Search Results (1,035)

Background/Objectives: The neonatal Fc receptor (FcRn) is a heterodimeric protein composed of a heavy α-chain with an MHC class I-like fold and β₂-microglobulin. It plays a crucial role in maintaining the homeostasis and pharmacokinetics of immunoglobulin G (IgG) and albumin through pH-dependent recycling. The production of soluble recombinant FcRn is technically challenging due to its heterodimeric structure and the presence of a transmembrane domain. This study aimed to develop a polycistronic construct enabling the co-expression of FcRn subunits from a single transcript and to evaluate the functional activity of the resulting protein in CHO-K1 cells. Methods: Integration vectors (pComV-FcRn-B2M) were designed to encode FcRn and β₂-microglobulin linked via self-cleaving 2A peptides (P2A, E2A, F2A, T2A). Stable producer cell lines were generated using the Sleeping Beauty transposon system. The purified proteins were characterized by SDS-PAGE, Western blotting, and size-exclusion chromatography (SEC). Functional activity was assessed by ELISA and bio-layer interferometry (BLI). Results: Electrophoretic and chromatographic analyses confirmed the expected subunit composition and demonstrated that over 95% of the recombinant protein was monomeric. Functional assays revealed pH-dependent IgG binding, with strong interaction at pH 6.0 and negligible binding at pH 7.5. BLI measurements showed high affinity consistent with native FcRn function (KD = 3.15 nM at pH 6.0). Conclusions: The developed polycistronic construct containing a P2A peptide with a GSG linker enabled efficient production of functional FcRn in CHO-K1 cells (yield up to 2.23 mg/mL). The P2A variant demonstrated the highest efficiency and can serve as a reference system for screening Fc-engineered antibodies with optimized pharmacokinetic properties. Full article

Background: Mullegama–Klein–Martinez syndrome (MKMS; OMIM #301022) is an X-linked cohesinopathy caused by pathogenic variants in STAG2, which encodes a subunit of the cohesin complex responsible for chromosomal segregation and transcriptional regulation. Individuals typically present with developmental delay, microcephaly, dysmorphic features, and variable congenital anomalies, though complex cardiac malformations are uncommon. Case Presentation: We report a female infant presenting on the first day of life with complex congenital heart disease, including pulmonary atresia, double-outlet right ventricle, large subaortic ventricular septal defect, and patent ductus arteriosus. She exhibited intrauterine growth restriction, mild craniofacial dysmorphism, and left upper-extremity hypotonia. Stepwise genetic evaluation revealed a de novo likely pathogenic STAG2 frameshift variant, c.2972_2975dup (p.His992Glnfs*11), identified by rapid trio whole-exome sequencing. This variant truncates the C-terminal domain critical for cohesin binding. A 3D structural model generated by SWISS-MODEL demonstrated disruption of β-strand and loop conformations within this domain, consistent with loss of cohesin complex stability. Conclusions: This case expands the phenotypic spectrum of STAG2-related MKM and highlights the role of STAG2 in cardiac development. Recognition of such presentations supports the inclusion of STAG2 in the differential diagnosis for complex congenital heart disease and underscores the diagnostic utility of rapid trio exome sequencing in neonatal care. The utility of 3D protein modeling to illustrate structural consequences of truncating variants provides valuable insight into variant pathogenicity and supports precision diagnosis in cohesinopathies. Full article

Background/Objectives: Streptococcus suis is a zoonotic pathogen that causes infections in pigs and humans, leading to significant economic losses. S. suis can evade the immune system of hosts and induce persistent infections. Early detection and vaccination are crucial for controlling the disease in swine industries. This study aimed to investigate candidate recombinant protein for antibodies against S. suis detection and subunit vaccine development. Methods: The whole genome of S. suis BM407 was analyzed using bioinformatic tools to predict suitable proteins and genes for recombinant protein expression. Partial extracellular factor protein (epf) genes of S. suis serotype 2 DMST18783 were amplified. A 3301 bp amplicon was digested, and a specific 615 bp fragment was inserted into a pQE81L-KAN vector. Then, the constructed plasmid was cloned and expressed in Escherichia coli DH10β. Purified protein was analyzed using SDS-PAGE. In addition, translated amino acid sequences were analyzed for immune response properties, molecular docking, molecular dynamic simulation, and epitope prediction. Results: The amino acid sequence of recombinant extracellular factor protein (rEF) was revealed as a promising antigen containing putative protective regions as linear epitopes. Furthermore, the rEF was expressed as a histidine-tagged recombinant protein, and its properties were nearly similar to the predicted rEF using bioinformatic tools. Binding of the recombinant EF (rEF) protein was found to reduce fluctuations in the swine toll-like receptor 2. Furthermore, the rEF contained several regions that were predicted to be epitopes for both B-cells and T-cells. Conclusions: This study indicates that the recombinant EF fragment is a promising candidate for detecting antibodies against S. suis and as a component of a subunit vaccine. Full article

Background/Objectives: Muscle aging is a complex, dynamic process that impairs overall metabolism and physiological function. The molecular mechanisms underlying declines in muscle performance and metabolic efficiency remain poorly understood, largely due to the time and resource demands of traditional model organisms. The hawk moth Manduca sexta offers a promising alternative, with a short adult lifespan (~10 days) and notable similarities to vertebrate muscle systems, making it well-suited for time-course molecular dissection of muscle aging. Methods: In this study, we performed high-resolution temporal analysis of muscle tissues from aging M. sexta, spanning the physiomuscular aging process from middle age to advanced age. Results: We observed decreased expression of genes involved in fatty acid β-oxidation, ATP synthase subunits, superoxide dismutase, glutathione S-transferases, and heat shock proteins. In contrast, genes associated with proteolysis, catabolic processes, insulin signaling, akirin, titin, high-affinity choline transporters, and vesicular acetylcholine transporters were increased in expression. Conclusions: These changes suggest a shift toward increased proteolysis and protein catabolism with age. Our findings support the use of M. sexta as a complementary model for muscle aging research. However, it remains unclear whether the observed gene expression changes are driven by intrinsic, sex-specific age-related muscle aging or confounded by potential starvation effects in older males. Full article

Background: Epithelial–mesenchymal transition (EMT) is a fundamental process that drives invasion and metastasis in patients with diffuse-type gastric cancer (DGC). The role of SMARCD3, a subunit of the SWI/SNF chromatin remodeling complex, in this process is largely unknown. The aim of this study is to elucidate the molecular mechanism through which SMARCD3 integrates with the PI3K-AKT and WNT/β-catenin signaling pathways to promote EMT and gastric cancer progression. Methods: Stable SMARCD3-overexpressing MKN45 and MKN74 cell lines were established. RNA sequencing (RNA-seq) was performed to investigate signaling alterations. Western blot analysis confirmed the expression of EMT markers (Snail and Slug) and the phosphorylation of AKT (Ser473) and GSK3β (Ser9). PI3K dependency was tested using the inhibitor LY294002. Cooperative effects were examined by activating the WNT pathway with WNT3A. Results: SMARCD3 overexpression upregulated PI3K-AKT and WNT signaling, which correlated with increased Snail/Slug expression and increased AKT/GSK3β phosphorylation. GSK3β inactivation (pSer9) stabilizes Snail, driving EMT. LY294002 treatment suppressed Snail/Slug expression, attenuated AKT activation, and reversed the mesenchymal phenotype. Furthermore, WNT3A treatment synergistically increased nuclear Snail accumulation. Conclusions: SMARCD3 acts as a critical epigenetic regulator that promotes EMT in patients with gastric cancer through the integration of the PI3K-AKT and WNT/β-catenin pathways. Targeting this SMARCD3-mediated mechanism offers a promising therapeutic strategy to inhibit metastasis and improve outcomes for patients with gastric cancer. Full article

Hepatocellular carcinoma (HCC), the most common primary liver malignancy worldwide, is strongly associated with chronic Hepatitis B Virus (HBV) infection, a significant risk factor. The ubiquitin–proteasome system, central to protein degradation, cellular homeostasis, and cell cycle regulation, has been implicated in the pathogenesis of several cancers, including HCC. Despite this, the specific expression patterns of proteasomal subunits during HBV infection and HBV-induced HCC, as well as the association between mRNA expression of proteasomal subunits and proteasomal activity, remain poorly defined. To address this critical knowledge gap, we analyzed mRNA expression profiles of proteasomal subunits in HBV-infected humanized mouse models to uncover HBV-specific molecular alterations. Our findings revealed that the chymotrypsin-like activity (β5) subunit of the proteasome (PSMB5) is consistently overexpressed following HBV infection. Functional studies demonstrated that β5 deficiency decreases MHC I levels on the cell surface and leads to the accumulation of ubiquitinated proteins, establishing a direct link between β5 overexpression and increased proteasomal activity. Concordantly, HBV-infected patient livers—regardless of HCC status—displayed elevated β5 mRNA/protein levels and enhanced chymotrypsin-like activity. Additionally, analysis of Protein Atlas data revealed that elevated β5 mRNA expression correlates with poor clinical prognosis in HCC patients. In summary, this study highlights how HBV infection induces significant alterations in proteasome function by elevating β5 expression and activity in human and mouse livers. These findings underscore the critical role of proteasomal dysregulation in HBV-associated liver pathology and provide new insights into its involvement in HCC development. Understanding the interplay between HBV infection and proteasome dynamics offers a valuable avenue for the identification of novel therapeutic targets and biomarkers in HCC. Full article

The proteasome is a central proteolytic complex that maintains protein homeostasis by eliminating damaged, misfolded, and regulatory proteins. Beyond this quality control role, it generates bioactive peptides that contribute to immune surveillance, intracellular signaling, neuronal communication, and antimicrobial defense. Proteolysis is mediated by the catalytic β1, β2, and β5 subunits, traditionally defined by caspase-like, trypsin-like, and chymotrypsin-like activities. However, these sites display overlapping and flexible specificities, enabling cleavage after nearly all amino acids. This review focuses on proteasome catalytic activity, with particular emphasis on the biochemical and structural features of the catalytic subunits that define cleavage selectivity. We first provide a historical overview of the discovery of proteolytic activities and trace the evolutionary diversification of subunits that gave rise to specialized variants such as the immunoproteasome, thymoproteasome, intermediate proteasomes, and the spermatoproteasome. We then highlight how advances in computational modeling and structural biology have refined our understanding of cleavage preferences. In addition, we examine how regulatory particles, post-translational modifications, and physiological conditions, including inflammation, oxidative stress, and aging, modulate proteolytic activity. Finally, we discuss the development of selective inhibitors targeting individual catalytic sites, emphasizing their therapeutic potential in cancer, autoimmunity, and infectious disease, and outline future directions for the field. Full article

Mycobacteria possess carbon monoxide dehydrogenase (CO-DH) to utilize CO as an energy source and to resist host defense mechanisms. The expression of the CO-DH gene is regulated by CutR, a LysR-type transcriptional regulator (LTTR) that exhibits unique characteristics, suggesting that it functions as a dimer rather than the typical tetramer. Size-exclusion chromatography revealed that CutR forms a stable dimer. Electrophoretic mobility shift assays demonstrated that dimeric CutR specifically binds to an inverted repeat sequence (IR1) containing T-n12-A motifs located upstream of the cutB gene, which encodes the medium subunit of CO-DH. Crystal structure determination at 1.8 Å resolution revealed that CutR consists of an N-terminal DNA-binding domain with a winged helix-turn-helix motif and a C-terminal ligand-binding domain comprising two regulatory subdomains (RD1 and RD2), forming a unique two-fold symmetrical homodimer. This dimer is stabilized through four interfaces, including an extensive 12-stranded antiparallel β-sheet formed between RD1 subdomains via intertwining C-terminal β11 strands. This represents the first symmetric dimeric LTTR structure with tightly associated ligand-binding domains. The recognition helices are spaced closer together than they are in typical DNA-bound LTTRs, despite binding longer T-n12-A sequences, suggesting that a conformational change is required to enhance DNA-binding affinity. A putative ligand-binding site was identified between the RD1 and RD2 subdomains, where glycerol binding induced local conformational changes. Comparative genomic analysis revealed conservation of CutR and the IR1 sequence across Mycobacterium species, supporting the dimeric regulatory mechanism and providing new insights into LTTR diversity. Full article

Eleutheroside E (EE), a natural compound, shows promise in mitigating cellular senescence—a key factor in skin aging—though its mechanisms remain incompletely understood. This study integrated network pharmacology, molecular docking, and cellular experiments to explore the protective effects and mechanistic basis of EE against D-galactose (D-gal)-induced senescence in human skin fibroblasts (HSFs). Network pharmacology analyses suggested EE’s involvement in inflammation-related pathways, especially phosphatidylinositol 3-kinase and protein kinase B (PI3K-AKT) and hypoxia-inducible factor 1 (HIF-1) signaling, which were corroborated by molecular docking revealing strong binding affinities between EE and key targets such as hypoxia-inducible factor 1-alpha (HIF1A), AKT serine/threonine kinase 1 (AKT1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PI3Kγ), and interleukin-6 (IL-6). Cellular assays showed that EE markedly lowered oxidative stress markers, including reactive oxygen species (ROS) and malondialdehyde (MDA), reduced senescence-associated beta-galactosidase (SA-β-gal) activity, and boosted antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Additionally, EE dose-dependently inhibited apoptosis and downregulated PI3K/AKT phosphorylation as well as the B-cell lymphoma 2-associated X protein/B-cell lymphoma-2 (Bax/Bcl-2) ratio. These findings suggest that EE alleviates cellular senescence in HSFs mainly via the PI3K/AKT pathway by attenuating oxidative stress and apoptosis, highlighting its potential as a therapeutic agent for anti-aging strategies. Full article

This study aimed to investigate the effects of synergistic K⁺ immersion-induced salting-out on the rheological properties, microstructure, molecular interactions, and swallowing adaptability of soy protein isolate (SPI)/λ-carrageenan composite gels under different enzymatic pretreatment times (0, 10, 20, 30, 60, and 120 min) using Flavourzyme. The results showed that enzymatic hydrolysis increased the degree of hydrolysis of SPI from 1.11% to 11.46%, gradually degraded the 7S subunit, and reached the highest surface hydrophobicity at 30 min of moderate hydrolysis. After KCl immersion treatment, the K-λ/SPI gels exhibited lower water holding capacity and higher whiteness compared to those before immersion. Among them, the K-λ/SPH30 group demonstrated the best rheological properties. Moderate enzymatic hydrolysis synergistically promoted the formation of a dense network in K-λ/SPI gels. This process enhanced the stability of the composite gel through hydrophobic interactions, electrostatic interactions, and hydrogen bonds while simultaneously increasing the proportion of β-structures (reaching a maximum of 62.05%). The expanded binding sites from moderate enzymatic hydrolysis, combined with the dense network and enhanced interactions, collectively strengthened the salting-out effect. This ultimately enabled K-λ/SPH30 to achieve the highest crystallinity (93.57%), the highest K⁺ content (4.80%), and the optimal swallowing performance (IDDSI level 5). This study not only deepens the understanding of the synergistic mechanism between protein hydrolysates and salting-out but also provides an innovative solution for designing foods for dysphagia diets. Full article

Background: β-casomorphin-10 (CM-10), a peptide fragment derived from milk casein with the sequence YPFPGPIPNS, has demonstrated notable anxiolytic activity in BALB/c mice. Yet, its cellular responses and mechanistic pathways remain largely uncharacterized. Methods: We performed RNA-seq analysis to profile gene expression changes in δ-opioid receptor-expressing HEK293 cells (DOR-HEK), comparing CM-10-treated and untreated conditions. Results: CM-10 exposure led to differential expression of 1714 genes in DOR-HEK cells, with 34 upregulated (>1.4-fold) (1.9%) and 1680 downregulated (<0.71-fold) (98.1%), based on a predicted p-value threshold of <0.05. Notably, we identified 10 clusters that were associated with reduced cyclic AMP (cAMP) in DOR-HEK cells following CM-10 treatment. These clusters particularly involved genes related to regulatory subunits of cAMP-dependent protein kinases, such as PRKAR2A, cAMP-responsive element-binding pathway, circadian rhythms, such as CLOCK, ARNT1, CRY2, PER1, and PER2, and anxiety and depression, such as NOTCH1, NOTCH2 and ANK2. A network with these selected genes was confirmed by STRING analysis. Conclusions: These findings indicate that CM-10 may activate DOR-mediated signaling by suppressing cAMP levels, implicating a distinct molecular cascade in HEK293 cells. Full article

Salt-tolerance improvement of tomatoes is largely a task of modern selection and plant molecular genetics because of cultivation on dry and irrigated lands under salt stress. To reveal the salt resistance gene, we need quantitative real-time polymerase chain reaction (qRT-PCR) normalization through reference genes analysis. Sometimes, housekeeping gene expression changes in response to various stress factors, especially salinity. In this manuscript, we evaluated expression changes of elongation factor 1α X53043.1 (EF1α), actin BT013707.1 (ACT), ubiquitin NM_001346406.1 (UBI), nuclear transcript factor XM_026030313.2 (NFT-Y), β-tubulin NM_001247878.2 (TUB), glyceraldehyde-3 phosphate dehydrogenase NM_001247874.2 (GAPDH), phosphatase 2A catalytic subunit NM_001247587.2 (PP2a), and phosphoglycerate kinase XM_004243920.4 (PGK) in salt-sensitive Solanum lycopersicum L. YaLF line and salt tolerance Rekordsmen cv. under 100 mM NaCl. We also suggested potential correlations between relative water content (RWC), ion accumulation, and reference gene expression in tomato genotypes with contrasting salinity tolerance. We used geNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder algorithms to establish a set of the most reliable tomato candidate genes. The most stable genes for YaLF tomatoes were ACT, UBI, TUB, and PP2a. Despite differences in ranks, the NFT-Y was present in Rekordsmen’s stable set. Full article

Gamma-aminobutyric acid type A receptors (GABA_ARs) are pentameric ligand-gated ion channels essential for inhibitory neurotransmission in the central nervous system. Subtype-specific expression patterns of GABA_AR subunits underlie their diverse roles in regulating anxiety, motor function, and sedation. While non-selective GABA_AR positive allosteric modulators (PAMs), such as benzodiazepines, are clinically effective anxiolytic drugs, their non-selective activity across α1/2/3/5 subunit-containing GABA_ARs leads to sedation, cognitive impairment, and risk of dependence. To address this, we evaluated ENX-102, a novel GABA_AR PAM, which exhibits selectivity for α2/3/5 subunits. In rodents, ENX-102 demonstrated dose-dependent anxiolytic-like activity following acute and sub-chronic administration, without sedation. ENX-102 exhibited a dose-dependent quantitative electroencephalography (qEEG) spectral signature in rodents that was distinct from that of benzodiazepines. In a double-blind, placebo-controlled, multiple-ascending dose study in healthy human volunteers, ENX-102 was evaluated using the NeuroCart, a CNS test battery including saccadic peak velocity (SPV), adaptive tracking, pupillometry, body sway, the Bond and Lader Visual Analog Scale (VAS), the Visual Verbal Learning Task (VVLT), and qEEG. ENX-102 produced reductions in SPV that were indicative of central target engagement, with minimal effects on alertness and motor coordination, which is consistent with subtype-selective GABA_AR targeting. Notably, qEEG revealed increased β-band power and decreased δ- and θ-band activity, which were distinct from the spectral profile of non-selective PAMs, supporting translational alignment with preclinical findings. Across dose levels, ENX-102 was well tolerated and exhibited favorable pharmacokinetics. These results support further clinical development of ENX-102 as a next-generation GABA_AR subtype-selective anxiolytic drug. Full article

Naringinase consists of two enzymes: α-L-rhamnosidase and β-D-glucosidase. The enzyme was immobilized on a carrier prepared from carob gum activated with polyethyleneimine. Cross-linking with dextran aldehyde was used to improve the stability of the immobilization. Knowledge of the characteristics of naringinase subunits is important for developing efficient and selective enzymatic reactions involving flavonoids. This study aimed to characterize two subunits of naringinase—α-L-rhamnosidase and β-D-glucosidase—free, immobilized on a magnetic polysaccharide carrier and cross-linked with dextran aldehyde. The characterization of free, immobilized, and stabilized naringinase, as well as α-L-rhamnosidase and β-D-glucosidase, included the effect of pH and temperature on enzyme activity, as well as the determination of their stability depending on the pH and temperature of the environment, and the determination of kinetic constants. Immobilization and subsequent stabilization of naringinase did not affect the optimal pH for the activity of α-L-rhamnosidase and β-D-glucosidase. Immobilization caused a change in the optimal temperature for the activity of α-L-rhamnosidase and β-D-glucosidase from 60 to 65°. Cross-linking of immobilized naringinase with dextran aldehyde increased the temperature stability of its subunits. Cross-linking also altered the pH stability profile of β-D-glucosidase. Immobilization and stabilization of naringinase slightly reduced the maximum reaction rate for α-L-rhamnosidase and β-D-glucosidase compared to the free enzyme. As a result of immobilization, the enzymes’ affinity for substrates for both subunits decreased. Full article

The fruiting stage of soybean (Glycine max L.) is critical for determining both its yield and quality, thereby influencing global production. While some studies have provided partial explanations for the occurrence of Fusarium species on soybean seeds and pods, the fungal diversity affecting soybean pods in Sichuan Province, a major soybean cultivation region in Southwestern China, remains inadequately understood. In this study, 182 infected pods were collected from a maize–soybean relay strip intercropping system. A total of 10 distinct pod-infecting fungal genera (132 isolates) were identified, and their pathogenic potential on soybean seeds and pods was evaluated. Using morphological characteristics and DNA barcode markers, we identified 43 Fusarium isolates belonging to 8 species, including F. verticillioides, F. incarnatum, F. equiseti, F. proliferatum, F. fujikuroi, F. oxysporum, F. chlamydosporum, and F. acutatum through the analysis of the translation elongation factor gene (EF1-α) and RNA polymerases II second largest subunit (RPB2) gene. Multi-locus phylogenetic analysis, incorporating the Internal Transcribed Spacer (rDNA ITS), β-tubulin (β-tubulin), Glyceraldehyde 3-phosphate dehydrogenase (GADPH), Chitin Synthase 1 (CHS-1), Actin (ACT), Beta-tubulin II (TUB2), and Calmodulin (CAL) genes distinguished 37 isolates as 6 Colletotrichum species, including C. truncatum, C. karstii, C. cliviicola, C. plurivorum, C. boninense, and C. fructicola. Among these, F. proliferatum and C. fructicola were the most dominant species, representing 20.93% and 21.62% of the isolation frequency, respectively. Pathogenicity assays revealed significant damage from both Fusarium and Colletotrichum isolates on soybean pods and seeds, with varying isolation frequencies. Of these, F. proliferatum, F. acutatum, and F. verticillioides caused the most severe symptoms. Similarly, within Colletotrichum genus, C. fructicola was the most pathogenic, followed by C. truncatum, C. karstii, C. cliviicola, C. plurivorum, and C. boninense. Notably, F. acutatum, C. cliviicola, C. boninense, and C. fructicola were identified for the first time as pathogens of soybean pods under the maize–soybean strip intercropping system in Southwestern China. These findings highlight emerging virulent pathogens responsible for soybean pod decay and provide a valuable foundation for understanding the pathogen population during the later growth stages of soybean. Full article