O-glycosylation in viruses: A sweet tango.

O-glycosylation is an ancient yet underappreciated protein posttranslational modification, on which many bacteria and viruses heavily rely to perform critical biological functions involved in numerous infectious diseases or even cancer. But due to the innate complexity of O-glycosylation, research techniques have been limited to study its exact role in viral attachment and entry, assembly and exit, spreading in the host cells, and the innate and adaptive immunity of the host. Recently, the advent of many newly developed methodologies (e.g., mass spectrometry, chemical biology tools, and molecular dynamics simulations) has renewed and rekindled the interest in viral-related O-glycosylation in both viral proteins and host cells, which is further fueled by the COVID-19 pandemic. In this review, we summarize recent advances in viral-related O-glycosylation, with a particular emphasis on the mucin-type O-linked α-N-acetylgalactosamine (O-GalNAc) on viral proteins and the intracellular O-linked ß-N-acetylglucosamine (O-GlcNAc) modifications on host proteins. We hope to provide valuable insights into the development of antiviral reagents or vaccines for better prevention or treatment of infectious diseases.

Co-expression network analysis and identification of core genes in the interaction between wheat and Puccinia striiformis f. sp. tritici.

The epidemic of stripe rust, caused by the pathogen Puccinia striiformis f. sp. tritici (Pst), would reduce wheat (Triticum aestivum) yields seriously. Traditional experimental methods are difficult to discover the interaction between wheat and Pst. Multi-omics data analysis provides a new idea for efficiently mining the interactions between host and pathogen. We used 140 wheat-Pst RNA-Seq data to screen for differentially expressed genes (DEGs) between low susceptibility and high susceptibility samples, and carried out Gene Ontology (GO) enrichment analysis. Based on this, we constructed a gene co-expression network, identified the core genes and interacted gene pairs from the conservative modules. Finally, we checked the distribution of Nucleotide-binding and leucine-rich repeat (NLR) genes in the co-expression network and drew the wheat NLR gene co-expression network. In order to provide accessible information for related researchers, we built a web-based visualization platform to display the data. Based on the analysis, we found that resistance-related genes such as TaPR1, TaWRKY18 and HSP70 were highly expressed in the network. They were likely to be involved in the biological processes of Pst infecting wheat. This study can assist scholars in conducting studies on the pathogenesis and help to advance the investigation of wheat-Pst interaction patterns.

A new multi-attribute group decision-making method based on Einstein Bonferroni operators under interval-valued Fermatean hesitant fuzzy environment.

Faced with the increasing complexity and uncertainty of decision-making information, interval-valued Fermatean hesitant fuzzy sets (IVFHFSs) were presented as a novel mathematical model that handled uncertain data more effectively. However, existing multi-attribute group decision-making (MAGDM) methods based on IVFHFSs do not thoroughly investigate the operational laws. Also, these existing MAGDM methods do not take into account the connections between attributes and are less flexible. To address these issues, this paper proposes a new MAGDM method based on Einstein Bonferroni operators under IVFHFSs. First, we thoroughly examine the operational laws of Einstein t-norms under the IVFHFSs to further extend the study of the operational laws. Then, we introduce the interval-valued Fermatean hesitant fuzzy Einstein Bonferroni mean operator and the interval-valued Fermatean hesitant fuzzy Einstein weighted Bonferroni mean operator under Einstein t-norms. Our suggested aggregation operators consider the relationship between attributes and are far more flexible in comparison to the current approaches. Later, a novel MAGDM method based on Einstein Bonferroni operators under the IVFHFSs is given. Finally, the practicality and validity of the proposed method are demonstrated by a cardiovascular disease diagnosis application.

Chaperone-mediated autophagy protects the bone formation from excessive inflammation through PI3K/AKT/GSK3ß/ß-catenin pathway.

Multiple regulatory mechanisms are in place to ensure the normal processes of bone metabolism, encompassing both bone formation and absorption. This study has identified chaperone-mediated autophagy (CMA) as a critical regulator that safeguards bone formation from the detrimental effects of excessive inflammation. By silencing LAMP2A or HSCA8, we observed a hindrance in the osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. To further elucidate the role of LAMP2A, we generated LAMP2A gene knockdown and overexpression of mouse BMSCs (mBMSCs) using adenovirus. Our results showed that LAMP2A knockdown led to a decrease in osteogenic-specific proteins, while LAMP2A overexpression favored the osteogenesis of mBMSCs. Notably, active-ß-catenin levels were upregulated by LAMP2A overexpression. Furthermore, we found that LAMP2A overexpression effectively protected the osteogenesis of mBMSCs from TNF-α, through the PI3K/AKT/GSK3ß/ß-catenin pathway. Additionally, LAMP2A overexpression significantly inhibited osteoclast hyperactivity induced by TNF-α. Finally, in a murine bone defect model, we demonstrated that controlled release of LAMP2A overexpression adenovirus by alginate sodium capsule efficiently protected bone healing from inflammation, as confirmed by imaging and histological analyses. Collectively, our findings suggest that enhancing CMA has the potential to safeguard bone formation while mitigating hyperactivity in bone absorption.

Positive role of plateau pika (Ochotona coronae) on environmental quality at low and moderate density on the Tibetan plateau: Evidence from a meta-analysis.

The roles of plateau pika (Ochotona coronae) in the Tibetan Plateau are often controversial, because it is often regarded as a destructive pest or an ecosystem engineer. Here a meta-analysis using 72 paired observations was conducted to examine whether the impacts of plateau pika on environmental quality (i.e., plant and soil properties) depend on population density in the Tibetan Plateau. Pika population density was used as a proxy for disturbance intensity. The pika disturbance intensity was divided into five groups based on the number of burrows, including low disturbance intensity (LD) (9-30 burrows per ha), medium disturbance intensity (MD) (31-100 burrows per ha), high disturbance intensity (HD) (101-170 burrows per ha), extreme disturbance intensity (ED) (171-240 burrows per ha) and uncontrolled (or excessive) disturbance intensity (UD) (>241 burrows per ha). Given that sample sizes in some of the groups are small (especially for the HD), we further pooled the disturbance groups including the LD-MD and HD-UD. Overall, relative to control (i.e., no disturbing), there was a great increase (80.3%) in aboveground biomass under the LD-MD, whereas a decrease of 41.1% occurred under the HD-UD. At the same time, plant coverage, species richness, height, and belowground biomass greatly decreased only in the HD-UD. Furthermore, the effect size of plant coverage, species richness, and aboveground biomass also declined with pika burrow density significantly. With regard to soil properties, there was a significant increase in soil organic carbon, ammonium nitrogen, and soil organic carbon stock under the LD-MD, whereas a decrease under the HD-UD. In addition, soil total nitrogen, total potassium, and nitrate nitrogen increased at the LD-MD and HD-UD. Nevertheless, the effect size of these soil properties (with >20 observations) was not related to pika burrow density. In summary, there is an implication that the low and moderate disturbance of pikas is beneficial to maintain and promote ecosystem functioning in the Tibetan grasslands. In the future pikas' eradication policy should be reconsidered in alpine grassland management.

Development and Application of a Selective Analytical Method for Indole Metabolites in Urine: Dietary Exposure Biomarkers for Broccoli Consumption.

The identification of dietary exposure biomarkers is crucial for advancing our understanding of the health benefits of specific foods. Broccoli, a vegetable with well-known anticancer properties, contains active ingredients, such as isothiocyanates with indole side chains. Hence, indole metabolites related to broccoli consumption have the potential to serve as biomarkers of dietary exposure. In this work, we developed a new analytical method for indole metabolites in urine using a poly(deep eutectic solvents)-molecularly imprinted polymer/vinyl-functionalized graphene oxide (PDESs-MIP/VGO) in miniaturized centrifugal pipet-tip solid-phase extraction (CPT-SPE) coupled with liquid chromatography. This method integrates the strengths of PDESs-MIP/VGO, including rich adsorption interactions, high adsorption capacity, and excellent selectivity, with the simplicity and cost-effectiveness of CPT-SPE. The proposed method demonstrated low limits of quantification (1.2-2.5 ng mL-1), high accuracy (91.7-104.8%), and good precision (relative standard deviation ≤4.4%). By applying this method to analyze indole metabolites in urine, our results suggested that indole-3-carbinol and indole-3-acetonitrile have the potential to emerge as reliable dietary exposure biomarkers for broccoli intake. Furthermore, highly selective analytical methods based on molecular imprinting technology are advantageous for precise screening and analysis of dietary exposure biomarkers associated with food consumption.

Enhancing post-harvest quality of tomato fruits with chitosan oligosaccharide-zinc oxide nanocomposites: A study on biocompatibility, quality improvement, and carotenoid enhancement.

In this study, a new composite with combination of chitosan oligosaccharide (COS) and zinc oxide nanoparticles (ZnO NPs), termed Chitosan Oligosaccharide-Zinc Oxide Nanocomposites (COS-ZnO NC), was designed to enhance the quality of tomato fruits during postharvest storage. SEM analysis showed a uniform distribution of COS-ZnO NC films on tomato surfaces, indicating high biocompatibility, while the FTIR spectrum confirmed the interaction of COS and ZnO NPs via hydrogen bonds. The COS-ZnO NC exerts positive effects on post-harvest quality of tomato fruits, including significantly reduced water loss, fewer skin wrinkles, increased sugar-acid ratio, and enhanced vitamin C and carotenoids accumulation. Furthermore, COS-ZnO NC induces transcription of carotenoid biosynthesis genes and promotes carotenoids storage in the chromoplast. These results suggest that the COS-ZnO NC film can significantly improve the quality traits of tomato fruits, and therefore is potential in post-harvest storage of tomato fruits.

High-enhancement photoluminescence of monolayer MoS2 in hybrid plasmonic systems.

Monolayer molybdenum disulfide (M o S 2) has a weak light-matter interaction due to ultrathin thickness, which limits its potential application in lasing action. In this study, we propose a hybrid structure consisting of a nanocavity and Au nanoparticles to enhance the photon emission efficiency of monolayer M o S 2. Numerical simulations show that photoluminescence (PL) emission is significantly enhanced by introducing localized surface plasmon resonance (LSPR) to the proposed structure. Furthermore, an exciton energy band system is proposed to elucidate the physical mechanism of the PL process. By optimizing the spacer thickness, a high Purcell enhancement factor of 95 can be achieved. The results provided by this work pave the way to improve the PL efficiency of two-dimensional (2D) material, which constitutes a significant step towards the development of nanodevices such as nanolasers and sensors.

DeepSeeded: Volumetric Segmentation of Dense Cell Populations with a Cascade of Deep Neural Networks in Bacterial Biofilm Applications.

Accurate and automatic segmentation of individual cell instances in microscopy images is a vital step for quantifying the cellular attributes, which can subsequently lead to new discoveries in biomedical research. In recent years, data-driven deep learning techniques have shown promising results in this task. Despite the success of these techniques, many fail to accurately segment cells in microscopy images with high cell density and low signal-to-noise ratio. In this paper, we propose a novel 3D cell segmentation approach DeepSeeded, a cascaded deep learning architecture that estimates seeds for a classical seeded watershed segmentation. The cascaded architecture enhances the cell interior and border information using Euclidean distance transforms and detects the cell seeds by performing voxel-wise classification. The data-driven seed estimation process proposed here allows segmenting touching cell instances from a dense, intensity-inhomogeneous microscopy image volume. We demonstrate the performance of the proposed method in segmenting 3D microscopy images of a particularly dense cell population called bacterial biofilms. Experimental results on synthetic and two real biofilm datasets suggest that the proposed method leads to superior segmentation results when compared to state-of-the-art deep learning methods and a classical method.

High-Temperature-Induced Differential Expression of miRNA Mediates Liver Inflammatory Response in Tsinling Lenok Trout (Brachymystax lenok tsinlingensis).

High-temperature stress poses a significant environmental challenge for aquatic organisms, including tsinling lenok trout (Brachymystax lenok tsinlingensis). This study aimed to investigate the role of microRNAs (miRNAs) in inducing liver inflammation in tsinling lenok trout under high-temperature stress. Tsinling lenok trout were exposed to high-temperature conditions (24 °C) for 8 h, and liver samples were collected for analysis. Through small RNA sequencing, we identified differentially expressed miRNAs in the liver of high-temperature-stressed tsinling lenok trout compared to the control group (maintained at 16 °C). Several miRNAs, including novel-m0105-5p and miR-8159-x, showed significant changes in expression levels. Additionally, we conducted bioinformatics analysis to explore the potential target genes of these differentially expressed miRNAs. Our findings revealed that these miRNA target genes are involved in inflammatory response pathways, such as NFKB1 and MAP3K5. The downregulation of novel-m0105-5p and miR-8159-x in the liver of high-temperature-stressed tsinling lenok trout suggests their role in regulating liver inflammatory responses. To validate this, we performed a dual-luciferase reporter assay to confirm the regulatory relationship between miRNAs and target genes. Our results demonstrated that novel-m0105-5p and miR-8159-x enhance the inflammatory response of hepatocytes by promoting the expression of NFKB1 and MAP3K5, respectively. In conclusion, our study provides evidence that high-temperature stress induces liver inflammation in tsinling lenok trout through dysregulation of miRNAs. Understanding the molecular mechanisms underlying the inflammatory response in tsinling lenok trout under high-temperature stress is crucial for developing strategies to mitigate the negative impacts of environmental stressors on fish health and aquaculture production.

Polymer Engineering Enables High Linear Capacity Fiber Electrodes by Microenvironment Regulation.

Unlike bulky and rigid traditional power systems, 1D fiber batteries possess appealing features such as flexibility and adaptability, which are promising for use in wearable electronic devices. However, the performance and energy density fiber batteries are limited by the contradiction between ionic transfer and robust structure of fiber electrodes. Herein, these problems are addressed via polymer engineering to regulate the microenvironment in electrodes, realizing high-linear-capacity thick fiber electrodes with excellent cycling performance. The porosity of the electrodes is regulated using polymer crosslink networks designed with various components, and lithium-ion transfer is optimized through ether-abundant polymer chains. Furthermore, reinforced covalent bonding with carbon nanotube networks is established based on the modified functional groups of polymer networks. The multiscale optimizations of the porous structure, ionic transportation, and covalent bonding network enhance the lithium-ion dynamics property and structural stability. Therefore, ultrahigh linear-capacity fiber electrodes (17.8 mAh m-1) can be fabricated on a large scale and exhibit excellent stability (92.8% after 800 cycles), demonstrating obvious superiority among the reported fiber electrodes. Moreover, this study highlights the high effectiveness of polymer regulation in fiber electrodes and offers new avenues for designing next-generation wearable energy-storage systems.

"Flexible-strong" polylactic acid porous membrane via tailored polymerization degree of lactic acid side-chains grafting for passive daytime radiative cooler.

Aerogel possesses the advantages of high specific surface area, low density, and high porosity, which have shown great application in thermal regulation due to its efficient light scattering capability. However, traditional polymer-based aerogels have poor mechanical properties and lack ductility in outdoor applications, the cooling efficiency of the material is easily affected by damage during transportation, installation, and environmental factors. In this work, combining the porous nature of aerogels and the high ductility of membranes, a polylactic acid-based porous membrane cooler was developed by combining a regular honeycomb surface porous structure design and physical/chemical modification to enhance flexibility, using a simple non-solvent induced phase separation method. This porous membrane exhibits both super-flexibility (116 % elongation at break) and porous characteristics. It achieves a sub-ambient temperature decrease of 4-6 °C under direct sunlight. The optimized porous membrane demonstrates high solar reflectance (94 % of peak reflectivity, 90 % of average reflectivity) and strong infrared emissivity (96 % of peak emissivity, 91 % of average emissivity), it also maintains a solar peak reflectivity of 91 % under 100 % tensile strain and 1000 bending cycles, the cooler still maintains a cooling effect of 2-5 °C below ambient temperature. This work paves the way for developing mechanical flexible and strong radiative coolers for thermal regulation.

High-Mobility Compensated Semimetals, Orbital Magnetization, and Umklapp Scattering in Bilayer Graphene Moiré Superlattices.

Twist-controlled moiré superlattices (MSs) have emerged as a versatile platform for realizing artificial systems with complex electronic spectra. The combination of Bernal-stacked bilayer graphene (BLG) and hexagonal boron nitride (hBN) can give rise to an interesting MS, which has recently featured a set of unexpected behaviors, such as unconventional ferroelectricity and the electronic ratchet effect. Yet, the understanding of the electronic properties of BLG/hBN MS has, at present, remained fairly limited. Here, we combine magneto-transport and low-energy sub-THz excitation to gain insights into the properties of this MS. We demonstrate that the alignment between BLG and hBN crystal lattices results in the emergence of compensated semimetals at some integer fillings of the moiré bands, separated by van Hove singularities where the Lifshitz transition occurs. A particularly pronounced semimetal develops when eight holes reside in the moiré unit cell, where coexisting high-mobility electron and hole systems feature strong magnetoresistance reaching 2350% already at B = 0.25 T. Next, by measuring the THz-driven Nernst effect in remote bands, we observe valley splitting, indicating an orbital magnetization characterized by a strongly enhanced effective gv-factor of 340. Finally, using THz photoresistance measurements, we show that the high-temperature conductivity of the BLG/hBN MS is limited by electron-electron umklapp processes. Our multifaceted analysis introduces THz-driven magnetotransport as a convenient tool to probe the band structure and interaction effects in van der Waals materials and provides a comprehensive understanding of the BLG/hBN MS.

Efficacy and safety of mesenchymal stem cell therapy for ovarian ageing in a mouse model.

BACKGROUND: Ovarian ageing is one of the major issues that impacts female fertility. Mesenchymal stem cell (MSC)-based therapy has made impressive progress in recent years. However, the efficacy and safety of MSCs, as nonautologous components, remain to be further verified. METHODS: Two common sources of MSCs, umbilical cord-derived MSCs (UC-MSCs) and adipose tissue-derived MSCs (AD-MSCs), were orthotopically transplanted into a mouse model of ovarian ageing to evaluate their therapeutic effects. The safety of the treatment was further evaluated, and RNA sequencing was performed to explore the underlying mechanisms involved. RESULTS: After orthotopic transplantation of MSCs into the ovary, the oestrous cycle, ovarian weight, number and proportion of primary follicles, granulosa cell proliferation, and angiogenesis were improved. The effects of AD-MSCs were superior to those of UC-MSCs in several indices, such as post-transplant granulosa cell proliferation, ovarian weight and angiogenesis. Moreover, the tumorigenesis, acute toxicity, immunogenicity and biodistribution of MSCs were evaluated, and both AD-MSCs and UC-MSCs were found to possess high safety profiles. Through RNA sequencing analysis, enhancement of the MAPK cascade was observed, and long-term effects were mainly linked to the activation of immune function. CONCLUSIONS: Orthotopic transplantation of MSCs displays significant efficacy and high safety for the treatment of ovarian ageing in mice.

Comparison of treatment performance and microbial community evolution of typical dye wastewater by different combined processes.

The degradation of typical dye wastewater is a focus of research in the printing and dyeing industry. In this study, a combined micro-electrolysis and microbial treatment method was established to treat refractory dye wastewater, and the pivotal factors in the microbial treatment were optimized. In the series and coupled modes, the removal rates of chroma reached 98.75% and 92.50%, and the removal rates of chemical oxygen demand (COD) reached 96.17% and 82.29%, respectively. The high-throughput sequencing results showed that the microbial communities in the microbial system varied at different treatment stages. From the culture stage to the domestication stage, the dominant phylum was Proteobacteria; however, the community abundance of microorganisms decreased. A combination of micro-electrolysis and biological methods can alter the characteristics of the microbial community, increase the number of dominant phyla, and increase the abundance of microorganisms. The degradation effect of the series mode and the overall strengthening effect of micro-electrolysis on the microorganisms were better than those of the coupled mode. In actual wastewater, the maximum removal rates of chroma, COD, total nitrogen (TN), ammonia nitrogen (NH3-N), and total phosphorus (TP) are 97.50%, 98.90%, 94.35%, 93.95%, and 91.17%, respectively. Three-dimensional fluorescence spectrum analysis showed that microbial processes could significantly degrade fluorescent components in wastewater, and methanogenic active enzymes in anaerobic processes could continue to react. The combined process can realize the efficient treatment of toxic dye wastewater by reducing the toxicity of wastewater and efficiently degrading organic matter, which has important guiding significance for the treatment of refractory dye wastewater.

TransVFS: A spatio-temporal local-global transformer for vision-based force sensing during ultrasound-guided prostate biopsy.

Robot-assisted prostate biopsy is a new technology to diagnose prostate cancer, but its safety is influenced by the inability of robots to sense the tool-tissue interaction force accurately during biopsy. Recently, vision based force sensing (VFS) provides a potential solution to this issue by utilizing image sequences to infer the interaction force. However, the existing mainstream VFS methods cannot realize the accurate force sensing due to the adoption of convolutional or recurrent neural network to learn deformation from the optical images and some of these methods are not efficient especially when the recurrent convolutional operations are involved. This paper has presented a Transformer based VFS (TransVFS) method by leveraging ultrasound volume sequences acquired during prostate biopsy. The TransVFS method uses a spatio-temporal local-global Transformer to capture the local image details and the global dependency simultaneously to learn prostate deformations for force estimation. Distinctively, our method explores both the spatial and temporal attention mechanisms for image feature learning, thereby addressing the influence of the low ultrasound image resolution and the unclear prostate boundary on the accurate force estimation. Meanwhile, the two efficient local-global attention modules are introduced to reduce 4D spatio-temporal computation burden by utilizing the factorized spatio-temporal processing strategy, thereby facilitating the fast force estimation. Experiments on prostate phantom and beagle dogs show that our method significantly outperforms existing VFS methods and other spatio-temporal Transformer models. The TransVFS method surpasses the most competitive compared method ResNet3dGRU by providing the mean absolute errors of force estimation, i.e., 70.4 ± 60.0 millinewton (mN) vs 123.7 ± 95.6 mN, on the transabdominal ultrasound dataset of dogs.

Genome assembly of autotetraploid Actinidia arguta highlights adaptive evolution and enables dissection of important economic traits.

Actinidia arguta, the most widely distributed Actinidia species and the second cultivated species in the genus, can be distinguished from the currently cultivated Actinidia chinensis on the basis of its small and smooth fruit, rapid softening, and excellent cold tolerance. Adaptive evolution of tetraploid Actinidia species and the genetic basis of their important agronomic traits are still unclear. Here, we generated a chromosome-scale genome assembly of an autotetraploid male A. arguta accession. The genome assembly was 2.77 Gb in length with a contig N50 of 9.97 Mb and was anchored onto 116 pseudo-chromosomes. Resequencing and clustering of 101 geographically representative accessions showed that they could be divided into two geographic groups, Southern and Northern, which first diverged 12.9 million years ago. A. arguta underwent two prominent expansions and one demographic bottleneck from the mid-Pleistocene climate transition to the late Pleistocene. Population genomics studies using paleoclimate data enabled us to discern the evolution of the species' adaptation to different historical environments. Three genes (AaCEL1, AaPME1, and AaDOF1) related to flesh softening were identified by multi-omics analysis, and their ability to accelerate flesh softening was verified through transient expression assays. A set of genes that characteristically regulate sexual dimorphism located on the sex chromosome (Chr3) or autosomal chromosomes showed biased expression during stamen or carpel development. This chromosome-level assembly of the autotetraploid A. arguta genome and the genes related to important agronomic traits will facilitate future functional genomics research and improvement of A. arguta.

Discovery of RORγ Allosteric Fluorescent Probes and Their Application: Fluorescence Polarization, Screening, and Bioimaging.

Retinoic acid receptor-related orphan receptor γ (RORγ) acts as a crucial transcription factor in Th17 cells and is involved in diverse autoimmune disorders. RORγ allosteric inhibitors have gained significant research focus as a novel strategy to inhibit RORγ transcriptional activity. Leveraging the high affinity and selectivity of RORγ allosteric inhibitor MRL-871 (1), this study presents the design, synthesis, and characterization of 11 allosteric fluorescent probes. Utilizing the preferred probe 12h, we established an efficient and cost-effective fluorescence polarization-based affinity assay for screening RORγ allosteric binders. By employing virtual screening in conjunction with this assay, 10 novel RORγ allosteric inhibitors were identified. The initial SAR studies focusing on the hit compound G381-0087 are also presented. The encouraging outcomes indicate that probe 12h possesses the potential to function as a powerful tool in facilitating the exploration of RORγ allosteric inhibitors and furthering understanding of RORγ function.

Dissecting the chiral recognition of TLR4/MD2 with Neoseptin-3 enantiomers by molecular dynamics simulations.

Toll-like receptor 4 (TLR4) is a pivotal innate immune recognition receptor that regulates intricate signaling pathways within the immune system. Neoseptin-3 (Neo-3), a recently identified small-molecule agonist for mouse TLR4/MD2, exhibits chiral recognition properties. Specifically, the L-enantiomer of Neo-3 (L-Neo-3) effectively activates the TLR4 signaling pathway, while D-Neo-3 fails to induce TLR4 activation. However, the underlying mechanism by which TLR4 enantioselectively recognizes Neo-3 enantiomers remains poorly understood. In this study, in silico simulations were performed to investigate the mechanism of chiral recognition of Neo-3 enantiomers by TLR4/MD2. Two L-Neo-3 molecules stably resided within the cavity of MD2 as a dimer, and the L-Neo-3 binding stabilized the (TLR4/MD2)2 dimerization state. However, the strong electrostatic repulsion between the hydrogen atoms on the chiral carbon of D-Neo-3 molecules caused the relative positions of two D-Neo-3 molecules to continuously shift during the simulation process, thus preventing the formation of D-Neo-3 dimer as well as their stable interactions with the surrounding residues in (TLR4/MD2)2. Considering that L-Neo-3 could not sustain a stable dimeric state in the bulk aqueous environment, it is unlikely that L-Neo-3 entered the cavity of MD2 as a dimeric unit. Umbrella sampling simulations revealed that the second L-Neo-3 molecule entering the cavity of MD2 exhibited a lower binding energy (-25.75 kcal mol-1) than that of the first L-Neo-3 molecule (-14.31 kcal mol-1). These results imply that two L-Neo-3 molecules enter the cavity of MD2 sequentially, with the binding of the first L-Neo-3 molecule facilitating the entry of the second one. This study dissects the binding process of Neo-3 enantiomers, offering a comprehensive understanding of the atomic-level mechanism underlying TLR4's chiral recognition of Neo-3 molecules.