Adiponectin receptor 1 (AdipoR1) and Adiponectin receptor 2 (AdipoR2) can bind to adiponectin (AdipoQ) secreted by adipose tissue to participate in various physiological functions of the body. In order to explore the role of AdipoR1 and AdipoR2 in amphibians infected by Aeromonas hydrophila (Ah), the genes adipor1 and adipor2 of Rana dybowskii were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by bioinformatics. The tissue expression difference of adipor1 and adipor2 was analyzed by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and an inflammatory model of R. dybowskii infected by Ah was constructed. The histopathological changes were observed by hematoxylin-eosin staining (HE staining); the expression profiles of adipor1 and adipor2 after infection were dynamically detected by qRT-PCR and Western blotting. The results show that AdipoR1 and AdipoR2 are cell membrane proteins with seven transmembrane domains. Phylogenetic tree also shows that AdipoR1 and AdipoR2 cluster with the amphibians in the same branch. qRT-PCR and Western blotting results show that adipor1 and adipor2 were up-regulated at different levels of transcription and translation upon Ah infection, but the response time and level were different. It is speculated that AdipoR1 and AdipoR2 participate in the process of bacterial immune response, providing a basis for further exploring the biological functions of AdipoR1 and AdipoR2 in amphibians.
Animals ; Receptors, Adiponectin/metabolism* ; Phylogeny ; Adiponectin/metabolism* ; Cloning, Molecular ; Ranidae/genetics*

Peptide dual agonists toward both glucagon-like peptide 1 receptor (GLP-1R) and glucagon receptor (GCGR) are emerging as novel therapeutics for the treatment of type 2 diabetes mellitus (T2DM) patients with obesity. Our previous work identified a Xenopus GLP-1-based dual GLP-1R/GCGR agonist termed xGLP/GCG-13, which showed decent hypoglycemic and body weight lowering activity. However, the clinical utility of xGLP/GCG-13 is limited due to its short in vivo half-life. Inspired by the fact that O-GlcNAcylation of intracellular proteins leads to increased stability of secreted proteins, we rationally designed a panel of O-GlcNAcylated xGLP/GCG-13 analogs as potential long-acting GLP-1R/ GCGR dual agonists. One of the synthesized glycopeptides 1f was found to be equipotent to xGLP/GCG-13 in cell-based receptor activation assays. As expected, O-GlcNAcylation effectively improved the stability of xGLP/GCG-13 in vivo. Importantly, chronic administration of 1f potently induced body weight loss and hypoglycemic effects, improved glucose tolerance, and normalized lipid metabolism and adiposity in both db/db and diet induced obesity (DIO) mice models. These results supported the hypothesis that glycosylation is a useful strategy for improving the in vivo stability of GLP-1-based peptides and promoted the development of dual GLP-1R/GCGR agonists as antidiabetic/antiobesity drugs.
Mice ; Animals ; Glucagon-Like Peptide 1/metabolism* ; Receptors, Glucagon/therapeutic use* ; Xenopus laevis/metabolism* ; Diabetes Mellitus, Type 2/drug therapy* ; Glycopeptides/therapeutic use* ; Obesity/drug therapy* ; Hypoglycemic Agents/pharmacology* ; Peptides/pharmacology*

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-κB, IKKα and IKKβ mRNA were all significantly upregulated (P < 0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.
Animals ; Cloning, Molecular ; DNA, Complementary ; Gene Expression Profiling ; Gene Expression Regulation ; Mammals/metabolism* ; NF-kappa B/genetics* ; Phylogeny ; RNA, Messenger/genetics* ; Ranidae/genetics*

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.
Animals ; Artificial Intelligence ; Cryoelectron Microscopy ; Nuclear Pore/ultrastructure* ; Oocytes/metabolism* ; Xenopus laevis

Ambystoma mexicanum/immunology* ; Amputation ; Animals ; Biomarkers/metabolism* ; Blastomeres/immunology* ; Cell Lineage/immunology* ; Connective Tissue Cells/immunology* ; Epithelial Cells/immunology* ; Forelimb ; Gene Expression ; High-Throughput Nucleotide Sequencing ; Humans ; Immunity ; Peroxiredoxins/immunology* ; Regeneration/immunology* ; Regenerative Medicine/methods* ; Single-Cell Analysis/methods*

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.
Amino Acid Sequence ; Animals ; Mice ; Phylogeny ; Pore Forming Cytotoxic Proteins ; Protein Structure, Secondary ; Ranidae

The aim of this study was to investigate the expression of MHCⅠ gene in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila (Ah), and to provide evidence for revealing the anti-infective immune response mechanism of amphibians. The experimental animal model of Aeromonas hydrophila infection was first constructed, and the pathological changes were observed by HE staining. The MHCⅠ gene α1+α2 peptide binding region of Rana dybowskii was cloned by RT-PCR and analyzed by bioinformatics. Real-time PCR was used to detect the transcription level of MHCⅠ in different tissues under Ah stress. After Ah infection, the skin, liver and muscle tissues showed signs of cell structure disappearance and texture disorder. The MHCⅠ gene α1+α2 peptide binding region fragment was 494 bp, encoding 164 amino acids, and homology with amphibians. Above 77%, the homology with mammals was as low as 14.96%, indicating that the α1+α2 region of MHC gene was less conserved among different species. The results of real-time PCR show that the liver, spleen and kidney of the experimental group were under Ah stress. The transcript levels of MHCⅠ gene in skin and muscle tissues were higher than those in the control group at 72 h, but the time to peak of each tissue was different (P<0.01), indicating that the response time of MHCⅠ gene in different tissues was different under Ah stress. This study provides a reference for further exploring the immune function of MHC molecules in anti-infection.
Aeromonas hydrophila ; Animals ; Gene Expression Profiling ; Gene Expression Regulation ; immunology ; Gram-Negative Bacterial Infections ; immunology ; Liver ; metabolism ; Ranidae ; genetics ; immunology ; microbiology ; Skin ; metabolism

OBJECTIVE: To quantitatively investigate the effects of Ringer's solution with different concentrations of alcohol (1%～80%) on biphasic compound action potentials (AP) from frog sciatic nerve trunk, and their recoveries from alcohol effects. METHODS: Individual segments of frog sciatic nerve trunk with a length of 6 to 8 cm were prepared. Ringer's solution with different concentrations of alcohol (0%, 1%, 2%, 4%, 8%, 16%, 32%, 48%, 64% and 80%) was applied onto the segment of the trunk between the stimulus and ground electrodes via an agent reservoir which was newly armed in a nerve trunk shielded chamber for 5 minutes. The nerve trunk was respectively electro-stimulated to generate the biphasic compound AP which was recorded using the experimental system of BL-420F. This was followed by 5 times washout plus 5 min administration with Ringer's solution before recovery recording of AP. RESULTS: Compared to normal Ringer's solution, Ringer's solution with alcohol at ≤4% did not have dramatic impacts on the AP amplitude and conduction velocity, while Ringer's solution with alcohol at ≥8% there was significant decrease in these two parameters. Ringer's solution with alcohol at the conentrations of 16%, 32% and ≥48% could prevent a small proportion (30%), a large proportion (90%) and all (100%) of sciatic nerve trunks, respectively, from generating AP. Washout with normal Ringer's solution after alcohol application at the concentration of ≤32%, AP could totally recover to normal status. While alcohol at the concentration of 48%, 64% and 80%, the probabilities to regenerate APs were 90%, 40% and 0%, and the AP amplitudes were decreased to 60%, 36% and 0%, respectively. After washout, AP conduction velocity showed no difference with alcohol at the concentration of ≤8% when compared with that before washout, while it could not be recovered to normal under alcohol at ≥16%. CONCLUSION Ringer's solution with different concentrations of alcohol exerts different effects on biphasic compound AP amplitude and conduction velocity. Hopefully, our findings could be helpful for the alcoholic usage and its recovery from alcoholic damage.
Action Potentials ; Animals ; Anura ; Ethanol ; pharmacology ; Ringer's Solution ; pharmacology ; Sciatic Nerve ; drug effects

Peritoneal dialysis (PD)-related peritonitis is a major cause of injury and technique failure in patients undergoing PD. Aeromonas hydrophila is ubiquitous in the environment, and is a Gram-negative rod associated with infections in fish and amphibians in most cases; however, it can also cause opportunistic infections in immunocompromised patients. We report a case of A. hydrophila peritonitis in a 56-year-old male on automated PD. Peritonitis may have been caused by contamination of the Set Plus, a component of the automated peritoneal dialysis device. Although Set Plus is disposable, the patient reused the product by cleansing with tap water. He was successfully treated with intraperitoneally-administered ceftazidime and has been well without recurrence for more than 2 years.
Aeromonas hydrophila ; Aeromonas ; Amphibians ; Ceftazidime ; Humans ; Immunocompromised Host ; Male ; Middle Aged ; Opportunistic Infections ; Peritoneal Dialysis ; Peritonitis ; Recurrence ; Water

K⁺ channels are key components of the primary and secondary basolateral Cl- pump systems, which are important for secretion from the salivary glands. Paroxetine is a selective serotonin reuptake inhibitor (SSRI) for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. We studied the effects of paroxetine on a human K⁺ channel, human ether-a-go-go-related gene (hERG), expressed in Xenopus oocytes and on action potential in guinea pig ventricular myocytes. The hERG encodes the pore-forming subunits of the rapidly-activating delayed rectifier K⁺ channel (I(Kr)) in the heart. Mutations in hERG reduce I(Kr) and cause type 2 long QT syndrome (LQT2), a disorder that predisposes individuals to life-threatening arrhythmias. Paroxetine induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The inhibition was concentration-dependent and time-dependent, but voltage-independent during each voltage pulse. In guinea pig ventricular myocytes held at 36℃, treatment with 0.4 µM paroxetine for 5 min decreased the action potential duration at 90% of repolarization (APD₉₀) by 4.3%. Our results suggest that paroxetine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects of clinical administration of paroxetine.
Action Potentials ; Animals ; Arrhythmias, Cardiac ; Guinea Pigs ; Heart ; Humans ; Long QT Syndrome ; Muscle Cells ; Oocytes ; Paroxetine ; Salivary Glands ; Serotonin ; Tail ; Torsades de Pointes ; Xenopus