Search Results (11,964)

Membrane proteins remain the most challenging targets for structural characterization, yet their elucidation provides valuable insights into protein function, disease mechanisms, and drug specificity. Structural biology platforms have advanced rapidly in recent years, notably through the development and implementation of nanodiscs—discoidal lipid–protein complexes that encapsulate and solubilize membrane proteins within a controlled, native-like environment. While nanodiscs have become powerful tools for studying membrane proteins, faithfully reconstituting the compositional asymmetry intrinsic to nearly all biological membranes has not yet been achieved. Proper membrane leaflet lipid distribution is critical for accurate protein folding, stability, and insertion. Here, we share a protocol for reconstituting tailored compositional asymmetry within nanodiscs through membrane extraction from giant unilamellar vesicles (GUVs) treated with a leaflet-specific methyl-β-cyclodextrin (mβCD) lipid exchange. Nanodisc asymmetry is verified through a geometric approach: biotin-DPPE-preloaded mβCD engages in lipid exchange with the outer leaflet of POPC GUVs solubilized by the lipid-free membrane scaffold protein (MSP) Δ49ApoA-I to form nanodisc structures. Once isolated, nanodiscs are introduced to the biotin-binding bacterial protein streptavidin. High-speed atomic force microscopy imaging depicts nanodisc–dimer complexes, indicating that biotin-DPPE was successfully reconstituted into a single leaflet of the nanodiscs. This finding outlines the first step toward engineering tailored nanodisc asymmetry and mimicking the native environment of integral proteins—a potentially powerful tool for accurately reconstituting and structurally analyzing integral membrane proteins whose functions are modulated by lipid asymmetry. Full article

The basic/helix-loop-helix (bHLH) transcription factors are crucial regulators of plant development and stress responses. In this study, we conducted a genome-wide analysis of the bHLH family in Rosa roxburghii, an economically important fruit crop. A total of 89 non-redundant RrbHLHs were identified and unevenly distributed across the seven chromosomes. Phylogenetic analysis classified them into 23 subfamilies and 7 Arabidopsis subfamilies were absent, indicating lineage-specific evolutionary trajectories. Conserved motif and gene structure analyses showed that members within the same subfamily generally shared similar architectures, yet subfamily-specific variations were evident, suggesting potential functional diversification. Notably, key residues involved in DNA-binding and dimerization were highly conserved within the bHLH domain. Promoter analysis identified multiple cis-acting elements related to hormone response, stress adaptation, and tissue-specific regulation, hinting at broad regulatory roles. Expression profiling across fruit developmental stages and in response to GA₃ treatment revealed dynamic expression patterns. Furthermore, 21 duplicated gene pairs (17 segmental and 4 tandem duplicated pairs) were identified, with most evolving under purifying selection. Detailed analysis of these pairs revealed that segmental duplication, coupled with structural variations such as exon indels, dissolution/joining, and exonization/pseudoexonization, substantially contributed to their functional divergence during evolution. Our results provide a basis for understanding the evolution and potential functions of the RrbHLHs. Full article

The blood–brain barrier (BBB) restricts therapeutic delivery to the central nervous system (CNS), hindering the treatment of neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, brain cancers, and stroke. Aptamers, short single-stranded DNA or RNA oligonucleotides that can fold into unique 3D shapes and bind to specific target molecules, offer high affinity and specificity, low immunogenicity, and promising BBB penetration via receptor-mediated transcytosis targeting receptors such as the transferrin receptor (TfR) and low-density lipoprotein receptor-related protein 1 (LRP1). This review examines aptamer design through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and its variants, mechanisms of BBB crossing, and applications in CNS disorders. Recent advances, including in silico optimization, in vivo SELEX, BBB chip-based MPS-SELEX, and nanoparticle–aptamer hybrids, have identified brain-penetrating aptamers and enhanced the brain delivery efficiency. This review highlights the potential of aptamers to transform CNS-targeted therapies. Full article

Bacteria use cholesterol-dependent cytolysins (CDCs) to damage eukaryotes. While well-studied in mammals, the mechanisms by which CDCs bind to and kill protozoans remain unclear. CDCs bind to the human pathogen Leishmania major but only kill in the absence of sphingolipids. The contribution of other leishmanial membrane components to CDC binding and cytotoxicity remains unknown. Here, we used genetic knockouts and inhibitors to determine the contribution of key membrane components to CDC binding and killing in L. major. We analyzed toxin binding and killing using flow cytometry and Western blotting. Loss of the virulence factor GP63 enhanced toxicity of perfringolysin O but not streptolysin O. Plasmenylethanolamine and lipophosphoglycan had minimal contributions to CDC binding and cytotoxicity. Removal of sterols protected cells from CDCs yet failed to reduce binding. We used CDCs defective in engaging glycans or cholesterol to confirm that CDCs deficient in sterol binding, but not glycan binding, could bind to L. major. Thus, in non-mammalian systems, CDCs may rely on glycans for binding, while using sterols for pore formation. This suggests that CDCs may not be sterol-specific probes in some non-mammalian systems. We conclude that early-branching eukaryotes use distinct mechanisms from mammals to limit CDC pore formation and killing. Full article

DNA double-strand breaks (DSBs) can be induced by cellular byproducts or genotoxic agents. Improper processing of these lesions leads to increased genome instability, which constitutes a hallmark of pathological conditions and fuels carcinogenesis. DSBs are primarily repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) and the proper balance between these two pathways is finely modulated by specific molecular events. Here, we report that the histone chaperone DAXX plays a fundamental role in the response to DSBs. Indeed, in human cells, DSBs induce ATM/ATR-dependent phosphorylation of DAXX on serine 424 and 712 and promote its binding to chromatin and the deposition of the histone variant H3.3 in proximity to DNA breaks. Enrichment of H3.3 at DSBs promotes 53BP1 recruitment to these lesions and the repair of DNA breaks by HR pathways. Moreover, H3.3-specific post translational modifications, particularly K36 tri-methylation, play a key role in these processes. Altogether, these findings indicate that DAXX and H3.3 mutations may contribute to tumorigenesis-enhancing genome instability. Full article

The landscape of first-line treatment for metastatic non-small cell lung cancer (NSCLC) without actionable driver mutations is rapidly evolving, currently dominated by pembrolizumab-based regimens. This review discusses the unique molecular characteristics of cemiplimab, a newer anti-PD-1 antibody, and defines its optimal positioning against established standards. Cemiplimab is a fully human IgG4 monoclonal antibody distinguished by two key features: an engineered hinge-region mutation that prevents Fab-arm exchange, ensuring exceptional molecular stability which minimizes anti-drug antibody (ADA) risks associated with unstable molecules; and a unique interaction with PD-1 glycosylation sites, potentially enhancing binding efficacy. These structural advantages may be particularly relevant in histologies like squamous NSCLC, where accumulating somatic mutations drive high neoantigen loads and heightened immune responses, creating an environment historically prone to ADA formation. Based on data from the pivotal EMPOWER-Lung program, we highlight cemiplimab’s exceptional promise in specific populations. Firstly, in the EMPOWER-Lung 1 trial, cemiplimab monotherapy demonstrated extraordinary survival benefits in a pre-specified analysis of the distinct “ultra-high” PD-L1 expression subgroup (TPS ≥90%), potentially surpassing historical benchmarks. Secondly, cemiplimab displays consistent, robust efficacy in challenging-to-treat squamous histology, both as monotherapy for patients with high PD-L1 expression and in combination with chemotherapy for patients with PD-L1 < 50%. In conclusion, cemiplimab establishes a unique therapeutic niche for patients with squamous histology and ultra-high PD-L1 expression, likely driven by its distinct structural stability and reduced immunogenicity. Full article

Over the past two decades, pharmaceutical peptides have emerged as a powerful alternative to traditional small molecules, offering high potency, specificity, and low toxicity. However, most computational drug discovery tools remain optimized for small molecules and need to be entirely adapted to peptide-based compounds. Molecular docking algorithms, commonly employed to rank drug candidates in early-stage drug discovery, often fail to accurately predict peptide binding poses due to their high conformational flexibility and scoring functions not being tailored to peptides. To address these limitations, we present PepScorer::RMSD, a novel machine learning-based scoring function specifically designed for pose selection and enhancement of docking power (DP) in virtual screening campaigns targeting peptide libraries. The model predicts the root-mean-squared deviation (RMSD) of a peptide pose relative to its native conformation using a curated dataset of protein–peptide complexes (3–10 amino acids). PepScorer::RMSD outperformed conventional, ML-based, and peptide-specific scoring functions, achieving a Pearson correlation of 0.70, a mean absolute error of 1.77 Å, and top-1 DP values of 92% on the evaluation set and 81% on an external test set. Our PLANTS-based workflow was benchmarked against AlphaFold-Multimer predictions, confirming its robustness for virtual screening. PepScorer::RMSD and the curated dataset are freely available in Zenodo Full article

Bacterial and viral diseases significantly reduce soybean (Glycine max) yield and quality. RNA modifications, particularly N⁶-methyladenosine (m⁶A), are increasingly recognized as having a regulatory role in plant–pathogen interactions, but the m⁶A methylome of soybean during viral and bacterial infection has not yet been characterized. Here, we performed transcriptome sequencing and MeRIP-seq (methylated RNA immunoprecipitation followed by high-throughput sequencing) of soybean leaves infected with Soybean mosaic virus (SMV) and/or Pseudomonas syringae pv. glycinea (Psg). In general, m⁶A peaks were highly enriched near stop codons and in 3′-UTR regions of soybean transcripts, and m⁶A methylation was negatively correlated with transcript abundance. Multiple genes showed differential methylation between infected and control plants: 1122 in Psg-infected plants, 539 in SMV-infected plants, and 2269 in co-infected plants; 195 (Psg), 84 (SMV), and 354 (Psg + SMV) of these transcripts were both differentially methylated and differentially expressed. Interestingly, viral infection was predominantly associated with hypermethylation and downregulation, whereas bacterial infection was predominantly associated with hypomethylation and upregulation. GO and KEGG enrichment analysis revealed shared processes likely affected by changes in m⁶A methylation during bacterial and viral infection, including ATP-dependent RNA helicase activity, RNA binding, and endonuclease activity, as well as specific processes affected by only one pathogen. Our findings shed light on the role of m⁶A modifications during pathogen infection and highlight potential targets for epigenetic editing to increase the broad-spectrum disease resistance of soybean. Full article

HDAC8 is a histone deacetylase enzyme that plays a key role in the development of various diseases in humans, including cancers, neurodegenerative diseases, and alcohol use disorder. Although HDAC8 is classified as a Zn²⁺-dependent metalloenzyme, available data regarding the affinity of other biologically relevant ions, such as Fe²⁺, Ni²⁺, Co²⁺, and Mg²⁺, for the HDAC8 enzyme active site remain unclear and contradictory. The mechanism by which these ions compete with Zn²⁺ for the HDAC8 active site is not well understood. In this study, we performed density functional theory (DFT) calculations at the B3LYP/6-31+G(d) level of theory, combined with polarizable continuum model computations (PCM) in water (ε = 78) and methanol (ε = 32). The results show that Zn²⁺ remains the thermodynamically preferred cofactor across all modeled reactions. Although Fe²⁺ and Co²⁺ gain partial stabilization upon increasing coordination number, the associated entropic and desolvation penalties prevent them from outcompeting Zn²⁺ under physiologically relevant conditions. Only a limited number of substitution reactions for Fe²⁺ and Co²⁺ yield ∆G values near thermodynamic neutrality, and only in specific coordination states. In contrast, all modeled Ni²⁺ substitution reactions are unfavorable, and Mg²⁺ is strongly excluded from the HDAC8 active site in all reactions. The resulting metal preference hierarchy—Zn²⁺ > Co²⁺ ≈ Fe²⁺ > Ni²⁺ > Mg²⁺—supports experimental observations and clarifies the intrinsic selectivity of the HDAC8 enzyme towards Zn²⁺. These insights provide a molecular basis for understanding HDAC8 metallo-regulation and may guide the rational design of novel, isoform-specific HDACi with improved binding properties. Full article

Iron-containing alcohol dehydrogenases (Fe-ADHs) from hyperthermophiles represent a distinct class of oxidoreductases characterized by exceptional thermostability, catalytic versatility, and unique metal-dependent properties. Despite considerable sequence diversity, Fe-ADHs share conserved motifs and a two-domain architecture essential for iron coordination and NAD(P)H cofactor binding. Physiologically, these enzymes are predicted to function primarily in aldehyde detoxification and redox homeostasis, with some also participating in fermentative alcohol production. Their remarkable stability and catalytic efficiency highlight their potential as robust biocatalysts for high-temperature industrial bioprocesses. This review presents a comprehensive comparative analysis of the biophysical, biochemical, and kinetic properties of Fe-ADHs, focusing on their thermostability, metal ion specificity, and catalytic mechanisms, as well as highlighting their potential for industrial biocatalytic applications. Full article

While the international influence of the Programme for International Student Assessment (PISA) on education policy debates is well recognized, the degree to which PISA findings drive actual policy reforms and classroom practices remain debated. Using PISA as a case, this article examines how educational research is translated into policy responses and practices in German federalism, focusing specifically on immigrant students—a key group within German education reform discourse. It analyzes the reflection of PISA findings from the 2000, 2018, and 2022 assessments on immigrant student performance in the resolutions of the Standing Conference of Ministers of Education and Cultural Affairs, the process of implementation by the federal states (Länder), and the effect on school-level practice. Framed by research knowledge mobilization theory, the article investigates the relationships among research production, mediation, and usage, clarifying the interplay between educational research, policy, and practice in Germany’s federal system. Historical analysis exposes consistent gaps between research-derived recommendations and binding, actionable change at both policy and practice levels, often due to challenges in developing evidence-based and consistently applied policy measures across the Länder. The article concludes with practical recommendations for improving the impact of interdisciplinary, policy-oriented research on policy and practice, considering the complexities of Germany’s federal governance. Full article

Multidrug resistance (MDR) is a central cause of chemotherapy failure and tumor recurrence and metastasis, and its mechanism involves enhanced drug efflux, target mutation, upregulation of DNA repair and remodeling of the tumor microenvironment. ABC transporter protein (P-gp, MRP, and BCRP)-mediated efflux of drugs is the most intensively researched aspect of the study, but the first three generations of small-molecule reversal agents were stopped in the clinic because of toxicity or pharmacokinetic defects. Natural products are considered as the fourth generation of MDR reversal agents due to their structural diversity, multi-targeting and low toxicity. In this paper, we systematically summarize the inhibitory activities of monoterpenes, sesquiterpenes, diterpenes and triterpenes against ABC transporter proteins in in vitro and in vivo models and focus on the new mechanism of reversing drug resistance by blocking efflux pumps, modulating signaling pathways such as PI3K-AKT, Nrf2, NF-κB and remodeling the tumor microenvironment. For example, Terpenoids possess irreplaceable core advantages over traditional multidrug resistance (MDR) reversers: Compared with the first three generations of synthetic reversers, natural/semisynthetic terpenoids integrate low toxicity (mostly derived from edible medicinal plants, half-maximal inhibitory concentration IC₅₀ > 50 μM), high target specificity (e.g., oleanolic acid specifically inhibits the ATP-binding cassette (ABC) transporter subtype ABCC1 without cross-reactivity with ABCB1), and multi-mechanistic synergistic effects (e.g., β-caryophyllene simultaneously mediates the dual effects of “ABCB1 efflux inhibition + apoptotic pathway activation”). These unique characteristics enable terpenoids to effectively circumvent key limitations of traditional synthetic reversers, such as high toxicity and severe drug–drug interactions. Among them, lupane-type derivative BBA and euphane-type sooneuphanone D (triterpenoids), as well as dihydro-β-agarofuran-type compounds and sesquiterpene lactone Conferone (sesquiterpenoids), have emerged as the core lead compounds with the greatest translational potential in current MDR reverser research, attributed to their potent in vitro and in vivo MDR reversal activity, low toxicity, and excellent druggable modifiability. At the same time, we point out bottlenecks, such as low bioavailability, insufficient in vivo evidence, and unclear structure–activity relationship and put forward a proposal to address these bottlenecks. At the same time, the bottlenecks of low bioavailability, insufficient vivo evidence and unclear structure–activity relationship have been pointed out, and future research directions such as nano-delivery, structural optimization and combination strategies have been proposed to provide theoretical foundations and potential practical pathways for the clinical translation research of terpenoid compounds, whose clinical application still requires further in vivo validation and translational research support. Full article

MicroRNAs (miRNAs) are small non-coding RNA molecules approximately 20–22 nucleotides in length that regulate gene expression at the post-transcriptional level. By binding to target messenger RNAs (mRNAs), miRNAs inhibit translation or induce degradation, thus influencing a wide array of biological processes including development, inflammation, apoptosis, and tissue remodeling. Owing to their remarkable stability and tissue specificity, miRNAs have emerged as promising biomarkers in both clinical and forensic settings. In recent years, increasing evidence has demonstrated their utility in cardiovascular diseases, where they may serve as diagnostic, prognostic, and therapeutic tools. This systematic review aims to comprehensively summarize the role of miRNAs in cardiovascular pathology, focusing on their diagnostic potential in myocardial infarction, sudden cardiac death (SCD), and cardiomyopathies, and their applicability in post-mortem investigations. Following PRISMA guidelines, we screened PubMed, Scopus, and Web of Science databases for studies up to December 2024. The results highlight several miRNAs—including miR-1, miR-133a, miR-208b, miR-499a, and miR-486-5p—as robust markers for ischemic injury and sudden death, even in degraded or formalin-fixed autopsy samples. The high stability of miRNAs under extreme post-mortem conditions reinforces their potential as molecular tools in forensic pathology. Nevertheless, methodological heterogeneity and limited standardization currently hinder their routine application. Future studies should aim to harmonize analytical protocols and validate diagnostic thresholds across larger, well-characterized cohorts to fully exploit miRNAs as reliable molecular biomarkers in both clinical cardiology and forensic medicine. Full article

Avian influenza viruses are predominantly associated with waterfowl and shorebirds, and are rarely detected in other avian hosts in nature. In 2021, an H1N1 virus was isolated from a Fieldfare Turdus pilaris in Zaporizhzhia Oblast, Ukraine. A phylogenetic analysis revealed that all eight gene segments belonged to the Eurasian low-pathogenic avian influenza lineages. The highest nucleotide identity of the HA gene was observed with viruses detected in Georgia, Sweden, and Ukraine (99.11%), while the NA gene showed the greatest identity to viruses from Western Europe (99.14–99.57%). Genetic analysis of the HA cleavage site showed a sequence (PSIQSR↓GLF) that contained a single basic amino acid. No deletions were detected in the stalk region of NA gene, and no specific mutations in PB2 protein were found. However, several amino acid substitutions were identified in the HA gene (D204E, S207T, and D239G) that may affect the binding affinity to specific antibodies. The occurrence of this virus in a wild, seemingly healthy thrush indicate that additional surveillance in poorly studied ecological groups such as Passeriformes is warranted. Full article

Background/Objectives: Infantile epileptic spasms syndrome (IESS) is a severe epilepsy of infancy. Corticotropin (ACTH) and vigabatrin are the only FDA-approved therapies. The efficacy of ACTH together with the strong convulsant effects of corticotropin-releasing hormone (CRH) suggests that excess CRH, secondary to impaired ACTH feedback, may contribute to spasms. We therefore hypothesized that CRH receptor 1 (CRHR1) antagonists would suppress spasms in a route- and drug-dependent manner. Methods: Using our validated rat model of IESS, in which prenatal priming with betamethasone was followed by postnatal triggering of spasms with N-methyl-D-aspartic acid (NMDA), we tested two CRHR1 antagonists, CP376395 and SN003, delivered intracranially (via intracerebroventricular or intraparenchymal infusion) or systemically. Results: Intracerebroventricular infusion of both antagonists suppressed spasms, with CP376395 providing more consistent effects. Intraparenchymal administration into the hypothalamic arcuate nucleus also reduced spasms, whereas misses into the mammillary bodies were ineffective, highlighting site specificity. Systemic administration yielded divergent results: SN003 robustly suppressed spasms, whereas CP376395 unexpectedly exacerbated them. No sex differences were observed. Conclusions: These findings demonstrate that CRHR1 blockade modifies experimental spasms in a route- and drug-specific manner and implicates discrete hypothalamic circuits, particularly those including the arcuate nucleus, in spasm generation. The divergent systemic responses between CP376395 and SN003 likely reflect differences in CRHR1 engagement (competitive and non-competitive antagonism, respectively) as well as differences in binding properties that may include differential network interactions beyond local CRH signaling or duration of receptor occupancy. In conclusion, SN003 may be a better option than CP376395 for further development as a CRHR1-targeted therapy pending additional pharmacokinetic/pharmacodynamic studies. Further work should explore dosing paradigms of CP376395 to determine if a therapeutic range for CP376395 exists. Full article