Lactic acid bacteria can use an electron transport system to pass reducing equivalents to extracellular mediators. This redox flexibility decouples carbon and electron balances and presents the opportunity to use electrodes to engineer fermentations. We show that growth with an anode may be a two-edged sword for the fitness of the model lactic acid bacterium Lactiplantibacillus plantarum grown in a chemically defined medium supplemented with quinones, depending on substrate and cultivation regime. In batch culture on glucose, the anode slows growth despite yielding an over four-fold higher ratio of acetate to lactate than the open-circuit control, a fermentation profile associated with higher ATP yield. In batch culture on the more reduced substrate mannitol, the anode facilitates growth by enabling the dissipation of reducing equivalents. However, when acetate is also present as an alternate electron sink, the anode prolongs the lag phase while maintaining a growth rate similar to the open-circuit control, despite the expected ATP cost of acetate-to-ethanol reduction. Under semi-continuous cultivation on mannitol in the absence of acetate, anode polarization reduces growth yield relative to open-circuit conditions. Although the anode promotes more energetically favorable fermentation patterns during batch growth on both glucose and mannitol, these are likely counterbalanced by prophage induction to affect population growth, as confirmed by transcriptomic analysis, extracellular DNA measurement, and transmission electron microscopy. Together, these results indicate that extracellular electron transfer associated with an anode is not necessarily beneficial for fermenters and prompt further inquiry into the context-dependent nature of this style of metabolism. A hybrid metabolism has been described in widespread fermenters where energy conservation through substrate level phosphorylation is coupled with electron transfer to external electron acceptors via extracellular redox mediators. This mechanism shapes interactions in microbial systems and presents opportunities in biotechnology. The variable physiological effects observed here of using an electrode as an electron sink highlight both the potential and challenges of applying electrodes to regulate fermentations. We show that polarization of an anode can hinder Lactiplantibacillus plantarum growth and is associated with induction of prophages, increasing the rate of lysis and thereby possibly counteracting the benefits of metabolic flexibility and more energetic fermentation patterns. These results make a new connection between electron balancing and a mobile genetic element and suggest a dynamic, context-dependent role of these mediators as public goods.
Antarctica hosts a highly endemic and diverse benthic marine fauna. Despite this biodiversity, the Antarctic marine food web remains structurally simple, rendering the ecosystem particularly vulnerable to environmental stressors. Benthic organisms, due to their sedentary nature, long lifespans, and close interaction with the sediment-water interface, are widely regarded as effective sentinels of ecological change. In this study, we extended a previously validated QuEChERS-based extraction protocol, originally developed for Adamussium colbecki organisms, to assess its applicability across additional Antarctic benthic taxa, including Sphaerotylus antarcticus, Odontaster validus, Trematomus bernacchii, and Laternula elliptica. The extraction method was used in combination with LC-MS/MS analysis for the determination of emerging contaminants in both targeted and suspect screening modes. Method performance was evaluated for 23 targeted emerging contaminants (ECs), yielding recovery rates of 58-116% and matrix effects between 62 and 108% for most compounds, confirming the method's suitability for taxonomically diverse matrices. Samples collected during Antarctic expeditions from 2018 to 2022 revealed the presence of multiple ECs, including perfluorooctanoic acid (PFOA), caffeine, pharmaceuticals and personal care products (PPCPs), and UV filters. Complementarily, a preliminary suspect screening via high-resolution mass spectrometry was attempted, revealing the potential presence of a broader spectrum of drugs, PPCPs, and lifestyle-related compounds in all studied species. This work represents one of the first applications of a QuEChERS-based analytical framework for ECs detection in Antarctic marine fauna, offering a reliable approach for long-term contaminant monitoring in one of the planet's most fragile ecosystems.
Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder that significantly predisposes individuals to delirium and dementia through multifaceted neurobiological pathways. The essence of this neurocognitive decline involves mechanisms such as central insulin resistance, chronic low-grade inflammation, and mitochondrial dysfunction. While metformin remains the cornerstone of T2DM management, its impact on the central nervous system exhibits a "double-edged sword" nature, balancing intrinsic neuroprotective properties against the potential neurotoxicity associated with vitamin B12 deficiency. This review aims to systematically synthesize epidemiological and clinical evidence linking metformin to neurocognitive outcomes, contrasting its efficacy with newer glucose-lowering agents such as GLP-1 receptor agonists and SGLT2 inhibitors. In addition, it sheds light on the reciprocal connectivity between systemic metabolic regulation and direct CNS modulation, specifically elucidating AMPK activation, the autophagy-lysosome axis, and the gut-brain and liver-brain axes. We review these molecular mechanisms to delineate the delicate trade-off between neuroprotection and risk, providing a framework for precision pharmacotherapy and biomarker-guided stratification in high-risk T2DM populations.
Iron-containing oxygenases play key roles in nature as part of the biosynthesis of natural products as well as the biodegradation of xenobiotics. The biosynthesis of natural products often takes place with high stereo-, chemo-, and regioselectivity, which makes these enzymes of interest to biotechnological applications. Interestingly, iron-containing enzymes appear in biology in a variety of coordination environments, including octahedral and trigonal bipyramidal geometries. To understand the structure, spectroscopic properties, and reactivity of these short-lived enzymatic intermediates, biomimetic models have been created. These models lack the secondary coordination sphere environment of proteins but explain the structural, functional, and spectroscopic properties of the metal coordination and the mechanistic features of the reaction. In this perspective, an overview is given on the first-coordination sphere environment of iron-(IV)-oxo intermediates, which are highly reactive oxidants linked to enzymatic reaction mechanisms. In particular, the effect of the axial and equatorial perturbations on the structure, spin-state ordering, and reactivity of these iron-(IV)-oxo complexes is discussed. Although systems with a porphyrinoid ligand environment show a considerable axial ligand effect due to mixing of the axial ligand orbitals with those on the porphyrin scaffold, by contrast, in nonheme iron systems, the axial ligand contribution is much less dominant due to weaker interactions of the axial ligand with equatorial ligands. However, several recent examples have shown that nonheme iron-(IV)-oxo systems are influenced by equatorial perturbations. Overall, we show that biomimetic porphyrinoid and nonheme iron-(IV)-oxo oxidants are versatile catalysts that can be engineered for stereoselective and regiospecific reaction processes.
An eco-friendly green synthesis approach was employed to produce copper nanoparticles (CuNPs) using a polyherbal extract derived from two medicinally important plant species, Hygrophila auriculata (Schumach.) Heine and Leucas aspera (Willd.) Link. The plant extracts were initially subjected to phytochemical screening to identify bioactive constituents potentially involved in nanoparticle synthesis. The synthesized CuNPs were characterized using UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), field-emission scanning electron microscopy coupled with energy-dispersive X-ray analysis (FESEM-EDAX), X-ray diffraction (XRD), and thin-layer chromatography (TLC). UV-visible spectroscopy revealed a characteristic absorption peak at 233.6 nm. FTIR analysis indicated the presence of functional groups associated with nanoparticle reduction and stabilization, whereas FESEM imaging showed predominantly spherical particles with sizes ranging 63-68 nm. Elemental composition was confirmed using EDAX analysis. XRD analysis demonstrated polycrystalline nature of the CuNPs, with an average crystallite size of 11.5 nm. GC-MS analysis and phytochemical screening further confirmed the presence of bioactive compounds, whereas TLC analysis revealed differences in mobility between the plant extract and synthesized CuNPs. Antibacterial activity of the synthesized CuNPs was evaluated using the agar well diffusion method against clinically relevant bacterial strains, including those of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Streptococcus pyogenes. The polyherbal-derived CuNPs produced larger inhibition zones than the individual plant extracts, particularly against multidrug-resistant pathogens such as P. aeruginosa and S. aureus. Additionally, the nanoparticles exhibited concentration-dependent antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl assay at concentrations ranging 10-50 mg/mL, with radical scavenging activity increasing from 29.9% to 76.5% and a corresponding decrease in absorbance from 0.698 to 0.234 (p < 0.05). Cytotoxic evaluation in HepG2 cells after 48 h of exposure demonstrated dose-dependent morphological changes and reduced cell viability. These findings suggest that polyherbal-derived CuNPs possess antibacterial, antioxidant, and cytotoxic properties with potential relevance for biomedical applications.
Enzyme cascades enable streamlined multi-step biocatalysis in one-pot systems, offering remarkable efficiency, selectivity, and sustainability compared to traditional chemical synthesis. This review highlights recent advances across three major directions: (i) efficient synthesis of bulk and chiral chemicals, including alcohol-to-amine conversion, polymer precursor biosynthesis, and non-canonical amino acid production; (ii) generation of complex pharmaceutical building blocks, exemplified by the synthesis of nucleotides, alkaloids, isoprenoids and their analogues; and (iii) integration of new-to-nature enzymatic reactions into metabolic pathways exemplified for engineered carbene-transferring P450s, artificial metalloenzymes, and photocatalytic active enzymes within microbial cell factories. These advances, driven by reaction route design, key enzyme engineering, and pathway/cell optimization, position enzyme cascades as transformative and versatile platforms for sustainable biomanufacturing across pharmaceuticals, chemicals, and materials.
Neuroligin (Nlgn) is a post-synaptic adhesion molecule that regulates synaptic maturation through trans-synaptic interactions with pre-synaptic neurexins. The extracellular domain (ECD) of Nlgns is known to form dimers that are critical for their functions. However, the dynamic nature of this dimerization remains poorly understood due to technical limitations of conventional methods. In this study, we employed mass photometry to quantitatively evaluate the oligomerization states of recombinant human Nlgn1, Nlgn2, and Nlgn3 ECDs. Unlike size exclusion chromatography-multi angle light scattering, which confirmed a predominant dimeric state at the μM concentrations, MP detected transient Nlgn monomers in the nM range. Our results demonstrate that the Nlgn ECD exists in a concentration-dependent equilibrium between monomeric and dimeric forms. Notably, we identified differences in dimerization affinities among Nlgn family members. Among the isoforms tested, Nlgn3 exhibited the highest homodimerization affinity, followed by Nlgn2 and Nlgn1. These findings suggest that Nlgn dimerization is a dynamic process governed by isotype-specific affinities, likely arising from sequence variations at the dimer interface. Furthermore, this study highlights the utility of mass photometry in quantifying the equilibrium of protein oligomerization.
The direct arylation of fluoroarenes is a powerful but sustainability-limited transformation, as most established protocols rely on organic solvents and stoichiometric silver additives. Here, we report the first silver-free palladium-catalyzed C-H arylation of fluoroarenes in water containing self-assembled surfactant structures (micelles) as reaction medium. Using the designer surfactant PS-750-M, this sustainable method enables the coupling of diverse pyridines and fluoroarenes under mild conditions, displaying broad functional-group tolerance while fully eliminating organic co-solvents. Mechanistic studies, including kinetics, FT-IR spectroscopy, dynamic light scattering (DLS), and freeze-fracture combined transmission electron microscopy (FF-TEM), reveal the nature of interactions between the catalyst, substrates and distinct micellar domains, providing insight into microenvironmental effects on reactivity and selectivity. The robustness of the catalytic system further allows tandem one-pot sequences, such as C-H arylation/SN Ar reactions, without intermediate workup or adding an additional catalyst. This work demonstrates how merging micellar catalysis with C-H activation unlocks a practical, environmentally responsible platform for cross-coupling chemistry, expanding the utility of aqueous media for sustainable synthesis.
This review proposes a tripartite regulatory framework for the Skin Extracellular Microenvironment (SEM), which the authors conceptualize as three cross-talked functional domains: hydration, nutritional, and signaling microenvironments. It systematically elaborates on the core functional architectures of each component-for instance, Hyaluronic Acid (HA) as the foundational element of the hydration microenvironment, collagen-based transport pathways in the nutritional microenvironment, and multi-axis signal networks (inflammatory, biochemical, mechanical) in the signaling microenvironment. Based on this, age-related dynamic alterations within each domain were further delineated: the hydration microenvironment undergoes progressive HA degradation and reduced biosynthesis, leading to decreased skin water retention, increased transepidermal water loss, and clinical manifestations like dryness and fine lines; the nutritional microenvironment exhibits microvascular degeneration, disrupted nutrient transport, and advanced glycation end-product accumulation, impairing extracellular matrix metabolic homeostasis; the signaling microenvironment suffers from chronic inflammation, TGF-β/Smad pathway imbalance, and mechanical signaling dysfunction, triggering a cascade of "inflammatory activation-uncontrolled degradation- mechanical disruption." Cross-regulatory interactions among the three microenvironments (e.g., hydration-signaling coupling via TGF-β/HAS2) are summarized to highlight their integrated regulatory nature. Additionally, targeted anti-aging strategies are discussed: exogenous HA supplementation and endogenous activation for the hydration microenvironment, metabolic substrate supply (e.g., proline, trace elements) for the nutritional microenvironment, and multi-pathway modulation (e.g., anti-inflammatory agents, platelet-rich plasma, collagen stimulants) for the signaling microenvironment, with particular attention to the synergistic effects observed in combination approaches such as mesotherapy-based cocktails and exosome therapy. Overall, this framework integrates previously fragmented SEM research and provides a coherent theoretical basis for evidence-based clinical anti-aging strategies and future precision interventions targeting skin aging.
Monkeypox virus (MPXV) is emerging as a global public health concern due to its nature of spread. There are limited treatment options, as the sole drug for treatment is lacking, highlighting the need for new therapeutic options. The use of computer-aided drugs discovery such as molecular docking, molecular dynamic (MD) simulations and post-simulation analysis are important tools in identifying potential compounds that can target specific proteins of the virus, such as DNA polymerase to stop virus replication. This study employed molecular docking and molecular simulation with the aim to identify potential inhibitors for MPXV treatment from the ZINC Database. Molecular docking was performed using PyRx 0.8 version after virtual screening of the ZINC database using the Tranches tool; then, toxicity prediction of the selected compounds was performed using the ProTox-3.0 web server. Molecular dynamics simulation was conducted using GROMACS version 4.5 to evaluate the structural stability and dynamic behavior of the protein-ligand complex for the best interacting compound. Furthermore, post-simulation analysis was conducted using standard GROMACS utilities for visualizing time-dependent properties from MD simulations. A total of 16 compounds were shortlisted based on their molecular docking scores and interaction profiles with the monkeypox virus DNA polymerase (PDB ID: 8HG1). The leading compound, ZINC000019418450, demonstrated strong binding affinity (-7.4 kcal/mol). According to post-simulation analysis, all top compounds formed between one and five hydrogen bonds and up to eleven hydrophobic contacts with residues within the active site, thus providing strong geometric and energetic evidence for binding stability. Notably, our identification of ZINC000104288636 as a Class 6 compound with an LD50 of 23,000 mg/kg adds translational value by highlighting candidates with low predicted acute toxicity. Overall, this study lays a solid foundation for the rational design of next-generation monkeypox antiviral therapeutics. Future work is needed for experimental validation of prioritized compounds to assess their biochemical efficacy and pharmacological potential.
The membrane lipids of archaea differ from those of bacteria and eukarya in backbone stereochemical configuration, chemical linkage type, and hydrophobic chain structure, a fundamental dichotomy termed the lipid divide. Synthetic biology and metabolic engineering have enabled reconstruction of archaeal lipid biosynthesis pathways in bacterial and eukaryotic hosts, yielding production levels reaching 30% of membrane phospholipids in Escherichia coli and 6.5% of total cellular lipids in Saccharomyces cerevisiae. These achievements required coordinated expression of core archaeal enzymes combined with enhanced isoprenoid precursor supply. A recurring finding is that bacterial and eukaryotic enzymes exhibit remarkable substrate promiscuity toward archaeal lipid precursors, enabling biosynthesis of archaetidylglycerol, archaetidylethanolamine, and archaetidylinositol without requiring archaeal enzymes. This review examines the biosynthetic pathways, host systems, and engineering strategies underlying these advances. We consider how heterologous reconstitution informs longstanding questions about membrane evolution and the nature of the last universal common ancestor. Engineered strains with hybrid archaeal-bacterial membranes not only remain viable but also exhibit enhanced stress tolerance, demonstrating that the lipid divide does not preclude membrane coexistence while enabling biotechnological applications from stress-tolerant cell factories to archaeosome-based delivery systems.
The protease TMPRSS2 facilitates coronavirus infections, yet its mechanism of viral glycoprotein recognition remains unclear. Here we show that, following ACE2 engagement of the SARS-CoV-2 spike (S) inducing the early fusion intermediate conformation (E-FIC), TMPRSS2 cleaves the R815 S2' site and promotes fusogenic conformational changes leading to viral entry. We unveil TMPRSS2 recognition of S2', identify key residues modulating binding specificity and demonstrate that S2' site-directed broadly neutralizing antibodies target E-FIC and inhibit viral entry by blocking TMPRSS2 access. We computationally designed stabilized E-FIC as a vaccine candidate, overcoming the transient nature of this state. We describe a TMPRSS2-directed monoclonal antibody inhibiting several coronaviruses, including SARS-CoV-2 variants and protecting mice against SARS-CoV-2 challenge. These results outline the mechanistic role of TMPRSS2 and S2' site-directed antibodies in coronavirus entry.
d-tagatose is a highly valuable rare sugar with significant application potential. However, its widespread use is severely limited by high production costs. One-step conversion from d-fructose represents an economically attractive bioprocess; nevertheless, the low catalytic efficiency of natural tagatose 4-epimerases (T4Es) toward d-fructose remains a major bottleneck for industrial application. Therefore, developing highly efficient T4Es is essential in this field. In this study, a structure-guided and evolution-assisted engineering strategy was employed to enhance the catalytic performance of a tagatose 4-epimerase (KoT4E) from Kosmotoga sp. The superior mutant (M6) exhibited 5.6-fold specific activity and 9.6-fold catalytic efficiency (kcat/Km) of the wild-type, respectively. Using 500 g/L d-fructose, 28% d-tagatose yield was achieved with a relatively low enzyme loading of 1 mg/mL within 12 h, substantially outperforming all currently known T4Es. Molecular dynamics simulations revealed that reduced flexibility of a key loop in the substrate-binding channel stabilized substrate binding and facilitated catalysis. The industrial applicability of M6 was further demonstrated, suggesting its potential for promoting the low-cost and high-efficiency large-scale bio-manufacturing of d-tagatose and offering insights for engineering of epimerases lacking in nature.
The primary goal of the study was to develop folic acid-conjugated eugenol-loaded PLGA nanoparticles for the treatment of breast cancer. Eugenol is reported to have potent anticancer activity. Entrapment of eugenol in folic acid-conjugated polymeric nanoparticles is expected to enhance its availability at the cancer site and improve overall efficacy in breast cancer treatment. Eugenol was isolated by the column chromatography technique. The isolated bioactive fraction was characterized by IR and NMR analyses. Polymeric NPs were prepared by the solvent emulsification-diffusion method and conjugated with FA by the EDC coupling method. The in vitro release profile for FA-conjugated eugenol-loaded PLGA NPs was evaluated by the dialysis membrane technique. Further in vitro anti-inflammatory activity, in vitro antioxidant assays, and cytotoxicity studies were carried out. NPs exhibiting particle size ranging from 444.2 to 928.3 nm, zeta potential ranging from -32.0 to -37.7 mV, entrapment efficiency ranging from 76.97% to 87.51%, and percent conjugation were found to be 72.59%-79.68%. The in vitro drug release profiles of the formulations were most effectively described by the Higuchi kinetic model, indicating that the mechanism governing drug release is primarily attributed to diffusion. The FTIR study indicated that there is no chemical modification of the drug, confirming its compatibility with other excipients. The morphology of the NPs was analyzed by FESEM analysis. The particulate nature of NPs showed homogenous, spherical shapes of NPs. The cell cytotoxicity studies on MDA-MB-231 cell lines exhibited enhanced cytotoxicity of the NPs. In conclusion, it was found that FA-conjugated PLGA NPs can be a suitable platform for the targeted administration of eugenol for BC treatment.
Colorectal cancer (CRC) is one of the most significant global health concerns, necessitating innovative therapeutic strategies for its effective management. Despite advances in treatment therapies, chemotherapy remains the mainstay of CRC treatment, with 5-Fluorouracil (5-FU) as a standard first-line agent. However, its clinical effectiveness is hindered by drug resistance, rapid clearance and systemic toxicity, underscoring the need for innovative drug delivery strategies. In this context, the current work involves engineering of a bioinspired nanocomplex (NX) comprising zein and a biological macromolecule, such as chitosan, using a Quality by Design (QbD) approach. The resulting NX was characterized for particle size (186.13 ± 8.61 nm), polydispersity index (0.194 ± 0.03), and %entrapment of 5-FU (54.39 ± 3.1%) and silibinin (97.44 ± 1.16%), respectively. SEM and TEM analysis revealed the smooth and spherical nature of NX. Thermal analysis was performed using TGA and DSC and XRD was employed for structural characterization. Subsequently, spectroscopic investigations were carried out using FTIR, Raman and fluorescence spectroscopy to examine the potential interactions between the drugs and polymers used in the formulation of the NX system. In vitro studies confirmed controlled drug release with Weibull release kinetic model. The dual-drug-loaded-NX exhibited a significant increase in cytotoxicity compared to individual 5-FU and silibinin, and achieving nearly a 5-fold increase in cytotoxicity compared to silibinin. The NX demonstrated apoptosis induction, S/G2 cell cycle arrest, and improved cellular uptake compared to control group. The current investigation suggests that QbD-engineered zein-chitosan-based-NX could be a promising therapeutic strategy for managing CRC.
Mutualistic symbioses are potentially vulnerable to exploitation, particularly in hosts that acquire symbionts from the environment, where harmful exploiters inhabit. The independent evolution and persistence of intricate partner-choice mechanisms in many symbioses testify the threat by specialized exploiters of mutualisms, although only few have been documented in nature. We report here a lethal "Trojan horse" pathogen, Burkholderia sp. SJ1, exploiting the stinkbug-Caballeronia gut symbiosis. This bacterium resembles symbionts by using wrapping motility to traverse the host's sorting organ, inducing symbiotic organ morphogenesis and colonizing it. Unlike mutualists, however, it resists host digestion for nutrient acquisition, breaches the gut epithelium, and causes sepsis, rapidly killing the host. Colonization of the symbiotic organ is essential for its lethality. This case shows how pathogens can exploit mutualisms, highlighting the evolutionary pressures shaping partner-choice mechanisms and the fragility of even highly specialized mutualisms.
Cancer remains one of the leading causes of death worldwide, and the limitations of conventional therapies such as surgery, chemotherapy, and radiotherapy underscore the urgent need for innovative therapeutic strategies. While advances in early detection and treatment have improved outcomes in some regions, challenges such as micrometastasis, tumor relapse, and multidrug resistance continue to hinder long-term success. The multifactorial nature of cancer-driven by complex genetic mutations, diverse tumor microenvironments, and adaptive cancer cell behavior- demands more precise and effective solutions. Recent breakthroughs in molecular biology and genetic engineering have led to the emergence of genome editing technologies that offer promising avenues for targeted cancer therapy. This review highlights the evolution and application of key genome editing platforms, including meganucleases, zinc finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs), and the CRISPR/CAS9 system. Meganucleases were among the earliest tools with site-specific cutting ability, but limited versatility. ZFNs and TALENs offered greater modularity and target specificity through protein-DNA interactions. The CRISPR/CAS9 system revolutionized genome editing with its RNA-guided targeting, allowing for higher efficiency, simplicity, and flexibility in gene modification. These tools have enabled researchers to disrupt oncogenes, repair tumor suppressor genes, and manipulate signalling pathways involved in tumor progression, resistance, and metastasis. Moreover, ongoing advancements in delivery systems and gene repair mechanisms have further enhanced their therapeutic potential. We also discuss their translational potential from bench to bedside and explore future perspectives on how these technologies may revolutionize precision oncology, ultimately contributing to more effective treatment outcomes.
Bacterial stem and root rot (BSRR), caused by Dickeya dadantii, poses a severe threat to global sweetpotato production, yet the genetic architecture underlying resistance remains elusive. To dissect these mechanisms, we conducted a high-resolution genome-wide association study (GWAS) on 135 diverse accessions, integrating two-year field phenotyping with best linear unbiased prediction (BLUP) and 6.8 million single-nucleotide polymorphism (SNP) markers. This approach mapped nine quantitative trait loci (QTLs) exhibiting significant allelic dosage-dependent effects, with the major locus, qBSRR.6.1 was the primary discriminator between resistant and susceptible genotypes. Crucially, transcriptomic profiling within these loci revealed distinct expression patterns: IbTCP5 and IbERF003 (located in qBSRR.5.1 and qBSRR.6.2) were highly expressed in the susceptible cultivar 'Xinxiang' but suppressed in the resistant 'Guangshu87'. Furthermore, BSRR challenge identified IbPUB4, IbKCS5, and IbLig1 as priority candidate genes involved in defense, with expression patterns suggesting roles in ubiquitin-mediated protein turnover, cuticular wax biosynthesis, and DNA repair, respectively. In stark contrast, IbPUB25 was constitutively upregulated in 'Xinxiang', potentially acting as a negative regulator of immunity via degradation of target proteins. These findings elucidate the polygenic, dosage-sensitive nature of BSRR resistance and prioritize specific targets for future functional characterization. Pyramiding favorable alleles of positive candidates while silencing potential negative regulators like IbPUB25 offers a promising avenue for developing durable, high-resistance sweetpotato varieties.
Interleukin-1β (IL-1β) plays a central role in driving vascular inflammation and endothelial dysfunction, key processes in the development of atherosclerosis. While biologic therapies targeting IL-1β have shown clinical benefit, their high cost, injectable nature, and potential side effects limit their broader use. Therefore, there is a need to explore more accessible alternatives. In this study, we aimed to identify repurposed small-molecule inhibitors that can effectively modulate IL-1β signaling and protect endothelial function. We used an integrated strategy combining computational and experimental approaches. Virtual screening, molecular docking, molecular dynamics simulations, and MM-PBSA analyses were performed to identify potential inhibitors targeting IL-1R1. The most promising candidates were then evaluated in vitro using endothelial cell models (HUVEC and EA.hy.926). Their effects were assessed through functional assays, including transendothelial electrical resistance (TEER), VE-cadherin immunofluorescence, and cell viability measurements. Two FDA-approved drugs, radotinib and lomitapide, emerged as strong candidates with high binding affinity and stability toward IL-1R1, outperforming the reference inhibitor anakinra in computational analyses. Experimental validation showed that both compounds effectively reduced IL-1β-induced endothelial dysfunction. They restored barrier integrity, improved TEER values, and maintained VE-cadherin expression and localization. Importantly, both compounds exhibited low cytotoxicity and mitigated IL-1β-driven increases in endothelial permeability. Our findings highlight radotinib and lomitapide as promising repurposed small-molecule inhibitors of IL-1β signaling. By preserving endothelial integrity and dampening inflammatory responses, these compounds may serve as cost-effective and orally available alternatives to current biologic therapies. Further in vivo and mechanistic studies are needed to advance their potential clinical application.
The development of artificial enzymes through incorporation of new-to-nature catalytic functionality into protein scaffolds has emerged as a powerful approach to expand the biocatalytic repertoire. Inspired by the success of Lactococcal multidrug resistance regulator (LmrR), a transcriptional regulator protein, whose unique scaffold has been used for the design of a range of artificial enzymes, we performed a bioinformatics study in an effort to expand the scope of protein scaffolds for artificial enzyme design with other LmrR-like proteins. LmrR belongs to the phenolic acid decarboxylase transcriptional regulator (PadR) subfamily 2 (PadR-s2) and exhibits an unusual open pore with promiscuous binding capabilities. Using genome mining and homology modeling, we identified six previously uncharacterized PadR-s2 proteins and experimentally evaluated them as protein scaffolds for the design of artificial Friedel-Crafts (FC) alkylases. Two of the candidates, Lactococcus fujiensis (LCf) PadR and Brachyspirahampsonii (Bh) PadR, could be applied in the iminium-promoted FC-alkylation using genetically incorporated noncanonical amino acids p-aminophenylalanine or 3-aminotyrosine as catalytic residues. Interestingly, contrary to homology models, AlphaFold predictions of the PadR-s2 candidates and X-ray crystallography of BhPadR and a variant incorporating 3-aminotyrosine revealed closed-pore structures. Our findings thus demonstrate that an open-pore structure like LmrR is not a prerequisite for designing artificial FC-alkylases and introduce two new PadR-s2 scaffolds for future application.