Search Results (590)

Equine lutropin hormone/choriogonadotropin receptor (LH/CGR) is a G protein-coupled receptor that binds to both luteinizing hormone and choriogonadotropin, with multiple potential N-linked glycosylation sites in the long extracellular domain region. The roles of these glycosylation sites in hormone binding have been widely studied; however, their relationships with cyclic adenosine monophosphate (cAMP) activation, loss of cell surface receptors, and phosphorylated extracellular signal-regulated kinases1/2 (pERK1/2) expression are unknown. We used site-directed mutagenesis with the substitution of Asn for Gln to alter the consensus sequences for N-linked glycosylation, and cAMP signaling was analyzed in the mutants. Specifically, the N174Q and N195Q mutants exhibited markedly reduced expression levels, reaching approximately 15.3% and 2.5%, respectively, of that observed for wild-type equine LH/CGR. Correspondingly, the cAMP EC₅₀ values were decreased by 7.6-fold and 5.6-fold, respectively. Notably, the N195Q mutant displayed an almost complete loss of cAMP activity, even at high concentrations of recombinant eCG, suggesting a critical role for this glycosylation site in receptor function. Despite these alterations, Western blot analysis revealed that pERK1/2 phosphorylation peaked at 5 min following agonist stimulation across all mutants, indicating that the ERK1/2 signaling pathway remains functionally intact. This study demonstrates that the specific N-linked glycosylation site, N195, in equine LH/CGR is indispensable for cAMP activity but is normally processed in pERK1/2 signaling. Thus, we suggest that in equine LH/CGR, agonist treatment induces biased signaling, differentially activating cAMP signaling and the pERK1/2 pathway. Full article

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by tissue damage in multiple organs caused by autoantibodies and the resulting immune complexes. One possible way for complement system contribution to onset of autoimmune disorder could be realized by the impairment of C1q-mediated apoptotic clearance as part of human homeostasis. The capacity of C1q to bind early apoptotic cells could be decreased or even lost in the presence of anti-C1q antibodies. A monoclonal anti-idiotypic single-chain (scFv) antibody was selected from the phage library Griffin1” to recognize anti-C1q autoantibodies, purified from sera of lupus nephritis patients. Lupus-prone MRL/lpr mice were injected weekly with scFv A1 fragment-binding anti-C1q antibodies. The number of in vitro and ex vivo studies with collected cells, sera, and organs from the treated animals was performed. scFv treatment changed the percentage of different B-, T-, and NK-cell subpopulations as well as plasma cells and plasmablasts in the spleen and bone marrow. An increase in the levels of splenocyte proliferation, anti-C1q antibodies, and the number of plasma cells producing anti-dsDNA and anti-C1q antibodies were also observed in scFv-treated animals. High levels of proteinuria and hematuria combined with unstable levels of IL10 and IFNγ promote the development of severe lupus and shorten the survival of treated MRL/lpr mice. Therapy with the scFv A1 antibody resulted in BCR recognition on the surface of anti-C1q-specific B-cells and had a disease progression effect, enhancing lupus symptoms in the MRL/lpr mouse model of SLE. Full article

Background/Objectives: Beak and feather disease virus (BFDV) is the causative agent of psittacine beak and feather disease (PBFD), affecting psittacine birds. There is currently no commercial vaccine or treatment for this disease. This study developed a novel BFDV coat protein mRNA vaccine encapsidated by TMV coat protein to form pseudovirions (PsVs) and tested its immunogenicity alongside BFDV coat protein (CP) subunit and DNA vaccine candidates. Methods: mRNA and BFDV CP subunit vaccine candidates were produced in Nicotiana benthamiana and subsequently purified using PEG precipitation and gradient ultracentrifugation, respectively. The DNA vaccine candidate was produced in E. coli cells harbouring a plasmid with a BFDV1.1mer pseudogenome. Immunogenicity of the vaccine candidates was evaluated in African grey parrot chicks. Results: Successful purification of TMV PsVs harbouring the mRNA vaccine, and of the BFDV-CP subunit vaccine, was confirmed by SDS-PAGE and western blot analysis. TEM analyses confirmed formation of TMV PsVs, while RT-PCR and RT-qPCR cDNA amplification confirmed encapsidation of the mRNA vaccine candidate within TMV particles. Restriction digests verified presence of the BFDV1.1mer genome in the plasmid. Four groups of 5 ten-week-old African grey parrot (Psittacus erithacus) chicks were vaccinated and received two boost vaccinations 2 weeks apart. Blood samples were collected from all four groups on day 14, 28 and 42, and sera were analysed using indirect ELISA, which showed that all vaccine candidates successfully elicited specific anti-BFDV-CP immune responses. The subunit vaccine candidate showed the strongest immune response, indicated by higher binding titres (>6400), followed by the mRNA and DNA vaccine candidates. Conclusions: The candidate vaccines present an important milestone in the search for a protective vaccine against PBFD, and their inexpensive manufacture could considerably aid commercial vaccine development. Full article

Bombyx mori, a key lepidopteran model with economic importance, is highly susceptible to environmental heavy metal pollution. This study investigated the mechanisms of Pb toxicity and the associated detoxification and metabolic defense responses in silkworms, employing transcriptome sequencing, enzyme activity assays, and histopathological analysis. Pb exposure caused significant histopathological changes and apoptosis in the fat body, marked by structural disorganization, swollen adipocytes, and degraded extracellular matrix. Molecular analysis showed activation of antioxidant defenses, with superoxide dismutase (SOD) and catalase (CAT) activities significantly elevated (p < 0.05), while peroxidase (POD) activity declined (p < 0.05). Levels of malondialdehyde (MDA) and glutathione (GSH) also decreased. In detoxification responses, carboxylesterase (CarE) activity was reduced, whereas cytochrome P450 (P450) and glutathione S-transferase (GST) activities increased (p < 0.05). Transcriptome sequencing revealed 1,418 differentially expressed genes (DEGs), with notable upregulation of key detoxification genes (p < 0.05), including six cytochrome P450s (CYPs), five uridine diphosphate-glycosyltransferases (UGTs), three glutathione S-transferases (GSTs), and six ATP-binding cassette transporters (ABCs). KEGG enrichment analysis highlighted the involvement of these DEGs in drug metabolism, glutathione metabolism, and ABC transporter pathways (p < 0.05). Functional validation showed that knocking down Cap ‘n’ Collar C (CncC) significantly suppressed key detoxification genes (CYP18A1, CYP332A1, GSTd3, GSTt1, UGT33D8; p < 0.05). qRT-PCR and Western blot analyses confirmed that the Caspase-3 pathway mediates Pb-induced apoptosis, with increased cleaved Caspase-3 and Caspase-4 levels following CncC silencing. Overall, our findings elucidate the mechanisms of Pb toxicity in silkworms and identify CncC as a critical regulator of detoxification and defense against heavy metal stress in lepidopteran insects. Full article

We used molecular docking as a computational tool to predict the binding affinities and interactions of quercetin 3-O-malonylglucoside (Q3MG) with vascular target proteins. First, the proteins 1M9M (human endothelial nitric oxide synthase; eNOS) and 6ND0 (human large-conductance voltage- and calcium-activated K⁺ channels; BK_Ca) were downloaded from the Protein Data Bank and submitted to molecular docking studies, revealing Q3MG binding affinities for both proteins. The vascular effect of Q3MG was investigated in the perfused mesenteric vascular beds (MVBs) of spontaneously hypertensive rats. In preparations with functional endothelium, Q3MG dose-dependently reduced the perfusion pressure in MVBs. Removal of the endothelium or inhibition of the nitric oxide synthase enzyme by L-NAME blocked the vasodilation induced by Q3MG. Perfusion with a physiological solution containing high KCl or the use of a non-selective blocker of K⁺ channels, as well as perfusion with iberiotoxin, completely abolished the vasodilatory effects of Q3MG. The data obtained suggest that the vascular effects of Q3MG involve the activation of the NO/cGMP pathway followed by the opening of BK_Ca. Full article

Background: The bone morphogenetic protein (BMP) signaling pathway is essential for lung development. BMP4, a key regulator, binds to type I (BMPR1) and type II (BMPR2) receptors to initiate downstream signaling. While the inactivation of Bmpr1a and Bmpr1b leads to tracheoesophageal fistulae, the role of BMPR2 mutations in lung epithelial development remains unclear. Methods: We generated induced pluripotent stem cells (iPSCs) from a patient carrying a BMPR2 mutation (c.631C>T), and gene-corrected isogenic controls were created using CRISPR/Cas9. These iPSCs were differentiated into lung progenitor cells and subsequently cultured to generate alveolar and airway organoids. The differentiation efficiency and epithelial lineage specification were assessed using immunofluorescence, flow cytometry, and qRT-PCR. Results: BMPR2-mutant iPSCs showed no impairment in forming a definitive or anterior foregut endoderm. However, a significant reduction in lung progenitor cell differentiation was observed. Further, while alveolar epithelial differentiation remained largely unaffected, airway organoids derived from BMPR2-mutant cells exhibited impaired goblet and ciliated cell development, with an increase in basal and club cell markers, indicating skewing toward undifferentiated airway cell populations. Conclusions: BMPR2 dysfunction selectively impairs late-stage lung progenitor specification and disrupts airway epithelial maturation, providing new insights into the developmental impacts of BMPR2 mutations. Full article

The APETALA2/ethylene response factor (AP2/ERF) is a class of plant-specific transcription factors, among which the dehydration-responsive element-binding protein (DREB) subfamily has been widely reported to enhance plant resistance to abiotic stresses. A high-temperature-related gene, Apium graveolens DREBA6b (AgDREBA6b; accession number: OR727346), was previously cloned from a heat-tolerant celery variety. In this study, we transformed this gene into Arabidopsis thaliana using an Agrobacterium rhizogenes-mediated method to explore its function. The results showed that overexpressing AgDREBA6b in Arabidopsis thaliana significantly improved plant growth under high-temperature stress (38 °C) compared to the dreb mutant and wild-type (WT) plants. The anatomical structure of the leaves revealed that the number and degree of stomatal openings in the overexpressed plants were significantly higher than those in the WT and dreb plants, suggesting that AgDREBA6b enhances stomatal opening. Additionally, the chlorophyll content, chlorophyll fluorescence properties, proline (Pro), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities were higher in the transgenic plants, indicating better stress tolerance. qPCR analysis showed that four heat tolerance-related genes (AtHSP98.7, AtHSP70-1, AtAPX1, and AtGOLS1) were upregulated in the transgenic plants, with higher expression levels than in WT and mutant plants. This study provides valuable genetic resources for understanding the molecular mechanisms of celery’s heat tolerance and offers insights for breeding heat-tolerant celery varieties. Full article

Background:Salmonella Typhimurium is a major zoonotic pathogen, in which type 1 fimbriae play a crucial role in intestinal colonization and immune modulation. This study aimed to improve the protective immunity of a previously developed growth-deficient strain—a double auxotroph for D-glutamate and D-alanine—by engineering the inducible expression of type 1 fimbriae. Methods: P_tetA-driven expression of the fim operon was achieved by λ-Red mutagenesis. fimA expression was quantified by qRT-PCR, and fimbriation visualized by transmission electron microscopy. Adhesive properties were evaluated through FimH sequence analysis, yeast agglutination, mannose-binding/inhibition assays, and HT-29 cell adherence. BALB/c mice were immunized orogastrically with IRTA ΔΔΔ or IRTA ΔΔΔ P_tetA::fim. Safety and immunogenicity were assessed by clinical monitoring, bacterial load, fecal shedding, ELISA tests, and adhesion/blocking assays using fecal extracts. Protection was evaluated after challenging with wild-type and heterologous strains. Results: IRTA ΔΔΔ P_tetA::fim showed robust fimA expression, dense fimbrial coverage, a marked mannose-sensitive adhesive phenotype and enhanced HT-29 attachment. Fimbrial overexpression did not alter intestinal colonization or translocation to mesenteric lymph nodes (mLNs). Immunization elicited a mixed IgG1/IgG2a, significantly increased IgA and IgG against type 1 fimbriae-expressing Salmonella, and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ P_tetA::fim reduced infection burden in the cecum (−1.46/1.47-log), large intestine (−1.35/2.17-log), mLNs (−1.32/0.98-log) and systemic organs more effectively than IRTA ΔΔΔ. Conclusions: Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation Salmonella vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery. Full article

Tieguanyin tea, celebrated as one of China’s top ten famous teas, is highly regarded for its unique flavor and taste. However, recent intensification of global warming has escalated the occurrence of abiotic stresses, posing significant threats to the growth, development, yield, and quality of Tieguanyin tea plants. DOF (DNA-binding one zinc finger protein), a plant-specific transcription factor, plays a critical role in plant development and stress response. In this study, we identified and analyzed 58 CsDOF genes across the whole genome, which were found to be randomly and unevenly distributed across 15 chromosomes. A phylogenetic tree was constructed using DOF genes from Arabidopsis thaliana and Tieguanyin, categorizing these genes into 10 subgroups. Collinearity analysis revealed homologous gene pairs between CsDOF and OsDOF(19 pairs), StDOF (101 pairs), and ZmDOF (24 pairs). Cis-acting element analysis indicated that CsDOF genes contain elements related to both stress and hormone responses. Heat map analysis demonstrated that subfamily C2 predominantly regulates the growth and development of roots, stems, and leaves in Tieguanyin. Tertiary structure analysis of CsDOF proteins revealed diverse structures, underscoring the functional variability within the CsDOF gene family. Furthermore, qRT-PCR analysis was employed to assess the expression profiles of 13 CsDOF genes under high-temperature and drought conditions. Notably, CsDOF51 and CsDOF12 exhibited significant expression changes under drought and high-temperature stress, respectively, while CsDOF44 showed significant changes under both conditions. This study provides foundational knowledge of the CsDOF gene family and offers novel insights for enhancing the drought and heat tolerance of Tieguanyin tea. Full article

Microbial contamination is the leading cause of foodborne diseases and spoilage in food and personal care products. Previous studies by our group have demonstrated that vine tea extract (VTE) and dihydromyricetin (DMY) inhibit the growth of Escherichia coli. In this study, we further explored the inhibitory mechanisms of VTE and DMY against E. coli through a label-free proteomics approach. The proteomic analysis detected 130 and 81 differentially expressed proteins (DEPs) in E.coli following VTE and DMY treatment, respectively. The analysis indicated that VTE and DMY inhibit bacterial growth through multiple-target mechanisms. Specifically, they inhibit E. coli growth by disrupting the cationic antimicrobial peptide resistance pathway, amino acid biosynthesis and metabolism, and nucleotide metabolism. Additionally, VTE disrupts various secondary metabolic pathways, while DMY interferes with E. coli ribosome assembly and function, and disrupts cell membrane lipid homeostasis by interfering with fatty acid metabolism. RT-qPCR validation confirmed transcriptional alterations in genes encoding key target proteins. Molecular docking results indicated that DMY may affect bacterial protein synthesis, cationic antimicrobial peptide resistance, and transcriptional regulation by binding to target proteins such as RplB, RplV, LpxA, and YafC. In conclusion, this study systematically deciphered the multi-target inhibitory mechanisms of VTE and DMY against E. coli, providing a theoretical basis for developing plant-derived antimicrobial agents. Full article

In traditional Chinese medicine, Marsdenia tenacissima is employed to prevent and treat lung cancer. The anti-tumor properties are further amplified by the fermentation product of Ganoderma lucidum and Marsdenia tenacissima (MGF). Nevertheless, the efficacy of the chemical components in combating lung cancer and the potential therapeutic targets for treating the disease remain ambiguous. UPLC-Q-TOF/MS was used to identify 19 components, all of which are unique C₂₁ steroidal saponins found in MGF. The analysis of network pharmacology indicated that the active targets of these components were significantly concentrated in lung cancer and had a strong connection with cell proliferation. The bioinformatics analysis was conducted on data from TCGA and DisGeNET to identify a total of 28 biomarkers. Furthermore, our findings showed that the 19 targets connected to the active ingredients of Marsdenia tenacissima demonstrated significant enrichment in both the EGFR and apoptosis signaling pathways. Molecular docking technology was utilized to confirm the binding interactions of the primary constituents with the designated target. Full article

Background/Objectives: Camellia nitidissima Chi (C. nitidissima), a traditional Chinese “food and medicine homology” plant, has demonstrated potential anti-tumor properties. However, its mechanisms of anti-lung cancer activity via ferroptosis remain unclear. This study aimed to construct an integrated research system of “natural product extraction-purification, bioactivity evaluation, and computational drug screening” to explore the bioactive compounds in C. nitidissima leaves targeting HMOX-1-mediated ferroptosis and their anti-lung cancer mechanisms. Methods: Active fractions were prepared using ethanol extraction combined with polyamide column chromatography. The anti-lung cancer activity was evaluated using the NCI-H1975 cell model. Ferroptosis was verified via transmission electron microscopy (TEM), biochemical indicators, a PCR Array, and immunofluorescence. The bioactive compounds were identified using UPLC-Q Exactive MS, and their binding affinity to HMOX-1 was evaluated via molecular docking and dynamics simulations, followed by cellular validation. Results: The 95% F1 fraction from the extracts of C. nitidissima leaves exhibited the strongest anti-lung cancer activity, which could be significantly reversed by Ferrostatin-1. Furthermore, it induced typical ferroptosis-related structural damage in mitochondria, including shrinkage and a reduction in size, increased membrane density, and a reduction or even the disappearance of cristae structures. At the molecular level, this fraction significantly increased the levels of oxidative stress markers (ROS↑, MDA↑, Fe²⁺↑, and GSH↓) and upregulated the expression of key ferroptosis-related genes, including HMOX-1, CHAC1, and NOX1. Using UPLC-Q Exactive MS combined with computational simulation methods, four bioactive compounds with high affinity for HMOX1 were successfully identified, including isochlorogenic acid A (−8.4 kcal/mol), isochlorogenic acid C (−8.4 kcal/mol), apigenin (−7.8 kcal/mol), and chrysin (−7.3 kcal/mol). Cellular experiments validated that these compounds exhibited dose-dependent anti-proliferative effects. Conclusions: The leaves of C. nitidissima induce anti-lung cancer effects via HMOX-1-mediated ferroptosis. Isochlorogenic acid A/C, apigenin, and chrysin were identified as key bioactive components. These findings lay the foundation for the development of natural ferroptosis-targeted drugs. Full article

Hyaluronan (HA) is essentially secreted by every cell and plays a critical role in maintaining normal cell physiology. While the structure and function of HA have been extensively investigated, questions regarding the sizes and conformation of HA under physiological and inflamed conditions, in relevance to its functions, remain elusive. In this article, we update our knowledge of the HA functional properties, including binding proteins and their signaling networks, as well as matrix formation, which can potentially induce phase separation and affect the mobility and behavior of small molecules, proteins, and cells. We detail the striking differences regarding the biological outcomes of signaling pathways for HA and membrane receptors versus HA and GPI-linked hyaluronidase Hyal-2. We describe: (1) the native, large-sized HA is not proapoptotic but signals with an overexpressed HYAL-2/WWOX/SMAD4 complex to induce apoptosis, which is likely to occur in an inflamed microenvironment; (2) HA-binding proteins are connected via signal pathway networks. The competitive binding of HA and TGF-β to the membrane HYAL-2 and the downstream HYAL-2/WWOX/SMAD4 signaling is addressed; (3) the phase-separated proteins or small molecules in the HA matrices may contribute to the aberrant interactions, leading to inflammation and disease progression; (4) the role of HA and complement C1q in Alzheimer’s disease via connection with a risk factor for Alzheimer’s disease WWOX is also discussed; (5) a hidden function is the inducible HA conformational changes that confer cancer suppression and, probably, retardation of neurodegeneration. Full article

Immunoassays for cardiac troponin, such as the Elecsys^® hs-TnT, have become the gold standard for myocardial infarction diagnostics. While various protein/chemical factors affecting the troponin complex and thus its diagnostic accuracy have been investigated, the role of coding single-nucleotide polymorphisms remains underexplored. To evaluate potential cSNP-induced interference with antibody binding in the Elecsys^® hs-TnT immunoassay, we applied ITEM-FOUR, a mass spectrometry-based method that quantifies changes in antibody binding upon amino acid substitutions in epitope peptides. Candidate cSNPs were selected from the dbSNP database and were mapped to human cardiac troponin T by molecular modeling. Consuming micromolar antibody concentrations and microliter sample volumes, two wild-type and 17 cSNP-derived variant epitope peptides—six for monoclonal antibody M7 and eleven for monoclonal antibody M11.7—were investigated to reveal the binding motifs “V₁₃₁-K₁₃₄-E₁₃₈-A₁₄₂” for M7 and “E₁₄₆-I₁₅₀-R₁₅₄-E₁₅₇” for M11.7. Loss of binding to M11.7 was observed for substitutions Q148R (rs730880232), R154W (rs483352832), and R154Q (rs745632066), whereas the E138K (rs730881100) exchange disrupted binding of M7. Except for cSNP Q148R, they are associated with cardiomyopathies, placing affected individuals at risk of both underlying heart disease and false-negative hs-TnT assay results in cases of myocardial infarction. Our results highlight the need to account for cSNP-related interferences in antibody-based diagnostics. ITEM-FOUR offers a powerful approach for tackling this challenge, fostering next-generation assay development. Full article

NAC transcription factors are a kind of plant specific transcription factor widely distributed in plants, and they play an important role in the process of plant growth and development. According to the transcriptome data, a transcription factor with typical NAC characteristics was isolated from Larix olgensis (common name “Dahurian larch”), that we named LoNAC5. The length of the coding sequence (CDS) was 1164 bp, encoding 387 amino acids. The LoNAC5 protein harbors a NAM (NAC family) domain at the 14–139 aa region of its N-terminus and an activation domain at the 324–364 aa region of the C-terminus. Phylogenetic tree analysis revealed that LoNAC5 belonged to the ATNAC3 subgroup. Cis-acting element analysis showed that there were multiple plant stress-resistance-related elements on the promoter of LoNAC5, including hormone and light responsiveness elements. LoNAC5 was localized in the nucleus by injection transformation of tobacco leaves. Results suggested that the LoNAC5 protein is active as a homodimer and that it binds to the GATGTG motif. The results of RT-qPCR showed that LoNAC5 is a highly expressed gene in L. olgensis, and the expression level is highest in 180-day needles. LoNAC5 responded to various hormone treatments and was induced by drought and salt stress. The yeast phenotype test showed that overexpression of LoNAC5 could make yeast grow better under drought and salt stress. It was speculated that LoNAC5 might act in L. olgensis as a positive regulator of drought and salt tolerance. Full article