Ligand recognition and G protein coupling of the human itch receptor MRGPRX1.

MRGPRX1, a Mas-related GPCR (MRGPR), is a key receptor for itch perception and targeting MRGPRX1 may have potential to treat both chronic itch and pain. Here we report cryo-EM structures of the MRGPRX1-Gi1 and MRGPRX1-Gq trimers in complex with two peptide ligands, BAM8-22 and CNF-Tx2. These structures reveal a shallow orthosteric pocket and its conformational plasticity for sensing multiple different peptidic itch allergens. Distinct from MRGPRX2, MRGPRX1 contains a unique pocket feature at the extracellular ends of TM3 and TM4 to accommodate the peptide C-terminal "RF/RY" motif, which could serve as key mechanisms for peptidic allergen recognition. Below the ligand binding pocket, the G6.48XP6.50F6.51G6.52X(2)F/W6.55 motif is essential for the inward tilting of the upper end of TM6 to induce receptor activation. Moreover, structural features inside the ligand pocket and on the cytoplasmic side of MRGPRX1 are identified as key elements for both Gi and Gq signaling. Collectively, our studies provide structural insights into understanding itch sensation, MRGPRX1 activation, and downstream G protein signaling.

Characteristics of intestinal microbiota in children with idiopathic short stature: a cross-sectional study.

Idiopathic short stature (ISS) accounts for more than 70% of childhood short stature cases, with an undefined etiology and pathogenesis, leading to limited treatment. However, recent studies have shown that intestinal microbiota may be associated with ISS. This study aimed to characterize the intestinal microbiota in children with ISS, effect of treatment with growth hormones, and association between specific bacterial species and ISS. This study enrolled 55 children, comprising 40 diagnosed with ISS at Jinhua Hospital, Zhejiang University, and 15 healthy controls. The subjects with ISS were divided into the untreated ISS group (UISS group, 22 children who had not been treated with recombinant human growth hormone [rhGH]), treated ISS group (TISS group, 18 children treated with rhGH for 1 year), and control group (NC group, 15 healthy children). High-throughput sequencing was used to determine the intestinal microbiota characteristics. Higher abundances of Bacteroides, Prevotella, Alistipes, Parabacteroides, Agathobacter and Roseburia were found in the UISS and TISS groups than in the control group, whereas Bifidobacterium, Subdoligranulum, and Romboutsia were less abundant. The composition of intestinal microbiota in the UISS and TISS groups was almost identical, except for Prevotella. The TISS group had significantly lower levels of Prevotella than did the UISS group, which were closer to those of the NC group. Receiver operating characteristic curve analysis revealed that the abundances of Prevotella, Bifidobacterium, Bacteroides, and Subdoligranulum were effective in differentiating between the UISS and NC groups. CONCLUSION: Alterations in intestinal microbiota may be associated with ISS. Specific bacterial species, such as Prevotella, may be potential diagnostic markers for ISS. WHAT IS KNOWN: • ISS is associated with the GH-IGF-1 axis. • Recent studies indicated an association between the GH-IGF-1 axis and intestinal microbiota. WHAT IS NEW: • Children with ISS showed alterations in intestinal microbiota, with a relative increase in the abundance of gut inflammation-related bacteria. • The relative abundances of Prevotella, Bacteroides, Bifidobacterium, and Subdoligranulum may serve as potential diagnostic markers.

Machine learning approach to screen new diagnostic features of adamantinomatous craniopharyngioma and explore personalised treatment strategies.

Background: Adamantinoma craniopharyngioma (ACP) is a non-malignant tumour of unknown pathogenesis that frequently occurs in children and has malignant potential. The main treatment options are currently surgical resection and radiotherapy. These treatments can lead to serious complications that greatly affect the overall survival and quality of life of patients. It is therefore important to use bioinformatics to explore the mechanisms of ACP development and progression and to identify new molecules. Methods: Sequencing data of ACP was downloaded from the comprehensive gene expression database for differentially expressed gene identification and visualized by Gene Ontology, Kyoto Gene, and gene set enrichment analyses (GSEAs). Weighted correlation network analysis was used to identify the genes most strongly associated with ACP. GSE94349 was used as the training set and five diagnostic markers were screened using machine learning algorithms to assess diagnostic accuracy using receiver operating characteristic (ROC) curves, while GSE68015 was used as the validation set for verification. Results: Type I cytoskeletal 15 (KRT15), Follicular dendritic cell secreted peptide (FDCSP), Rho-related GTP-binding protein RhoC (RHOC), Modulates negatively TGFB1 signaling in keratinocytes (CD109), and type II cytoskeletal 6A (KRT6A) (area under their receiver operating characteristic curves is 1 for both the training and validation sets), Nomograms constructed using these five markers can predict progression of ACP patients. Whereas ACP tissues with activated T-cell surface glycoprotein CD4, Gamma delta T cells, eosinophils and regulatory T cells were expressed at higher levels than in normal tissues, which may contribute to the pathogenesis of ACP. According to the analysis of the CellMiner database (Tumor cell and drug related database tools), high CD109 levels showed significant drug sensitivity to Dexrazoxane, which has the potential to be a therapeutic agent for ACP. Conclusions: Our findings extend understandings of the molecular immune mechanisms of ACP and suggest possible biomarkers for the targeted and precise treatment of ACP.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

Novel prognostic features and personalized treatment strategies for mitochondria-related genes in glioma patients.

Background: Gliomas are the most common intracranial nervous system tumours that are highly malignant and aggressive, and mitochondria are an important marker of metabolic reprogramming of tumour cells, the prognosis of which cannot be accurately predicted by current histopathology. Therefore, Identify a mitochondrial gene with immune-related features that could be used to predict the prognosis of glioma patients. Methods: Gliomas data were downloaded from the TCGA database and mitochondrial-associated genes were obtained from the MITOCARTA 3.0 dataset. The CGGA, kamoun and gravendeel databases were used as external datasets. LASSO(Least absolute shrinkage and selection operator) regression was applied to identify prognostic features, and area and nomograms under the ROC(Receiver Operating Characteristic) curve were used to assess the robustness of the model. Single sample genomic enrichment analysis (ssGSEA) was employed to explore the relationship between model genes and immune infiltration, and drug sensitivity was used to identify targeting drugs. Cellular studies were then performed to demonstrate drug killing against tumours. Results: COX assembly mitochondrial protein homolog (CMC1), Cytochrome c oxidase protein 20 homolog (COX20) and Cytochrome b-c1 complex subunit 7 (UQCRB) were identified as prognostic key genes in glioma, with UQCRB, CMC1 progressively increasing and COX20 progressively decreasing with decreasing risk scores. ROC curve analysis of the TCGA training set model yielded AUC (Area Under The Curve) values >0.8 for 1-, 2- and 3-year survival, and the model was associated with both CD8+ T cells and immune checkpoints. Finally, using cellMiner database and molecular docking, it was confirmed that UQCRB binds covalently to Amonafide via lysine at position 78 and threonine at position 82, while cellular assays showed that Amonafide inhibits glioma migration and invasion. Conclusion: Our three mitochondrial genomic composition-related features accurately predict Survival in glioma patients, and we also provide glioma chemotherapeutic agents that may be mitochondria-related targets.

Machine learning screening for Parkinson's disease-related cuproptosis-related typing development and validation and exploration of personalized drugs for cuproptosis genes.

Background: Parkinson's disease (PD) is a common, degenerative disease of the nervous system that is characterized by the death of dopaminergic neurons in the substantia nigra densa (SNpc). There is growing evidence that copper (Cu) is involved in myelin formation and is involved in cell death through modulation of synaptic activity as well as neurotrophic factor-induced excitotoxicity. Methods: This study aimed to explore potential cuproptosis-related genes (CRGs) and immune infiltration patterns in PD and the development of Cu chelators relevant for PD treatment. The PD datasets GSE7621, GSE20141, and GSE49036 were downloaded from the Gene Expression Omnibus (GEO) database. The consensus clustering method was used to classify the specimens of PD. Using weighted gene co-expression network analysis (WGCNA) and random forest (RF) tree model, support vector machine (SVM) learning model, extreme gradient boosting (XGBoost) model, and general linear model (GLM) algorithms to screen disease progression-related models, the column charts were created to verify the accuracy of these CRGs in predicting PD progression. Single sample genomic enrichment analysis (ssGSEA) was used to estimate the correlation between genes associated with copper poisoning and genes associated with immune cells and immune function. Molecular docking was used to verify interactions with copper chelating agents associated with cuproptosis for PD treatment. Results: Through ssGSEA, we identified three copper poisoning related genes ATP7A, NFE2L2 and MTF1, which are related to immune cells in PD. We also verified that LAGASCATRIOL can bind to NFE2L2 through molecular docking. Consistent cluster analysis identified two subtypes, among which C2 subtype was just enriched in PD. And to more accurately diagnose PD progression, patients can benefit from a feature map based on these genes. Conclusions: CRGs such as NFE2L2, MTF1, and ATP7B were identified to be associated with the pathogenesis of PD and provide a possible new direction for the treatment of PD, which needs further in-depth study.

A molecular mechanism of UDCA engagement with GPBAR and subsequent G protein interaction revealed by scattered alanine scanning.

As the most known therapeutic component of bear bile acids, ursodeoxycholic acid (UDCA) is an FDA-approved drug for the treatment of primary biliary cirrhosis (PBC), the dissolution of cholesterol gallstones. UDCA produces many beneficial effects on metabolism and immune responses via its interaction with the membrane G protein-coupled bile acid receptor (GPBAR); however, how UDCA interacts with GPBAR and its selective cellular effects remain elusive. In this study, we delineated the interaction of UDCA with GPBAR and activation mechanism of GPBAR by scattered alanine scanning and molecular docking. Our results indicated that transmembrane helix 2 (TM2), TM3, TM5 and TM6 of GPBAR contribute to the interaction of UDCA in GPBAR binding pocket. Moreover, we predicted that the engagement of the 3-OH of UDCA with phenolic oxygen of Y2406.51 in GPBAR plays a key role in GPBAR activation. Unexpectedly, in addition to the well-known roles of intracellular loop2 (ICL2) residues, we identified that ICL3 residues play an important role in G protein coupling to GPBAR in response to UDCA binding. Our study provides a preliminary molecular mechanism of how GPBAR recognizes UDCA and subsequent activation and G protein interaction, which may facilitate the development of new bile acid derivatives as therapeutics.

Structure, function and pharmacology of human itch receptor complexes.

In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals1,2. As an MRGPR, MRGPRX2 is known to sense basic secretagogues (agents that promote secretion) and is involved in itch signals and eliciting pseudoallergic reactions3-6. MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2-Gi1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2-Gi1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp6.48 to Gly6.48 (superscript annotations as per Ballesteros-Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the Gi trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions.

Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1-Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway.

Acute kidney injury (AKI) is a serious disease characterized by a rapid decline in kidney function. Oxidative stress is the primary pathogenesis of AKI. Salvianolic acid B (SalB), a water-soluble compound extracted from Salvia miltiorrhiza, possesses a potent antioxidant activity. Here, we investigated the protective effect of SalB against renal ischemia-reperfusion injury (I/R) in mice. Briefly, by analyzing renal function, oxidative stress markers and inflammatory biomarkers, we found that SalB could improve kidney damage, reduce oxidative stress and inflammatory factor levels. Interestingly, the expression of the NLR family pyrin domain-containing 3 (NLRP3), caspase-1, pyroptosis related proteins gasdermin D (GSDMD) and interleukin (IL)-1ß, which were significantly upregulated in the kidney tissues of I/R group, was effectively reversed by SalB. Meanwhile, renal tubular epithelial cells hypoxia and reoxygenation model was used to explore pyroptosis of caspase-1-dependent. Further mechanism study showed that the SalB pretreatment could promote the increase of nuclear factor erythroid-2 related factor 2 (Nrf2) nuclear accumulation, which significantly suppressed oxidative stress, proinflammatory cytokines, NLRP3 inflammasome activation and pyroptosis. These results indicate that SalB can inhibit caspase-1/GSDMD-mediated pyroptosis by activating Nrf2/NLRP3 signaling pathway, resulting in alleviating I/R injury in mice.

Zhen-Wu-Tang Protects IgA Nephropathy in Rats by Regulating Exosomes to Inhibit NF-κB/NLRP3 Pathway.

Immunoglobulin A nephropathy (IgAN) is one of the most frequent kinds of primary glomerulonephritis characterized by IgA immune complexes deposition and glomerular proliferation. Zhen-wu-tang (ZWT), a well-known traditional Chinese formula has been reported to ameliorate various kidney diseases. However, its pharmacological mechanism remains unclear. Exosomes have been described in diverse renal diseases by mediating cellular communication but rarely in the IgAN. The purpose of the present study is to explore whether the underlying mechanisms of the effect of ZWT on IgAN is correlated to exosomes. Our results demonstrated that in human renal tubular epithelial cells (HK-2) stimulated by lipopolysaccharide, exosomes are obviously released after ZWT-containing serum treatment especially with 10% ZWT. In addition, once released, HK-2-derived exosomes were uptaked by human mesangial cells (HMC), which impeded the activation of NF-κB/NLRP3 signaling pathway to exert anti-inflammatory effects in a lipopolysaccharide induced proliferation model. Moreover, IgAN rat model was established by bovine serum albumin, CCL4 mixed solution and LPS. We found that 10% ZWT could significantly promote the release of exosomes from HK-2 and inhibit HMC proliferation to improve inflammation. Thus HK-2-derived exosomes treated with 10% ZWT (ZWT-EXO) were administered to the rats by tail vein injection. Our results showed that ZWT-EXO decreased the levels of 24 h proteinuria, urinary erythrocyte, IgA deposition in glomerulus and renal pathological injury which ameliorated the kidney damage. In addition, ZWT was able to dramatically promote secretion of exosomes in renal tissues while blocked NF-κB nuclear translocation as well as activation of NLRP3 inflammasome, leading to the inhibition of IL-1ß and caspase-1. In conclusion, our study reveal that ZWT has protective effects on IgAN by regulating exosomes secretion to inhibit the activation of NF-κB/NLRP3 pathway, thereby attenuating the renal dysfunction. These findings may provide a new therapeutic target for the treatment of IgAN.

Integrated Fecal Microbiome and Serum Metabolomics Analysis Reveals Abnormal Changes in Rats with Immunoglobulin A Nephropathy and the Intervention Effect of Zhen Wu Tang.

Immunoglobulin A nephropathy (IgAN), an autoimmune renal disease with complicated pathogenesis, is one of the principal reasons for end-stage renal disease in the clinic. Evidence has linked apparent alterations in the components of the microbiome and metabolome to renal disease in rats. However, thus far, there is insufficient evidence that supports the potential relationship between gut microbiome, circulating metabolites, and IgAN. This study was designed to probe the effects of IgAN on intestinal microecology and metabolic phenotypes and to understand the possible underlying mechanisms. Fecal and serum samples were collected from IgAN rats. Composition of the gut microbiota and biochemical changes in the metabolites was analyzed using 16S rDNA sequencing and untargeted metabolomics. The IgAN rats exhibited renal insufficiency and increased concentration of 24-h urine protein, in addition to deposition of IgA and IgG immune complexes in the kidney tissues. There was a disturbance in the balance of gut microbiota in IgAN rats, which was remarkably associated with renal damage. Marked changes in microbial structure and function were accompanied by apparent alterations in 1,403 serum metabolites, associated with the disorder of energy, carbohydrate, and nucleotide metabolisms. Administration of Zhen Wu Tang ameliorated microbial dysbiosis and attenuated the renal damage. Besides, treatment with Zhen Wu Tang modulated the metabolic phenotype perturbation in case of gut microbiota dysbiosis in IgAN rats. In conclusion, these findings provided a comprehensive understanding of the potential relationship between the intestinal microbiota and metabolic phenotypes in rats with IgAN. Elucidation of the intestinal microbiota composition and metabolic signature alterations could identify predictive biomarkers for disease diagnosis and progression, which might contribute to providing therapeutic strategies for IgAN.

[Change of peripheral blood appetite regulation factor of anorexia children and infect of child anorexia granule].

Study the infect of child anorexia granule on serum ghrelin and leptin of anorexia children and its clinical efficacy. Selected 81 cases of anorexia children aged 1-6 years old into treatment group (42 cases) and control group (39 cases), in addition, 30 case healthy children as healthy control group. The control group children were treated with domperidone suspension 0.3 mg x kg(-1) x d(-1), tid, orally 30 minutes before meals. Treatment group were treated with child anorexia granule, 1-3 years 1 package, bid; 4-6 years 1 package, tid; po, 4 weeks as a course of treatment. Study the change of serum ghrelin and leptin before and after therapy. The study demonstrates that before treatment, the serum ghrelin level of disease group was lower than healthy group (P < 0.01), and the serum leptin level was higher than healthy group (P < 0.01). After treatment, the serum ghrelin level both increase, and the serum leptin decline. And the change of treatment group was significantly different with control group (P < 0.01). And the clinical effective rate are 95.23% and 74.35% (P < 0.01). After 6 months of follow-up visit, the children weight significantly increase in treatment group (P < 0.01). Results indicate that child anorexia granule can facilitate secretion of ghrelin, and inhibit secretion of leptin, so as to work up an appetite. And the molecular mechanism is its infect on serum ghrelin, leptin.