BACKGROUND Anti-leucine-rich glioma inactivated protein 1(anti-LGI1) encephalitis is an infrequent type of autoimmune encephalitis(AE) characterized by acute or subacute cognitive and psychiatric disturbance, facio-br...BACKGROUND Anti-leucine-rich glioma inactivated protein 1(anti-LGI1) encephalitis is an infrequent type of autoimmune encephalitis(AE) characterized by acute or subacute cognitive and psychiatric disturbance, facio-brachial dystonic seizures(FBDSs), and hyponatremia. Anti-LGI1 AE has increasingly been considered a primary form of AE. Early identification and treatment of this disease are clearly very important.CASE SUMMARY Here, we report that a male patient developed severe anti-LGI1 encephalitis, which was initially misdiagnosed as a sleep disturbance. He was hospitalized for epileptic seizures and typical FBDSs half a month after he developed sleep disturbances. LGI1 antibodies were detected in his cerebrospinal fluid and serum(1:100 and 1:3.2, respectively), which led to the diagnosis of classic anti-LGI1 AE. No obvious abnormality was observed on brain computed tomography images. T2-weighted fluid-attenuated inversion recovery and T2-weighted scans of brain magnetic resonance imaging(MRI) showed slightly elevated signals within the left basal ganglia area. No tumor was detected within the brain of this patient using MRI. After hormone and antiepileptic drug treatment, the patient’s symptoms improved significantly.CONCLUSION Anti-LGI1 antibody-associated encephalitis has characteristic clinical manifestations, such as cognitive impairment, psychiatric symptoms, seizures, sleep disorders, hyponatremia, and FBDSs. LGI1 antibodies are present in the serum and/or cerebrospinal fluid, but their production is sensitive to immunosuppressants, and this disease has a relatively good prognosis. In particular, we should be aware of the possibility of anti-LGI1 antibody-associated encephalitis in adolescents with sleep disorders to avoid missed diagnoses and misdiagnoses.展开更多
BACKGROUND F-box and leucine-rich repeat 6(FBXL6)have reportedly been associated with several cancer types.However,the role and mechanisms of FBXL6 in gastric cancer(GC)require further elucidation.AIM To investigate t...BACKGROUND F-box and leucine-rich repeat 6(FBXL6)have reportedly been associated with several cancer types.However,the role and mechanisms of FBXL6 in gastric cancer(GC)require further elucidation.AIM To investigate the effect of FBXL6 in GC tissues and cells and the underlying mechanisms.METHODS TCGA and GEO database analysis was performed to evaluate the expression of FBXL6 in GC tissues and adjacent normal tissues.Reverse transcription-quantitative polymerase chain reaction,immunofluorescence,and western blotting were used to detect the expression of FBXL6 in GC tissue and cell lines.Cell clone formation,5-ethynyl-2’-deoxyuridine(EdU)assays,CCK-8,transwell migration assay,and wound healing assays were performed to evaluate the malignant biological behavior in GC cell lines after transfection with FBXL6-shRNA and the overexpression of FBXL6 plasmids.Furthermore,in vivo tumor assays were performed to prove whether FBXL6 promoted cell proliferation in vivo.RESULTS FBXL6 expression was upregulated more in tumor tissues than in adjacent normal tissues and positively associated with clinicopathological characteristics.The outcomes of CCK-8,clone formation,and Edu assays demonstrated that FBXL6 knockdown inhibited cell proliferation,whereas upregulation of FBXL6 promoted proliferation in GC cells.Additionally,the transwell migration assay revealed that FBXL6 knockdown suppressed migration and invasion,whereas the overex pression of FBXL6 showed the opposite results.Through the subcutaneous tumor implantation assay,it was evident that the knockdown of FBXL6 inhibited GC graft tumor growth in vivo.Western blotting showed that the effects of FBXL6 on the expression of the proteins associated with the epithelial-mesenchymal transition-associated proteins in GC cells.CONCLUSION Silencing of FBXL6 inactivated the EMT pathway to suppress GC malignancy in vitro.FBXL6 can potentially be used for the diagnosis and targeted therapy of patients with GC.展开更多
BACKGROUND It has been suggested that serum leucine-richα-2 glycoprotein(LRG)could be a novel monitoring biomarker for the assessment of disease activity in inflammatory bowel disease.In particular,the relationship b...BACKGROUND It has been suggested that serum leucine-richα-2 glycoprotein(LRG)could be a novel monitoring biomarker for the assessment of disease activity in inflammatory bowel disease.In particular,the relationship between LRG levels and the endoscopically assessed activity of ulcerative colitis(UC)has become a matter of interest.AIM To clarify appropriate LRG cut-off values for the prediction of endoscopic and histologic remission in Japanese patients with UC.METHODS This was a cross-sectional,single-center,observational study of Japanese patients with UC.Among 213 patients with UC,in whom LRG was measured from September 2020 to February 2022,we recruited 30 patients for whom a total colonoscopy and measurements of LRG and C-reactive protein(CRP)were performed on the same day.We retrospectively analyzed correlations between the LRG and CRP levels and endoscopic indices,including the Mayo endoscopic subscore and UC endoscopic index of severity.RESULTS Correlations between the LRG values and the Mayo endoscopic subscore or UC endoscopic index of severity were significant(r=0.754,P<0.0001;r=0.778,P<0.0001,respectively).There were also significant correlations between CRP levels and Mayo endoscopic subscore or UC endoscopic index of severity(r=0.599,P=0.0005;r=0.563,P=0.0012,respectively),although the correlation coefficients were higher for LRG.The LRG cutoff value for predicting endoscopic remission was 13.4μg/mL for a Mayo endoscopic subscore of 0[area under the curve(AUC):0.871;95%confidence interval(CI):0.744-0.998],and 13.4μg/mL for an UC endoscopic index of severity of 0 or 1(AUC:0.904;95%CI:0.792-1.000).CONCLUSION LRG may be a surrogate marker for endoscopic activity in UC,with a cut-off value of around 13.4μg/mL for endoscopically inactive disease.展开更多
BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens ...BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeatcontaining G protein-coupled receptor 5(LGR5)-expressing stem cells,these socalled“tumour-initiating”cells,reminiscent in their properties of the normal intestinal stem cells(ISCs),may explain the apparent heterogeneity of colon cancer cell lines.Also,colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5.Furthermore,in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines(Caco-2 and LoVo).These cells were then characterized according to their proliferation capacity,gene expression signatures of ISC markers,and their tumorigenic properties in vivo and in vitro.RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2,but not from the LoVo,cell lines,in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog(+4 crypt)stem cells.This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny.Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein(EYFP)from this cell line exhibit tumorigenic-like properties in vivo and in vitro.In contrast,cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties.Thus,cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells,but not LoVo cells.CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines.Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data.We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.展开更多
Leucine-rich repeat kinase 1 (LRRK1) plays a critical role in regulating cytoskeletal organization, osteoclast activity, and bone resorption with little effect on bone formation parameters. Deficiency of Lrrk1 in mice...Leucine-rich repeat kinase 1 (LRRK1) plays a critical role in regulating cytoskeletal organization, osteoclast activity, and bone resorption with little effect on bone formation parameters. Deficiency of Lrrk1 in mice causes a severe osteopetrosis in the metaphysis of the long bones and vertebrae bones, which makes LRRK1 an attractive alternative drug target for the treatment of osteoporosis and other high-turnover bone diseases.This review summarizes recent advances on the functions of the Lrrk1-related family members, Lrrk1deficiency-induced skeletal phenotypes, LRRK1 structure–function, potential biological substrates and interacting proteins, and the mechanisms of LRRK1 action in osteoclasts.展开更多
BACKGROUND Cancer stem cells(CSCs)are a subpopulation of cancer cells with the potential of self-renewal and differentiation.CSCs play critical roles in tumorigenesis,recurrence,metastasis,radiation tolerance and chem...BACKGROUND Cancer stem cells(CSCs)are a subpopulation of cancer cells with the potential of self-renewal and differentiation.CSCs play critical roles in tumorigenesis,recurrence,metastasis,radiation tolerance and chemoresistance.AIM To assess the expression patterns and clinical potential of doublecortin-like kinase 1(DCLK1)and leucine-rich repeat-containing G-protein-coupled receptor 5(Lgr5),as prognostic CSC markers of colorectal cancer(CRC).METHODS The expression of DCLK1 and Lgr5 in CRC tissue sections from 92 patients was determined by immunohistochemistry.Each case was evaluated using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-3).The final staining score was calculated as the intensity score multiplied by the proportion score.Low expression of DCLK1 and Lgr5 was defined as a score of 0-3;high expression of DCLK1 and Lgr5 was defined as a score of≥4.Specimens were categorized as either high or low expression,and the correlation between the expression of DCLK1 or Lgr5 and clinicopathological factors was investigated.RESULTS DCLK1 and Lgr5 expression levels were significantly positively correlated.CRC patients with high DCLK1,Lgr5 and DCLK1/Lgr5 expressions had poorer progression-free survival and overall survival.Moreover,high expression of DCLK1 was an independent prognostic factor for recurrence and overall survival in patients with CRC by multivariate analysis(P=0.026 and P=0.049,respectively).CONCLUSION DCLK1 may be a potential CSC marker for the recurrence and survival of CRC patients.展开更多
Leucine rich repeats (LRRs) are present in over 14,000 proteins that have been identified in viruses, bacteria, archaea, and eukaryotes. Two to sixty-two LRRs occur in tandem forming an overall arc shaped domain. Ther...Leucine rich repeats (LRRs) are present in over 14,000 proteins that have been identified in viruses, bacteria, archaea, and eukaryotes. Two to sixty-two LRRs occur in tandem forming an overall arc shaped domain. There are eight classes of LRRs. Plant specific LRRs (class: PS-LRR) had previously been recognized in only plant proteins. However, we find that PS-LRRs are also present in proteins from bacteria. We investigated the origin of bacterial PS-LRR domains. PSLRR proteins are widely distributed in most plants;they are found in only a few bacterial species. There are no PS-LRR proteins from archaea. Bacterial PS-LRRs in twenty proteins from eleven bacterial species (in the three phyla: Proteobacteria, Cyanobacteria, and Bacteroidetes) are significantly more similar to the PS-LRR class than to the other seven classes of LRR proteins. Not only amino acid sequences but also nucleotide sequences of the bacterial PS-LRR domains show highly significant similarity with those of many plant proteins. The program, EGID (Ensemble algorithm for Genomic Island Detection), predicts that Synechococcus sp. CYA_ 1022 came from another organism. Four bacterial PS-LRR proteins contain AhpC-TSA, IgA peptidase M64, the immunoglobulin domain, the Calx-b domain, and the He_PIG domain;these domains show no similarity with any eukaryotic (plant) proteins, in contrast to the similarities of their respective PS-LRRs. The present results indicate that horizontal gene transfer (HGT) of genes/gene fragments encoding PS-LRR domains occurred between bacteria and plants, and HGT among the eleven bacterial species, of the three phyla, as opposed to descent from a common ancestor. There is the possibility of the occurrence of one HGT event from plant to bacteria. A series of HGTs might then have occurred recently and rapidly among these eleven species of bacteria.展开更多
Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibi...Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.展开更多
Identification of immunity-associated leucine-rich repeat receptor-like protein kinases(LRR-RLK) is critical to elucidate the LRR-RLK mediated mechanism of plant immunity.Here,we reported the map-based cloning of a no...Identification of immunity-associated leucine-rich repeat receptor-like protein kinases(LRR-RLK) is critical to elucidate the LRR-RLK mediated mechanism of plant immunity.Here,we reported the map-based cloning of a novel rice SPOTTED-LEAF 41(Os SPL41) encoding a putative LRR-RLK protein(Os LRR-RLK41/Os SPL41) that regulated disease responses to the bacterial blight pathogen Xanthomonas oryzae pv.oryzae(Xoo).An 8-bp insertion at position 865 bp in a mutant spotted-leaf 41(spl41) allele led to the formation of purple-brown lesions on leaves.Functional complementation by the wild type allele(Os SPL41) can rescue the mutant phenotype,and the complementary lines showed similar performance to wild type in a number of agronomic,physiological and molecular indices.Os SPL41 was constitutively expressed in all tissues tested,and Os SPL41 contains a typical transmembrane domain critical for its localization to the cell membrane.The mutant exhibited an enhanced level of resistance to Xoo in companion of markedly up-regulated expression of pathogenesis-related genes such as Os PR10a,Os PAL1 and Os NPR1,while the level of salicylic acid was significantly increased in spl41.In contrast,the over-expression lines exhibited a reduced level of H_(2)O_(2) and were much susceptible to Xoo with down-regulated expression of pathogenesis-related genes.These results suggested that Os SPL41 might negatively regulate plant immunity through the salicylic acid signaling pathway in rice.展开更多
The multifunctional secondary metabolites known as cyclic lipopeptides(CLPs),which are produced by a large variety of bacteria,have become a key category of plant immunity elicitors.Pseudomonas-CLPs(PsCLPs)are extreme...The multifunctional secondary metabolites known as cyclic lipopeptides(CLPs),which are produced by a large variety of bacteria,have become a key category of plant immunity elicitors.Pseudomonas-CLPs(PsCLPs)are extremely diverse in structure and biological activity.However,an understanding of CLP-plant structure–function interactions currently remains elusive.Here,we identify medpeptin,a novel CLP from Pseudomonas mediterranea that consists of 22 amino acids.Medpeptin is synthesized by a non-ribosomal peptide synthase(NRPS)gene cluster and regulated by a quorum-sensing system.Further research indicates that medpeptin does not exhibit antimicrobial activity;instead,it induces plant cell death immunity and confers resistance to bacterial infection.Comparative transcriptome analysis and virus-induced gene silencing(VIGS)reveal a set of immune signaling candidates involved in medpeptin perception.Silencing of a cell-wall leucine-rich repeat extensin protein(NbLRX3)or a receptor-like protein kinase(NbRLK25)—but not BAK1 or SGT1—compromises medpeptin-triggered cell death and resistance to pathogen infection in Nicotiana benthamiana.Our findings point to a noncanonical mechanism of CLP sensing and suggest perspectives for the development of plant disease resistance.展开更多
AIM:To investigate the differential expression of leu-cine-rich repeat-containing G protein-coupled receptor5(LGR5)in gastric cancer tissues and its significance related to tumor growth and spread.METHODS:Formalin-fix...AIM:To investigate the differential expression of leu-cine-rich repeat-containing G protein-coupled receptor5(LGR5)in gastric cancer tissues and its significance related to tumor growth and spread.METHODS:Formalin-fixed biopsy specimens of intestinal metaplasia(n=90),dysplasia(n=53),gastric adenocarcinoma(n=180),metastases in lymph nodes and the liver(n=15),and lesion-adjacent normal gastric mucosa(controls;n=145)were obtained for analysis from the Peking University Cancer Hospital’s Department of Pathology and Gastrointestinal Surgery tissue archives(January 2003 to December 2011).The biopsied patients’demographic and clinicopathologic data were retrieved from the hospital’s medical records database.Each specimen was subjected to histopathological typing to classify the tumor node metastasis(TNM)stage and to immunohistochemistry staining to detect the expression of the cancer stem cell marker LGR5.The intergroup differences in LGR5 expression were assessed by Spearman’s rank correlation analysis,and the relationship between LGR5 expression level and the patients’clinicopathological characteristics was evaluated by theχ2test or Fisher’s exact test.RESULTS:Significantly more gastric cancer tissues showed LGR5+staining than normal control tissues(all P<0.01),with immunoreactivity detected in 72.2%(65/90)and 50.9%(27/53)of intestinal metaplasia and dysplasia specimens,respectively,52.8%(95/180)of gastric adenocarcinoma specimens,and 73.3%%(11/15)of metastasis specimens,but 26.9%(39/145)of lesion-adjacent normal gastric mucosa specimens.Comparison of the intensity of LGR5+staining showed an increasing trend that generally followed increasing dedifferentiation and tumor spread(normal tissue<dysplasia,<gastric adenocarcinoma<metastasis;all P<0.001),with the exception of expression level detected in intestinal metaplasia which was higher than that in normal gastric tissues(P<0.001).Moreover,gastric cancer-associated enhanced expression of LGR5 was found to be signifcantly associated with age,tumor differentiation,Lauren type and TNM stage(Ⅰ+ⅡvsⅢ+Ⅳ)(all P<0.05),but not with sex,tumor site,location,size,histology,lymphovascular invasion,depth of invasion,lymph node metastasis or distant metastasis.Patients with LGR5+gastric cancer specimens and without signs of metastasis from the original biopsy experienced more frequent rates of recurrence or metastasis during follow-up than patients with LGR5-specimens(P<0.05).CONCLUSION:Enhanced LGR5 is related to progressive dedifferentiation and metastasis of gastric cancer,indicating the potential of this receptor as an early diagnostic and prognostic biomarker.展开更多
Obstructive sleep apnea can worsen the prognosis of subarachnoid hemorrhage.Howeve r,the underlying mechanism remains unclear.In this study,we established a mouse model of subarachnoid hemorrhage using the endovascula...Obstructive sleep apnea can worsen the prognosis of subarachnoid hemorrhage.Howeve r,the underlying mechanism remains unclear.In this study,we established a mouse model of subarachnoid hemorrhage using the endovascular perforation method and exposed the mice to intermittent hypoxia for 8 hours daily for 2 consecutive days to simulate sleep apnea.We found that sleep apnea aggravated brain edema,increased hippocampal neuron apoptosis,and worsened neurological function in this mouse model of subarachnoid hemorrhage.Then,we established an in vitro HT-22 cell model of hemin-induced subarachnoid hemorrhage/intermittent hypoxia and found that the cells died,and lactate dehydrogenase release increased,after 48 hours.We further investigated the underlying mechanism and found that sleep apnea increased the expression of hippocampal neuroinflammatory factors interleukin-1β,interleukin-18,inte rleukin-6,nuclear factorκB,pyro ptosis-related protein caspase-1,pro-caspase-1,and NLRP3,promoted the prolife ration of astrocytes,and increased the expression of hypoxia-inducible factor 1αand apoptosis-associated speck-like protein containing a CARD,which are the key proteins in the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.We also found that knockdown of hypoxia-inducible factor 1αexpression in vitro greatly reduced the damage to HY22 cells.These findings suggest that sleep apnea aggravates early brain injury after subarachnoid hemorrhage by aggravating neuroinflammation and pyroptosis,at least in part through the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.展开更多
AIM:To investigate the potential effect and mechanism of leucine-richα-2-glycoprotein-1(LRG1)on corneal angiogenesis and lymphangiogenesis.METHODS:Corneal neovascularization and lymphatics were induced by establishin...AIM:To investigate the potential effect and mechanism of leucine-richα-2-glycoprotein-1(LRG1)on corneal angiogenesis and lymphangiogenesis.METHODS:Corneal neovascularization and lymphatics were induced by establishing alkali burn mouse model.Immunofluorescence staining was performed to detect the location of LRG1 in cornea tissues and to verify the source of LRG1-positive cells.Corneal whole-mount staining for CD31(a panendothelial cell marker)and lymphatic endothelial hyluronan receptor-1(LYVE-1;lymphatic marker)was performed to detect the growth of blood and lymphatic vessels after local application of exogenous LRG1 protein or LRG1 si RNA.In addition,expressions of the proangiogenic vascular endothelial growth factor(VEGF)related proteins were detected using Western blot analysis.RESULTS:LRG1 was dramatically increased in alkali burned corneal stroma in both the limbal and central areas.LRG1-positive cells in the corneal stroma were mainly derived from Vimentin-positive cells.Local application ofexogenous LRG1 protein not only aggravated angiogenesis but also lymphangiogenesis significantly(P<0.01).LRG1 group upregulated the levels of VEGF and the vascular endothelial growth factor receptor(VEGFR)family when compared with the phosphate-buffered saline(PBS)control group.We also found that LRG1-specific si RNA could suppress corneal angiogenesis and lymphangiogenesis when compared with the scramble si RNA-treated group(P<0.01).CONCLUSION:LRG1 can facilitate corneal angiogenesis and lymphangiogenesis through heightening the stromal expression of VEGF-A,B,C,D and VEGFR-1,2,3;LRG1-specific si RNA can suppress corneal angiogenesis and lymphangiogenesis in corneal alkali burn mice.展开更多
The G2019S mutation of the leucine-rich repeat kinase 2(LRRK2)is the most common genetic cause of Parkinson's disease (PD).However,the molecular mechanisms of LRRK2 mutation contributing to the onset and progressi...The G2019S mutation of the leucine-rich repeat kinase 2(LRRK2)is the most common genetic cause of Parkinson's disease (PD).However,the molecular mechanisms of LRRK2 mutation contributing to the onset and progression of PD have not been fully illustrated.We generated HEK293 cells stably transfected with α-synuclein and investigated the effect of LRRK2 G2019S mutation on the degradation of α-synuclein.The lysosomal activity was assessed by the protein degradation of glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A.It was found that α-synuclein was mainly degraded in lysosomes.LRRK2G2019S inhibited the degradation of α-synuclein,and promoted its aggregation.LRRK2G 2019S also decreased the activities of lysosomal enzymes including cathepsin B and cathepsin L.Furthermore,the inhibitory effect of LRRK2 G2019S on lysosomal functions did not depend on its kinase activity.These findings indicated that the inhibitory effect of LRRK2 G2019S on α-synuclein degradation could underlie the pathogenesis of aberrant α-synuclein aggregation in PD with LRRK2 mutation.展开更多
BACKGROUND Patients with mesenteric ischemia frequently suffer from bowel necrosis even after revascularization.Hydrogen gas has showed promising effects for ischemiareperfusion injury by reducing reactive oxygen spec...BACKGROUND Patients with mesenteric ischemia frequently suffer from bowel necrosis even after revascularization.Hydrogen gas has showed promising effects for ischemiareperfusion injury by reducing reactive oxygen species in various animal and clinical studies.We examined intestinal tissue injury by ischemia and reperfusion under continuous initiation of 3%hydrogen gas.AIM To clarify the treatment effects and target cells of hydrogen gas for mesenteric ischemia.METHODS Three rat groups underwent 60-min mesenteric artery occlusion(ischemia),60-min reperfusion following 60-min occlusion(reperfusion),or ischemiareperfusion with the same duration under continuous 3%hydrogen gas inhalation(hydrogen).The distal ileum was harvested.Immunofluorescence staining with caspase-3 and leucine-rich repeat-containing G-protein-coupled 5(LGR5),a specific marker of intestinal stem cell,was conducted to evaluate the injury location and cell types protected by hydrogen.mRNA expressions of LGR5,olfactomedin 4(OLFM4),hairy and enhancer of split 1,Jagged 2,and Neurogenic locus notch homolog protein 1 were measured by quantitative polymerase chain reaction.Tissue oxidative stress was analyzed with immunostaining for 8-hydroxy-2'-deoxyguanosine(8-OHdG).Systemic oxidative stress was evaluated by plasma 8-OHdG.RESULTS Ischemia damaged the epithelial layer at the tip of the villi,whereas reperfusion induced extensive apoptosis of the cells at the crypt base,which were identified as intestinal stem cells with double immunofluorescence stain.Hydrogen mitigated such apoptosis at the crypt base,and the LGR5 expression of the tissues was higher in the hydrogen group than in the reperfusion group.OLFM4 was also relatively higher in the hydrogen group,whereas other measured RNAs were comparable between the groups.8-OHdG concentration was high in the reperfusion group,which was reduced by hydrogen,particularly at the crypt base.Serum 8-OHdG concentrations were relatively higher in both reperfusion and hydrogen groups without significance.CONCLUSION This study demonstrated that hydrogen gas inhalation preserves intestinal stem cells and mitigates oxidative stress caused by mesenteric ischemia and reperfusion.展开更多
Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor grow...Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor growth. This study investigated the interaction between human LRIG1 and EGFR and attempted to delineate the functions of as well as the mechanisms used by the extracellular(ECD) and cytoplasmic(CPD) domains of the human LRIG1 protein to downregulate human EGFR signaling activity.Methods Two constructed chimeric eukaryotic expression vectors, pIRES2-EGFP-3XFLAG-LRIG1-ET and p3FLAG-LRIG1-TC, encoding the extracellular and transmembrane regions(LRIG1-ET) and the transmembrane and cytoplasmic regions(LRIG1-TC), respectively, and the plasmid p3XFLAG-CMV-9-LRIG1 encoding full-length LRIG1(LRIG1-FL) were transfected into the human glioma cell line U251 or primary astrocytoma cells by using liposomes. The number and affinity of cell surface EGFR on transfected cells was determined by ^(125)I-EGF binding assay. Results The dissociation constant(KD) values for EGFR were higher, and the maximum increase was observed in the cells transfected into LRIG1-ET(1.36 folds). The number of maximal binding sites(Bmax) of the receptors was decreased in all transfected cells; the maximum decrease was noted in the cells transfected into LRIG1-FL(40.05%).Conclusion Both the ECD and CPD of LRIG1 are important to negate EGFR signaling. The ECD may interfere with the binding between EGFR and its ligand and facilitate the functions of CPD. The CPD may, when brought in proximity to EGFR, enhance receptor degradation. These two mechanisms can contribute to the downregulation of EGFR-mediated signaling by LRIG1.展开更多
Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-...Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana benthamiana.We used the cell-death phenotype caused by INF1,an oomycete elicitin that induces pattern-triggered immunity,as an indicator.By screening 33 small LRR-RLKs(≤6 LRRs)of unknown function,we identified ELICITIN INSENSITIVE RLK 1(NbEIR1)as a positive regulator of INF1-induced immunity and oomycete resistance.Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly compromised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phytophthora capsici.NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1(NbBAK1)and a downstream component,BRASSINOSTEROIDSIGNALING KINASE 1(NbBSK1).NbBSK1 also contributes to INF1-induced defense and P.capsici resistance.Upon INF1 treatment,NbEIR1 was released from NbBAK1 and NbBSK1 in vivo.Moreover,the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1.We also showed that NbEIR1 regulates flg22-induced immunity and associates with its receptor,FLAGELLIN SENSING 2(NbFLS2).Collectively,our results suggest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity.NbBSK1-NbEIR1 association is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.展开更多
Large-conductance Ca^(2+)-activated K+channels(BK channels)constitute an key physiological link between cellular Ca^(2+) signaling and electrical signaling at the plasma membrane.Thus these channels are critical to th...Large-conductance Ca^(2+)-activated K+channels(BK channels)constitute an key physiological link between cellular Ca^(2+) signaling and electrical signaling at the plasma membrane.Thus these channels are critical to the control of action potential firing and neurotrans-mitter release in several types of neurons,as well as the dynamic control of smooth muscle tone in resis-tance arteries,airway,and bladder.Recent advances in our understanding of K^(+) channel structure and function have led to new insight toward the molecular mecha-nisms of opening and closing(gating)of these chan-nels.Here we will focus on mechanisms of BK channel gating by Ca^(2+),transmembrane voltage,and auxiliary subunit proteins.展开更多
Plant intracellular nucleotide binding leucine-rich repeat (NLR) immune receptors play critical roles in pathoge n surveillance. Most plant NLRs characterized so far were found to use a single domain/sensor to recogni...Plant intracellular nucleotide binding leucine-rich repeat (NLR) immune receptors play critical roles in pathoge n surveillance. Most plant NLRs characterized so far were found to use a single domain/sensor to recognize pathogen effectors. Here we report that the Sw-5b NLR immune receptor uses two distinct domains to detect the viral movement protein NSm encoded by tospovirus. In addition to its leucine-rich repeat (LRR) domain that has been previously reported, the N-terminal Solanaceae domain (SD) of Sw- 5b also interacts with NSm and a conserved 21-amino-acid region of NSm (NSm^21). The specific interaction between Sw-5b SD and NSm is required for releasing the inhibitory effect of coiled-coil domain on the NBARC- LRR region. Furthermore, we found that the binding of NSm affects the nucleotide binding activity of the NB-ARC-LRR in vitro, while Sw-5b NB-ARC-LRR is activated only when NSm and NSm^21 levels are high. Interestingly, Sw-5b SD could significantly enhanee the ability of the NB-ARC-LRR to detect low levels of NSm effector and facilitate its activation and induction of defense response. An Sw-5b SD mutant that is disrupted in NSm recognition failed to enhance the ability of the NB-ARC-LRR to sense low levels of NSm and NSm^21 . Taken together, our results suggest that Sw-5b SD functions as an extra sensor and the NB-ARC-LRR as an activator, and that Sw-5b NLR adopts a two-step recog nition mechanism to enhance viral effector perception.展开更多
Brassinosteroids,a group of plant steroid hormones,reg-ulate many aspects of plant growth and development.We and other have previously solved the crystal structures of BRI1(LRR)in complex with brassinolide,the most ac...Brassinosteroids,a group of plant steroid hormones,reg-ulate many aspects of plant growth and development.We and other have previously solved the crystal structures of BRI1(LRR)in complex with brassinolide,the most active brassinosteroid identifi ed thus far.Although these studies provide a structural basis for the recognition of brassi-nolide by its receptor BRI1,it still remains poorly under-stood how the hormone differentiates among its con-served receptors.Here we present the crystal structure of the BRI1 homolog BRL1 in complex with brassinolide.The structure shows that subtle differences around the brassinolide binding site can generate a striking effect on its recognition by the BRI1 family of receptors.Structural comparison of BRL1 and BRI1 in their brassinolide-bound forms reveals the molecular basis for differential binding of brassinolide to its different receptors,which can be used for more effi cient design of plant growth regulators for agricultural practice.On the basis of our structural studies and others’data,we also suggest possible mech-anisms for the activation of BRI1 family receptors.展开更多
文摘BACKGROUND Anti-leucine-rich glioma inactivated protein 1(anti-LGI1) encephalitis is an infrequent type of autoimmune encephalitis(AE) characterized by acute or subacute cognitive and psychiatric disturbance, facio-brachial dystonic seizures(FBDSs), and hyponatremia. Anti-LGI1 AE has increasingly been considered a primary form of AE. Early identification and treatment of this disease are clearly very important.CASE SUMMARY Here, we report that a male patient developed severe anti-LGI1 encephalitis, which was initially misdiagnosed as a sleep disturbance. He was hospitalized for epileptic seizures and typical FBDSs half a month after he developed sleep disturbances. LGI1 antibodies were detected in his cerebrospinal fluid and serum(1:100 and 1:3.2, respectively), which led to the diagnosis of classic anti-LGI1 AE. No obvious abnormality was observed on brain computed tomography images. T2-weighted fluid-attenuated inversion recovery and T2-weighted scans of brain magnetic resonance imaging(MRI) showed slightly elevated signals within the left basal ganglia area. No tumor was detected within the brain of this patient using MRI. After hormone and antiepileptic drug treatment, the patient’s symptoms improved significantly.CONCLUSION Anti-LGI1 antibody-associated encephalitis has characteristic clinical manifestations, such as cognitive impairment, psychiatric symptoms, seizures, sleep disorders, hyponatremia, and FBDSs. LGI1 antibodies are present in the serum and/or cerebrospinal fluid, but their production is sensitive to immunosuppressants, and this disease has a relatively good prognosis. In particular, we should be aware of the possibility of anti-LGI1 antibody-associated encephalitis in adolescents with sleep disorders to avoid missed diagnoses and misdiagnoses.
基金Supported by the Key Research and Development Program of Anhui Province,No.202104J07020029.
文摘BACKGROUND F-box and leucine-rich repeat 6(FBXL6)have reportedly been associated with several cancer types.However,the role and mechanisms of FBXL6 in gastric cancer(GC)require further elucidation.AIM To investigate the effect of FBXL6 in GC tissues and cells and the underlying mechanisms.METHODS TCGA and GEO database analysis was performed to evaluate the expression of FBXL6 in GC tissues and adjacent normal tissues.Reverse transcription-quantitative polymerase chain reaction,immunofluorescence,and western blotting were used to detect the expression of FBXL6 in GC tissue and cell lines.Cell clone formation,5-ethynyl-2’-deoxyuridine(EdU)assays,CCK-8,transwell migration assay,and wound healing assays were performed to evaluate the malignant biological behavior in GC cell lines after transfection with FBXL6-shRNA and the overexpression of FBXL6 plasmids.Furthermore,in vivo tumor assays were performed to prove whether FBXL6 promoted cell proliferation in vivo.RESULTS FBXL6 expression was upregulated more in tumor tissues than in adjacent normal tissues and positively associated with clinicopathological characteristics.The outcomes of CCK-8,clone formation,and Edu assays demonstrated that FBXL6 knockdown inhibited cell proliferation,whereas upregulation of FBXL6 promoted proliferation in GC cells.Additionally,the transwell migration assay revealed that FBXL6 knockdown suppressed migration and invasion,whereas the overex pression of FBXL6 showed the opposite results.Through the subcutaneous tumor implantation assay,it was evident that the knockdown of FBXL6 inhibited GC graft tumor growth in vivo.Western blotting showed that the effects of FBXL6 on the expression of the proteins associated with the epithelial-mesenchymal transition-associated proteins in GC cells.CONCLUSION Silencing of FBXL6 inactivated the EMT pathway to suppress GC malignancy in vitro.FBXL6 can potentially be used for the diagnosis and targeted therapy of patients with GC.
文摘BACKGROUND It has been suggested that serum leucine-richα-2 glycoprotein(LRG)could be a novel monitoring biomarker for the assessment of disease activity in inflammatory bowel disease.In particular,the relationship between LRG levels and the endoscopically assessed activity of ulcerative colitis(UC)has become a matter of interest.AIM To clarify appropriate LRG cut-off values for the prediction of endoscopic and histologic remission in Japanese patients with UC.METHODS This was a cross-sectional,single-center,observational study of Japanese patients with UC.Among 213 patients with UC,in whom LRG was measured from September 2020 to February 2022,we recruited 30 patients for whom a total colonoscopy and measurements of LRG and C-reactive protein(CRP)were performed on the same day.We retrospectively analyzed correlations between the LRG and CRP levels and endoscopic indices,including the Mayo endoscopic subscore and UC endoscopic index of severity.RESULTS Correlations between the LRG values and the Mayo endoscopic subscore or UC endoscopic index of severity were significant(r=0.754,P<0.0001;r=0.778,P<0.0001,respectively).There were also significant correlations between CRP levels and Mayo endoscopic subscore or UC endoscopic index of severity(r=0.599,P=0.0005;r=0.563,P=0.0012,respectively),although the correlation coefficients were higher for LRG.The LRG cutoff value for predicting endoscopic remission was 13.4μg/mL for a Mayo endoscopic subscore of 0[area under the curve(AUC):0.871;95%confidence interval(CI):0.744-0.998],and 13.4μg/mL for an UC endoscopic index of severity of 0 or 1(AUC:0.904;95%CI:0.792-1.000).CONCLUSION LRG may be a surrogate marker for endoscopic activity in UC,with a cut-off value of around 13.4μg/mL for endoscopically inactive disease.
基金We thank Professor McGuckin M(MMRI,Brisbane)for providing human colon cancer cell lines(Caco-2,LoVo,and SW480)Dr.Rolfe B(AIBN,Brisbane)for providing mouse NSC-34 cells.
文摘BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeatcontaining G protein-coupled receptor 5(LGR5)-expressing stem cells,these socalled“tumour-initiating”cells,reminiscent in their properties of the normal intestinal stem cells(ISCs),may explain the apparent heterogeneity of colon cancer cell lines.Also,colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5.Furthermore,in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines(Caco-2 and LoVo).These cells were then characterized according to their proliferation capacity,gene expression signatures of ISC markers,and their tumorigenic properties in vivo and in vitro.RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2,but not from the LoVo,cell lines,in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog(+4 crypt)stem cells.This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny.Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein(EYFP)from this cell line exhibit tumorigenic-like properties in vivo and in vitro.In contrast,cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties.Thus,cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells,but not LoVo cells.CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines.Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data.We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.
基金supported by National Institutes of Health grant AR066831-01ASBMR GAP grant to Weirong R Xingsupported by a senior research career scientist award from the Department of Veteran’s Affairs
文摘Leucine-rich repeat kinase 1 (LRRK1) plays a critical role in regulating cytoskeletal organization, osteoclast activity, and bone resorption with little effect on bone formation parameters. Deficiency of Lrrk1 in mice causes a severe osteopetrosis in the metaphysis of the long bones and vertebrae bones, which makes LRRK1 an attractive alternative drug target for the treatment of osteoporosis and other high-turnover bone diseases.This review summarizes recent advances on the functions of the Lrrk1-related family members, Lrrk1deficiency-induced skeletal phenotypes, LRRK1 structure–function, potential biological substrates and interacting proteins, and the mechanisms of LRRK1 action in osteoclasts.
基金Supported by Sanming Project of Shenzhen,No.SZSM201612041Shenzhen Science and Technology Innovation Commission Project,No.GJHZ20180420180754917 and No.ZDSYS20190902092855097Postdoctoral Science Foundation of China,No.2018M633095.
文摘BACKGROUND Cancer stem cells(CSCs)are a subpopulation of cancer cells with the potential of self-renewal and differentiation.CSCs play critical roles in tumorigenesis,recurrence,metastasis,radiation tolerance and chemoresistance.AIM To assess the expression patterns and clinical potential of doublecortin-like kinase 1(DCLK1)and leucine-rich repeat-containing G-protein-coupled receptor 5(Lgr5),as prognostic CSC markers of colorectal cancer(CRC).METHODS The expression of DCLK1 and Lgr5 in CRC tissue sections from 92 patients was determined by immunohistochemistry.Each case was evaluated using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-3).The final staining score was calculated as the intensity score multiplied by the proportion score.Low expression of DCLK1 and Lgr5 was defined as a score of 0-3;high expression of DCLK1 and Lgr5 was defined as a score of≥4.Specimens were categorized as either high or low expression,and the correlation between the expression of DCLK1 or Lgr5 and clinicopathological factors was investigated.RESULTS DCLK1 and Lgr5 expression levels were significantly positively correlated.CRC patients with high DCLK1,Lgr5 and DCLK1/Lgr5 expressions had poorer progression-free survival and overall survival.Moreover,high expression of DCLK1 was an independent prognostic factor for recurrence and overall survival in patients with CRC by multivariate analysis(P=0.026 and P=0.049,respectively).CONCLUSION DCLK1 may be a potential CSC marker for the recurrence and survival of CRC patients.
文摘Leucine rich repeats (LRRs) are present in over 14,000 proteins that have been identified in viruses, bacteria, archaea, and eukaryotes. Two to sixty-two LRRs occur in tandem forming an overall arc shaped domain. There are eight classes of LRRs. Plant specific LRRs (class: PS-LRR) had previously been recognized in only plant proteins. However, we find that PS-LRRs are also present in proteins from bacteria. We investigated the origin of bacterial PS-LRR domains. PSLRR proteins are widely distributed in most plants;they are found in only a few bacterial species. There are no PS-LRR proteins from archaea. Bacterial PS-LRRs in twenty proteins from eleven bacterial species (in the three phyla: Proteobacteria, Cyanobacteria, and Bacteroidetes) are significantly more similar to the PS-LRR class than to the other seven classes of LRR proteins. Not only amino acid sequences but also nucleotide sequences of the bacterial PS-LRR domains show highly significant similarity with those of many plant proteins. The program, EGID (Ensemble algorithm for Genomic Island Detection), predicts that Synechococcus sp. CYA_ 1022 came from another organism. Four bacterial PS-LRR proteins contain AhpC-TSA, IgA peptidase M64, the immunoglobulin domain, the Calx-b domain, and the He_PIG domain;these domains show no similarity with any eukaryotic (plant) proteins, in contrast to the similarities of their respective PS-LRRs. The present results indicate that horizontal gene transfer (HGT) of genes/gene fragments encoding PS-LRR domains occurred between bacteria and plants, and HGT among the eleven bacterial species, of the three phyla, as opposed to descent from a common ancestor. There is the possibility of the occurrence of one HGT event from plant to bacteria. A series of HGTs might then have occurred recently and rapidly among these eleven species of bacteria.
基金a Ph D fellowship by FCT-Fundacao para a Ciência Tecnologia (SFRH/BD/135868/2018)(to SSC)。
文摘Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.
基金supported by the National Natural Science Foundation of China(Grant No.32072049)the Central Public-Interest Scientific Institution Basal Research Fund, China(Grant No.CPSIBRF-CNRRI-202203)。
文摘Identification of immunity-associated leucine-rich repeat receptor-like protein kinases(LRR-RLK) is critical to elucidate the LRR-RLK mediated mechanism of plant immunity.Here,we reported the map-based cloning of a novel rice SPOTTED-LEAF 41(Os SPL41) encoding a putative LRR-RLK protein(Os LRR-RLK41/Os SPL41) that regulated disease responses to the bacterial blight pathogen Xanthomonas oryzae pv.oryzae(Xoo).An 8-bp insertion at position 865 bp in a mutant spotted-leaf 41(spl41) allele led to the formation of purple-brown lesions on leaves.Functional complementation by the wild type allele(Os SPL41) can rescue the mutant phenotype,and the complementary lines showed similar performance to wild type in a number of agronomic,physiological and molecular indices.Os SPL41 was constitutively expressed in all tissues tested,and Os SPL41 contains a typical transmembrane domain critical for its localization to the cell membrane.The mutant exhibited an enhanced level of resistance to Xoo in companion of markedly up-regulated expression of pathogenesis-related genes such as Os PR10a,Os PAL1 and Os NPR1,while the level of salicylic acid was significantly increased in spl41.In contrast,the over-expression lines exhibited a reduced level of H_(2)O_(2) and were much susceptible to Xoo with down-regulated expression of pathogenesis-related genes.These results suggested that Os SPL41 might negatively regulate plant immunity through the salicylic acid signaling pathway in rice.
基金funded by the National Key R&D Program of China(2022YFD1901300)the National Natural Science Foundation of China(31901932)+2 种基金the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-ZDRW202308 and Y2022PT12)the Beijing Innovation Consortium of Agriculture Research System(BAIC04-2022)the Science and Technology Programs of the Zunyi Tobacco(2021XM03)。
文摘The multifunctional secondary metabolites known as cyclic lipopeptides(CLPs),which are produced by a large variety of bacteria,have become a key category of plant immunity elicitors.Pseudomonas-CLPs(PsCLPs)are extremely diverse in structure and biological activity.However,an understanding of CLP-plant structure–function interactions currently remains elusive.Here,we identify medpeptin,a novel CLP from Pseudomonas mediterranea that consists of 22 amino acids.Medpeptin is synthesized by a non-ribosomal peptide synthase(NRPS)gene cluster and regulated by a quorum-sensing system.Further research indicates that medpeptin does not exhibit antimicrobial activity;instead,it induces plant cell death immunity and confers resistance to bacterial infection.Comparative transcriptome analysis and virus-induced gene silencing(VIGS)reveal a set of immune signaling candidates involved in medpeptin perception.Silencing of a cell-wall leucine-rich repeat extensin protein(NbLRX3)or a receptor-like protein kinase(NbRLK25)—but not BAK1 or SGT1—compromises medpeptin-triggered cell death and resistance to pathogen infection in Nicotiana benthamiana.Our findings point to a noncanonical mechanism of CLP sensing and suggest perspectives for the development of plant disease resistance.
基金Supported by A grant from the Beijing Municipal Science and Technology Commission’s NOVA Program,No.2009BG-02Beijing Municipal Health System Special funds of High-Level Medical Personnel Construction,No.2013-3-082
文摘AIM:To investigate the differential expression of leu-cine-rich repeat-containing G protein-coupled receptor5(LGR5)in gastric cancer tissues and its significance related to tumor growth and spread.METHODS:Formalin-fixed biopsy specimens of intestinal metaplasia(n=90),dysplasia(n=53),gastric adenocarcinoma(n=180),metastases in lymph nodes and the liver(n=15),and lesion-adjacent normal gastric mucosa(controls;n=145)were obtained for analysis from the Peking University Cancer Hospital’s Department of Pathology and Gastrointestinal Surgery tissue archives(January 2003 to December 2011).The biopsied patients’demographic and clinicopathologic data were retrieved from the hospital’s medical records database.Each specimen was subjected to histopathological typing to classify the tumor node metastasis(TNM)stage and to immunohistochemistry staining to detect the expression of the cancer stem cell marker LGR5.The intergroup differences in LGR5 expression were assessed by Spearman’s rank correlation analysis,and the relationship between LGR5 expression level and the patients’clinicopathological characteristics was evaluated by theχ2test or Fisher’s exact test.RESULTS:Significantly more gastric cancer tissues showed LGR5+staining than normal control tissues(all P<0.01),with immunoreactivity detected in 72.2%(65/90)and 50.9%(27/53)of intestinal metaplasia and dysplasia specimens,respectively,52.8%(95/180)of gastric adenocarcinoma specimens,and 73.3%%(11/15)of metastasis specimens,but 26.9%(39/145)of lesion-adjacent normal gastric mucosa specimens.Comparison of the intensity of LGR5+staining showed an increasing trend that generally followed increasing dedifferentiation and tumor spread(normal tissue<dysplasia,<gastric adenocarcinoma<metastasis;all P<0.001),with the exception of expression level detected in intestinal metaplasia which was higher than that in normal gastric tissues(P<0.001).Moreover,gastric cancer-associated enhanced expression of LGR5 was found to be signifcantly associated with age,tumor differentiation,Lauren type and TNM stage(Ⅰ+ⅡvsⅢ+Ⅳ)(all P<0.05),but not with sex,tumor site,location,size,histology,lymphovascular invasion,depth of invasion,lymph node metastasis or distant metastasis.Patients with LGR5+gastric cancer specimens and without signs of metastasis from the original biopsy experienced more frequent rates of recurrence or metastasis during follow-up than patients with LGR5-specimens(P<0.05).CONCLUSION:Enhanced LGR5 is related to progressive dedifferentiation and metastasis of gastric cancer,indicating the potential of this receptor as an early diagnostic and prognostic biomarker.
基金the Natural Science Foundation of Jiangsu Province(Youth Program),No.BK20190129National Scientific Program of Jiangsu Colleges and Universities of China,No.19KJB320012(both to LY)。
文摘Obstructive sleep apnea can worsen the prognosis of subarachnoid hemorrhage.Howeve r,the underlying mechanism remains unclear.In this study,we established a mouse model of subarachnoid hemorrhage using the endovascular perforation method and exposed the mice to intermittent hypoxia for 8 hours daily for 2 consecutive days to simulate sleep apnea.We found that sleep apnea aggravated brain edema,increased hippocampal neuron apoptosis,and worsened neurological function in this mouse model of subarachnoid hemorrhage.Then,we established an in vitro HT-22 cell model of hemin-induced subarachnoid hemorrhage/intermittent hypoxia and found that the cells died,and lactate dehydrogenase release increased,after 48 hours.We further investigated the underlying mechanism and found that sleep apnea increased the expression of hippocampal neuroinflammatory factors interleukin-1β,interleukin-18,inte rleukin-6,nuclear factorκB,pyro ptosis-related protein caspase-1,pro-caspase-1,and NLRP3,promoted the prolife ration of astrocytes,and increased the expression of hypoxia-inducible factor 1αand apoptosis-associated speck-like protein containing a CARD,which are the key proteins in the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.We also found that knockdown of hypoxia-inducible factor 1αexpression in vitro greatly reduced the damage to HY22 cells.These findings suggest that sleep apnea aggravates early brain injury after subarachnoid hemorrhage by aggravating neuroinflammation and pyroptosis,at least in part through the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.
基金Supported by the National Natural Science Foundation of China(No.81670828)the Shandong Provincial Key Research and Development Program(No.2017GSF18141).
文摘AIM:To investigate the potential effect and mechanism of leucine-richα-2-glycoprotein-1(LRG1)on corneal angiogenesis and lymphangiogenesis.METHODS:Corneal neovascularization and lymphatics were induced by establishing alkali burn mouse model.Immunofluorescence staining was performed to detect the location of LRG1 in cornea tissues and to verify the source of LRG1-positive cells.Corneal whole-mount staining for CD31(a panendothelial cell marker)and lymphatic endothelial hyluronan receptor-1(LYVE-1;lymphatic marker)was performed to detect the growth of blood and lymphatic vessels after local application of exogenous LRG1 protein or LRG1 si RNA.In addition,expressions of the proangiogenic vascular endothelial growth factor(VEGF)related proteins were detected using Western blot analysis.RESULTS:LRG1 was dramatically increased in alkali burned corneal stroma in both the limbal and central areas.LRG1-positive cells in the corneal stroma were mainly derived from Vimentin-positive cells.Local application ofexogenous LRG1 protein not only aggravated angiogenesis but also lymphangiogenesis significantly(P<0.01).LRG1 group upregulated the levels of VEGF and the vascular endothelial growth factor receptor(VEGFR)family when compared with the phosphate-buffered saline(PBS)control group.We also found that LRG1-specific si RNA could suppress corneal angiogenesis and lymphangiogenesis when compared with the scramble si RNA-treated group(P<0.01).CONCLUSION:LRG1 can facilitate corneal angiogenesis and lymphangiogenesis through heightening the stromal expression of VEGF-A,B,C,D and VEGFR-1,2,3;LRG1-specific si RNA can suppress corneal angiogenesis and lymphangiogenesis in corneal alkali burn mice.
基金the National Natura Science Foundation of China(NSFC)(No.81401051,No 81671051,and No.81501107).
文摘The G2019S mutation of the leucine-rich repeat kinase 2(LRRK2)is the most common genetic cause of Parkinson's disease (PD).However,the molecular mechanisms of LRRK2 mutation contributing to the onset and progression of PD have not been fully illustrated.We generated HEK293 cells stably transfected with α-synuclein and investigated the effect of LRRK2 G2019S mutation on the degradation of α-synuclein.The lysosomal activity was assessed by the protein degradation of glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A.It was found that α-synuclein was mainly degraded in lysosomes.LRRK2G2019S inhibited the degradation of α-synuclein,and promoted its aggregation.LRRK2G 2019S also decreased the activities of lysosomal enzymes including cathepsin B and cathepsin L.Furthermore,the inhibitory effect of LRRK2 G2019S on lysosomal functions did not depend on its kinase activity.These findings indicated that the inhibitory effect of LRRK2 G2019S on α-synuclein degradation could underlie the pathogenesis of aberrant α-synuclein aggregation in PD with LRRK2 mutation.
文摘BACKGROUND Patients with mesenteric ischemia frequently suffer from bowel necrosis even after revascularization.Hydrogen gas has showed promising effects for ischemiareperfusion injury by reducing reactive oxygen species in various animal and clinical studies.We examined intestinal tissue injury by ischemia and reperfusion under continuous initiation of 3%hydrogen gas.AIM To clarify the treatment effects and target cells of hydrogen gas for mesenteric ischemia.METHODS Three rat groups underwent 60-min mesenteric artery occlusion(ischemia),60-min reperfusion following 60-min occlusion(reperfusion),or ischemiareperfusion with the same duration under continuous 3%hydrogen gas inhalation(hydrogen).The distal ileum was harvested.Immunofluorescence staining with caspase-3 and leucine-rich repeat-containing G-protein-coupled 5(LGR5),a specific marker of intestinal stem cell,was conducted to evaluate the injury location and cell types protected by hydrogen.mRNA expressions of LGR5,olfactomedin 4(OLFM4),hairy and enhancer of split 1,Jagged 2,and Neurogenic locus notch homolog protein 1 were measured by quantitative polymerase chain reaction.Tissue oxidative stress was analyzed with immunostaining for 8-hydroxy-2'-deoxyguanosine(8-OHdG).Systemic oxidative stress was evaluated by plasma 8-OHdG.RESULTS Ischemia damaged the epithelial layer at the tip of the villi,whereas reperfusion induced extensive apoptosis of the cells at the crypt base,which were identified as intestinal stem cells with double immunofluorescence stain.Hydrogen mitigated such apoptosis at the crypt base,and the LGR5 expression of the tissues was higher in the hydrogen group than in the reperfusion group.OLFM4 was also relatively higher in the hydrogen group,whereas other measured RNAs were comparable between the groups.8-OHdG concentration was high in the reperfusion group,which was reduced by hydrogen,particularly at the crypt base.Serum 8-OHdG concentrations were relatively higher in both reperfusion and hydrogen groups without significance.CONCLUSION This study demonstrated that hydrogen gas inhalation preserves intestinal stem cells and mitigates oxidative stress caused by mesenteric ischemia and reperfusion.
基金Supported by the grants of the National Natural Science Foundation of China(No.30973073 and 81172402)
文摘Objective Leucine-rich repeats and immunoglobulin-like domains 1(LRIG1) is a newly identified human gene that inhibits the epidermal growth factor receptor(EGFR), which on combining with a ligand, can drive tumor growth. This study investigated the interaction between human LRIG1 and EGFR and attempted to delineate the functions of as well as the mechanisms used by the extracellular(ECD) and cytoplasmic(CPD) domains of the human LRIG1 protein to downregulate human EGFR signaling activity.Methods Two constructed chimeric eukaryotic expression vectors, pIRES2-EGFP-3XFLAG-LRIG1-ET and p3FLAG-LRIG1-TC, encoding the extracellular and transmembrane regions(LRIG1-ET) and the transmembrane and cytoplasmic regions(LRIG1-TC), respectively, and the plasmid p3XFLAG-CMV-9-LRIG1 encoding full-length LRIG1(LRIG1-FL) were transfected into the human glioma cell line U251 or primary astrocytoma cells by using liposomes. The number and affinity of cell surface EGFR on transfected cells was determined by ^(125)I-EGF binding assay. Results The dissociation constant(KD) values for EGFR were higher, and the maximum increase was observed in the cells transfected into LRIG1-ET(1.36 folds). The number of maximal binding sites(Bmax) of the receptors was decreased in all transfected cells; the maximum decrease was noted in the cells transfected into LRIG1-FL(40.05%).Conclusion Both the ECD and CPD of LRIG1 are important to negate EGFR signaling. The ECD may interfere with the binding between EGFR and its ligand and facilitate the functions of CPD. The CPD may, when brought in proximity to EGFR, enhance receptor degradation. These two mechanisms can contribute to the downregulation of EGFR-mediated signaling by LRIG1.
基金supported by the National Natural Science Foundation of China(32100155 and 32072507)the Natural Science Foundation of Jiangsu Province(BK20221000)+1 种基金the fellowship of China Postdoctoral Science Foundation(2021M701740)the Jiangsu Funding Program for Excellent Postdoctoral Talent(2022ZB343 and 2022ZB768)。
文摘Cell-surface-localized leucine-rich-repeat receptorlike kinases(LRR-RLKs)are crucial for plant immunity.Most LRR-RLKs that act as receptors directly recognize ligands via a large extracellular domain(ECD),whereas LRR-RLK that serve as regulators are relatively small and contain fewer LRRs.Here,we identified LRR-RLK regulators using high-throughput tobacco rattle virus(TRV)-based gene silencing in the model plant Nicotiana benthamiana.We used the cell-death phenotype caused by INF1,an oomycete elicitin that induces pattern-triggered immunity,as an indicator.By screening 33 small LRR-RLKs(≤6 LRRs)of unknown function,we identified ELICITIN INSENSITIVE RLK 1(NbEIR1)as a positive regulator of INF1-induced immunity and oomycete resistance.Nicotiana benthamiana mutants of eir1 generated by CRISPR/Cas9-editing showed significantly compromised immune responses to INF1 and were more vulnerable to the oomycete pathogen Phytophthora capsici.NbEIR1 associates with BRI1-ASSOCIATED RECEPTOR KINASE 1(NbBAK1)and a downstream component,BRASSINOSTEROIDSIGNALING KINASE 1(NbBSK1).NbBSK1 also contributes to INF1-induced defense and P.capsici resistance.Upon INF1 treatment,NbEIR1 was released from NbBAK1 and NbBSK1 in vivo.Moreover,the silencing of NbBSK1 compromised the association of NbEIR1 with NbBAK1.We also showed that NbEIR1 regulates flg22-induced immunity and associates with its receptor,FLAGELLIN SENSING 2(NbFLS2).Collectively,our results suggest that NbEIR1 is a novel regulatory element for BAK1-dependent immunity.NbBSK1-NbEIR1 association is required for maintaining the NbEIR1/NbBAK1 complex in the resting state.
基金supported in part by the National Institute of General Medical Science of the National Institutes of Health(Grant No.R01GM068523).
文摘Large-conductance Ca^(2+)-activated K+channels(BK channels)constitute an key physiological link between cellular Ca^(2+) signaling and electrical signaling at the plasma membrane.Thus these channels are critical to the control of action potential firing and neurotrans-mitter release in several types of neurons,as well as the dynamic control of smooth muscle tone in resis-tance arteries,airway,and bladder.Recent advances in our understanding of K^(+) channel structure and function have led to new insight toward the molecular mecha-nisms of opening and closing(gating)of these chan-nels.Here we will focus on mechanisms of BK channel gating by Ca^(2+),transmembrane voltage,and auxiliary subunit proteins.
基金the National Natural Science Foundation, China (31630062, 31801705 and 31870143)the National Program on Key Basic Research Project, China (973 Program, 2014CB138400)+4 种基金the Youth Talent Support Program, China and Distinguished Professor of Jiangsu Province, China to X.T.the Natural Science Foundation of Jiangsu Province, China (BK20180532)the Postdoctoral Science Foundation, China (2018M642269) to J.L.the National Science Foundation grants, United States (NSF-IOS-1354434 and NSF-IOS-1339185) to S.P.D.-K. The materials of tomato wild species were obtained from the UC Davis/C.Mmaintained by the Department of Plant Sciences, University of California, Davis, CA.
文摘Plant intracellular nucleotide binding leucine-rich repeat (NLR) immune receptors play critical roles in pathoge n surveillance. Most plant NLRs characterized so far were found to use a single domain/sensor to recognize pathogen effectors. Here we report that the Sw-5b NLR immune receptor uses two distinct domains to detect the viral movement protein NSm encoded by tospovirus. In addition to its leucine-rich repeat (LRR) domain that has been previously reported, the N-terminal Solanaceae domain (SD) of Sw- 5b also interacts with NSm and a conserved 21-amino-acid region of NSm (NSm^21). The specific interaction between Sw-5b SD and NSm is required for releasing the inhibitory effect of coiled-coil domain on the NBARC- LRR region. Furthermore, we found that the binding of NSm affects the nucleotide binding activity of the NB-ARC-LRR in vitro, while Sw-5b NB-ARC-LRR is activated only when NSm and NSm^21 levels are high. Interestingly, Sw-5b SD could significantly enhanee the ability of the NB-ARC-LRR to detect low levels of NSm effector and facilitate its activation and induction of defense response. An Sw-5b SD mutant that is disrupted in NSm recognition failed to enhance the ability of the NB-ARC-LRR to sense low levels of NSm and NSm^21 . Taken together, our results suggest that Sw-5b SD functions as an extra sensor and the NB-ARC-LRR as an activator, and that Sw-5b NLR adopts a two-step recog nition mechanism to enhance viral effector perception.
基金the State Key Program of National Natural Science of China(No.31130063)the National Basic Research Program(973 Program)(No.2010CB835300).
文摘Brassinosteroids,a group of plant steroid hormones,reg-ulate many aspects of plant growth and development.We and other have previously solved the crystal structures of BRI1(LRR)in complex with brassinolide,the most active brassinosteroid identifi ed thus far.Although these studies provide a structural basis for the recognition of brassi-nolide by its receptor BRI1,it still remains poorly under-stood how the hormone differentiates among its con-served receptors.Here we present the crystal structure of the BRI1 homolog BRL1 in complex with brassinolide.The structure shows that subtle differences around the brassinolide binding site can generate a striking effect on its recognition by the BRI1 family of receptors.Structural comparison of BRL1 and BRI1 in their brassinolide-bound forms reveals the molecular basis for differential binding of brassinolide to its different receptors,which can be used for more effi cient design of plant growth regulators for agricultural practice.On the basis of our structural studies and others’data,we also suggest possible mech-anisms for the activation of BRI1 family receptors.
