Search Results (26,243)

Hypoxia is a prevalent pathophysiological condition. Prolonged exposure to hypobaric hypoxia can lead to maladaptation, increasing the risk of chronic hypoxic diseases such as high-altitude polycythemia (HAPC). Dauricine, an alkaloid derived from the root of Menispermum dauricum DC, has been demonstrated to possess anti-hypoxic properties; however, its underlying molecular mechanisms remain elusive. In this study, a potential multi-target anti-hypoxic mechanism of dauricine was proposed and computationally evaluated using an integrated approach combining network pharmacology, molecular docking, and molecular dynamics simulations. Common targets between dauricine and hypoxia-related genes were identified through network pharmacology screening. A protein–protein interaction (PPI) network was constructed to identify core targets, followed by Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Molecular docking was subsequently employed to evaluate the binding affinities between dauricine and the candidate core targets, while molecular dynamics simulations were performed to assess the dynamic stability of the resulting complexes. Additionally, the drug-likeness and safety profiles of dauricine were assessed. The results suggest that dauricine may exert its anti-hypoxic effects by modulating candidate core targets, including ESR1, PIK3CA, and MTOR, and by acting on key signaling pathways such as PI3K-Akt, MAPK, and mTOR. This study provides a theoretical foundation for the further investigation of dauricine as a multi-target candidate for intervention in hypoxia and establishes a bioinformatics basis for subsequent experimental validation. Full article

Acaryochloris marina is a distinctive cyanobacterium that uses chlorophyll d as its primary photosynthetic pigment and possesses two major light-harvesting systems: membrane-integral chlorophyll-binding Pcb/CBP complexes and water-soluble phycobiliproteins. How these antenna systems respond at the transcriptome level to contrasting light environments remains incompletely characterized. Here, we re-analyzed a publicly available RNA-seq dataset for A. marina MBIC11017 (NCBI BioProject PRJNA1130970), comparing cells grown under low-irradiance far-red light (LL-FR; 1.5–2 µmol photons m⁻² s⁻¹, 710-nm peak) and high-irradiance white light (HL-WL; 30–35 µmol photons m⁻² s⁻¹). Because light quality and irradiance both differ in this experimental design, the two effects cannot be separated; all transcriptional changes are therefore interpreted as responses to the combined LL-FR versus HL-WL contrast rather than to far-red wavelength alone. Of 8439 expressed genes, 1810 (21.4%) were significantly differentially expressed (adjusted p < 0.05). Using GFF-verified locus tags which corrected mis-annotations propagated in earlier analyses, the PS-I core gene set showed a mean log₂ fold-change of +1.96 (3.9-fold; 11/11 loci significant), whereas the PS-II core gene set showed a mean log₂ fold-change of +1.10 (2.1-fold; 12/20 loci significant). Light-harvesting genes showed the strongest response: 17/18 phycobiliprotein-pathway genes in KEGG amr00196 were upregulated, together with multiple putative Pcb/CBP loci (mean antenna log₂FC = +3.51; 11.4-fold). Weighted gene co-expression network analysis placed the antenna-associate genes examined here within a module positively correlated with the LL-FR condition (r = 0.802, p = 0.017), and STRING analysis supported an enriched network of predicted or known protein associations (1115 nodes, 4763 edges; PPI enrichment p < 1.0 × 10⁻¹⁶). Recent matched-irradiance experiments indicate that, at equal photon flux, far-red wavelengths reduce phycobilisome content relative to white light. The transcriptional pattern reported here is therefore most parsimoniously interpreted as predominantly a low-irradiance response, with possible wavelength-associated CA5 contributions that cannot be isolated in the present design. Overall, the analysis reveals coordinated transcript-level changes across plasmid-encoded reacquired phycobiliprotein genes, chromosomal Pcb/CBP loci, chlorophyll biosynthesis genes, and photosystem core genes, consistent with coordinated regulation of light-harvesting components in A. marina. Full article

Lectin receptor-like kinases (LecRLKs) are a large subfamily of receptor-like kinases (RLKs), and their N-terminal lectin domain is predicted to reversibly bind to carbohydrates. Within this family, G-type LecRLKs represent a distinct subclass defined by an extracellular S-locus glycoprotein (SLG) domain, which was originally identified for its role in governing self-incompatibility in Brassica species. Emerging evidence suggests that G-type LecRLKs are involved in plant immunity; however, only a small fraction have been functionally characterized, leaving the roles of most family members largely unknown. In this study, we identified RK3 (Receptor Kinase 3) as the most strongly induced gene within the G-type LecRLK clade VI upon infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Through both gain- and loss-of-function analyses, we demonstrated that RK3 positively regulates flg22-induced immune signaling events, including oxidative burst and mitogen-activated protein kinase (MAPK) activation, as well as downstream responses such as defense gene expression and ethylene production. Remarkably, the immune-enhancing activity of RK3 does not require its kinase domain. Critically, both full-length RK3 and a kinase-deleted variant (RK3-ΔK) constitutively interact with FLS2 (Flagellin-Sensing 2) and BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1). This provides direct evidence that RK3 functions primarily as a co-regulatory component within the PRR complex, independent of its kinase activity. Moreover, ectopic expression of RK3 in tomato enhanced resistance to Pst DC3000, highlighting its potential utility in engineering disease resistance in crops. Thus, RK3 reveals a non-canonical, kinase-independent mechanism by which a G-type LecRLK potentiates plant immunity, expanding our understanding of RLK signaling complexity. Full article

Fibroblast Growth Factor 2 (FGF2) is a highly effective regulator of cell proliferation, differentiation, migration, and adhesion, suggesting a significant therapeutic potential as a tissue regeneration promoter both in acute and chronic tissue damage settings. Despite an extensive list of pathologies that lend themselves as viable targets for FGF2-based therapy (ranging from periodontics to burns to diabetic ulcers to coronary artery disease), the success record in the clinic remains modest, with no FDA approvals obtained so far. The inferior stability of this protein is frequently cited as the most significant factor behind its disappointing performance as a biotherapeutic. Multiple strategies have been designed and tested in an effort to ameliorate this problem, but the success remains elusive. We investigate the aggregation propensity of a recombinantly produced FGF2 using native mass spectrometry (MS) to identify conditions favoring formation of small soluble oligomers, which are considered precursors to larger aggregates. Tandem MS of proteolytic fragments produced by digestion of the oligomeric species allows the formation of external disulfide bonds to be identified as the process leading to oligomerization. Specifically, Cys-31 (one of the two unpaired cysteine residues in intact FGF2) appears to be a particularly active promoter of oligomerization by forming external disulfide bonds. As a high-pI protein, FGF2 readily associates with heparin, and molecular modeling identifies a positive charge basin proximal to Cys-31 as a potential heparin binding site, which can readily accommodate a synthetic heparin mimetic fondaparinux. Adding an equimolar amount of the latter to the FGF2 solution not only leads to formation of a stable protein/polyanion complex (as revealed by native MS), but also inhibits formation of FGF2 oligomers (presumably via a combination of steric hindrance and electrostatic repulsion). These findings advance our understanding of FGF2 stability, which will be invaluable for optimizing its formulation, storage, and administration. Full article

ATP-binding cassette (ABC) transporters are one of the largest superfamilies of membrane proteins, but little is known about the structural and evolutionary features of their non-domain regions. To clarify the diversity of these non-canonical regions across evolutionary lineages, we performed an analysis of intrinsically disordered regions, site-specific selection and predicted post-translational modification (PTM) sites among five architectural classes involving 1581 prokaryotic and eukaryotic sequences. Linker and flanking regions were more disordered than transmembrane and nucleotide-binding domains in all architectures. Disorder fraction was significantly different between region types after phylogenetic correction (Pagel’s λ ≈ 0.97). Predicted PTM sites are enriched in disordered non-domain segments, with N-linked glycosylation and phosphoserine showing the strongest positive enrichment. A total of 140 sites satisfied a tiered conservation criterion (MusiteDeep score ≥ 0.5; cross-species conservancy ≥ 30%), including 40 high-confidence or moderate-confidence sites (conservancy ≥ 50%) as well as novel phosphotyrosine candidates in half transporters and NBD-only proteins. Site-specific selection analyses showed pervasive purifying selection across domain cores and architecture-dependent enrichment of episodic positive selection in non-domain regions, with significant non-domain enrichment in full reverse and half forward transporters (Fisher’s exact, BH-adjusted p < 0.05). In summary, these findings establish that non-canonical regions of ABC transporters are evolutionarily dynamic and contain conserved predicted modification sites, supporting the idea that these regions are evolutionary dynamic segments that deserve experimental characterization as candidate regulatory interfaces. Full article

Background/Objectives: Autism spectrum disorder (ASD) is a neurodevelopmental disease with both clinical and genetic heterogeneity. Several loss-of-function variants in the chromodomain helicase DNA-binding protein 8 (CHD8) gene have been identified in individuals with ASD and/or developmental delay/intellectual disability. These are associated with specific clinical manifestations, including overgrowth, macrocephaly, sleep disturbance, and gastrointestinal problems. Methods: We performed clinical exome sequencing in a female patient with ASD and macrocephaly. RNA analysis from peripheral blood was carried out to investigate the functional effect of the identified variants. Results: We identified a novel maternally inherited CHD8 variant (c.5390+2T>C). Transcript analysis demonstrated that this variant disrupts the canonical splice donor in intron 30, causing splicing anomalies in the CHD7-binding domain of the CHD8 protein, resulting in a truncated inactive protein. Conclusions: In conclusion, this study identified a novel splice-site variant in the CHD8 gene with experimentally confirmed pathogenic effects on RNA splicing, expanding the mutational spectrum of CHD8-related neurodevelopmental disorders. The considerable intrafamilial phenotypic variability associated with CHD8 haploinsufficiency supports the presence of reduced penetrance and highlights the influence of modifying factors on the clinical expression of CHD8-related disorders. Full article

Primula nutans Georgi, a medicinal herb used in Mongolian and Tibetan medicine for treating respiratory ailments, is a natural agent with antiobesity potential. We investigated the antiobesity and insulin-sensitizing effects of P. nutans Georgi extract (PGE) using in vitro and in vivo models. In 3T3-L1 preadipocytes, PGE inhibited adipocyte differentiation and lipid accumulation without cytotoxicity, accompanied by the reduced expression of adipogenic transcription factors (PPARG, C/EBPA, and adiponectin) and lipogenic genes (FASN, SCD1, and ACC), particularly during the early stages of adipogenesis. Similar effects were observed in primary stromal vascular cells derived from mouse inguinal white adipose tissue. PGE upregulated C/EBP homologous protein and C/EBPB and was associated with altered cell cycle progression, increased G2/M phase distribution, and the potential disruption of mitotic clonal expansion during early adipogenesis. In HFD-induced obese mice, intraperitoneal administration of PGE (10 or 30 mg/kg) significantly reduced body weight gain, white adipose tissue mass, and hepatic steatosis, independent of food intake. PGE downregulated lipogenic and proinflammatory gene expression in adipose and hepatic tissues and increased AMPK phosphorylation in white adipose tissue. PGE improved glucose tolerance and was associated with enhanced insulin sensitivity, as evidenced by reduced areas under the curve in the glucose tolerance and insulin tolerance tests and increased circulating adiponectin levels. Feature-based molecular networking identified 61 compounds from PGE. Network pharmacology analysis revealed several antiobesity targets, including PPARG and AKT1. Molecular docking analyses suggested favorable binding affinities between major compounds and metabolic regulators. Collectively, these findings suggest that PGE may suppress adipogenesis and improve metabolic parameters in obese mice, supporting its potential as a natural candidate for obesity and related metabolic disorders. Full article

The tumor suppressor protein phosphatase 2A (PP2A) plays a crucial role in regulating oncogenic signaling. Its inactivation, specifically through inhibitory phosphorylation at Tyr307 mediated by SET and CIP2A, contributes to breast cancer (BC) progression. Modulation of these interactions represents a promising pharmacological strategy to restore PP2A function. We integrated computational approaches with experimental validation to analyse SET/CIP2A mechanisms and explore how PP2A reactivation suppresses tumor progression. Molecular docking and dynamics simulations showed that the SET inhibitor/FTY-720 forms stable hydrogen bond networks with SET, disrupting its interaction with PP2A. In contrast, CIP2A suppressor/erlotinib interacts with CIP2A through weaker hydrophobic and π-interactions. Protein–protein interaction analyses indicate reduced SET/CIP2A binding to PP2A upon treatment, supporting a structural basis for PP2A reactivation. Gene expression analyses revealed upregulation of PP2A, SET, CIP2A, and cytoskeletal markers in tumor and metastatic tissues. Studies on Triple Negative Breast Cancer (TNBC) cells showed that FTY-720 and erlotinib significantly reduce PP2A-Tyr307 phosphorylation, restoring its activity. Additionally, both compounds decreased c-Myc levels and inhibited Src/FAK/paxillin/PAK1 and ERK signaling, attenuating migratory and proliferative pathways. Our findings identify the SET/CIP2A–PP2A axis as a pharmacological target for the design of next-generation PP2A activators, highlighting the potential of inhibition as a therapeutic strategy to counteract TNBC progression. Full article

Background/Objectives: Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers and is a leading cause of cancer-related deaths worldwide. Pharmacological targeting of the p53–MDM2 interaction to activate wild-type p53 is a promising strategy for treating NSCLC that retain functional p53 (approximately 50% of all cases). Methods: We screened 33 ethnomedicinal Vietnamese plant extracts for their anticancer effects using p53-expressing and p53-null NSCLC cell models, as well as two non-cancerous cell lines for control. We used an array of different experimental approaches including NMR spectroscopy; molecular docking; an MTT test; cell cycle analysis; apoptosis analysis; wound healing, migration, and invasion assays; Real-Time PCR; immunoblotting; and Seahorse energy profiling to characterize and study the effects of these bioactive compounds on NSCLC cells. Results: Ethanol extract of Zanthoxylum nitidum stems and twigs demonstrated potent and selective activity by inducing p53-dependent cell cycle arrest and apoptosis. Phytochemical analysis identified several benzophenanthridine alkaloids as active constituents. Molecular docking revealed their strong in silico binding to MDM2. Notably, nitidine was the most promising compound among the molecules tested. Unlike nutlin, but similar to SP141 (two well-known MDM2 inhibitors), nitidine strongly stabilized p53 while concomitantly attenuating MDM2 at the protein level. Surprisingly, this effect was p53-independent. Additionally, nitidine suppressed the EMT master regulator Snail, and hence disrupted cellular bioenergetics and inhibited migration and invasion of NSCLC cells. Conclusions: Our findings identify Z. nitidum and nitidine as promising sources for developing novel MDM2-targeting therapeutics against NSCLC irrespective of the p53 status. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, which has recently been found to be closely associated with neuroinflammation. As an anti-inflammatory drug, triptolide (TP), a natural diterpenoid from Tripterygium wilfordii, was selected in the current study for treating PS19 (tau^P301S transgenic) mice, tauopathy AD mice. In addition, we have previously found that TP had the ability to reduce the level of cholesterol. However, the roles and mechanisms of TP in the above processes are not clear. To this end, we found that elevated cholesterol in serum and brain tissues upregulated the expression of apolipoprotein E (APOE) and sialic acid-binding Ig-like lectin 3 (CD33), leading to the activation of SH2-containing protein tyrosine phosphatase 1 (SHP-1). The activation of SHP-1 inhibits the signaling pathways of Janus kinase 1 (JAK1) and signal transducer and activator of transcription 6 (STAT6), which results in inhibition of the M2 polarization of microglia, which exacerbates neuroinflammation and cognitive decline in high-cholesterol diet (HCD)-fed mice. Conversely, TP treatment significantly inhibited the hepatic sterol regulatory element-binding protein 2 (SREBP2)/3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) pathway, which reduced the cholesterol levels in the serum and brain. By depressing the levels of cholesterol, the axis of CD33 and SHP-1 was suppressed, which resulted in restoration of the activity of JAK1 and STAT6 pathways, leading to the transition of microglia from the M1 to the M2 phenotype. Of note, these observations demonstrate that TP alleviates the cognitive impairment of PS19 mice via depressing neuroinflammation. Altogether, our results revealed the mechanisms of TP in treating AD via CD33/SHP-1/JAK1/STAT6 pathways in a cholesterol-dependent manner. Full article

Although mango is not classified among the nine major allergenic foods reported by the Food and Drug Administration (FDA), the increasing global and domestic consumption of mango has been accompanied by a growing number of reported cases of mango allergy. Currently, reports on cross-reactive allergens and cross-reactive linear epitopes in mango are limited. This study employed BLASTp (version 2.11.0+) to predict potential allergens that may cross-react with mango protein allergens and other food protein allergens. Subsequently, cross-reactive allergens were identified using sera from mango-allergic patients. Furthermore, similar sequences of the identified cross-reactive allergens were predicted by BLAST. These similar sequences were then synthesized by the solid-phase peptide synthesis method. Finally, the cross-reactive linear epitopes were determined by assessing their IgE-binding capacity using serum IgE from the same patient cohort. The results demonstrated that the sera from mango-allergic patients exhibited IgE-binding cross-reactivity with those from peanut, wheat, cashew, pistachio, and hazelnut, particularly with IgE-binding cross-reactivity to wheat and hazelnut, which has not been previously reported. The following novel cross-reactive linear epitopes were identified: the AA80–88 sequence of mango chitinase with the AA37–45 sequence of wheat Tri a 27 and the AA15–22 sequence of mango profilin with the AA65–72 sequence of pistachio Pis v 1. Furthermore, multiple cross-reactive epitopes were mapped between mango profilin and peanut Ara h 5, corresponding to the sequences AA31–51/AA31–50, AA50–65/AA52–65, AA76–96/AA76–96, and AA103–117/AA104–117, respectively. Full article

Hereditary sensorineural hearing loss (SNHL) represents a significant global public health burden. DFNX2, an X-linked form of non-syndromic SNHL, is caused by pathogenic variants in the POU3F4 gene. This study aimed to identify a novel POU3F4 mutation and characterize its functional consequences to elucidate the molecular pathogenesis of DFNX2. A three-generation Chinese family with X-linked deafness was recruited. Targeted next-generation sequencing was used to screen candidate variants, which were validated by Sanger sequencing for co-segregation analysis. Functional assays, including subcellular localization, dual-luciferase reporter assay, Western blotting, and homology modeling, were performed to assess the mutation’s effects. A novel frameshift mutation, c.670_673dupGGTA (p.(Asn225Argfs*2)), was identified and showed complete co-segregation with the deafness phenotype. The mutant protein exhibited cytoplasmic mislocalization, and dual-luciferase assays revealed a severe reduction in transcriptional activation capacity, whereas Western blot confirmed stable expression of the truncated protein. Structural modeling predicted the loss of both the POUS and POUH DNA-binding subdomains. Collectively, this study expands the mutational spectrum of POU3F4 and supports previously reported mechanisms underlying DFNX2 pathogenesis. Full article

Tropomyosins (Tpm) are the family of actin-binding proteins encoded by four genes in humans. Missense mutations in the TPM1 gene associated with cardiomyopathies have been studied in the sarcomeric isoform Tpm1.1. The cardiomyopathy-causing mutations E40K and E54K are located in exon 2b of the TPM1 gene and may be expressed in non-muscle cytoplasmic Tpm isoforms, including Tpm1.7, which is associated with early tissue development. In the present work, we investigate the effects of mutations E40K and E54K on the properties of Tpm1.7. The E40K and E54K mutations caused destabilization of the Tpm1.7 molecule at the N- and C-termini parts. Neither mutation affected the Tpm1.7 affinity for filamentous actin (F-actin). The bending stiffness of F-actin/Tpm1.7 E40K filaments was lower compared to F-actin/Tpm1.7 WT (wild-type). The interplay of Tpm1.7 and motor proteins was studied in an in vitro motility assay with skeletal myosin. Tpm1.7 WT reduced the sliding velocity of F-actin by half; the velocity of F-actin with Tpm1.7 E54K did not differ from that of bare F-actin; and Tpm1.7 E40K decreased the F-actin velocity by approximately threefold. While Tpm1.7 E40K did not affect the protective effect of Tpm1.7 against F-actin severing by cofilin-1, the E54K mutation enhanced protection against cofilin-1. Thus, cardiomyopathic mutations in the TPM1 gene can affect the properties of non-muscle Tpm isoforms, which indicates that this should be taken into account when studying the molecular mechanisms of the pathogenesis of these diseases. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly impacted public health due to high rates of transmissibility and mutation during the COVID-19 pandemic. Macrodomain 1 (Mac1) of non-structural protein 3 remained well conserved across variants and is critical to suppression of host immune response to infection, making Mac1 a promising target for therapeutic development. Mac1 binds and cleaves the post-translational modification ADP-ribose and is hypothesized to have a downstream effect on the host interferon response, but the exact cellular targets of Mac1 are still unknown. Characterizing the substrates of Mac1 ADP-ribosyl hydrolase activity using a catalytically inactive mutant N40D can reveal critical virus–host interactions to identify protein targets of Mac1 and reveal mechanisms of host interferon suppression. Here, we performed affinity enrichment with WT Mac1 and Mac1 N40D in HEK293T and A549 cells and quantified changes in protein interactions by TMT-multiplexed tandem mass spectrometry. We identified interactions between Mac1 and ADP-ribosylated substrates involved in DNA damage response, cytoskeletal components, and cell cycle regulation. Additionally, several members of the TRiC complex involved in protein folding were selectively enriched with mutant Mac1 from A549 cells. These findings suggest a novel role of Mac1 in regulating host protein folding. Full article

Pregabalin (PGB) exerts its therapeutic effects by binding to the α₂δ auxiliary subunits of voltage-gated calcium channels and modulates synaptic transmission in the brain. However, its influence on cerebellar parallel fiber–Purkinje cell (PF–PC) synaptic transmission remains unclear. In the present study, we investigated the effects of PGB on PF–PC synaptic transmission using whole-cell patch-clamp recording, glutamate fluorescence imaging, immunohistochemistry, co-immunoprecipitation, Western blotting, and pharmacological approaches. Micro-application of PGB to the cerebellar molecular layer induced a concentration-dependent inhibition of PF–PC excitatory postsynaptic currents (EPSCs), accompanied by an increased paired-pulse ratio. The inhibitory effect of PGB on PF–PC EPSCs was abolished by extracellular blockade of N-methyl-D-aspartate receptors (NMDAR) or their GluN2A subtype, as well as by disruption of α₂δ-1–NMDAR complexes, but not by intracellular NMDAR inhibition. Glutamate sensor imaging further showed that PGB markedly reduced the fluorescence intensity of glutamate release evoked by PF stimulation. In the presence of tetrodotoxin (TTX) and a gamma-aminobutyric acid type A (GABAA) receptor antagonist, PGB reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude. The PGB-induced reduction in mEPSC frequency was fully abolished by extracellular blockade of GluN2A-containing NMDARs or disruption of α₂δ-1–NMDAR complexes. Similarly, the inhibitory effects of PGB on PF–PC EPSCs and mEPSCs were eliminated by extracellular PKA inhibition, but not by intracellular protein kinase A (PKA) inhibition. Western blot analysis showed that PGB significantly increased PKA phosphorylation in the molecular layer of the cerebellar cortex. Immunoreactivity for GluN2A and α₂δ-1 subunits was colocalized within the molecular layer and abundantly distributed around the dendrites and somata of PCs. Co-immunoprecipitation further verified that α₂δ-1 was co-precipitated with GluN1 in cerebellar molecular layer tissue samples. The results indicate that PGB depresses glutamate release from parallel-fiber terminals in the mouse cerebellar cortex through the presynaptic α₂δ-1-coupled GluN2A-containing NMDAR/PKA signaling pathway, thereby attenuating PF–PC synaptic transmission. Full article