ACS Omega

Members of the transforming growth factor-{beta} (TGF-{beta}) superfamily, including inhibins and activins, are structurally related glycoprotein dimers that regulate reproductive and endocrine signaling. Their high degree of molecular similarity presents challenges for label-free analytical discrimination. To evaluate the ability of Raman spectroscopy to distinguish closely related TGF-{beta} superfamily proteins based on intrinsic vibrational fingerprints. Raman spectra of recombinant human Inhibin -subunit, Inhibin B ({beta}B homodimer), and Activin A ({beta}A--{beta}A) were acquired using confocal Raman microscopy with 532 nm excitation. Spectra were baseline-corrected, area-normalized, and analysed using principal component analysis (PCA). Distinct spectral signatures were observed across the 500--1800 cm-1 region. Differences within the S--S stretching region (500--550 cm-1) were consistent with variations in disulfide-bond environments, with the Inhibin -subunit exhibiting the highest relative intensity in this region. Variations in the amide I band (1600--1700 cm-1) suggested differences in protein secondary structure, while aromatic amino acid vibrations provided additional discriminatory features. PCA revealed clear clustering and separation of all three protein classes based on their Raman fingerprints. Raman spectroscopy enables label-free differentiation of structurally related endocrine glycoproteins and demonstrates potential for the structural characterization and classification of inhibin and activin proteins within the TGF-{beta} superfamily.

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II' (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

Introduction: Thromboinflammation, which represents the pathological interplay between inflammation and thrombosis, is a leading cause of global mortality. Current therapies are frequently associated with an increased risk of bleeding and do not adequately address the inflammatory component of the disease. The African tree Guibourtia tessmannii represents a promising source of natural anti-inflammatory compounds. This study aimed to synthesize and characterize silver nanoparticles using an aqueous bark extract of G. tessmannii (GT-AgNPs) and to evaluate their anti-inflammatory and anticoagulant properties. Methods: GT-AgNPs were synthesized by reducing silver nitrate with an aqueous extract of G. tessmannii bark. The nanoparticles were comprehensively characterized using UV-Vis spectroscopy, FTIR spectroscopy, powder X-ray diffraction, and scanning electron microscopy. In vitro anti-inflammatory activity was evaluated through inhibition of bovine serum albumin denaturation, whereas in vivo anti-inflammatory activity was assessed using the carrageenan-induced rat paw edema model. Anticoagulant activity was investigated by measuring activated partial thromboplastin time (aPTT) and prothrombin time (PT), corresponding to the intrinsic and extrinsic coagulation pathways, respectively. Results: The synthesis successfully produced GT-AgNPs with an average particle size of approximately 20 nm. Both the aqueous extract and GT-AgNPs exhibited marked anti-inflammatory activity. The nanoparticles achieved 95% inhibition of protein denaturation in vitro and 95% inhibition of carrageenan-induced paw edema in vivo at a dose of 0.4 mg/kg body weight after 5 h. Furthermore, both the extract and GT-AgNPs demonstrated dose-dependent anticoagulant activity. Conclusion: The study demonstrated that GT-AgNPs, synthesized from the bark of G. tessmannii, possess significant anti-inflammatory and anticoagulant properties. These findings highlight the potential of GT-AgNPs as nanotherapeutic candidates for the management of thrombo-inflammatory disorders.

Amyloid fibrils are traditionally associated with protein misfolding disorders; however, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils (FFs) and rigid fibrils (RFs), and their antibacterial activities were evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli (Top10) and Salmonella Typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. FFs and RFs consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with FFs exhibiting slightly stronger activity against S. Typhimurium. At concentrations of 600-800 M, FFs achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH-SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for FFs, and substantially greater toxicity for RFs, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies FFs as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

Marine macroalgae are valuable sources of bioactive compounds. In this study, we thus investigated the chemical composition and biological activity of an extract from the green seaweed Ulva lacinulata, composed of small bioactive compounds. Comprehensive compositional analyses and high-resolution mass spectrometry revealed its diverse molecular profile composed in particular of peptides/amino acid derivatives, saccharides, low-chain fatty diacids, oxylipins and minerals. Its anti-inflammatory activity was assessed after 6 h pre-treatment in LPS-stimulated cultured RAW 264.7 macrophages, showing that it significantly and dose-dependently reduced the expression and/or secretion of pro-inflammatory cytokines such as TNF-alpha; and IL-6, and targeted the NF-kB signaling cascade. It modulated the SIRT1-AMPK signaling axis and increased the LC3-II/LC3-I ratio, supporting the activation of a controlled autophagic response. This work highlighted the potential of this marine-derived extract as a safe and effective functional ingredient for the development of functional food and/or dietary supplements targeting chronic low-grade inflammation.

Predicting the location of metal-binding sites in proteins is crucial for fundamental biological questions and biotechnological applications. Over the past decade, the rise in metal-bound protein structures in the Protein Data Bank, combined with advanced statistical models such as deep learning, has accelerated the development of metal-binding site prediction tools. Several approaches are now available, offering high-quality benchmarks and predictive performance. Our initial development in this area is BioMetAll, whose first version was based on backbone pre-organization. Here, we introduce its second version, featuring two major updates: 1) metal-specific scoring functions and 2) prediction using backbone geometry alone or in combination with first coordination sphere descriptors. Apart from demonstrating metal sensitivity and yielding better benchmarking results, this new version allows the assessment of the influence of considering the metals first coordination sphere versus backbone pre-organization on how metallic species bind to proteins.

Antimycin A (AA) is widely used as an inhibitor of the mitochondrial respiratory chain, targeting the Qi site of cytochrome bc1 (complex III). In photosynthetic organisms, AA is also well known to inhibit the photosynthetic PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron flow around photosystem I (CEF-PSI). Although AA is frequently used as a specific inhibitor of PGR5-dependent CEF-PSI in photosynthetic reactions, we recently clarified that some of the major components of AA, which is typically a mixture of closely related compounds, also exert direct inhibitory effects on photosystem II (PSII). Nevertheless, the binding site and binding mode of AA in PSII remain largely unexplored. Structurally, AA consists of a salicylic acid moiety connected via an amide bond to a hydrophobic dilactone ring moiety. To identify important structural factors of AA for exhibiting inhibitory effects on PSII (assessed by QA- reoxidation measurements), we here investigated the relationship between structure and inhibitory potency using 38 AA-like compounds (AALCs), including commercial compounds and a series of synthetic AA analogs. Some AALCs exhibited substantially stronger impacts on PSII than natural AA. High acidity of the phenolic OH and the presence of a free amide NH of the salicylamide moiety were critical for the effects on PSII. In contrast, while the dilactone ring moiety also affected the inhibitory activity, this was replaceable with certain hydrophobic structures. Based on our results, together with the known structure-activity relationship and binding mode of AA in complex III, we propose tentative binding models for AA in PSII. HighlightsO_LIStructure-activity relationship of AA-like compounds on PSII is examined C_LIO_LISeveral AA-like compounds more potent than AA against PSII are identified C_LIO_LIPhenolic OH acidity and free amide NH of salicylamide moiety are key for AA effects C_LIO_LIThe dilactone ring moiety is replaceable with certain hydrophobic structures C_LIO_LITentative binding models for AA in PSII are proposed C_LI

Human aquaporins (AQPs) are essential membrane channels, yet their inherent hydrophobicity complicates structural and functional studies. We present the systematic application of the QTY code to human AQPs, integrating it with AlphaFold 3 structure prediction to design and validate that four-representative human AQPs (AQP1, AQP3, AQP4, AQP7) can be converted into water-soluble analogs while maintaining their conformation. This approach features a novel platform for editing challenging membrane proteins. The QTY code was applied to the transmembrane regions of the selected four AQPs. Subsequently, the water-soluble QTY analogs of the four AQPs were predicted using AlphaFold 3. The predicted structures were superposed with CyroEM- or X-ray-determined native structures in PyMOL. Further analyses included root-mean-square deviation (RMSD) calculations, visualization of hydrophobic surface reduction, and inspection of conserved protein-ligand binding ability. After applying the QTY code, sequence changes between native AQPs and their QTY analogs was significant (42.86-48.80%). Nevertheless, their structures superposed well in analyses, with only slight deviations (RMSD < 0.6 [A]). In addition, the surface hydrophobicity of all QTY-edited AQPs was significantly reduced. Importantly, molecular contacts between the cholesterol ligand and protein were largely preserved for both native AQP1 and its QTY analog. Finally, all AlphaFold3-predicted structures for AQPs have high confidence values (pLDDT > 90; pTM ~0.83), supporting the reliability of the predicted structures. The findings demonstrate that membrane protein hydrophobicity can be edited and reduced without compromising fold integrity or functional architecture. Integration of the QTY code with AlphaFold 3 affords a high-throughput platform for designing water-soluble, structurally faithful analogs of challenging membrane proteins. Such a strategy can provide a potent platform for detergent-free biochemical studies and water-soluble analogs for therapeutic monoclonal antibody discoveries, thus advancing research of this pharmacologically important protein family.

Culture conditions were optimized for the production of melanin nanoparticle by the bacterial strain Pseudomonas stutzeri BTCZ 305. Response surface methodology was employed for determining the most significant fermentation conditions using variables including, pH, temperature and L-tyrosine concentration identified through one-factor-at-a time approach. Box-behnken design consisting of 17 different combinations of all these factors were performed. Using this methodology, a quadratic regression model was built and the optimal combinations of media constituents for maximum melanin production 1192.27 microg/mL were determined as temperature (32.5 degreeC), pH (8.5) and L-tyrosine concentration (7 g/L). Melanin production was obtained experimentally coincident with the predicted value and the model was proven to be adequate. The nanostructural distribution, its stability in colloidal suspension and particle size were also characterized with the help of TEM, particle size analysis and Zeta potential. The potent applicability of this molecule in anti-inflammation and wound healing was also elucidated.

Past few decades witnessed a boom in pharmaceutical and bioproduct industry with the help of bioprocess technology. Industrially important bioproducts can be produced in large scale for commercialization with the help of fermenters. Here in, pharmaceutically valuable bioproduct melanin, synthesized from Pseudomonas stutzeri strain BTC109 by using two different sized bioreactors. Under controlled conditions the bacteria were allowed to synthesis melanin nanoparticles. The important parameters to be monitored here are pH, dissolved oxygen, agitation, aeration, melanin production and cell biomass concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/737634v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d92fe5org.highwire.dtl.DTLVardef@d76c07org.highwire.dtl.DTLVardef@f5516forg.highwire.dtl.DTLVardef@1b5864b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPharmaceutical and bioproduct development industries witnessed a shoot up due to bioprocess technology. C_LIO_LIIndustrially important bioproduct like melanin can be produced in large scale with the help of industrial fermentation technology. C_LIO_LIThe product thus obtained was found to be nano sized and it can be commercialized. C_LI

Small molecule inhibitors of cell death have wide-ranging potential applications, both as tool compounds in the laboratory and as clinical modulators of pathologic cell death. Previous screening efforts have identified candidate compounds targeting the pro-apoptotic, pore-forming BCL-2 family proteins BAX and BAK, but the complex interactions of these proteins at the mitochondrial outer membrane (with other proteins and the membrane itself) present challenges for compound screening. Although no inhibitors of BAX or BAK have advanced to clinical testing to date, candidate inhibitors have thus far been identified via screening of membrane-containing systems such as liposomes and isolated mitochondria. To address some of the challenges of chemical screening for apoptosis inhibitors, we conducted a small molecule screen utilizing BH3 profiling, a method that quantifies mitochondrial outer membrane permeabilization (MOMP) upon treatment with pro-apoptotic peptides derived from BCL-2 family proteins. Of over 40,000 compounds screened, we identified a series of compounds that prevent MOMP in response to pro-apoptotic peptides. The most potent of these, CDL36, binds to BAX and prevents MOMP at early timepoints. In longer term viability assays, the cytoprotective effect of CDL36 is most potent against death induced by doxorubicin, a widely used chemotherapeutic agent that causes dose-limiting cardiovascular toxicity. Our results elucidate the mechanism of action of new and existing cell death inhibitors, providing a foundation for further development of these inhibitors and potential insights into the mechanisms mediating doxorubicin toxicity in patients.

Background: Natural essential oils exhibit antimicrobial and wound-healing properties, but their therapeutic application is limited by poor water solubility, volatility, and instability. This study developed and characterized a nanoemulsion of Ocimum gratissimum essential oil (OGNe) and evaluated its physicochemical properties, dermal safety, antibacterial activity, and wound-healing potential. Methods: Essential oil was obtained by hydrodistillation and formulated into nanoemulsions by high-speed stirring emulsification. Physicochemical properties, including pH, droplet size, polydispersity index, and storage stability, were determined. Acute dermal toxicity was assessed in Wistar rats following OECD Test Guideline 402. Antibacterial activity was evaluated using broth microdilution, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays. Wound-healing efficacy was investigated using an excision wound model over 21 days using distilled water and trolamine serving as controls. Results: OGNe exhibited a stable milky appearance, near-neutral pH, and droplet sizes ranging from 26 to 224 nm. No signs of dermal toxicity or behavioral abnormalities were observed after topical administration. The nanoemulsion showed selective antibacterial activity, with the highest susceptibility against Acinetobacter baumannii (MIC = 1.125 L/mL), whereas Escherichia coli remained resistant. Time-kill assays demonstrated concentration-dependent bacteriostatic activity. In vivo, OGNe significantly accelerated wound contraction from day 3 onward (p < 0.0001), achieving healing rates comparable to or exceeding those of trolamine during the inflammatory and proliferative phases. Conclusion: Ocimum gratissimum nanoemulsions represent stable, biocompatible topical formulations that combine selective antibacterial activity with enhanced wound healing, supporting their potential as phytopharmaceutical nanoformulations for the management of acute skin wounds.

Antifreeze proteins (AFPs) found in various cold-adapted organisms inhibit ice growth and are of interest for applications in food products, cryopreservation, agriculture, and materials science. Although high-resolution structures are available for several AFPs, the amino acids required for full antifreeze activity remain incompletely defined, and the development of AFP variants with properties such as enhanced solubility, high expression yield, and improved thermostability may further facilitate applications. Here, we used the deep learning model ProteinMPNN to redesign the globular fish antifreeze protein AFPIII, keeping the previously reported ice-binding residues fixed. We readily obtained sequences confidently predicted to adopt AFPIIIs structure and we selected five designed variants for expression, all of which expressed efficiently in E. coli. Circular dichroism spectroscopy showed that two of these variants retained secondary structure elements consistent with AFPIII, whereas the other three exhibited structural differences. One design was predicted and experimentally confirmed to have increased thermostability. All five variants displayed measurable thermal hysteresis activity. However, none reached the activity of wild-type AFPIII, suggesting that maintaining the currently established set of ice-binding residues is not sufficient to fully preserve this AFPs function; other, unidentified residues can also impact its activity. Our findings highlight the value of deep learning-based protein design methods both for generating AFP variants with desirable properties and for uncovering gaps in existing knowledge of well-characterized AFPs.

We present a comparative study of the inner membrane of three Gram-positive bacterial strains, namely S. aureus, S. epidermidis and N. lacusekhoensis. A lipidomics study is used to obtain the lipid architecture and composition for S. epidermidis found in the skin microbiome and N. lacusekhoensis, an extremophile present in halophilic and alkophilic environments. Differences between the strains arise from both the lipid architecture and the cardiolipin content varying from 5% in S. aureus to 85% in N. lacusekhoensis. We develop coarse grained (CG) Martini-3 membrane models which reproduce structural properties such as membrane area, thickness, density distributions as well as ion-correlations with all-atom models. Inter-lipid correlations reveal a homogeneous distribution of lipids in the membranes despite the wide variation in lipid types and composition. Mechanical properties such as the area stretch modulus increased with cardiolipin content, however the bending modulus has a more complex dependence on membrane charge and lipid type. Using the CG models we evaluate the insertion free energies for four widely used antimicrobial molecules. Entry barriers for thymol and methylparaben arise from the charge density modulation at the membrane headgroups due to counterion condensation. The entry mechanisms of the antimicrobial peptide cecropin-melittin-15 (CM15) and the preservative molecule ethyl-lauroyl-arginate (ELAR) are found to be similar across all three strains. We also illustrate the manner in which the extremophilic strain, N. lacusekhoensis with its high cardiolipin content, modulates the partitioning kinetics of the antimicrobial molecule thymol with pH and salt. Our study reveals that membrane properties are largely conserved across the three model membranes. The molecular models and insights emerging from the present work should aid in the development of novel antimicrobials against Gram-positive strains.

BackgroundThe interaction between the anti-apoptotic protein Bcl-xL and the BH3-only sensitizer BAD represents a critical regulatory checkpoint in the intrinsic apoptotic pathway. Although this interaction is known to influence mitochondrial fate, its dynamic regulation and structural determinants in living cells remain poorly understood. Here, we developed a fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET) platform to visualize and quantify Bcl-xL-BAD interactions in real-time. MethodsWe developed a quantitative fluorescence lifetime-based FRET (FLIM-FRET) approach to visualize and measure Bcl-xL-BAD interactions in single living glioblastoma cells. Stable GFP/Venus-Bcl-xL and mCherry-BAD FRET pairs were created, followed by acceptor photobleaching FRET, FLIM-FRET, Annexin V-BFP-based apoptosis assays, pharmacological perturbation using BH3 mimetics, and molecular dynamics simulations with MM/GBSA analysis. Statistical significance was assessed using appropriate parametric tests across multiple independent experiments. ResultsUsing this platform, we observed that apoptotic stress markedly enhances the engagement of Bcl-xL and BAD. Increased FRET efficiency coincided with Annexin V positivity and nuclear condensation, indicating that maximal BAD binding reflects a higher level of apoptotic commitment. Structure-function analysis using targeted Bcl-xL mutants revealed distinct binding requirements: disruption of the core hydrophobic groove (Y101K) abolished BAD binding and impaired BH3 mimetic sensitivity, whereas mutation within the BH1 domain (G138A) preserved BAD interaction and sensitivity to BH3 mimetics. Molecular dynamics simulations corroborated these observations by revealing preserved BAD-binding energetics in the G138A mutant, but destabilization in the Y101K mutant. ConclusionsTogether, these findings demonstrate the utility of a live-cell FLIM-FRET platform for resolving protein-protein interactions involving apoptotic proteins at the single-cell level. By linking interaction dynamics, structural determinants, and functional outcomes, this approach provides a broadly applicable framework for studying apoptotic priming, structural tolerance at BCL-2 family interfaces, and cellular responses to BH3-mimetic therapies.

Glyceraldehyde (GA) contributes to the development of various diseases, such as diabetes and Alzheimer's disease via protein glycation and the formation of advanced glycation end products (AGEs); however, effective strategies for neutralizing GA are limited. Carnosine (Car), an imidazole dipeptide (IDP) that is abundant in meat, suppresses protein glycation by scavenging reactive aldehydes. There are only a few reports on the antiglycation activity of Car against GA. For other IDPs, such as anserine, balenine (Bal), and homocarnosine, there are almost no reports on their antiglycation activity. In this study, we demonstrated the antiglycation activity of four types of IDPs and 2-oxocarnosine (2-oxo-Car), an oxidized form of Car, against GA-induced intracellular protein glycation and neuronal cytotoxicity. Car and Bal exhibited significantly higher reactivity with GA compared with other IDPs and 2-oxo-Car. An in silico analysis suggested that the difference in reactivity is dependent upon intramolecular hydrogen bond formation and the conformation of each IDP. Although there were differences in reactivity with GA, LC-MS analysis revealed that all of the IDPs and 2-oxo-Car reacted with two molecules of GA to form adducts containing pyridinium rings. Car and Bal exhibited high reactivity with GA and markedly suppressed GA-induced cytotoxicity in SH-SY5Y cells. Western blot and qPCR analyses revealed that IDPs suppressed GA-induced protein glycation and the upregulation of endoplasmic reticulum and oxidative stress response genes. Our results indicate that IDPs represent a novel preventive approach to AGE-related diseases and provide a foundation for the development of strategies to treat GA-related neurotoxicity.

Numerous advances in quantum and computational chemistry over the last decades, well as the development of computer science, allowed utilisation of more precise and complex models, which can be now applied to much bigger systems than in the past. The authors used Gaussian, coupled with theoretical methods, to predict a new way of peptide bond formation, which could have taken place in prebiotic conditions. To better tackle this difficult task, the properties of substrates (glycine-derived radicals) were extensively analysed, using the aforementioned tool - Gaussian, paired with taking resonance and hybridisation into account, to better understand the stereochemistry and the very nature of processes taking place. The result is a series of reactions, which without any sophisticated catalysts and with relatively low energy thresholds ({inverted exclamation}20 kcal/mol) can lead to formation of dipeptides (and further, oligopeptides). The authors also hope, the other predicted properties of the investigated molecules can be of use to any researcher, who would like to utilise them in their experiments. Author summaryOur goal was to investigate a way first peptide bonds in prebiotic conditions could have been formed. This is an extremely important step in research into the beginning of life on Earth. We found a very promising series of reactions, which uses atomic hydrogen as its only catalyst and confirmed our expectations with theoretical calculations, using Gaussian. There are two radicals derived from glycine, which perform major roles in the process, so we investigated their properties with Gaussian and verified that the results are in agreement with our own theoretical considerations. This involved checking for possible geometric isomers and conformers and creating models which could explain their properties. We are well aware that such calculations have limitations and there is no model, which is 100% accurate, so our results should be further confirmed by empirical data in the future. However, we still to be as thorough as possible in how we approached the subject.

Discovering small molecules targeting proteins represents a major effort in drug development. RNA, however, as a class of macromolecule that carrying out important regulatory roles in the cell as drug target, only received attention recently. Although several methods have been proposed, an easy to operate, fast and robust method is still lacking. We designed a generic florescence screening method by fusing the target RNA with a florescent aptamer (fusion RNA) and then carried out screening using high-throughput format (Fluorescent Aptamer Screening, FAS). In this work, we chose SL5 on SARS-Cov-2 5’UTR as the test target. SL5 is a conserved motif across several corona virus family members whose core is not prone to mutation. We screened 9528 compounds, successfully identified four molecules (Sertraline (hydrochloride), Samuraciclib (hydrochloride), Minocycline (hydrochloride), JG-98 bind direct to the full-length SL5 at micromolar or higher affinity. The design of FAS could be easily adapted to structured RNA motifs without prior knowledge of its 3D structural information. In addition, this work showed the possibility of developing generic drugs for RNA virus by targeting the conserved viral RNA genome and paved a new way for the discovery of small molecule drugs in combating human diseases.

Background: Cutaneous melanoma is the most aggressive malignant skin tumor, and metastasis represents the primary cause of patient mortality. Bisphenol S (BPS) has an unclear influence on melanoma metastasis and its underlying molecular mechanisms. Methods: Potential BPS targets were predicted using the SEA, SwissTargetPrediction, and SuperPred databases. Based on TCGA-SKCM transcriptomic data, differential expression analysis was performed, and Weighted Gene Co-expression Network Analysis (WGCNA) was employed to construct a gene co-expression network. Candidate genes were obtained by integrating BPS-related targets, differentially expressed genes (DEGs), module genes, and univariate Cox regression genes, followed by Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network construction. Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression was applied to screen core prognostic genes and construct a risk prediction model. Further analyses included network construction, molecular docking, and 100 ns molecular dynamics (MD) simulation. Results: Integration of BPS-related targets, DEGs, WGCNA module genes, and Cox regression results yielded 13 candidate genes enriched in kinase activity regulation and melanoma-related pathways. LASSO-Cox regression ultimately identified three core prognostic genes--ABCB1, PIM2, and TSHR--all significantly upregulated in metastatic tissues, with area under the curve (AUC) values of approximately 0.7. High-expression patients exhibited significantly better overall survival than low-expression patients (P < 0.05). A nomogram incorporating the three genes and clinical parameters demonstrated good calibration performance. Within the ceRNA network, MALAT1 and hsa-miR-155-5p were identified as key regulatory molecules, and 37 potential transcription factors were predicted, including CEBPA, JUN, and STAT3. Molecular docking revealed strong binding affinities of BPS toward ABCB1 , PIM2, and TSHR, and MD simulations confirmed the structural stability of all three complexes. Conclusion: ABCB1, PIM2, and TSHR are the core target genes through which BPS influences melanoma metastasis via multidrug resistance, kinase signaling, and receptor-mediated signal transduction. The prognostic model based on these three genes demonstrates good clinical applicability, and the ceRNA and transcription factor regulatory networks provide a systematic molecular basis for understanding the association between BPS exposure and melanoma metastasis.

Silicones are elastomers that have a wide variety of uses, including biomedical applications such as the coating of biomedical devices and as implants. Soft silicones with mechanical properties similar to those of biological tissues have particularly gained use as substrates for cell culture in mechanobiology studies. In this context, it would be desirable to be able to alter their surface mechanical properties with a relatively simple physical treatment. While deep ultraviolet (deep UV) or ultraviolet C (UV-C) treatment has been previously used as a surface treatment method for stiffer silicones formulations, the effect of this treatment on soft silicones relevant for mechanobiology applications is still uncharacterized. We first used nanoindentation to determine the Youngs modulus of two types of soft silicones, Qgel and GEL-8100/Syl (GEL-8100 with Sylgard-184 crosslinker), both with initial moduli in the kilopascal range. We show that nanoindentation in the presence of 1% sodium dodecyl sulfate avoids adhesion between the nanoindentation glass probe and the soft silicones. After deep UV exposure in the presence of air, nanoindentation revealed that the apparent Youngs moduli of the soft silicones Qgel and GEL-8100/Syl increased by 70% and 33%, respectively. The bulk rheology of the soft silicones were not affected, suggesting that this corresponds to a surface stiffening effect with a topical stiffening of at least several hundred kilopascals. Energy-dispersive X-ray spectroscopy results show an increase in the mole fraction of oxygen, consistent with oxidation of the surface. Attenuated Total Reflectance Fourier-Transform Infrared spectra show evidence of Si-OH group formation in GEL-8100/Syl and silicon sub-oxide formation in Qgel. Consistent with this, water contact angle measurements show enhanced hydrophilicity after deep UV treatment. Our results have implications for using soft silicones as substrates in mechanobiology studies and in processes where deep UV light is used in the surface treatment of soft silicones.