Search Results (4,549)

Pediatric tumors such as neuroblastoma are characterized by a genome-wide ‘transcriptional burden’, surmising the involvement of multiple alterations of gene expression. Search for master regulators of transcription whose inactivation is lethal for tumor cells identified the non-POU domain-containing octamer-binding protein (NONO), a member of the Drosophila Behavior/Human Splicing family known for the ability to form complexes with macromolecules. NONO emerges as an essential mechanism in normal neurogenesis as well as in tumor biology. In particular, NONO interactions with RNAs, largely with long non-coding MYCN transcripts, have been attributed to the aggressiveness of neuroblastoma. Broadening its significance beyond MYCN regulation, NONO guards a subset of transcription factors that comprise a core regulatory circuit, a self-sustained loop that maintains transcription. As a component of protein–protein complexes, NONO has been implicated in the control of cell cycle progression, double-strand DNA repair, and, generally, in cell survival. Altogether, the pro-oncogenic roles of NONO justify the need for its inactivation as a therapeutic strategy. However, considering NONO as a therapeutic target, its druggability is a challenge. Recent advances in the inactivation of NONO and downstream signaling with small molecular weight compounds make promising the development of pharmacological antagonists of NONO pathway(s) for neuroblastoma treatment. Full article

BACH1 (BTB And CNC Homology 1) and NRF2 (Nuclear Factor Erythroid 2-related Factor 2) are transcription factors that regulate antioxidant and iron metabolism genes by competing for binding to cis-regulatory Maf-binding elements (CsMBEs) as heterodimers with small Maf proteins (sMafs). To dissect the mechanisms underlying this competition, we developed a chimeric tethering system where the DNA-binding domains of BACH1 or NRF2 were covalently linked to sMafG via a flexible, cleavable linker. This design enables efficient heterodimer formation on DNA and circumvents kinetic barriers to partner exchange in the solution. The site-specific fluorescent labelling of proteins allowed for the tracking of complex compositions by electrophoretic mobility shift assays. Both BACH1/sMafG and NRF2/sMafG heterodimers bind CsMBEs with similar affinities. Notably, DNA binding by BACH1 was impaired in a C574-dependent, redox-sensitive manner and promoted the exchange of heterodimer partners. Competition assays demonstrated that BACH1 and NRF2 can displace each other from preformed DNA-bound complexes, with greater efficiency when presented as preassembled heterodimers with sMafG. These findings reveal a redox-sensitive mechanism for regulating transcriptional switches at CsMBEs and highlight how preformed heterodimers facilitate the rapid displacement at target promoters. Full article

Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples. Full article

The production of green hydrogen by microalgae is a promising strategy to convert energy of sun light into a carbon-free fuel. Many problems must be solved before large-scale industrial applications. One solution is to find a microalgal species that is easy to grow, easy to manipulate, and that can produce hydrogen open-air, thus in the presence of oxygen, for periods of time as long as possible. In this work we investigate by means of predictive computational models, the [FeFe] hydrogenase enzyme of Nannochloropsis salina, a promising microcalga already used to produce high-value products in salt water. Catalysis of water reduction to hydrogen by [FeFe] hydrogenase occurs in a peculiar iron-sulfur cluster (H-cluster) contained into a conserved H-domain, well represented by the known structure of the single-domain enzyme in Chlamydomonas reinhardtii (457 residues). By combining advanced deep-learning and molecular simulation methods we propose for N. salina a two-domain enzyme architecture hosting five iron-sulfur clusters. The enzyme organization is allowed by the protein size of 708 residues and by its sequence rich in cysteine and histidine residues mostly binding Fe atoms. The structure of an extended F-domain, containing four auxiliary iron-sulfur clusters and interacting with both the reductant ferredoxin and the H-domain, is thus predicted for the first time for microalgal [FeFe] hydrogenase. The structural study is the first step towards further studies of the microalga as a microorganism producing pure hydrogen gas. Full article

Background/Objectives: Wilson’s disease (WD) is an autosomal recessive disorder characterized by pathological copper accumulation, primarily in the liver and brain. Severe hepatic involvement can be effectively treated with liver transplantation (LT). Geographic variation in ATP7B mutations suggests the presence of regional patterns that may impact disease presentation and management. This study aims to investigate the genetic basis of WD in patients from a major LT center in Iran. Methods: A retrospective analysis was conducted on clinical, biochemical, and pathological data from patients suspected of WD who underwent evaluation for LT between May 2020 and June 2025 at Shiraz University of Medical Sciences. Genetic testing was carried out on 20 patients at the Shiraz Transplant Research Center (STRC). Direct mutation analysis of ATP7B was performed for all patients, and the results correlated with clinical and demographic information. Results: In total, 20 WD patients who underwent liver transplantation (15 males, 5 females) carried 25 pathogenic or likely pathogenic ATP7B variants, 21 of which were previously unreported. Fifteen patients were homozygous, and five were compound-heterozygous; all heterozygous combinations occurred in the offspring of second-degree consanguineous unions. Recurrent changes included p.L549V, p.V872E, and p.P992S/L, while two nonsense variants (p.E1293X, p.R1319X) predicted truncated proteins. Variants were distributed across copper-binding, transmembrane, phosphorylation, and ATP-binding domains, and in silico AlphaMissense scores indicate damaging effects for most novel substitutions. Post-LT follow-up showed biochemical normalization in the majority of recipients, with five deaths recorded during the study period. Conclusions: This single-center Iranian study reveals a highly heterogeneous ATP7B mutational landscape with a large proportion of novel population-specific variants and underscores the benefit of comprehensive gene sequencing for timely WD diagnosis and family counseling, particularly in regions with prevalent consanguinity. Full article

Background: B7-H3, an immunoregulatory protein of the B7 family, has been associated with both anti-cancer immunity and tumor promotion, with its expression commonly correlated with poor prognosis. Although it is frequently expressed across cancers, its heterogeneity may limit the effectiveness of B7-H3-targeted therapies. Consequently, a sensitive and non-invasive method is needed to assess B7-H3 expression for patient selection and stratification. Single-domain antibody fragments (sdAbs) offer a promising platform for developing such a diagnostic tool. Methods: To generate B7-H3 sdAbs, two Ilamas were immunized with the recombinant human B7-H3 protein. Positive clones were selected through Phage biopanning and characterized for thermal stability, binding specificity, and affinity to human and murine B7-H3 proteins. Selected sdAbs were radiolabeled with Technetium-99m (^99mTc) and evaluated for B7-H3 detection in two xenograft tumor models using micro-SPECT/CT imaging and dissection studies. Results: Sixteen purified sdAbs bound specifically to recombinant B7-H3 proteins and cells expressing native B7-H3 antigens, with nanomolar affinities. The four best-performing sdAbs bound promiscuously to tested mouse and human B7-H3 isoforms. Lead sdAb C51 labeled with ^99mTc displayed specific accumulation across two human B7-H3⁺ tumor models, achieving high contrast with a tumor-to-blood ratio of up to 10 ± 3.16, and a tumor uptake of up to 4.96 ± 1.4%IA/g at 1.5 h post injection. Conclusions: The lead sdAb enabled rapid, specific, and non-invasive imaging of human B7-H3⁺ tumors. Its isoform promiscuity supports broad applicability across cancers expressing different human B7-H3 isoforms. These results support further development for clinical translation to enable patient selection and improved B7-H3-targeted therapies. Full article

Drought severely threatens wheat production. Under drought conditions, root system architecture (DRSA)-related traits in common wheat significantly affect wheat production. In China, Zhoumai16 is a high-yield winter wheat variety in the Huang-Huai wheat region. It is suitable for high-fertilizer and high-water cultivation and has moderate drought tolerance. DK171 is a newly developed high-yield and stress-tolerant variety, with higher drought tolerance. Thus, identifying genetic loci associated with DRSA-related traits from DK171 and developing available molecular markers are of great importance for enhancing wheat stress tolerance breeding. In this study, DRSA-related traits, including the total root dry weight (DDRW), total root length (DTRL), total root area (DTRA), and the number of root tips (DNRT) under drought stress, were assessed using the hydroponic system in Zhoumai16/DK171 recombinant inbred lines (RIL) population. A total of five quantitative trait loci (QTL) for DRSA-related traits were identified, e.g., QDDRW.daas-1BL, QDTRS.daas-4AL, QDNRT.daas-4DS, QDTRL.daas-3AL, and QDDRW.daas-5D, and explained 6.1% to 18.9% of the phenotypic variances, respectively. Among these, QDTRS.daas-4AL and QDTRL.daas-3AL were consistent with previous reports, whereas the QDDRW.daas-1BL, QDNRT.daas-4DS, and QDDRW.daas-5D are novel. The favorable alleles of QDTRS.daas-4AL and QDNRT.daas-4DS were inherited from Zhoumai16, whereas the favorable alleles for QDDRW.daas-1BL, QDTRL.daas-3AL, and QDDRW.daas-5D were contributed by DK171. Furthermore, five kompetitive allele-specific PCR (KASP) markers, Kasp_1BL_DTRS (QDDRW.daas-1BL), Kasp_3AL_DTRS (QDTRL.daas-3AL), Kasp_4A_DTRS (QDTRA.daas-4A), Kasp_5D_DDRW (QDDRW.daas-5D), and Kasp_4D_DNRT (QDNRT.daas-4D), were developed and validated in a diverse panel with 108 wheat varieties mainly from China. Additionally, eight candidate genes related to plant hormone regulation, ABC transporters, and calcium-dependent lipid-binding domain proteins were identified. This study offers new loci, candidate genes, and available KASP markers for wheat drought tolerance breeding and facilitating progress in developing drought-tolerant wheat cultivars. Full article

Thiol isomerases are a family of enzymes that participate in oxidative protein folding. They contain highly reactive vicinal thiols in a CXXC motif within their catalytic domains to mediate thiol-disulfide switching as part of their reductase, oxidase, and isomerase activity. In addition, they participate in chaperone function by binding to partially folded or misfolded proteins and preventing aggregation, thereby facilitating correct protein folding. The CXXC motif is conducive to oxidative influence based on the sulfur nucleophilicity. Redox modification of the CXXC motif may influence the enzymatic function. In this review we briefly discuss the family of thiol isomerases as it relates to thrombotic disorders. We then discuss the chemical mechanisms of making and breaking disulfides by the enzymes. Enzymatic and chemical models of oxidizing the CXXC motif are proposed. Lastly, we highlight evidence that natural galloylated polyphenols can inhibit both the coronavirus main protease Mpro and thiol isomerases, supporting a therapeutic strategy for COVID-19-associated coagulopathy and thrombosis by targeting the CXXC motif with these anti-oxidative compounds. Full article

Background/Objectives: Orofacial clefts (OFCs) are among the most common birth defects globally, sometimes exacerbated by adverse drug reactions (ADRs) from corticosteroids and antiepileptics. Comprehending the pharmacogenomic and pharmacogenetic elements that lead to ADRs is essential for enhancing precision medicine and clinical outcomes. This study examines rare genetic variants in drug-metabolizing and drug-transporting genes among Ghanaian and Nigerian families with a history of OFCs, intending to assess their pathogenicity and functional implications. Methods: We recruited 104 Ghanaian families and 26 Nigerian families, generating whole-genome sequencing (WGS) data from 390 individuals (130 case-parent trios). DNA isolated from saliva and buccal swab samples underwent WGS, and subsequent WGS data were analyzed through extensive bioinformatics analyses. Variants were called and annotated using the GATK workflow. The HOPE in silico modeling tool evaluated the structural impact of genetic variants on encoded proteins, while molecular docking using PyRx examined alterations in ligand binding affinity. Results: Our study revealed pathogenic variants in vital genes associated with drug metabolism and transport, specifically CYP1A2, CYP2C18, CYP27A1, CYP2B6, SLC6A2, and ABCC3. Structural modeling research demonstrated substantial size, charge, conformation, and hydrophobicity variations between wildtype and mutant proteins. Variants positioned near conserved regions or within functional domains were anticipated to be deleterious, potentially compromising protein function and ligand interactions. Molecular docking studies verified changes in binding affinities between wildtype and mutant proteins for common ligands. The identified variations were linked to the metabolism of frequently used pharmaceuticals in Africa, such as caffeine, ketoconazole, efavirenz, carbamazepine, and artemether. Conclusions: These findings highlight the need for pharmacogenetic screening to inform personalized medicine, diminish ADRs, and enhance the clinical care of OFCs in Sub-Saharan Africa. Full article

Since the appearance of the Severe Acute Respiratory Syndrome (SARS) virus, there has been increased interest in understanding the role of bats in the maintenance and circulation of coronaviruses. This study aimed to describe the phylogenetic and evolutionary relationships and antigenic architecture of a new coronavirus detected in bats in the Department of Córdoba. In a surveillance study of pathogens of interest to public health, a bat Phyllostomus hastatus was captured. Rectal swabs samples were collected from the bats, and RNA was extracted and sequenced using NGS with MGI-G50 equipment. The results were analyzed using bioinformatics software. A contig of 28,619 nucleotides associated with the Coronaviridae family was obtained. Phylogenetic and molecular clock analyses of the ORF1ab gene revealed a novel divergent Alphacoronavirus that originated directly from an ancestral node. The analysis of the spike (S) protein and receptor-binding domain (RBD) is similar to that of humans (HCoV-229E) and porcine coronaviruses. In silico analysis suggests potential RBD interaction sites with human and pig cellular receptor aminopeptidase N. There is a possible risk of interspecies jumping of the new AlphaCoV/P. hastatus in humans and pigs. This is the first study to perform phylogenetic, evolutionary, and antigenic characterization of bat coronaviruses in Colombia. Full article

RNA polymerases are essential enzymes that catalyze DNA transcription into RNA, vital for protein synthesis, gene expression regulation, and cellular responses. Non-template-dependent RNA polymerases, which synthesize RNA without a template, are valuable in biological research due to their flexibility in producing RNA without predefined sequences. However, their substrate polymerization mechanisms are not well understood. This study examines Poly (A) polymerase (PAP), a nucleotide transferase superfamily member, to explore its substrate selectivity using computational methods. Previous research shows PAP’s polymerization efficiency for nucleoside triphosphates (NTPs) ranks ATP > GTP > CTP > UTP, though the reasons remain unclear. Using 500 ns Gaussian accelerated molecular dynamics simulations, stability analysis, secondary structure analysis, MM-PBSA calculations, and Markov state modeling, we investigate PAP’s differential polymerization efficiencies. Results show that ATP binding enhances PAP’s structural flexibility and increases solvent-accessible surface area, likely strengthening protein–substrate or protein–solvent interactions and affinity. In contrast, polymerization of other NTPs leads to a more open conformation of PAP’s two domains, facilitating substrate dissociation from the active site. Additionally, ATP binding induces a conformational shift in residues 225–230 of the active site from a loop to an α-helix, enhancing regional rigidity and protein stability. Both ATP and GTP form additional π–π stacking interactions with PAP, further stabilizing the protein structure. This theoretical study of PAP polymerase’s substrate selectivity mechanisms aims to clarify the molecular basis of substrate recognition and selectivity in its catalytic reactions. These findings offer valuable insights for the targeted engineering and optimization of polymerases and provide robust theoretical support for developing novel polymerases for applications in drug discovery and related fields. Full article

Lactate is a key metabolite with applications ranging from monitoring training efficiency to early sepsis detection and monitoring the metabolic state of cell cultures. In this study, a paper-based lactate sensor utilizing a fluorescent readout was developed. Unlike common lactate dehydrogenase (LDH)-based methods, these sensors use a green fluorescent protein (GFP) or mApple-coupled lactate binding domain, which provides a fluorescent readout upon lactate binding. We demonstrate that immobilizing these proteins on paper does not affect their ability to bind lactate and produce a fluorescent readout, by monitoring lactate levels in the cell culture supernatant applying different cell culture conditions. Full article

Novel therapies to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of respiratory coronavirus disease 2019 (COVID-19), would be of great clinical value to combat the current and future pandemics. Two viral proteases, papain-like protease (PL^pro) and the main protease 3-chymotrypsin-like protease (3CL^pro), are vital in processing the SARS-CoV-2 polyproteins (pp1a and pp1ab) and in releasing 16 nonstructural proteins, making them attractive antiviral drug targets. In this study, we investigated the degradation of the SARS-CoV-2 main protease 3CL^pro by matrix metalloproteinase-14 (MMP14). MMP14 is known to recognize over 10 distinct substrate cleavage sequences. Through sequence analysis, we identified 17 and 10 putative MMP14 cleavage motifs within the SARS-CoV-2 3CL^pro and PL^pro proteases, respectively. Despite the presence of potential sites in both proteins, our in vitro proteolysis assays demonstrated that MMP14 selectively binds to and degrades 3CL^pro, but not PL^pro. This selective proteolysis by MMP14 results in the complete loss of 3CL^pro enzymatic activity. In addition, SARS-CoV-2 pseudovirus replication was inhibited in 293 T cells when either full-length MMP14 or its catalytic domain (cat-MMP14) were overexpressed, presumably due to 3CL^pro degradation by MMP14. Finally, to prevent MMP14 from degrading off-target proteins, we propose a new recombinant pro-PL-MMP14 construct that can be activated only by another SARS-CoV-2 protease, PL^pro. These findings could open the potential of an alternative therapeutic strategy against SARS-CoV-2 infection. Full article

Background: Mycobacterium avium (Mav) is a leading cause of pulmonary disease among non-tuberculous mycobacteria (NTMs) due to its extensive antibiotic resistance profile. The essential Rel protein is a bifunctional enzyme, which is sensitive to environmental stress and regulates cellular guanosine-3′,5′-bispyrophosphate ((p)ppGpp). Increased levels of the alarmone thereby initiate a survival response, contributing to bacterial persistence and virulence. Objectives: MavRel harbors an unusual extension at the N-terminal domain (NTD), which we aim to characterize its possible regulatory role in maintaining (p)ppGpp homeostasis. We also studied whether the TGS domain retains its regulation capacity in MavRel and the binding propensity of the ACT domain to valine. Methods: Molecular dissection of MavRel was performed to generate a series of truncates to quantify the synthetase and hydrolase activities. Binding experiments with tRNA and valine were carried out via tryptophan quenching assay and NMR, respectively. Results: Bi-catalytic regulation of MavRel was found to be predominantly governed by the residues 37–50 at the NTD extension in its free state. The TGS domain was shown to harbor the capacity to bind with deacylated tRNA and represses synthetase activity to a lower degree compared to the NTD extension. We also characterized the dimeric Mav ACT-domain and the interacting residues contributing to its affinity with valine to function as a nutrient sensor. Conclusions: The mapping of the unique NTD regulatory element of MavRel reveals its functional relevance to coordinate the catalytic states of synthetase and hydrolase, hence underscores the prospect to drive inhibitor development targeting this novel site against Mav infections. Full article

Background/Objectives: Intrinsically disordered protein regions (IDRs) are difficult to study due to their flexible nature and transient interactions. Computational folding using AlphaFold may offer one way to explore potential folding of these regions under various conditions. Human DNA topoisomerase IIα (TOP2A) is an essential enzyme involved in regulating DNA topology during replication and cell division. TOP2A has an IDR at the C-terminal domain (CTD) that has been shown to be important for regulating TOP2A function, but little is known about potential conformations that it may undertake. Methods: Utilizing the AlphaFold 3 (AF3) model by way of AlphaFold Server, TOP2A was folded as a dimer first without and then with 29 literature-supported post-translational modifications (PTMs) and DNA to observe whether there is predicted folding. Results: TOP2A CTD does not fold in the absence of PTMs. With the addition of PTMs, however, the CTD is predicted to fold into a globular bundle of loops and α-helices. While DNA alone did not induce folding, in the presence of PTMs, DNA ligands increased helicity of the folded CTD and caused it to interact at different core domain interfaces. In addition, DNA is predicted to enable folding of the TOP2A CTD in the presence of fewer PTMs when compared to the absence of DNA. Conclusions: AF3 predicts the folding of TOP2A CTD in the presence of specific PTMs, and this folding appears to shift to allow binding to DNA in functionally relevant regions. These studies provide predicted folding patterns that can be tested by biochemical approaches. AF3 may support the development of testable hypotheses regarding IDRs and enables researchers to model protein-DNA interactions. Full article