ABSTRACT
BACKGROUND: Adductor canal block and periarticular infiltration analgesia (PIA) have been shown to relieve pain in total knee arthroplasty (TKA) effectively. However, their analgesic effectiveness has some limitations. Thus, we considered a novel blocking site that could achieve analgesia without affecting the muscle strength of the lower limbs. METHODS: Seventy-two patients undergoing primary unilateral total knee arthroplasty were randomized into two groups. One group was treated with adductor canal and popliteal plexus (APB) combined with interspace between the popliteal artery and posterior capsule of the knee (iPACK) and local infiltration anesthesia (LIA) and the other was treated with PIA. The primary outcomes included postoperative pain, as assessed by the visual analog scale (VAS), and the consumption of oral tramadol. Secondary outcomes included functional recovery and daily ambulation distance. Tertiary outcomes included postoperative adverse effects. RESULTS: The APB group had lower VAS scores after surgery at rest and during motion. Compared with the PIA group, the walking distance of the APB group on the second day was greater. The muscle strength of the APB group was lower than that of the PIA group at the early stage. Patients in the APB group also consumed less tramadol than those in the PIA group. There was no difference in the incidence of adverse events between the two groups. CONCLUSIONS: APB combined with iPACK and LIA is a novel block for TKA, and it can reduce postoperative pain sooner after TKA without affecting postoperative functional recovery or increasing complications.
ABSTRACT
Protein-protein interactions (PPIs) stabilization with molecular glues plays a crucial role in drug discovery, albeit with significant challenges. In this study, we propose a dual-site approach, targeting the PPI region and its dynamic surroundings. We conduct molecular dynamics simulations to identify critical sites on the PPI that stabilize the cyclin-dependent kinase 12 - DNA damage-binding protein 1 (CDK12-DDB1) complex, resulting in further cyclin K degradation. This exploration leads to the creation of LL-K12-18, a dual-site molecular glue, which enhances the glue properties to augment degradation kinetics and efficiency. Notably, LL-K12-18 demonstrates strong inhibition of gene transcription and anti-proliferative effects in tumor cells, showing significant potency improvements in MDA-MB-231 (88-fold) and MDA-MB-468 cells (307-fold) when compared to its precursor compound SR-4835. These findings underscore the potential of dual-site approaches in disrupting CDK12 function and offer a structural insight-based framework for the design of cyclin K molecular glues.
Subject(s)
Cyclin-Dependent Kinases , Protein Binding , Humans , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinases/metabolism , Cyclins , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , Molecular Dynamics SimulationABSTRACT
Chemoresistance is a main cause of chemotherapy failure and tumor recurrence. The effects of global protein SUMOylation on chemoresistance in colorectal cancer (CRC) remains to be investigated. Herein, we have proposed that the elevated SUMO2/3-modified proteins confer 5-fluorouracil (5-FU) chemoresistance acquisition in CRC. The SUMOylation levels of global proteins in CRC cell lines were elevated compared with normal colon cell line NCM460. 5-FU treatment obviously reduced SUMOylation of global proteins in 5-FU-sensitive CRC cells including HT29, HCT116 and HCT-8. However, in 5-FU-resistant HCT-8/5-FU cells, the expression level of SUMO2/3-modified proteins was increased under 5-FU exposure in a concentration-dependent manner. 5-FU treatment combined with SUMOylation inhibitor ML-792 significantly increased the sensitivity of 5-FU-resistant cells to 5-FU and reduced colony formation numbers in HCT-8/5-FU cells. And UBC9-mediated SUMOylation elevation contributes to 5-FU resistance in HCT116 cells. Moreover, we also identified RREB1 as a regulator of SUMOylation profiling of global cellular proteins via directly binding to the promoter of UBC9. Overexpression of RREB1 promoted 5-FU resistance in CRC, which was partially abolished by treatment of inhibitor ML-792. In conclusion, RREB1-enhanced protein SUMOylation contributes to 5-FU resistance acquisition in CRC.
ABSTRACT
In this work, we design a new approach for digital counting-based protein kinase activity assay by using plasmonic nanoparticle-assisted single-molecule dynamic binding. This method breaks the concentration-dependent limitation in single-molecule detection and displays good selectivity and sensitivity with a detection limit as low as 0.0001 U mL-1 for the protein kinase A (PKA) assay, which would find broad applications in drug screening and medical investigations.
Subject(s)
Metal Nanoparticles , Metal Nanoparticles/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/chemistry , Gold/chemistry , Limit of DetectionABSTRACT
Stomata in leaves regulate gas (carbon dioxide and water vapor) exchange and water transpiration between plants and the atmosphere. SLow Anion Channel 1 (SLAC1) mediates anion efflux from guard cells and plays a crucial role in controlling stomatal aperture. It serves as a central hub for multiple signaling pathways in response to environmental stimuli, with its activity regulated through phosphorylation via various plant protein kinases. However, the molecular mechanism underlying SLAC1 phosphoactivation has remained elusive. Through a combination of protein sequence analyses, AlphaFold-based modeling and electrophysiological studies, we unveiled that the highly conserved motifs on the N- and C-terminal segments of SLAC1 form a cytosolic regulatory domain (CRD) that interacts with the transmembrane domain(TMD), thereby maintaining the channel in an autoinhibited state. Mutations in these conserved motifs destabilize the CRD, releasing autoinhibition in SLAC1 and enabling its transition into an activated state. Our further studies demonstrated that SLAC1 activation undergoes an autoinhibition-release process and subsequent structural changes in the pore helices. These findings provide mechanistic insights into the activation mechanism of SLAC1 and shed light on understanding how SLAC1 controls stomatal closure in response to environmental stimuli.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Plant Stomata , Signal Transduction , Phosphorylation , Plant Stomata/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Arabidopsis/metabolism , Arabidopsis/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , Protein Domains , MutationABSTRACT
BACKGROUND: The range of Glires is influenced by human activities and climate change. However, the extent to which human activities and environmental changes have contributed to this relationship remains unclear. We examined alterations in the distribution changes and driving factors of the Himalayan marmot, plateau pika, and plateau zokor on the Qinghai-Tibet Plateau (QTP) using the maximum entropy (MaxEnt) model and a geographical detector (Geodetector). RESULTS: The MaxEnt model showed that the contribution rates of the human footprint index (HFI) to the distribution patterns of the three types of Glires were 46.70%, 58.70%, and 59.50%, respectively. The Geodetector results showed that the distribution pattern of the Himalayan marmot on the QTP was influenced by altitude and the normalized difference vegetation index (NDVI). The distribution patterns for plateau pikas and plateau zokors were driven by HFI and NDVI. Climate has played a substantial role in shaping suitable habitats for these three Glires on the QTP. Their suitable area is expected to decrease over the next 30-50 years, along with their niche breadth and overlap. Future suitable habitats for the three Glires tended to shift toward higher latitudes on the QTP. CONCLUSION: These findings underscore the impacts of environmental and human factors on the distribution of the three Glires on the QTP. They have enhanced our understanding of the intricate relationships between Glires niches and environments. This can aid in identifying necessary interventions for developing effective early warning systems and prevention strategies to mitigate Glires infestations and plague epidemics on the QTP. © 2024 Society of Chemical Industry.
ABSTRACT
The extraordinary adaptability and dispersal abilities have allowed Hyphantria cunea to expand its range, posing a great threat to urban landscapes and natural ecosystems. Searching for safe, efficient, and low-cost control methods may provide new strategies for pest management in H. cunea spread areas. In this study, based on the attraction of insects by preferred hosts, it was found that the response rates of virgin H. cunea female adults to Salix matsudana, Juglans mandshurica and Ulmus pumila were 89.17%, 97.92% and 93.98%, respectively. It was further found that this significant preference was mainly related to the volatiles m-xylene, o-xylene, dodecane and tetradecane found in the three species. Even though all four compounds at 10 µL/mL and 100 µL/mL had significant attractive effects on the virgin H. cunea female adults, m-xylene and dodecane at 100 µL/mL elicited significant EAG responses and tending behaviors by stimulating the olfactory receptor neurons (ORN A) of females, with response rates of 83.13% and 84.17%, while also having significant attractive effects on virgin male adults with rates of 65.74% and 67.51%. Therefore, both m-xylene and dodecane which at concentrations of 100 µL/mL had strong attractions to adults, could be used as the first choice of attractants for both sexes of H. cunea. This has important practical significance in reducing the frequency of H. cunea generations, limiting their population, controlling their spread range, and improving the efficiency of pest management in epidemic areas.
Subject(s)
Volatile Organic Compounds , Animals , Female , Male , Volatile Organic Compounds/pharmacology , JuglansABSTRACT
Mesenchymal stem cells (MSCs) have demonstrated potent immunomodulatory properties that have shown promise in the treatment of autoimmune diseases, including rheumatoid arthritis (RA). However, the inherent heterogeneity of MSCs triggered conflicting therapeutic outcomes, raising safety concerns and limiting their clinical application. This study aimed to investigate the potential of extracellular vesicles derived from human gingival mesenchymal stem cells (GMSC-EVs) as a therapeutic strategy for RA. Through in vivo experiments using an experimental RA model, our results demonstrate that GMSC-EVs selectively homed to inflamed joints and recovered Treg and Th17 cell balance, resulting in the reduction of arthritis progression. Our investigations also uncovered miR-148a-3p as a critical contributor to the Treg/Th17 balance modulation via IKKB/NF-κB signaling orchestrated by GMSC-EVs, which was subsequently validated in a model of human xenograft versus host disease (xGvHD). Furthermore, we successfully developed a humanized animal model by utilizing synovial fibroblasts obtained from patients with RA (RASFs). We found that GMSC-EVs impeded the invasiveness of RASFs and minimized cartilage destruction, indicating their potential therapeutic efficacy in the context of patients with RA. Overall, the unique characteristics - including reduced immunogenicity, simplified administration, and inherent ability to target inflamed tissues - position GMSC-EVs as a viable alternative for RA and other autoimmune diseases.
Subject(s)
Arthritis, Rheumatoid , Extracellular Vesicles , Mesenchymal Stem Cells , MicroRNAs , NF-kappa B , T-Lymphocytes, Regulatory , Th17 Cells , Arthritis, Rheumatoid/therapy , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Humans , Animals , Th17 Cells/immunology , Th17 Cells/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , NF-kappa B/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Mice , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/immunology , Extracellular Vesicles/metabolism , Extracellular Vesicles/transplantation , I-kappa B Kinase/metabolism , Signal Transduction , Disease Models, Animal , Gingiva/cytology , Gingiva/metabolism , Gingiva/pathology , Gingiva/immunology , Male , Fibroblasts/metabolismABSTRACT
BACKGROUND: The protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression. METHODS: The modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient's overall survival versus AnxA6 expression level was evaluated by the Kaplan-Meier method. RESULTS: Lys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients. CONCLUSIONS: AnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Humans , Mice , Annexin A6/genetics , Annexin A6/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Chromatography, Liquid , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/pathology , Proto-Oncogene Proteins c-akt/metabolism , rho GTP-Binding Proteins/metabolism , Sumoylation , Tandem Mass SpectrometryABSTRACT
The core clinical characteristics of autism, which is a neurodevelopmental disease, involve repetitive behavior and impaired social interactions. Studies have shown that the Notch and Neuregulin1 (NRG1) signaling pathways are abnormally activated in autism, but the mechanism by which these two signaling pathways interact to contribute to the progression of autism has not been determined. Our results suggest that the levels of Notch1, Hes1, NRG1, and phosphorylated ErbB4 in the cerebellum (CB), hippocampus (HC), and prefrontal cortex (PFC) were increased in rats with valproic acid (VPA)-induced autism compared to those in the Con group. However, 3, 5-difluorophenyl-L-alanyl-L-2-phenylglycine tert-butyl (DAPT), which is a Notch pathway inhibitor, ameliorated autism-like behavioral abnormalities and decreased the protein levels of NRG1 and phosphorylated ErbB4 in rats with VPA-induced autism; these results demonstrated that the Notch1/Hes1 pathway could participate in the pathogenesis of autism by regulating the NRG1/ErbB4 signaling pathway. Studies have shown that the Notch pathway regulates microglial differentiation and activation during the onset of neurological disorders and that microglia affect autism-like behavior via synaptic pruning. Therefore, we hypothesized that the Notch1/Hes1 pathway could regulate the NRG1/ErbB4 pathway and thus participate in the development of autism by regulating microglial functions. The present study showed that AG1478, which is an ErbB4 inhibitor, ameliorated the autism-like behaviors in a VPA-induced autism rat model, reduced abnormal microglial activation, and decreased NRG1 and Iba-1 colocalization; however, AG1478 did not alter Notch1/Hes1 activity. These results demonstrated that Notch1/Hes1 may participate in the microglial activation in autism by regulating NRG1/ErbB4, revealing a new mechanism underlying the pathogenesis of autism.
Subject(s)
Autistic Disorder , Quinazolines , Tyrphostins , Animals , Rats , Autistic Disorder/chemically induced , Neuregulin-1 , Microglia , Valproic Acid , Transcription Factor HES-1 , Receptor, Notch1ABSTRACT
As a pervasive neurodevelopmental disease, autism spectrum disorder (ASD) is caused by both hereditary and environmental elements. Research has demonstrated the functions of the Notch pathway and DNA methylation in the etiology of ASD. DNA methyltransferases DNMT3 and DNMT1 are responsible for methylation establishment and maintenance, respectively. In this study, we aimed to explore the association of DNA methyltransferases with the Notch pathway in ASD. Our results showed Notch1 and Hes1 were upregulated, while DNMT3A and DNMT3B were downregulated at the protein level in the prefrontal cortex (PFC), hippocampus (HC) and cerebellum (CB) of VPA-induced ASD rats compared with Control (Con) group. However, the protein levels of DNMT3A and DNMT3B were augmented after treatment with 3,5-difluorophenacetyl-L-alanyl-S-phenylglycine-2-butyl ester (DAPT), suggesting that abnormal Notch pathway activation may affect the expression of DNMT3A and DNMT3B. Besides, our previous findings revealed that the Notch pathway may participate in development of ASD by influencing autophagy. Therefore, we hypothesized the Notch pathway adjusts autophagy and contributes to ASD by affecting DNA methyltransferases. Our current results showed that after receiving the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-2'dc), the VPA + DAPT+5-Aza-2'dc (V + D + Aza) group exhibited reduced social interaction ability and increased stereotyped behaviors, and decreased expression of DNMT3A, DNMT3B and autophagy-related proteins, but did not show changes in Notch1 and Hes1 protein levels. Our results indicated that the Notch1/Hes1 pathway may adjust DNMT3A and DNMT3B expression and subsequently affect autophagy in the occurrence of ASD, providing new insight into the pathogenesis of ASD.
Subject(s)
Autism Spectrum Disorder , Valproic Acid , Rats , Animals , Valproic Acid/pharmacology , Autism Spectrum Disorder/chemically induced , Autism Spectrum Disorder/genetics , DNA Methylation , Signal Transduction , DNA Modification Methylases/metabolism , DNA/metabolism , Autophagy , Transcription Factor HES-1/genetics , Transcription Factor HES-1/metabolism , Receptor, Notch1/genetics , Receptor, Notch1/metabolismABSTRACT
BACKGROUND: The Annexin A6 (AnxA6) protein is known to inhibit the epidermal growth factor receptor (EGFR)-extracellular signal regulated kinase (ERK)1/2 signaling upon EGF stimulation. While the biochemical mechanism of AnxA6 inactivating phosphorylation of EGFR and ERK1/2 is not completely explored in cancer cells. METHODS: Cells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1 plasmids to enrich the SUMOylated AnxA6 by immunoprecipitation, and the modification level of AnxA6 by SUMO1 was detected by Western blot against SUMO1 antibody. The SUMOylation level of AnxA6 was compared in response to chemical SUMOylation inhibitor treatment. AnxA6 SUMOylation sites were further identified by LC-MS/MS and amino acid site mutation validation. AnxA6 gene was silenced through AnxA6 targeting shRNA-containing pLKO.1 lentiviral transfection in HeLa cells, while AnxA6 gene was over-expressed within the Lenti-Vector carrying AnxA6 or mutant AnxA6K299R plasmid in A431 cells using lentiviral infections. Moreover, the mutant plasmid pGFP-EGFRT790M/L858R was constructed to test AnxA6 regulation on EGFR mutation-induced signal transduction. Moreover, cell proliferation, migration, and gefitinib chemotherapy sensitivity were evaluated in HeLa and A431 cells under AnxA6 konckdown or AnxA6 overexpression by CCK8, colony form and wound healing assays. And tumorigenicity in vivo was measured in epithelial cancer cells-xenografted nude mouse model. RESULTS: AnxA6 was obviously modified by SUMO1 conjugation within Lys (K) residues, and the K299 was one key SUMOylation site of AnxA6 in epithelial cancer cells. Compared to the wild type AnxA6, AnxA6 knockdown and its SUMO site mutant AnxA6K299R showed less suppression of dephosphorylation of EGFR-ERK1/2 under EGF stimulation. The SUMOylated AnxA6 was prone to bind EGFR in response to EGF inducement, which facilitated EGFR-PKCα complex formation to decrease the EGF-induced phosphorylation of EGFR-ERK1/2 and cyclin D1 expression. Similarly, AnxA6 SUMOylation inhibited dephosphorylation of the mutant EGFR, thereby impeding EGFR mutation-involved signal transduction. Moreover, AnxA6 knockdown or the K299 mutant AnxA6K299R conferred AnxA6 inability to suppress tumor progression, resulting in drug resistance to gefitinib in epithelial cancer cells. And in epithelial cancer cells-xenografted nude mouse model, both the weight and size of tumors derived from AnxA6 knockdown or AnxA6K299R mutation-expressing cells were much greater than that of AnxA6-expressing cells. CONCLUSIONS: Besides EGFR gene mutation, protein SUMOylation modification of EGFR-binding protein AnxA6 also functions pivotal roles in mediating epithelial cancer cell growth and gefitinib drug effect. Video Abstract.
Subject(s)
ErbB Receptors , Lung Neoplasms , Humans , Animals , Mice , ErbB Receptors/metabolism , Gefitinib/pharmacology , Annexin A6/genetics , Annexin A6/metabolism , Genes, erbB-1 , HeLa Cells , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Sumoylation , Mice, Nude , Chromatography, Liquid , Epidermal Growth Factor/genetics , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Lung Neoplasms/pathology , Mutation , Tandem Mass SpectrometryABSTRACT
N-acetylcysteine (NAC) has been reported to improve social interaction behavior, irritability, self-injury, and anxiety-like behavior in autism. However, the molecular mechanism underlying the therapeutic roles of NAC in autism remains unknown. This study mainly aimed to investigate the therapeutic effect of NAC on valproic acid (VPA)-induced autism model and the underlying mechanisms. Our results showed that NAC ameliorated the deficits in sociability and the anxiety- and repetitive-like behaviors displayed by VPA-exposed rats. In addition, VPA exposure induced autophagic deficiency and enhanced Notch-1/Hes-1 pathway activity based on lowered Beclin-1 and LC3B levels, while increased expression of p62, Notch-1, and Hes-1 expression at the protein level. However, NAC recovered VPA-induced autophagic deficiency and reduced Notch-1/Hes-1 pathway activity in a VPA-exposed autism rat model and SH-SY5Y neural cells. The present results demonstrated that NAC improves autism-like behavioral abnormalities by inactivating Notch-1/Hes-1 signaling pathway and recovering autophagic deficiency. Taken together, this study helps to elucidate a novel molecular mechanism that underlies the therapeutic actions of NAC in autism and suggests its potential to ameliorate behavioral abnormalities in neurodevelopmental disorders.
Subject(s)
Autistic Disorder , Neuroblastoma , Prenatal Exposure Delayed Effects , Rats , Humans , Animals , Female , Autistic Disorder/drug therapy , Acetylcysteine/pharmacology , Behavior, Animal , Valproic Acid/adverse effects , Disease Models, Animal , Prenatal Exposure Delayed Effects/chemically inducedABSTRACT
The Notch signaling pathway is mainly involved in the regulation of neural stem cell proliferation, survival and differentiation during the development of the central nervous system. As a neurodevelopmental disorder, autism is associated with an abnormal increase in the number of microglia in several brain regions. These findings suggest that the pathogenesis of autism may be related to the Notch signaling pathway and microglia. In this review, we discuss how Notch pathway activity leads to behavioral abnormalities such as learning and memory impairment by influencing neuronal biological activities. An increase in microglial protein synthesis and abnormal autophagy can affect synaptic development and lead to behavioral abnormalities, and all of these changes can lead to autism. Furthermore, the Notch signaling pathway regulates the activation and differentiation of microglia and promotes inflammatory responses, leading to the occurrence of autism. When excessive reactive oxygen species (ROS) secreted by microglia cannot be cleared by autophagy in a timely manner, Notch signaling pathway activity is affected, possibly further increasing susceptibility to autism. This review reveals the mechanism underlying the role of the Notch signaling pathway, microglia and their interaction in the pathogenesis of autism and provides a theoretical reference for targeted clinical therapies for autism.
Subject(s)
Autistic Disorder , Microglia , Humans , Microglia/metabolism , Autistic Disorder/metabolism , Signal Transduction/physiology , Neurons , Central Nervous SystemABSTRACT
Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.
Subject(s)
Calcium Channels , Cryoelectron Microscopy , NLR Proteins , Plant Proteins , Receptors, Immunologic , Triticum , Arabidopsis/immunology , Arabidopsis/metabolism , Arginine , Calcium Channels/chemistry , Calcium Channels/immunology , Calcium Channels/metabolism , Cations/metabolism , Leucine , NLR Proteins/chemistry , NLR Proteins/immunology , NLR Proteins/metabolism , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Immunity , Plant Proteins/chemistry , Plant Proteins/immunology , Plant Proteins/metabolism , Receptors, Immunologic/chemistry , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism , Triticum/immunology , Triticum/metabolism , Amino Acid Motifs , Conserved Sequence , ElectrophysiologyABSTRACT
The poly-L-cysteine modified Au nanoparticles (Au@p-L-Cys) were constructed on electrode surface as a highly efficient chiral interface for tryptophan (Trp) enantiomers recognition via one step electropolymerization. With the aid of Cu2+, L-Cys residues and D-Trp target formed a sandwich complex D-Trp-Cu2+-L-Cys, while L-Trp was unable to form such complex due to the steric hindrance provided by the chiral interface, which was confirmed by the electrochemical and SEM results. With the introduction of ferricyanide probe, D-Trp produced significant current decrease while L-Trp produced a slight current increase, which implied the successful enantioselective recognition of Trp enantiomers (specifically D-Trp) in the true sense. This novel sensor showed a surprisingly wide linear range toward D-Trp of 6 × 10-7 M to 1 × 10-2 M, with a detection limit as low as 75 nM (S/N = 3). Moreover, the exclusive enantioselectivity toward D-Trp was discovered since other amino acids showed negligible interference to detection of D-Trp. The recovery of D-Trp in human serum was between 91.30 and 109.3%, which further verified the satisfying specificity and practicality of the proposed strategy. The coordination thermodynamics by UV-Vis spectroscopy and DFT simulation were also used to investigate the enantioselective mechanism. These results highlight the great potential of using Au@p-L-Cys to construct chiral interface for enantiomers recognition and hold the promise of practical application of electrochemical chiral sensors in fields like pharmaceutics and bioanalysis.
Subject(s)
Metal Nanoparticles , Tryptophan , Cysteine , Electrochemical Techniques/methods , Gold/chemistry , Humans , Stereoisomerism , Tryptophan/analysisABSTRACT
The surgical stress responses, surgeries, and anesthetics used during surgeries have effects on post-surgery complications and metastasis. Volatile and/or intravenous anesthetics are generally used for cancer curative surgeries. Therefore, appropriate selection of anesthetics should be considered for better clinical outcomes. The objectives of the study were to compare postoperative complications, the overall survival, and recurrence-free survival of patients who had received volatile anesthesia against those of patients who had received propofol-based total intravenous anesthesia for digestive tract cancer curative surgeries. Patients had received propofol-based total intravenous anesthesia (PA cohort, n = 120) or volatile anesthesia (VA cohort, n = 185) for elective digestive tract cancer curative surgeries. Patients with age > 50 years (P = .0399), body mass index ≥ 25 kg/m2 (P = .0423), cancer stage III (P = .0041), and cancer stage IV (P = .0189) were operated through volatile anesthesia. Females (P = .0346), disable patients (P = .0479), patients with Charlson Comorbidity Index 2 (P = .0449), patients with cancer stage 0 or I (P = .0141), and patients with cancer stage II (P = .0289) were operated through propofol-based total intravenous anesthesia. Postoperative complication(s) between patients of both cohorts were statistically same (P = .9217). After 3-years of the follow-up period, a total of 81 (44%) patients from the VA cohort and 63 (52%) patients from the PA cohort survived irrespective of any kind of disease(s) (P = .9918). Also, a total of 53 (29%) patients from the VA cohort and 42 (35%) patients from the PA cohort survived without progression of cancer (P = .9981) after 3-years. Age > 50 years (P = 0.0491), Charlson Comorbidity Index ≥ 3 (P = 0.0481), and cancer stage > II (P = .0412) were independent parameters for death of patients suffering from digestive tract cancer due to any reason(s) during 3-years of the follow-up period after surgeries. The selection of anesthetic agents for cancer curative surgeries does not affect survival during 3-years of follow-up and postoperative complication(s) of patients suffering from digestive tract cancer (Level of Evidence: III; Technical Efficacy Stage: 4).
Subject(s)
Anesthetics, Inhalation , Neoplasms , Propofol , Anesthesia, General , Anesthesia, Intravenous , Anesthetics, Intravenous , Female , Gastrointestinal Tract , Humans , Middle Aged , Neoplasms/drug therapy , Postoperative Complications/drug therapy , Postoperative Complications/epidemiology , Propofol/therapeutic use , Retrospective StudiesABSTRACT
Mutations in MYH9, the gene encoding the heavy chain of nonmuscle myosin IIa (NMII-A), cause MYH9-related disease (MYH9-RD), which is an autosomal-dominant thrombocytopenia with bleeding tendency. Previously, we showed that NMII-A in endothelial cells (ECs) is critical for hemostasis via regulating von Willebrand factor (VWF) release from Weibel-Palade bodies (WPBs). The aim of this study was to determine the role of the expression of MYH9 mutants in ECs in the pathogenesis of the MYH9-RD bleeding symptom. First, we expressed the 5 most common NMII-A mutants in ECs and found that E1841K mutant-expressing ECs secreted less VWF than the controls in response to a cyclic adenosine monophosphate (cAMP) signaling agonist. Then, we generated 2 knockin mouse lines, 1 with Myh9 E1841K in ECs and the other in megakaryocytes. Endothelium-specific E1841K mice exhibited impaired cAMP-induced VWF release and a prolonged bleeding time with normal platelets, whereas megakaryocyte-specific E1841K mice exhibited macrothrombocytopenia and a prolonged bleeding time with normal VWF release. Finally, we presented mechanistic findings that E1841K mutation not only interferes with S1943 phosphorylation and impairs the peripheral distribution of Rab27a-positive WPBs in Ecs under quiescent condition but also interferes with S1916 phosphorylation by disrupting the interaction with zyxin and CKIIα and reduces actin framework formation around WPBs and subsequent VWF secretion under the stimulation by a cAMP agonist. Altogether, our results suggest that impaired cAMP-induced endothelial VWF secretion by E1841K mutant expression may contribute to the MYH9-RD bleeding phenotype.
Subject(s)
Endothelial Cells , Hemostasis , Myosin Heavy Chains , Thrombocytopenia , von Willebrand Factor , Animals , Endothelial Cells/metabolism , Hemostasis/genetics , Hemostasis/physiology , Mice , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Thrombocytopenia/congenital , Thrombocytopenia/genetics , von Willebrand Factor/genetics , von Willebrand Factor/metabolismABSTRACT
Ear length (EL) is a key trait that contributes greatly to grain yield in maize (Zea mays). While numerous quantitative trait loci for EL have been identified, few causal genes have been studied in detail. Here we report the characterization of ear apical degeneration1 (ead1) exhibiting strikingly shorter ears and the map-based cloning of the casual gene EAD1. EAD1 is preferentially expressed in the xylem of immature ears and encodes an aluminum-activated malate transporter localizing to the plasma membrane. We show that EAD1 is a malate efflux transporter and loss of EAD1 leads to lower malate contents in the apical part of developing inflorescences. Exogenous injections of malate rescued the shortened ears of ead1. These results demonstrate that EAD1 plays essential roles in regulating maize ear development by delivering malate through xylem vessels to the apical part of the immature ear. Overexpression of EAD1 led to greater EL and kernel number per row and the EAD1 genotype showed a positive association with EL in two different genetic segregating populations. Our work elucidates the critical role of EAD1 in malate-mediated female inflorescence development and provides a promising genetic resource for enhancing maize grain yield.