov. Type strain is BT6-1-3T (= NBRC 115452T = CGMCC 1.60032T).

Single-Molecule Monitoring of Membrane Association of the Necroptosis Executioner MLKL with Discernible Anchoring and Insertion Dynamics.

The dynamics of membrane proteins that are well-folded in water and become functional after self-insertion into cell membranes is not well understood. Herein we report on single-molecule monitoring of membrane association dynamics of the necroptosis executioner MLKL. We observed that, upon landing, the N-terminal region (NTR) of MLKL anchors onto the surface with an oblique angle and then is immersed in the membrane. The anchoring end does not insert into the membrane, but the opposite end does. The protein is not static, switching slowly between water-exposed and membrane-embedded conformations. The results suggest a mechanism for the activation and function of MLKL in which exposure of H4 is critical for MLKL to adsorb on the membrane, and the brace helix H6 regulates MLKL rather than inhibits it. Our findings provide deeper insights into membrane association and function regulation of MLKL and would have impacts on biotechnological applications.

Ureteroscopic lithotripsy with pressure-measuring ureteral access sheath for large ureteral stones.

INTRODUCTION: To evaluate the safety and efficacy of ureteroscopic lithotripsy with pressure-measuring ureteral access sheath (PM-UAS) for large ureteral stones. MATERIAL AND METHODS: A total of 258 consecutive patients with large ureteral stones ≥15 mm was enrolled. They were treated by ureteroscopic lithotripsy with PM-UAS in the oblique supine lithotomy position. The technology can precisely monitor and automatically control cavity pressure. The cavity pressure control value was set at -15 mmHg∼-5 mmHg. The cavity pressure limit value was set at 30 mmHg. Infusion flow rate was set at 100-200 ml/min. Postoperative data such as stone-free rate and complications were analyzed. RESULTS: PM-UAS was successfully implanted in 225 patients at one stage. Eighteen cases of patients who had failed the first surgery were successfully treated with a second operation. Fifty-one cases with stones migrating up to the kidney were converted to flexible lithotripsy. The other 15 cases were converted to percutaneous nephrolithotomy due to significant ureteral stenosis. The operative time was 49.5 ± 11.2 min. The stone-free rates after one month and three months were 87.2% (212/243) and 94.2% (229/243), respectively. Complications from grade I to II were observed in 25(10.3%) patients. No other complications from grade III to V were noted. CONCLUSION: The ureteroscopic lithotripsy with PM-UAS is safe and efficacious for large ureteral stones.

LncR2metasta: a manually curated database for experimentally supported lncRNAs during various cancer metastatic events.

Mounting evidence has shown the involvement of long non-coding RNAs (lncRNAs) during various cancer metastatic events (abbreviated as CMEs, e.g. cancer cell invasion, intravasation, extravasation, proliferation, etc.) that may cooperatively facilitate malignant tumor spread and cause massive patient deaths. The study of lncRNA-CME associations might help understand lncRNA functions in metastasis and present reliable biomarkers for early dissemination detection and optimized treatment. Therefore, we developed a database named 'lncR2metasta' by manually compiling experimentally supported lncRNAs during various CMEs from existing studies. LncR2metasta documents 1238 associations between 304 lncRNAs and 39 CMEs across 54 human cancer subtypes. Each entry of lncR2metasta contains detailed information on a lncRNA-CME association, including lncRNA symbol, a specific CME, brief description of the association, lncRNA category, lncRNA Entrez or Ensembl ID, lncRNA genomic location and strand, lncRNA experiment, lncRNA expression pattern, detection method, target gene (or pathway) of lncRNA, lncRNA regulatory role on a CME, cancer name and the literature reference. An easy-to-use web interface was deployed in lncR2metasta for its users to easily browse, search and download as well as to submit novel lncRNA-CME associations. LncR2metasta will be a useful resource in cancer research community. It is freely available at http://lncR2metasta.wchoda.com.

Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation.

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

Diastereoselective Synthesis of Dihydrobenzofuran-Fused Spiroindolizidines via Double-Dearomative [3 + 2] Cycloadditions.

Spiroindolizidine oxindoles represent a kind of privileged scaffold in many biologically active natural alkaloids. 2,3-Dihydrobenzofuran derivatives exhibit significant bioactivities in a variety of pharmaceuticals. Herein, we assembled these two privileged fragments into a small molecule via double-dearomative [3 + 2] cycloadditions with pyridinium ylides and 2-nitrobenzofurans. This protocol features remarkable advantages including wide substrate scope, mild condition, high level of diastereoselectivities and yields. Thus, a collection of spiroindolizidine-fused dihydrobenzofurans/indolines were facilely produced efficiently.

A NIR fluorescent probe for dual imaging of mitochondrial viscosity and FA in living cells and zebrafish.

Formaldehyde (FA) and viscosity play multiple roles in human health and diseases, and viscosity has great regional differences due to the diversity of subcellular organelles. However, it is challenging to achieve dual detection of viscosity and FA in subcellular organelles. Herein, we developed a near infrared (NIR) fluorescent probe FA-Cy, which can simultaneously monitor the viscosity and FA concentration of mitochondria in living cells. The probe could detect mitochondrial viscosity and exogenous and endogenous FA in living cells and zebrafish.

The effect of different combinations of physical activity and natural environment videos on children's attention levels between class breaks.

BACKGROUND: Physical activity intervention and watching natural environment videos have been proven to improve young children's attention levels. However, evidence comparing the improvement effects of different combinations of the two activities has rarely been reported. By comparing the differences in the improvement effects of four combinations of physical activities and watching natural environment videos on young children's attention levels, this study can enrich the evidence in this research field and also provide a reference for arranging effective intervention methods for children's attention recovery between classes. METHOD: A total of 152 children aged 4 to 6 years were recruited and randomly divided into four intervention groups: (1) physical activity intervention first and thereafter watching a natural environment video group (activity + video group), (2) watching a natural environment video first and thereafter the physical activity intervention group (video + activity group), (3) physical activity-based group, and (4) natural environment video-based group. Physical activity involved 4 min of moderate-intensity basic physical fitness combination training. The subjects wore the Pico Neo pioneer version of the VR glasses all-in-one machine to watch a natural environment video. Thereafter, population sociological variables and daily physical activity levels were investigated. Auditory and visual sustained attention tests were performed before and after intervention in each group. RESULT: The auditory attention post-test scores of the four groups showed an improvement trend compared with the pretest scores. In particular, the activity + video group (F = 10.828; ɳp2 = 0.226; p = 0.002) and natural environment video-based group (F = 9.452; ɳp2 = 0.203; p = 0.004) have the best improvement effect. For visual attention, only the activity + video group showed a significant improvement trend (F = 4.287; ɳp2 = 0.104; p = 0.045), while the other three groups showed a downward trend in scores. CONCLUSIONS: Among the different intervention combinations, the physical activity intervention first and watching natural environment videos thereafter group has the best effect on improving children's attention during recess. Physical activity interventions at the end of recess adversely affect young children's visual attention levels at the beginning of the class. Therefore, this study recommends that children should not engage in physical activity interventions in the second half of the class break. Lastly, the current research recommends presenting the content of physical activity interventions first and further improving their attention thereafter by watching natural environment videos.

Novel harmine derivatives as potent acetylcholinesterase and amyloid beta aggregation dual inhibitors for management of Alzheimer's disease.

In this study, a series of potential ligands for the treatment of AD were synthesised and characterised as novel harmine derivatives modified at position 9 with benzyl piperazinyl. In vitro studies revealed that the majority of the derivatives exhibited moderate to potent inhibition against hAChE and Aß1 - 42 aggregation. Notably, compounds 13 and 17d displayed potent drug - likeness and ADMET properties, demonstrating remarkable inhibitory activities towards AChE (IC50 = 58.76 nM and 89.38 nM, respectively) as well as Aß aggregation (IC50 = 9.31 µM and 13.82 µM, respectively). More importantly, compounds 13 and 17d showed exceptional neuroprotective effects against Aß1 - 42-induced SH - SY5Y damage, while maintaining low toxicity in SH - SY5Y cells. Further exploration of the mechanism through kinetic studies and molecular modelling confirmed that compound 13 could interact with both the CAS and the PAS of AChE. These findings suggested that harmine derivatives hold great potential as dual - targeted candidates for treating AD.

Assessing plant uptake of organic contaminants by food crops tomato, wheat, and corn through sap concentration factor.

This study investigated uptake of two organic compounds including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and exogenous caffeine by tomato (Solanum lycopersicum L.), corn (Zea mays L.), and wheat (Triticum aestivum L.). The plants were grown in a growth chamber under recommended conditions and then were exposed to these compounds for 19 days. The uptake of the compounds was measured by sap concentration factor. The plant samples (stem transpiration stream) and solution in the exposure media were taken and analyzed by high performance liquid chromatography-tandem mass spectrometry. The plant stem samples were analyzed after a freeze-thaw centrifugation process. The average sap concentration factor for the RDX by tomato, wheat, and corn was 0.71, 0.67, and 0.65. The average sap concentration factor for the exogenous caffeine by tomato, wheat, and corn was 0.72, 0.50, and 0.34. These relatively high sap concentration factor values were expected as available predictive models offer high sap concentration factor values for moderately hydrophobic and hydrophilic compounds. The generated sap concentration factor values for the RDX and exogenous caffeine are important for improving the accuracy of previously developed machine learning models predicting the uptake and translocation of emerging contaminants.

The uptake of two organic compounds (RDX and exogenous caffeine) was examined in three crop plants (corn, wheat, and tomato). There have not been any uptake studies on exogenous caffeine and also we do not have good data for the uptake of RDX by these three crop plants. The estimated sap concentration factor from these experiments fills the gap in the data for developing predictive models for uptake of emerging contaminants. A novel rapid freeze­thaw/centrifugation extraction method followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the samples.

Enhanced Capacity Retention of Li3V2(PO4)3-Cathode-Based Lithium Metal Battery Using SiO2-Scaffold-Confined Ionic Liquid as Hybrid Solid-State Electrolyte.

Li3V2(PO4)3 (LVP) is one of the candidates for high-energy-density cathode materials matching lithium metal batteries due to its high operating voltage and theoretical capacity. However, the inevitable side reactions of LVP with a traditional liquid-state electrolyte under high voltage, as well as the uncontrollable growth of lithium dendrites, worsen the cycling performance. Herein, a hybrid solid-state electrolyte is prepared by the confinement of a lithium-containing ionic liquid with a mesoporous SiO2 scaffold, and used for a LVP-cathode-based lithium metal battery. The solid-state electrolyte not only exhibits a high ionic conductivity of 3.14 × 10-4 S cm-1 at 30 °C and a wide electrochemical window of about 5 V, but also has good compatibility with the LVP cathode material. Moreover, the cell paired with a solid-state electrolyte exhibits good reversibility and can realize a stable operation at a voltage of up to 4.8 V, and the discharge capacity is well-maintained after 100 cycles, which demonstrates excellent capacity retention. As a contrast, the cell paired with a conventional liquid-state electrolyte shows only an 87.6% discharge capacity retention after 100 cycles. In addition, the effectiveness of a hybrid solid-state electrolyte in suppressing dendritic lithium is demonstrated. The work presents a possible choice for the use of a hybrid solid-state electrolyte compatible with high-performance cathode materials in lithium metal batteries.

A lysosome-targeting fluorescent probe to visualize endogenous and exogenous methylglyoxal in live cells and zebrafish.

The development of a lysosome-targeting fluorescent probe to visualize endogenous and exogenous methylglyoxal (MGO) in live cells has important implications for associated diseases. Herein, a lysosome-targeting fluorescent probe MGO-Naph-A was designed and synthesized to detect MGO with high selectivity. The probe contained naphthalimide as the fluorescent group, o-phenylenediamine as the MGO recognition group, and morpholine as the lysosome targeting group. This fluorescent probe could detect endogenous and exogenous MGO in living cells by precisely targeting and staining lysosomes. It could also detect MGO in living zebrafish. The results showed that the probe MGO-Naph-A has the potential to visualize MGO in lysosomes.

The association between the health-related physical fitness and inhibitory control in preschool children.

BACKGROUND: Inhibitory control develops rapidly during the preschool stage, and development of inhibitory control in this period is significant for the healthy growth of the future. However, most studies paid more attention to children and adolescents in recent years, but less focus on preschool children. Therefore, the purpose of this study was to explore the association between the health-related physical fitness and inhibitory control in preschool children. METHODS: This cross-sectional study was based on a baseline data from randomized controlled trial by cluster sampling(including 128 preschoolers, 70 boys, 58girls).The health-related physical fitness T-score (HPFT) was obtained by adding standard scores of six indicators: body mass index, handgrip strength, standing long jump, one-leg balance, 2 × 10 m shuttle run test, and 20 m shuttle run test. Inhibitory control was assessed using the flanker task and reflected by reaction time and accuracy. RESULTS: A total of 120 preschoolers were included in the final statistical analysis. After adjusting the confounders, HPFT was associated with reaction time (ß=-2.49 ms, 95%CI: -4.22, -0.75). Similarly, a negative association was observed between one-leg balance and reaction time (ß=-12.04 ms, 95%CI: -18.94, -5.15), and between 20 m shuttle run test and reaction time (ß=-23.28 ms, 95%CI: -37.00, -9.56). Compared with the participants in the lowest tertile, HPFT (ß=-158.74, 95%CI: -309.63, -7.84), one-leg balance (ß=-267.25 ms, 95%CI: -420.71, -113.79) and 20 m shuttle run test (ß=-215.58 ms, 95%CI: -374.67, -56.49) were all negatively associated with reaction time of those in the highest tertile. CONCLUSIONS: Negative relationships between HPFT and RT of the inhibitory control were observed in preschoolers. To have better inhibitory control, it's suggested that HPFT of preschoolers should be at least 249. These findings are of great significance for the early improvement of HPFT and the development of inhibitory control in preschool children.

A Feature-Encoded Physics-Informed Parameter Identification Neural Network for Musculoskeletal Systems.

Identification of muscle-tendon force generation properties and muscle activities from physiological measurements, e.g., motion data and raw surface electromyography (sEMG), offers opportunities to construct a subject-specific musculoskeletal (MSK) digital twin system for health condition assessment and motion prediction. While machine learning approaches with capabilities in extracting complex features and patterns from a large amount of data have been applied to motion prediction given sEMG signals, the learned data-driven mapping is black-box and may not satisfy the underlying physics and has reduced generality. In this work, we propose a feature-encoded physics-informed parameter identification neural network (FEPI-PINN) for simultaneous prediction of motion and parameter identification of human MSK systems. In this approach, features of high-dimensional noisy sEMG signals are projected onto a low-dimensional noise-filtered embedding space for the enhancement of forwarding dynamics prediction. This FEPI-PINN model can be trained to relate sEMG signals to joint motion and simultaneously identify key MSK parameters. The numerical examples demonstrate that the proposed framework can effectively identify subject-specific muscle parameters and the trained physics-informed forward-dynamics surrogate yields accurate motion predictions of elbow flexion-extension motion that are in good agreement with the measured joint motion data.

Subnanometer-Precision Measurements of Transmembrane Motions of Biomolecules in Plasma Membranes Using Quenchers in Extracellular Environment.

Characterization of biomolecular dynamics at cellular membranes lags far behind that in solutions because of challenges to measure transmembrane trafficking with subnanometer precision. Herein, by introducing nonfluorescent quenchers into extracellular environment of live cells, we adopted Förster resonance energy transfer from one donor to multiple quenchers to measure positional changes of biomolecules in plasma membranes. We demonstrated the method by monitoring flip-flops of individual lipids and by capturing transient states of the host defense peptide LL-37 in plasma membranes. The method was also applied to investigate the interaction of the necroptosis-associated protein MLKL with plasma membranes, showing a few distinct depths of MLKL insertion. Our method is especially powerful to quantitate the dynamics of proteins at the cytosolic leaflets of plasma membranes which are usually not accessible by conventional techniques. The method will find wide applications in the systematic analysis of fundamental cellular processes at plasma membranes.