Search Results (192)

The barbel chub (Squaliobarbus curriculus) exhibits remarkable resistance to grass carp reovirus (GCRV), a devastating pathogen in aquaculture. To reveal the molecular basis of this resistance, we investigated complement factor D (DF)—a rate-limiting serine protease governing alternative complement pathway activation. Molecular cloning revealed that the barbel chub DF (ScDF) gene encodes a 1251-bp cDNA sequence translating into a 250-amino acid protein. Crucially, bioinformatic characterization identified a unique N-glycosylation site at Asn¹³⁹ in ScDF, representing a structural divergence absent in grass carp (Ctenopharyngodon idella) DF (CiDF). While retaining a conserved Tryp_SPc domain harboring the catalytic triad (His⁶¹, Asp¹⁰⁹, and Ser²⁰⁴) and substrate-binding residues (Asp¹⁹⁸, Ser²¹⁹, and Gly²²¹), sequence and phylogenetic analyses confirmed ScDF’s evolutionary conservation, displaying 94.4% amino acid identity with CiDF and clustering within the Cyprinidae. Expression profiling revealed constitutive ScDF dominance in the liver, and secondary prominence was observed in the heart. Upon GCRV challenge in S. curriculus kidney (SCK) cells, ScDF transcription surged to a 438-fold increase versus uninfected controls at 6 h post-infection (hpi; p < 0.001)—significantly preceding the 168-hpi response peak documented for CiDF in grass carp. Functional validation showed that ScDF overexpression suppressed key viral capsid genes (VP2, VP5, and VP7) and upregulated the interferon regulator IRF9. Moreover, recombinant ScDF protein incubation induced interferon pathway genes and complement C3 expression. Collectively, ScDF’s rapid early induction (peaking at 6 hpi) and multi-pathway coordination may contribute to barbel chub’s GCRV resistance. These findings may provide molecular insights into the barbel chub’s high GCRV resistance compared to grass carp and novel perspectives for anti-GCRV breeding strategies in fish. Full article

Lanthanide rare earth elements possess significant promise for material applications owing to their distinctive optical and magnetic characteristics, as well as their versatile coordination capabilities. This study introduced a lanthanide-functionalized magnetic nanocellulose composite (NNC@Fe₃O₄@La(OH)₃) for effective phosphorus/nitrogen (P/N) ligand separation. The hybrid material employs the adaptable coordination geometry and strong affinity for oxygen of La³⁺ ions to show enhanced DNA-binding capacity via multi-site coordination with phosphate backbones and bases. This study utilized cellulose as a carrier, which was modified through carboxylation and amination processes employing deep eutectic solvents (DES) and polyethyleneimine. Magnetic nanoparticles and La(OH)₃ were subsequently incorporated into the cellulose via in situ growth. NNC@Fe₃O₄@La(OH)₃ showed a specific surface area of 36.2 m²·g⁻¹ and a magnetic saturation intensity of 37 emu/g, facilitating the formation of ligands with accessible La³⁺ active sites, hence creating mesoporous interfaces that allow for fast separation. NNC@Fe₃O₄@La(OH)₃ showed a significant affinity for DNA, with adsorption capacities reaching 243 mg/g, mostly due to the multistage coordination binding of La³⁺ to the phosphate groups and bases of DNA. Simultaneously, kinetic experiments indicated that the binding process adhered to a pseudo-secondary kinetic model, predominantly dependent on chemisorption. This study developed a unique rare-earth coordination-driven functional hybrid material, which is highly significant for constructing selective separation platforms for P/N-containing ligands. Full article

The ZC4H2 gene is the site of congenital mutations linked to neurodevelopmental and musculoskeletal pathologies collectively termed ZARD (ZC4H2-Associated Rare Disorders). ZC4H2 consists of a coiled coil and a single novel zinc finger with four cysteines and two histidines, from which the protein obtains its name. Alpha Fold 3 confidently predicts a structure for the zinc finger but also for similarly sized random sequences, providing equivocal information on its folding status. We show using synthetic peptide fragments that the zinc finger of ZC4H2 is genuine and folds upon binding a zinc ion with picomolar affinity. NMR pH titration of histidines and UV–Vis of a cobalt complex of the peptide indicate its four cysteines coordinate zinc, while two histidines do not participate in binding. The experimental NMR structure of the zinc finger has a novel structural motif similar to RANBP2 zinc fingers, in which two orthogonal hairpins each contribute two cysteines to coordinate zinc. Most of the nine ZARD mutations that occur in the ZC4H2 zinc finger are likely to perturb this structure. While the ZC4H2 zinc finger shares the folding motif and cysteine-ligand spacing of the RANBP2 family, it is missing key substrate-binding residues. Unlike the NZF branch of the RANBP2 family, the ZC4H2 zinc finger does not bind ubiquitin. Since the ZC4H2 zinc finger occurs in a single copy, it is also unlikely to bind DNA. Based on sequence homology to the VAB-23 protein, the ZC4H2 zinc finger may bind RNA of a currently undetermined sequence or have alternative functions. Full article

Infections with cytomegalovirus (CMV) can result in increased morbidity and mortality in immunocompromised patients. The pUL97 kinase is a critical enzyme in the regulation of CMV replication. Although it does not phosphorylate deoxynucleosides, this enzyme is involved in the first phosphorylation step of ganciclovir (GCV), a viral DNA polymerase inhibitor. In contrast, maribavir (MBV) is a specific inhibitor of pUL97 kinase activity. In this paper, we analyzed the already-reported amino acid changes, conferring resistance to MBV and cross-resistance to GCV, in the pUL97 protein structure, predicted with AlphaFold2. Docking experiments suggest that MBV is a dual-site inhibitor, targeting ATP binding and substrate phosphorylation. Substitutions that confer resistance to MBV only may directly or indirectly alter the shape of the cavity in the vicinity of the invariant K355 in the putative ATP binding site, without affecting the viral growth. The most frequently encountered T409M substitution may correspond to a gatekeeper mutation. Substitutions that induce cross-resistance to MBV and GCV may directly or indirectly affect the environment of D456 and N461 residues in the catalytic loop, with reduced viral replicative capacity. These results have implications for the clinical use of MBV as well as for the design of novel pUL97 kinase inhibitors. Full article

Background: Monoclonal antibodies against the sodium-dependent phosphate transporter NaPi2b (SLC34A2) represent a promising approach in the treatment of ovarian and lung cancer. Of particular interest is the potential cancer-specific MX35 epitope of NaPi2b, as it serves as a target for monoclonal antibodies studied at various stages of preclinical and clinical trials. However, variations in the NaPi2b protein structure may limit the efficacy of therapeutic antibodies by affecting the accessibility of the MX35 epitope. Methods: An in silico analysis was performed using data from 101,562 tumor samples. Genomic DNA sequencing was conducted on blood samples from patients with ovarian carcinoma, breast cancer, and renal carcinoma to access the frequency of germline mutations in the SLC34A2 gene region encoding the MX35 epitope. To assess the impact of the selected mutation, we generated a model cell line through site-directed mutagenesis carrying the mutant NaPi2b variant. Results: Using in silico analysis, we identified 17 unique variants in the SLC34A2 gene leading to amino acid substitutions within the MX35 epitope of the NaPi2b. Among these, the most prevalent mutation, c.989C>T, resulting in p.T330M substitution, was detected in 5 out of 64 patients through genomic DNA sequencing. Using site-directed mutagenesis, we created the OVCAR-8/NaPi2b^p.T330M model cell line. L3 (28/1) monoclonal antibodies specific to the MX35 epitope failed to recognize the mutant NaPi2b^p.T330M variant compared to the wild-type of the NaPi2b in both Western blot and confocal microscopy experiments. Conclusions: The obtained data may serve as a basis for predicting the efficacy of monoclonal antibody-based targeted therapy binding to the MX35 epitope of NaPi2b in the treatment of oncological diseases. Full article

Thiamine, crucial for energy metabolism, is associated with various human diseases when deficient. We studied how variations in the SLC19A3 gene, encoding THTR2, a thiamine transporter, may influence type 2 diabetes (T2DM) and gout (arthritis urica, AU). We characterized the SLC19A3 gene variants using bioinformatics and analyzed DNA samples from controls, T2DM, and gout patients to explore associations with physical/laboratory parameters. In human cells, we used a luciferase reporter assay to assess how these variants affect gene expression. We examined four large haplotypes (H1–4) in this gene, identified lead SNPs for the minor variants (MV), and explored potential transcription factor binding sites. We found that in T2DM patients, H3-MV correlated significantly with impaired glucose metabolism (pHOMA = 0.0189, pHbA1c% = 0.0102), while H4-MV correlated with altered uric acid (p = 0.0008) and white blood cell levels (p = 0.0272). In AU patients, H3-MV correlated with increased basophil granulocyte levels (p = 0.0273). In model cell lines, H3-MV presence increased gene expression (p = 0.0351), influencing responses to thiamine depletion and metformin (p = 0.0016). Although H4-MV did not directly affect luciferase expression, thiamine and fedratinib co-treatment significantly enhanced gene expression in thiamine-depleted cells (p = 0.04854). Our results suggest a connection between selected SLC19A3 variants and the severity of metabolic diseases or their response to treatment. Full article

Abnormal expression of miRNAs is associated with the occurrence and progression of cancer and other diseases, making miRNAs essential biomarkers for disease diagnosis and prognosis. However, the intrinsic properties of miRNAs, such as short length, low abundance, and high sequence homology, represent great challenges for fast and accurate miRNA detection in clinics. Herein, we developed a novel hybridization chain reaction (HCR)-based electrochemical miRNAs chip (e-miRchip), featured with gold nanostructured electrodes (GNEs) and silver nanoparticle reporters (AgNRs), for sensitive and multiplexed miRNA detection. AgNRs were synthesized and applied on the e-miRchip to generate strong redox signals in the presence of miRNA. The stem–loop capture probe was covalently immobilized on the GNEs, and was opened upon miRNA hybridization to consequently trigger the HCR for signal amplification. The multiple long-repeated DNA helix generated by HCR provides the binding sites for the AgNRs, contributing to the amplification of the electrochemical signals of miRNA hybridization. To optimize the detection sensitivity, GNEs with three distinct structures were electroplated, in which flower-like GNEs were found to be the best electrode morphology for miRNAs analysis. Under optimal conditions, the HCR-based e-miRchip showed an excellent detection performance with an LOD of 0.9 fM and a linear detection range from 1 fM to 10 pM. Moreover, this HCR-based e-miRchip platform was able to effectively distinguish miRNAs from the one- or two-base mismatches. This HCR-based e-miRchip holds great potential as a highly efficient and promising miRNA detection platform for the diagnosis and prognosis of cancer and other diseases in the future. Full article

Zinc finger protein 687 (ZNF687), a transcription factor implicated in osteoblast/osteoclast differentiation and linked to Paget’s disease of bone, has unclear mechanisms in bone metabolism. Epigenetic disruptions can affect bone cell activity and contribute to bone-related diseases. This work aimed to elucidate the regulatory role of epigenetics in modulating Zfp687 expression throughout osteoblast differentiation and bone growth/aging in mice. Differentiation of the mouse-derived osteoblast precursor cell line (MC3T3-E1) showed increased expression of osteogenic markers and decreased Zfp687 expression. In the hindlimb bones of C57BL/6J mice, the expression of most bone-forming genes decreased from youth to adulthood, while Zfp687 and Runx2 expression was maintained, being only significantly reduced in old mice in comparison to young mice. Bisulfite sequencing revealed hypomethylation of the Zfp687 promoter during MC3T3-E1 differentiation and bone growth/aging. Bioinformatics predicted miR-142a-3p, miR-122b-5p, and miR-124-3p binding sites in Zfp687 3′UTR, and RT-qPCR analysis showed higher expression of these miRNAs in mature osteoblasts. Transfection of a miR-142-3p mimic reduced luciferase activity in the wildtype Zfp687 3′UTR but not the mutant 3′UTR and downregulated the Zfp687 gene and protein levels. In conclusion, miR-142a-3p directly targets the Zfp687 3′UTR, promoting its downregulation during osteoblastogenesis. Furthermore, DNA methylation does not appear to regulate Zfp687 during osteoblast differentiation or bone development in mice. Full article

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that are almost exclusively expressed in germ cells to silence harmful transposons to maintain genome stability. PIWIL4 is guided by its associated piRNAs to transposable elements, where it recruits the DNA methylation apparatus and instructs de novo DNA methylation. Herein, we identified a missense variant of PIWIL4 (c.805 C>T p.R269W) in two infertile males. Homozygous male mice carrying the orthologous knock-in variant displayed elevated transposable element expression and aberrant gene expression during the first wave of spermatogenesis, despite exhibiting normal sperm counts and morphology. Mechanistically, the mutated site altered the piRNA-binding ability of PIWIL4 and led to the derepression of endogenous LINE-1 elements. In summary, we identified a piRNA binding mutation in PIWIL4 that may be involved in human nonobstructive azoospermia. Full article

This study aims to design improved inhibitors targeting the thymidylate kinase (TMK) of Mycobacterium tuberculosis (Mtb), the causative agent of infectious disease tuberculosis that is associated with high morbidity and mortality in developing countries. TMK is an essential enzyme for the synthesis of bacterial DNA. We have performed computer-aided molecular design of MtbTMK inhibitors by modification of the reference crystal structures of the lead micromolar inhibitor TKI1 1-(1-((4-(3-Chlorophenoxy)quinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione bound to TMK of Mtb strain H37Rv (PDB entries: 5NRN and 5NR7) using the computational approach MM-PBSA. A QSAR model was prepared for a training set of 31 MtbTMK inhibitors with published inhibitory potencies (${\mathrm{I}\mathrm{C}}_{50}^{\mathrm{e}\mathrm{x}\mathrm{p}}$) and showed a significant correlation between the calculated relative Gibbs free energies of the MtbTMK–TKIx complex formation and the observed potencies. This model was able to explain approximately 95% of the variation in the in vitro inhibition data and validated our molecular model of MtbTMK inhibition for the subsequent design of new TKI analogs. Furthermore, we have confirmed the predictive capacity of this complexation QSAR model by generating a 3D QSAR PH4 pharmacophore-based model. A satisfactory correlation was also obtained for the validation PH4 model of MtbTMK inhibition (R² = 0.84). We have extended the hydrophobic m-chloro-phenoxyquinolin-2-yl group of TKI1 that can occupy the entry into the thymidine binding cleft of MtbTMK by alternative larger hydrophobic groups. Analysis of residue interactions at the enzyme binding site made it possible to select suitable building blocks to be used in the preparation of a virtual combinatorial library of 28,900 analogs of TKI1. Structural information derived from the complexation model and the PH4 pharmacophore guided the in silico screening of the library of analogs and led to the identification of new potential MtbTMK inhibitors that were predicted to be effective in the low nanomolar concentration range. The QSAR complexation model predicted an inhibitory concentration ${\mathrm{I}\mathrm{C}}_{50}^{\mathrm{p}\mathrm{r}\mathrm{e}}$ of 9.5 nM for the best new virtual inhibitor candidate TKI 13_1, which represents a significant improvement in estimated inhibitory potency compared to TKI1. Finally, the stability of the MtbTMK–inhibitor complexes and the flexibility of the active conformation of the inhibitors were assessed by molecular dynamics for five top-ranking analogs. This computational study resulted in the discovery of new MtbTMK inhibitors with predicted enhanced inhibitory potencies, which also showed favorable predicted pharmacokinetic profiles. Full article

Our previous study identified the apolipoprotein M (APOM) and cytochrome P450 family 7 subfamily A polypeptide 1 (CYP7A1) genes as candidates for milk traits in dairy cattle, which were significantly up-regulated in liver tissue of Holstein cows between the dry and lactation periods. The two genes play critical roles in the peroxisome proliferator-activated receptor (PPAR) pathway. In this study, we further confirmed whether the APOM and CYP7A1 genes had significant genetic impacts on milk production traits in a Chinese Holstein population. By dual-direction sequencing of the polymerase chain reaction (PCR) products of the complete coding sequences and 2000 bp of the 5′ and 3′ flanking regions on pooled DNA sample, seven and three single nucleotide polymorphisms (SNPs) were identified in APOM and CYP7A1, respectively. With SAS 9.2, phenotype-genotype association analysis revealed such SNPs were significantly associated with at least one of the milk production traits, including 305-day milk yield, milk fat yield, milk fat percentage, milk protein yield, and milk protein percentage in the first and second lactations (p = <0.01~0.04). With Haploview 4.2, we further found that six SNPs in APOM and thee SNPs in CYP7A1 formed one haplotype, respectively. The haplotypes were significantly associated with at least one of milk production traits as well (p = <0.01~0.02). Of note, we found the SNPs in the 5′ regulatory region, rs209293266 and rs110721287 in APOM and rs42765359 in CYP7A1, significantly impacted the gene transcriptional activity after mutation (p < 0.01) through changing the transcription factor binding sites by using luciferase assay experiments. Additionally, with RNAfold Web Server, rs110098953 and rs378530166 changed the mRNA secondary structures of APOM and CYP7A1 genes, respectively. In summary, our research is the first to demonstrate that APOM and CYP7A1 genes have significantly genetic effects on milk yield and composition traits, and the identified SNPs may serve as available genetic markers for genomic selection program in dairy cattle. Full article

Cisplatin-based platinum compounds are important clinical chemotherapeutic agents that participate in most tumor chemotherapy regimens. Through density-functional theory calculations, the formation and stability of the inorganic oxide carrier, the mechanisms of the hydrolysis reaction of the activated platinum compound, and its binding mechanism with DNA bases can be studied. The higher the oxidation state of Pt (II to IV), the more electrons transfer from the magnesia–gold composite material to the platinum compound. After adsorption on the composite carrier, 5d←2p coordination bonds of Pt-N are strengthened. For flat and oblique adsorption modes of cisplatin, there is no significant difference in the density of states of the gold and magnesium oxide film, indicating the maintenance of the heterojunction structural framework. However, there are significant changes in the electronic states of cisplatin itself with different adsorption configurations. In the flat configuration, the band gap width of cisplatin is larger than that of the oblique configuration. The Cl-Pt bond range in the Pt(III) compound shows a clear charge reduction on the magnesia film, indicating the Cl-Pt bond is an active site with the potential for decomposition and hydrolysis. The substitution of chloride ions by water can lead to hydrolysis products, enhancing the polarization of the composite and showing strong charge separation. The hydrolysis of the free platinum compound is endothermic by 0.309 eV, exceeding the small activation energy barrier of 0.399 eV, indicating that hydrolysis of this platinum compound is easily achievable. ADME (absorption, distribution, metabolism, and excretion) prediction parameters indicate that hydrolysis products have good ESOL (Estimated SOLubility) solubility and high gastrointestinal absorption, consistent with Lipinski’s rule. During the coordination reaction process, there are significant changes in the distribution of frontier molecular orbitals, with the HOMO (highest occupied molecular orbital) of the initial state primarily located on the purine base, providing the possibility for electron transfer to the empty orbitals of the platinum compound in the LUMO (lowest unoccupied molecular orbital). The HOMO and HOMO-1 of the transition state and product are mainly distributed on the platinum compound, indicating clear electron transfer and orbital rearrangement. The activation energy barrier for the purine coordination reaction with the hydrolysis products is reduced to 0.61 eV, and the dipole moment gradually decreases to 6.77 Debye during the reaction, indicating a reduction in the system’s charge separation and polarization. This contribution is anticipated to provide a new theoretical clue for developing inorganic oxide carriers of platinum compounds. Full article

Mycobacterium avium subsp. paratuberculosis (MAP) is known to cause paratuberculosis. One notable protein, MAP3773c, plays a critical role in iron metabolism as a transcription factor. This study aims to investigate the binding affinity of MAP3773c to the chromatin of the Ferroportin1 (FPN1) gene in murine macrophage J774 A.1. We conducted a sequence alignment to identify potential interaction sites for MAP3773c. Following this, we used in silico analysis to predict binding interactions, complemented by electrophoretic mobility shift assay (EMSA) to confirm in vitro binding of MAP3773c. The map3773c gene was cloned into the pcDNA3.1 vector, with subsequent expression analysis carried out via Western blotting and real-time PCR. Chromatin immunoprecipitation (CHiP) assays were performed on transfected macrophages to confirm binding in the native chromatin context. Our in silico and in vitro analysis indicated that MAP3773c interacts with two binding motifs within the FPN1 coding region. The ChiP results provided additional validation, demonstrating the binding of MAP3773c to the FPN1 chromatin through successful amplification of the associated chromatin fragment via PCR. Our study demonstrated that MAP3773c binds to FPN1 and provides insight into the role of MAP3773c and its effect on host iron transport. Full article

The lens epithelium derived growth factor of 75 kD (LEDGF/p75) is a transcription co-activator and epigenetic reader that has emerged as a stress oncoprotein in multiple human cancers. Growing evidence indicates that it promotes tumor cell survival against certain therapeutic drugs. The amino (N)-terminal region of LEDGF/p75 contains a PWWP domain that reads methylated histone marks, critical for recognizing transcriptionally active chromatin sites. Its carboxyl (C)-terminus has an integrase binding domain (IBD) that serves as the binding site for the HIV-1 integrase and multiple oncogenic transcription factors. Acting as hubs for protein-protein interactions, both domains facilitate the tethering of oncogenic transcription factors and regulators to active chromatin to regulate mRNA splicing, promote DNA repair, and enhance the expression of stress and cancer-related genes that contribute to tumor cell aggressiveness and chemoresistance. This review summarizes our current knowledge of the emerging roles of LEDGF/p75 in cancer biology and therapy resistance and discusses its potential as a novel oncotherapeutic target in combinatorial treatments. Full article

Background: Schwann cells (SCs) and their plasticity contribute to the peripheral nervous system’s capacity for nerve regeneration after injury. The Egr2/Krox20 promoter antisense RNA (Egr2-AS) recruits chromatin remodeling complexes to inhibit Egr2 transcription following peripheral nerve injury. Methods: RNA-seq and ATAC-seq were performed on control cells, Lenti-GFP-transduced cells, and cells overexpressing Egr2-AS (Lenti-AS). Egr2 AS-RNA was cloned into the pLVX-DsRed-Express2-N1 lentiviral expression vector (Clontech, Mountain View, CA, USA), and the levels of AS-RNA expression were determined. Ezh2 and Wdr5 were immunoprecipitated from rat SCs and RT-qPCR was performed against AS-Egr2 RNA. ChIP followed by DNA purification columns was used to perform qPCR for relevant promoters. Hi-C, HiC-DC+, R, Bioconductor, and TOBIAS were used for significant and differential loop analysis, identifications of COREs and CORE-promotor loops, comparisons of TF activity at promoter sites, and identification of site-specific TF footprints. OnTAD was used to detect TADs, and Juicer was used to identify A/B compartments. Results: Here we show that a Neuregulin-ErbB2/3 signaling axis mediates binding of the Egr2-AS to YY1^Ser184 and regulates its expression. Egr2-AS modulates the chromatin accessibility of Schwann cells and interacts with two distinct histone modification complexes. It binds to EZH2 and WDR5 and enables targeting of H3K27me3 and H3K4me3 to promoters of Egr2 and C-JUN, respectively. Expression of the Egr2-AS results in reorganization of the global chromatin landscape and quantitative changes in the loop formation and contact frequency at domain boundaries exhibiting enrichment for AP-1 genes. In addition, the Egr2-AS induces changes in the hierarchical TADs and increases transcription factor binding scores on an inter-TAD loop between a super-enhancer regulatory hub and the promoter of mTOR. Conclusions: Our results show that Neuregulin-ErbB2/3-YY1 regulates the expression of Egr2-AS, which mediates remodeling of the chromatin landscape in Schwann cells. Full article