From Proline to Chlorantraniliprole Mimics: Computer-Aided Design, Simple Preparation, and Excellent Insecticidal Profiles.

Chlorantraniliprole (CHL), a favored agricultural insecticide, is renowned for its high efficiency and broad-spectrum effectiveness against lepidoptera insects. However, the urgency for new insecticide development is underscored by the intricate multistep preparation process and modest overall yields of CHL, along with the escalating challenge of insect resistance. In response, we have crafted CHL mimics from proline employing computer-aided drug design. Molecular docking analysis of CHL's interactions with the ryanodine receptor (RyR) revealed that the nitrogen atom within the pyrazole moiety does not engage in pivotal interactions. Its removal may not abolish bioactivity entirely but could substantially simplify the synthetic process, thereby enhancing atom economy. This revelation prompted the exclusion of nitrogen and the subsequent formation of a pyrrole ring, enabling the meticulous design of synthetic pathways characterized by cost-effective precursors, streamlined synthesis, the avoidance of toxic reagents, minimal instrumentation, and high yields in the pursuit of innovative RyR modulators. Among these modulators, A1 and B1, obtained with yields exceeding 60%, showcased exceptional insecticidal potency, with LC50 values spanning from 0.12 to 1.47 mg L-1 against P. xylostella and M. separate. The inhibitory effects of these two compounds on insect detoxification enzymes imply a reduced likelihood of eliciting resistance in comparison to CHL, a finding further corroborated by their insecticidal potency against resistant pests. Moreover, molecular docking, MD simulations, and DFT calculations provided valuable structural insights, potentially unraveling the superior insecticidal activity of these two molecules, and thus paving the way for developing more potent insecticides.

Structure and function of Mycobacterium tuberculosis EfpA as a lipid transporter and its inhibition by BRD-8000.3.

EfpA, the first major facilitator superfamily (MFS) protein identified in Mycobacterium tuberculosis (Mtb), is an essential efflux pump implicated in resistance to multiple drugs. EfpA-inhibitors have been developed to kill drug-tolerant Mtb. However, the biological function of EfpA has not yet been elucidated. Here, we present the cryo-EM structures of EfpA complexed with lipids or the inhibitor BRD-8000.3 at resolutions of 2.9 Å and 3.4 Å, respectively. Unexpectedly, EfpA forms an antiparallel dimer. Functional studies reveal that EfpA is a lipid transporter and BRD-8000.3 inhibits its lipid transport activity. Intriguingly, the mutation V319F, known to confer resistance to BRD-8000.3, alters the expression level and oligomeric state of EfpA. Based on our results and the observation of other antiparallel dimers in the MFS family, we propose an antiparallel-function model of EfpA. Collectively, our work provides structural and functional insights into EfpA's role in lipid transport and drug resistance, which would accelerate the development of antibiotics against this promising drug target.

Case report: Asymmetric bilateral deep brain stimulation for the treatment of pantothenate kinase-associated neurodegeneration in a patient: a unique case of atypical PKAN with a novel heterozygous PANK2 mutation.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare autosomal recessive hereditary neurodegenerative disorder, usually caused by mutations in the pantothenate kinase 2 (PANK2) gene. We report a young female patient with atypical PKAN, harboring a novel heterozygous PANK2 mutation, diagnosed through clinical imaging and genetic analysis. The patient presented with dystonia and motor dysfunction after onset, but early brain MRI showed normal findings. Due to progressive symptom deterioration, her MRI was reevaluated and the characteristic "eye of the tiger" sign was identified. Further genetic testing revealed that she was a carrier of two heterozygous PANK2 mutations, one being a known pathogenic variant and the other unknown. Given the patient's clinical presentation, progressive symptoms, and poor response to medication, we boldly attempted asymmetric bilateral deep brain stimulation (abDBS). Postoperative outcomes showed significant symptom improvement. This study suggests that early brain MRI in PKAN patients may not exhibit typical radiological features, leading to potential diagnostic omissions. Furthermore, it highlights the potential therapeutic effect of abDBS in atypical PKAN, particularly in patients with novel heterozygous PANK2 mutations. Asymmetric bilateral deep brain stimulation may represent a promising treatment approach.

iP3T: an interpretable multimodal time-series model for enhanced gait phase prediction in wearable exoskeletons.

Introduction: Wearable exoskeletons assist individuals with mobility impairments, enhancing their gait and quality of life. This study presents the iP3T model, designed to optimize gait phase prediction through the fusion of multimodal time-series data. Methods: The iP3T model integrates data from stretch sensors, inertial measurement units (IMUs), and surface electromyography (sEMG) to capture comprehensive biomechanical and neuromuscular signals. The model's architecture leverages transformer-based attention mechanisms to prioritize crucial data points. A series of experiments were conducted on a treadmill with five participants to validate the model's performance. Results: The iP3T model consistently outperformed traditional single-modality approaches. In the post-stance phase, the model achieved an RMSE of 1.073 and an R2 of 0.985. The integration of multimodal data enhanced prediction accuracy and reduced metabolic cost during assisted treadmill walking. Discussion: The study highlights the critical role of each sensor type in providing a holistic understanding of the gait cycle. The attention mechanisms within the iP3T model contribute to its interpretability, allowing for effective optimization of sensor configurations and ultimately improving mobility and quality of life for individuals with gait impairments.

ß-Cyclodextrin-optimized supramolecular nanovesicles enhance the droplet/foliage interface interactions and inhibition of succinate dehydrogenase (SDH) for efficient treatment of fungal diseases.

BACKGROUND: Plant fungal diseases present a major challenge to global agricultural production. Despite extensive efforts to develop fungicides, particularly succinate dehydrogenase inhibitors (SDHIs), their effectiveness is often limited by poor retention of fungicide droplets on hydrophobic leaves. The off-target losses and unintended release cause fungal resistance and severe environmental pollution. RESULTS: To update the structure of existing SDHIs and synchronously realize the efficient utilization, we have employed a sophisticated supramolecular strategy to optimize a structurally novel SDH inhibitor (AoH25), creating an innovative supramolecular SDH fungicide (AoH25@ß-CD), driven by the host-guest recognition principle between AoH25 and ß-cyclodextrin (ß-CD). Intriguingly, AoH25@ß-CD self-assembles into biocompatible supramolecular nanovesicles, which reinforce the droplet/foliage (liquid-solid) interface interaction and the effective wetting and retention on leaf surfaces, setting the foundation for enhancing fungicide utilization. Mechanistic studies revealed that AoH25@ß-CD exhibited significantly higher inhibition of SDH (IC50 = 1.56 µM) compared to fluopyram (IC50 = 244.41 µM) and AoH25 alone (IC50 = 2.29 µM). Additionally, AoH25@ß-CD increased the permeability of cell membranes in Botryosphaeria dothidea, facilitating better penetration of active ingredients into pathogenic cells. Further experimental outcomes confirmed that AoH25@ß-CD was 88.5% effective against kiwifruit soft rot at a low-dose of 100 µg mL-1, outperforming commercial fungicides such as fluopyram (52.4%) and azoxystrobin (65.4%). Moreover, AoH25@ß-CD showed broad-spectrum bioactivity against oilseed rape sclerotinia, achieving an efficacy of 87.2%, outstripping those of fluopyram (48.7%) and azoxystrobin (76.7%). CONCLUSION: This innovative approach addresses key challenges related to fungicide deposition and resistance, improving the bioavailability of agricultural chemicals. The findings highlight AoH25@ß-CD as a novel supramolecular SDH inhibitor, demonstrating its potential as an efficient and sustainable solution for plant disease management.

Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris.

Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines.

The MADS-domain transcription factor DAL10 is a direct target of putative DAL1-mediated age pathway in conifers.

The optimal timing of the transition from vegetative growth to reproductive growth is critical for plant reproductive success, and the underlying regulatory mechanisms have been well studied in angiosperm model species, but relatively little in gymnosperms. DAL1, a MADS domain transcription factor (TF) that shows a conserved age-related expression profile in conifers, may be an age timer. However, how DAL1 mediates the onset of reproductive growth remains poorly understood. Here, we showed that PtDAL1 directly regulates PtDAL10 transcription by binding to its promoter region in vitro. Both in vitro and in Nicotiana benthamiana PtDAL1 forms ternary complexes with PtDAL10 and PtMADS11, two potential candidate regulators of the vegetative to reproductive transition in Chinese pine (Pinus tabuliformis). In new shoots PtDAL10 was progressively induced with age and was also expressed in male and female cones. Overexpression of PtDAL10 rescued the flowering of ft-10 and soc1-1-2 mutants in Arabidopsis. We provide insights into the molecular components associated with PtDAL1, which integrates the vegetative to reproductive phase transition into age-mediated progressive development of the whole plant in conifers.

Prognostic nomogram based on the gamma-glutamyl transpeptidase-to-platelet ratio for patients with compensated cirrhotic hepatocellular carcinoma after local ablation.

Background: Hepatocellular carcinoma (HCC) patients with compensated cirrhosis typically face a high prevalence and unfavorable prognosis. However, there is currently a deficiency in prediction models to anticipate the prognosis of these patients. Therefore, our study included the Gamma-glutamyl transpeptidase-to-platelet ratio (GPR) in analysis and aimed to develop a nomogram for HCC patients with compensated cirrhosis after local ablation. Methods: Enrolling 669 patients who underwent local ablation at Beijing You'an Hospital during the period from January 1, 2014, to December 31, 2022, this study focused on individuals with compensated cirrhotic HCC. In a ratio of 7:3, patients were allocated to the training cohort (n=468) and the validation cohort (n=201). Lasso-Cox regression was employed to identify independent prognostic factors for overall survival (OS). Subsequently, a nomogram was constructed using these factors and was validated through receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA). Results: GPR, age, and hemoglobin were identified by Lasso-Cox regression as independent prognostic factors of the nomogram. The area under the ROC curves (AUCs) for 3-, 5-, and 8-year OS (0.701, 0.755, and 0.768 for the training cohort; 0.684, 0.707, and 0.778 for the validation cohort), and C-indices (0.695 for training cohort; 0.679 for validation cohort) exhibited the excellent predictive ability of the nomogram. Calibration curves and DCA curves indicated favorable calibration performance and clinical utility. Patients were further stratified into two risk groups according to the median nomogram score. There existed an obvious distinction between the two groups both in the training cohort and validation cohort. Conclusion: In summary, this research established and validated a novel nomogram to predict OS, which had good predictive power for HCC patients with compensated cirrhosis after local ablation.

Ultrastructural insights into cellular organization, energy storage and ribosomal dynamics of an ammonia-oxidizing archaeon from oligotrophic oceans.

Introduction: Nitrososphaeria, formerly known as Thaumarchaeota, constitute a diverse and widespread group of ammonia-oxidizing archaea (AOA) inhabiting ubiquitously in marine and terrestrial environments, playing a pivotal role in global nitrogen cycling. Despite their importance in Earth's ecosystems, the cellular organization of AOA remains largely unexplored, leading to a significant unanswered question of how the machinery of these organisms underpins metabolic functions. Methods: In this study, we combined spherical-chromatic-aberration-corrected cryo-electron tomography (cryo-ET), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS) to unveil the cellular organization and elemental composition of Nitrosopumilus maritimus SCM1, a representative member of marine Nitrososphaeria. Results and Discussion: Our tomograms show the native ultrastructural morphology of SCM1 and one to several dense storage granules in the cytoplasm. STEM-EDS analysis identifies two types of storage granules: one type is possibly composed of polyphosphate and the other polyhydroxyalkanoate. With precise measurements using cryo-ET, we observed low quantity and density of ribosomes in SCM1 cells, which are in alignment with the documented slow growth of AOA in laboratory cultures. Collectively, these findings provide visual evidence supporting the resilience of AOA in the vast oligotrophic marine environment.

Baicalin Weakens the Virulence of Porcine Extraintestinal Pathogenic Escherichia coli by Inhibiting the LuxS/AI-2 Quorum-Sensing System.

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogenic bacterium that causes huge economic losses to the pig farming industry and considerably threatens human health. The quorum sensing (QS) system plays a crucial role in the survival and pathogenesis of pathogenic bacteria. Hence, it is a viable approach to prevent ExPEC infection by compromising the QS system, particularly the LuxS/AI-2 system. In this study, we investigated the effects of baicalin on the LuxS/AI-2 system of ExPEC. Baicalin at concentrations of 25, 50, and 100 µg/mL significantly diminished the survival ability of ExPEC in hostile environments and could inhibit the biofilm formation and autoagglutination ability in ExPEC. Moreover, baicalin dose-dependently decreased the production of AI-2 and down-regulated the expression level of luxS in PCN033. These results suggest that baicalin can weaken the virulence of PCN033 by inhibiting the LuxS/AI-2 system. After the gene luxS was deleted, AI-2 production in PCN033 was almost completely eliminated, similar to the effect of baicalin on the production of AI-2 in PCN033. This indicates that baicalin reduced the production of AI-2 by inhibiting the expression level of luxS in ExPEC. In addition, the animal experiment further showed the potential of baicalin as a LuxS/AI-2 system inhibitor to prevent ExPEC infection. This study highlights the potential of baicalin as a natural quorum-sensing inhibitor for therapeutic applications in preventing ExPEC infection by targeting the LuxS/AI-2 system.

Appearance comparison on social networking sites and body shame: The role of negative body talk and perceived sociocultural influences on body image.

Social media platforms play a significant role in the lives of young people. While the usage of these platforms has grown, research exploring the challenges of body image remains limited. This study investigated whether initiating negative body talk functioned as an indirect pathway between appearance comparison on social media and body shame and whether perceived sociocultural influences from parents, friends, and media on body image moderated this indirect effect. An online cross-sectional survey of 795 Chinese college students (Mage = 20.17, SD = 1.65; 60% female, 40% male) was conducted. Negative body talk was a partial indirect pathway in the association, and this indirect effect was significant among those experiencing higher sociocultural pressures from all three sources. This study highlights the need for health psychology in understanding and addressing the mental health consequences associated with digital media and sociocultural influences on body image perception.

Learning-Based Control for Soft Robot-Environment Interaction with Force/Position Tracking Capability.

Soft robotics promises to achieve safe and efficient interactions with the environment by exploiting its inherent compliance and designing control strategies. However, effective control for the soft robot-environment interaction has been a challenging task. The challenges arise from the nonlinearity and complexity of soft robot dynamics, especially in situations where the environment is unknown and uncertainties exist, making it difficult to establish analytical models. In this study, we propose a learning-based optimal control approach as an attempt to address these challenges, which is an optimized combination of a feedforward controller based on probabilistic model predictive control and a feedback controller based on nonparametric learning methods. The approach is purely data-driven, without prior knowledge of soft robot dynamics and environment structures, and can be easily updated online to adapt to unknown environments. A theoretical analysis of the approach is provided to ensure its stability and convergence. The proposed approach enabled a soft robotic manipulator to track target positions and forces when interacting with a manikin in different cases. Moreover, comparisons with other data-driven control methods show a better performance of our approach. Overall, this work provides a viable learning-based control approach for soft robot-environment interactions with force/position tracking capability.

Structure of a unique PSII-Pcb tetrameric megacomplex in a chlorophyll d-containing cyanobacterium.

Acaryochloris marina is a unique cyanobacterium using chlorophyll d (Chl d) as its major pigment and thus can use far-red light for photosynthesis. Photosystem II (PSII) of A. marina associates with a number of prochlorophyte Chl-binding (Pcb) proteins to act as the light-harvesting system. We report here the cryo-electron microscopic structure of a PSII-Pcb megacomplex from A. marina at a 3.6-angstrom overall resolution and a 3.3-angstrom local resolution. The megacomplex is organized as a tetramer consisting of two PSII core dimers flanked by sixteen symmetrically related Pcb proteins, with a total molecular weight of 1.9 megadaltons. The structure reveals the detailed organization of PSII core consisting of 15 known protein subunits and an unknown subunit, the assembly of 4 Pcb antennas within each PSII monomer, and possible pathways of energy transfer within the megacomplex, providing deep insights into energy transfer and dissipation mechanisms within the PSII-Pcb megacomplex involved in far-red light utilization.

Transcriptome and metabolome profiling to elucidate the mechanism underlying the poor growth of Streptococcus suis serotype 2 after orphan response regulator CovR deletion.

The deletion of orphan response regulator CovR reduces the growth rate of Streptococcus suis serotype 2 (S. suis 2). In this study, metabolome and transcriptome profiling were performed to study the mechanisms underlying the poor growth of S. suis 2 caused by the deletion of orphan response regulator CovR. By comparing S. suis 2 (ΔcovR) and S. suis 2 (SC19), 146 differentially accumulated metabolites (upregulated: 83 and downregulated: 63) and 141 differentially expressed genes (upregulated: 86 and downregulated: 55) were identified. Metabolome and functional annotation analysis revealed that the growth of ΔcovR was inhibited by the imbalance aminoacyl tRNA biosynthesis (the low contents of L-lysine, L-aspartic acid, L-glutamine, and L-glutamic acid, and the high content of L-methionine). These results provide a new insight into the underlying poor growth of S. suis 2 caused by the deletion of orphan response regulator CovR. Metabolites and candidate genes regulated by the orphan response regulator CovR and involved in the growth of S. suis 2 were reported in this study.

Phenolics from Dendrobium officinale Leaf Ameliorate Dextran Sulfate Sodium-Induced Chronic Colitis by Regulating Gut Microbiota and Intestinal Barrier.

The increasing incidence of colitis and the side effects of its therapeutic drugs have led to the search for compounds of natural origin, including phenolics, as new treatments for colitis. In this study, the potential mechanism of Dendrobium officinale leaf phenolics (DOP) on the relief of dextran sulfate sodium (DSS)-induced colitis was explored. The results showed that DOP treatment for 36 days reduced the symptoms of colitis caused by DSS, including reduction of the disease activity index and alleviation of colonic tissue damage. In addition, DOP downregulated the expression of key proteins of the TLR4/NF-κB signaling pathway and reduced the production of inflammatory cytokines. Furthermore, DOP could enhance the expression of tight junction proteins including ZO-1, Occludin, and Claudin-1 to restore intestinal mucosal barrier function. DOP also effectively regulates disordered intestinal flora and enhances the production of short-chain fatty acids, which is also beneficial in modulating gut internal environmental homeostasis, inhibiting inflammation, and restoring the intestinal barrier. These findings indicated that DOP can ameliorate DSS-induced chronic colitis by regulating gut microbiota, intestinal barrier, and inflammation, and it is a promising ingredient from D. officinale.

CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers.

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimized a bacterial CRISPR-hybrid system coupled with FACS, and identified novel high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allowed effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

Broad protective RBD heterotrimer vaccines neutralize SARS-CoV-2 including Omicron sub-variants XBB/BQ.1.1/BF.7.

SARS-CoV-2 variants with severe immune evasion are a major challenge for COVID-19 prevention, especially the circulating Omicron XBB/BQ.1.1/BF.7 strains. Thus, the next-generation of broad-spectrum vaccines are urgently needed. Previously, we developed a COVID-19 protein subunit vaccine, ZF2001, based on the RBD-homodimer as the immunogen. To adapt SARS-CoV-2 variants, we developed chimeric RBD-heterodimers to induce broad immune responses. In this study, we further explored the concept of tandem RBD homotrimer and heterotrimer. Prototype SARS-CoV-2 RBD-homotrimer, prototype-Delta-BA.1 (PDO) RBD-heterotrimer and Delta-BA.2-BA.5 (DBA2BA5) RBD-heterotrimer were designed. Biochemical and cryo-EM structural characterization demonstrated total epitope exposure of the RBD-trimers. In mouse experiments, PDO and DBA2BA5 elicited broad SARS-CoV-2 neutralization. Potent protection against SARS-CoV-2 variants was observed in challenge assays and was correlated with neutralizing antibody titer. This study validated the design strategy of tandem RBD-heterotrimers as multivalent immunogens and presented a promising vaccine candidate, DBA2BA5, eliciting broad-spectrum immune responses, including against the circulating XBB/BF.7/BQ.1.1.

An ethylene-induced NAC transcription factor acts as a multiple abiotic stress responsor in conifer.

The proper response to various abiotic stresses is essential for plants' survival to overcome their sessile nature, especially for perennial trees with very long-life cycles. However, in conifers, the molecular mechanisms that coordinate multiple abiotic stress responses remain elusive. Here, the transcriptome response to various abiotic stresses like salt, cold, drought, heat shock and osmotic were systematically detected in Pinus tabuliformis (P. tabuliformis) seedlings. We found that four transcription factors were commonly induced by all tested stress treatments, while PtNAC3 and PtZFP30 were highly up-regulated and co-expressed. Unexpectedly, the exogenous hormone treatment assays and the content of the endogenous hormone indicates that the upregulation of PtNAC3 and PtZFP30 are mediated by ethylene. Time-course assay showed that the treatment by ethylene immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), activated the expression of PtNAC3 and PtZFP30 within 8 hours. We further confirm that the PtNAC3 can directly bind to the PtZFP30 promoter region and form a cascade. Overexpression of PtNAC3 enhanced unified abiotic stress tolerance without growth penalty in transgenic Arabidopsis and promoted reproductive success under abiotic stress by shortening the lifespan, suggesting it has great potential as a biological tool applied to plant breeding for abiotic stress tolerance. This study provides novel insights into the hub nodes of the abiotic stresses response network as well as the environmental adaptation mechanism in conifers, and provides a potential biofortification tool to enhance plant unified abiotic stress tolerance.

Baicalin Weakens the Porcine ExPEC-Induced Inflammatory Response in 3D4/21 Cells by Inhibiting the Expression of NF-κB/MAPK Signaling Pathways and Reducing NLRP3 Inflammasome Activation.

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of death in pigs and has led to considerable economic losses for the pig industry. Porcine ExPEC infections often cause systemic inflammatory responses in pigs, characterized by meningitis, arthritis, pneumonia, and septicemia. Baicalin has been reported to possess potent anti-inflammatory activity, but its function in porcine ExPEC remains unknown. The aim of this study was to explore the protective effect and mechanism of baicalin against the porcine ExPEC-induced inflammatory responses in 3D4/21 cells. After treatment with baicalin, the effects on cell damage, the level of pro-inflammatory cytokines, the expression of nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) signaling pathways, and the activation of NOD-like receptor protein 3 (NLRP3) inflammasomes were examined. Our results show that baicalin significantly reduced the damage to 3D4/21 cells infected with porcine ExPEC PCN033. Further study showed that baicalin significantly reduced the transcription and expression of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and interleukin-8 (IL-8). Furthermore, baicalin inhibited the phosphorylation of proteins such as P65, nuclear factor κB inhibitor α (IκBα), extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and P38 and reduced the expression levels of proteins such as NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. These results reveal that baicalin reduced the damage to 3D4/21 cells by inhibiting the expression of NF-κB/MAPK signaling pathways and blocking NLRP3 inflammasome activation in 3D4/21 cells infected with porcine ExPEC. Taken together, these results suggest that baicalin may have potential as a medicine for the treatment of porcine ExPEC-infected pigs by regulating inflammatory responses. This study provides a novel potential pharmaco-therapeutic approach to preventing porcine ExPEC infection.

Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes.

SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed, and open. S-trimers adopting locked conformations are tightly packed featuring structural elements incompatible with RBD in the "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably because of their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures with other available spike structures of SARS-related CoVs to identify conserved features and discuss their possible functions.