Chemistry – A European Journal

Prasugrel is a prodrug, widely used in antiplatelet strategy for secondary prevention after acute coronary syndrome. The metabolism of prasugrel leads to the formation of the Prasugrel Active Metabolite (PAM), an irreversible P2Y12 receptor antagonist. Its mode of binding has not yet been fully established, although it is known that it binds covalently to P2Y12 by forming a disulfide bridge with cysteines and its sulfur moiety. PAM is a molecule with two chiral centers, resulting in four stereoisomers which appear to be stereoselective upon binding. A combination of different molecular modeling methods, such as molecular dynamics, ensemble docking, and Density Functional Theory (DFT), were used to rationalize these differences in antagonism observed in vitro and to elucidate the mode of binding of PAM to P2Y12. PAM is found to bind to the closed P2Y12 conformation in a preferential way. Although the four stereoisomers have comparable affinity, the location of the RS stereoisomer makes the formation of a disulfide bond with cysteines more favorable, particularly with cysteine 175. Compared to the RR stereoisomer, the RS stereoisomer interacts less deeply with the P2Y12 receptor, interacting in particular with the second and third extracellular loops, explaining the competition observed with cangrelor and an intermediate metabolite of prasugrel. Furthermore, DFT calculations have shown that the formation of a disulfide bridge is energetically more favorable with the RS stereoisomer than with the RR stereoisomer. The physical interactions and chemical reaction between the RS stereoisomer and the P2Y12 receptor are key factors in explaining the stereoselective binding of PAM to P2Y12.

Cationic polymers, which mimic the structure of antimicrobial peptides (AMPs), are increasingly recognized as promising antimicrobial materials. Here, we report the synthesis and evaluation of a new class of cationic lipid-terminated oligomers (CLOs), comprised of 2C18-hydrophobic lipid tails, and short oligomeric cationic chains synthesised via Cu(0)-mediated reversible-deactivation radical polymerization (RDRP). Two 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers with degrees of polymerization (DP) of 20 or 50 were synthesized using the lipid functional initiator (R)-3-((2-bromo-2-methylpropanoyl) oxy)propane-1,2-diyl dioctadecanoate (2C18-Br). Post-polymerization modification of the pendant oxazolone moieties was carried out using reactive amines, including N-Boc-ethylenediamine (BEDA) and N,N-dimethylethylenediamine (DMEN). Subsequent deprotection of the BEDA groups and quaternization of DMEN groups enabled the synthesis of six functional CLOs exhibiting distinct cationic functionalities. Antimicrobial assays against a panel of WHO bacterial and fungal priority pathogens (methicillin-resistant Staphylococcus aureus [MRSA], Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans) revealed that these CLOs exhibited potent and selective structure-dependent antibacterial activity, particularly against MRSA, with minimum inhibitory concentrations (MICs) in the clinically relevant range, below 4 {micro}g mL-1, comparable to antibiotics vancomycin and colistin. Among these, BEDA-functionalized CLOs demonstrated the strongest antimicrobial profile, which was significantly increased by increasing DP, as evidenced by a reduction in MIC values from 64 {micro}g mL-1 (for DP20) to [≤] 4 {micro}g mL-1 (for DP50) against A. baumannii. Biocompatibility assays against red blood cells and HEK293 cells indicated negligible toxicity, with haemolytic (HC50) and cytotoxic (CC50) values exceeding 512 {micro}g mL-1 across all CLOs. All CLOs displayed minimal activity against C. albicans (MIC [≥] 512 {micro}g mL-1). In contrast, activity against C. neoformans was influenced by both cationic functionality and DP, with DMEN-based CLOs exhibited superior antifungal activity at higher DP relative to their BEDA-based counterparts. Most CLOs displayed high selectivity (SI) toward MRSA (SI >128), while 2C18-O(BEDA)50 exhibited the broadest spectrum, showing potent antimicrobial activity and high selectivity against E. coli (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), A. baumannii (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), and MRSA (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), along with moderate activity against P. aeruginosa (MIC = 32 {micro}g mL-1, SI > 16). Taken together, these findings elucidate the combined influence of end-group lipidation, cationic functionality, and polymer length in modulating antimicrobial activity, thereby establishing 2C18-terminated CLOs as a rationally tunable and biocompatible platform for antimicrobial material development.

Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. Myo-inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. Scyllo-inositol is the second most abundant inositol isomer in the human brain and aberrant concentrations are associated with various diseases; however, its biological functions remain poorly understood. Here, the development and application of [13C6]scyllo-inositol as an isotopic tracer to study its metabolism is reported. A concise and robust synthetic route was established to obtain [13C6]scyllo-inositol from [13C6]myo-inositol in good yield. The uptake of [13C6]scyllo-inositol and responses of endogenous inositol isomers were measured in multiple cell lines by HILIC-MS/MS, showcasing the advantages of isotopic tracing. [13C6]scyllo-inositol proved to be a versatile isotopic tracer, when coupled with MS-based lipidomics and 2D NMR experiments. These experiments provide evidence that scyllo-inositol is incorporated into phosphatidylinositols in different cell lines. The results suggest a previously underappreciated role of scyllo-inositol in mammalian cells. The utilization of [13C6]scyllo-inositol will help to elucidate the role of scyllo-inositol metabolism in healthy and diseased states. SignificanceScyllo-inositol is a cyclic sugar alcohol found predominantly in the human brain. Changes in its concentration are associated with different diseases, and scyllo-inositol has been investigated as a potential drug against Alzheimers disease in clinical trials. However, its metabolic fate in mammalian cells is not well understood. We report here a synthetic strategy to obtain [13C6]scyllo-inositol and demonstrate, through isotopic tracing, its incorporation into phosphatidylinositols in different human-derived cell lines. This new stable isotopic tracer enables the investigation of the biological role of scyllo-inositol in mammals and beyond. HighlightsO_LIConcise synthesis of [13C6]scyllo-inositol C_LIO_LI[13C6]scyllo-inositol uptake and response of endogenous inositol isomers studied in multiple cell lines C_LIO_LIUse of [13C6]scyllo-inositol as an isotopic tracer in metabolomics and lipidomics experiments C_LIO_LIEvidence for scyllo-inositol incorporation into phosphatidylinositol in mammalian cells C_LI

Age-accompanied chronic, low-grade systemic inflammation (inflammaging) drives the onset and progression of neurodegenerative disorders like Parkinsons disease (PD). Currently, no disease-modifying therapies are available for PD. Exposure to environmental toxicants, including paraquat (PQ), rotenone, and neurotoxic metals, increases disease risk. Conversely, sustained consumption of dietary soft electrophiles, such as flavonoids, carotenoids, vitamin E vitamers, and essential fatty acids, has been associated with increased lifespan and delayed age-related neurological decline. Omega-3 and select omega-6 fatty acids also serve as precursors of lipid-derived specialized pro-resolving mediators (SPMs), which exert potent anti-inflammatory and inflammation-resolving activities. Here, we report the development of a robust analytical method to quantify pro-resolving oxylipins in a PQ-induced Drosophila melanogaster model of PD, enabling investigation of how dietary phytochemicals modulate anti-inflammatory and pro-resolving lipid metabolism in vivo. We hypothesized that plant-derived soft electrophiles promote active resolution of neuroinflammation by enhancing the production of pro-resolving oxylipins derived from essential fatty acids, and that their neuroprotective effects are linked to their soft electrophilic properties. Our results demonstrate that specific lipophilic plant-derived soft electrophiles significantly upregulate pro-resolving oxylipins in Drosophila heads following PQ exposure. We identify a subset of flavones and structurally related phytochemicals that selectively enhance SPM biosynthesis and show that this response involves the NF-{kappa}B orthologue relish. Additionally, feeding modality and sex-specific dimorphisms were found to influence oxylipin production. Collectively, these findings indicate that structurally related dietary soft electrophiles enhance endogenous pro-resolving lipid pathways, promote resolution of toxin-induced neuroinflammation, and have potential preventive and therapeutic relevance for neuroinflammation-associated neurodegenerative diseases. HighlightsO_LIQuantification of pro-resolving lipids in a Drosophila Parkinsons model. C_LIO_LISpecific structural features of phytochemicals contribute to in vivo bioactivity. C_LIO_LILipophilic soft electrophiles show therapeutic potential against neuroinflammation. C_LIO_LIFeeding modality and sexual dimorphism also regulate oxylipin production. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/714080v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@2088cforg.highwire.dtl.DTLVardef@1f5d026org.highwire.dtl.DTLVardef@134aa44org.highwire.dtl.DTLVardef@965e28_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intercalation of small molecules between DNA base pairs affects DNA conformation, disrupting essential cellular processes including replication, transcription, and repair. We investigated conformational changes in 18-mer DNA upon intercalation of doxorubicin, SYBR Gold and YOYO-1 using extensive MD simulations. Two main patterns for the intercalation were identified: RISE-type intercalation occurs between adjacent base pairs and extends the DNA helix with decreased twist angles, while OPEN-type intercalation proceeds through base-pair opening without significant DNA extension. Kinetic analysis revealed that association rates for intercalation followed the order: first YO-moiety (mono-intercalation) > SYBR Gold > doxorubicin > YOYO-1 (bis-intercalation). Free energy landscape showed that forces at DNA termini reached up to 117 pN during stretching. Notably, base pairs adjacent to intercalators were protected from strand separation, accompanied by additional helical unwinding. MM-PBSA/GBSA analysis revealed that the driving force for intercalation is the stacking energy, and the binding affinity was highest for minor groove binding. Persistence length decreased with single molecule binding but recovered with two molecules due to their electrostatic repulsion. Mechanical properties of intercalated DNA showed position-dependence, demonstrating that multiple intercalation modes coexist in solution. The heterogeneous nature of intercalation explains why experimental measurements reflect ensemble averages rather than single binding configurations.

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with cisplatin as the primary chemotherapy despite its limitations. Organotin(IV) dithiocarbamates have emerged as promising anticancer agents due to their potent cytotoxicity and stability. This study reports the successful synthesis of four novel organotin(IV) dithiocarbamates: dimethyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-1), diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-2), triphenyltin(IV) N-methyl-N-benzyldithiocarbamate (TriSn-3), and triphenyltin(IV) N-ethyl-N-benzyldithiocarbamate (TriSn-4). Their cytotoxicity against A549 lung carcinoma cells was evaluated via MTT assay, while Annexin V-FITC/PI staining determined the mode of cell death. DioSn-2, TriSn-3, and TriSn-4 exhibited potent cytotoxicity (IC: 0.52-1.86 M), whereas DioSn-1 was inactive (IC > 50 M). Apoptotic features such as cell shrinkage and membrane blebbing were observed, with apoptosis rates ranging from 58% to 91%. DioSn-2 was the most selective (SI = 6.45) and induced early DNA damage within 30 minutes, followed by mitochondrial depolarization and excessive ROS generation. Caspase-9 activation exceeded caspase-8, confirming intrinsic apoptosis. NAC treatment reduced apoptosis by 52%, highlighting oxidative stress as a key cytotoxic mechanism. These findings suggest DioSn-2 as a promising alternative to cisplatin for lung cancer therapy.

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

Angiogenesis, the process by which new blood vessels form from existing vasculature, is fundamental to tissue repair and regeneration but also underlies pathological conditions such as cancer progression. Targeting angiogenesis has thus become a promising approach for developing novel cancer therapeutics. While various phytochemicals have demonstrated anti-angiogenic effects, the role of 2-5(H)-Furanone, a naturally occurring lactone found in various plants and marine sources with diverse biological activities, remains insufficiently explored. In this study, we systematically evaluate the anti-angiogenic potential of 2-5(H)-Furanone using Human Umbilical Vein Endothelial Cells (HUVECs) as an in vitro model and zebrafish embryos as an in vivo model. Experimental findings demonstrated that treatment of HUVECs with increasing concentrations of 2-5(H)-Furanone led to significant, dose-dependent reductions in proliferation, invasion, migration, and tube formation. Analyses of gene expression revealed marked downregulation of key pro-angiogenic mediators, VEGF, and HIF-1. Complementing these in vitro results, in vivo studies in zebrafish embryos showed robust, dose-dependent inhibition of intersegmental vessel (ISV) formation, accompanied by suppression of critical angiogenesis-related genes. Molecular docking further supported these observations by indicating stable binding of 2-5(H)-Furanone to major angiogenic targets, including VEGFR2, MMP2, HIF-1, and PIK3CA. Collectively, our data demonstrate that 2-5(H)-Furanone potently inhibits angiogenesis, as evidenced in both HUVEC and zebrafish models, through functional and molecular mechanisms. These findings support the further development of 2-5(H)-Furanone as a promising anti-angiogenic therapy candidate.

Microorganisms in the human gut influence the efficacy and metabolism of host-targeted small molecule therapeutics, including the frontline Parkinsons disease drug levodopa (L-dopa). Previous work has identified a mechanism-based inhibitor of gut bacterial decarboxylases that degrade L-dopa, -fluoromethyltyrosine (AFMT). However, early experiments with AFMT in rodent models suggested undesirable in vivo metabolism by host tyrosine hydroxylase, producing a metabolite likely to worsen Parkinsons phenotypes and prevent application as an L-dopa co-treatment. Here, we demonstrate oxidation of AFMT in vitro by recombinant human tyrosine hydroxylase. We then develop AFMT analogs that retain activity against bacterial decarboxylases but have reduced susceptibility to host hydroxylation. Suitable arenes for inhibitor design were identified using assays with commercially available noncanonical amino acids, which revealed aryl difluorination as a promising modification. Difluoroaryl AFMT derivatives are less prone to degradation by tyrosine hydroxylase in vitro yet still inhibit L-dopa metabolism by bacterial decarboxylases. This work exemplifies how substrate reactivity can streamline design of mechanism-based enzyme inhibitors, as well as how constraints posed by the host can be incorporated during development of microbiome-targeted therapeutics. The compounds reported here are promising starting points for future studies in animal models and further exploration of gut bacterial effects on L-dopa treatment efficacy.

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

Nicotinamide adenine dinucleotide (NAD+) is crucial for cellular functions including DNA repair and metabolism. Nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymes catalyze the final step of NAD+ synthesis from NMN and ATP. There are three NMNAT isoforms: NMNAT1, NMNAT2, and NMNAT3, located in the nucleus, cytoplasm, and mitochondria, respectively. Nuclear NAD+ promotes disease progression in NAD+-dependent cancers, and it is hypothesized that targeting NMNAT1 with small-molecule inhibitors could be an effective therapeutic strategy. Here, we identify an NMNAT1 inhibitor from a bioactive compound screen and report its effects on NAD+ levels and the viability of NMNAT1-dependent cancer cell lines. The compound AMI-1 is a known inhibitor of Protein Arginine N-Methyltransferase 1, and we find that it also inhibits NMNAT1 with similar potency. Additionally, we determined a cryo-EM structure of NMNAT1 bound to AMI-1 and revealed its mechanism of inhibition. This provides proof of principle for inhibiting NMNAT1 to target NAD+ metabolism in dependent cancers, while also highlighting that caution is warranted when interpreting studies using AMI-1 as a PRMT1 inhibitor, given its effect on NAD+ through NMNAT1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/716846v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@59933borg.highwire.dtl.DTLVardef@d1298borg.highwire.dtl.DTLVardef@1fe902dorg.highwire.dtl.DTLVardef@1abb3cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

KRAS mutations drive multiple cancers and represent an important therapeutic target, together with other oncogenic regulators such as MYC, KIT, and BCL2 that are critically involved in pancreatic cancer. Here we describe a novel therapeutic strategy based on stable nucleolipid-modified G-quadruplexes (NLG4). Cell viability assays demonstrate that NLG4 strongly inhibit pancreatic cancer cell proliferation, whereas non-lipidic G-quadruplex sequences display minimal activity under comparable conditions. Owing to their distinctive physicochemical properties, including stabilization of parallel G-quadruplex structures and self-assembly into micellar aggregates, NLG4 efficiently internalize into cells and interact with key G-quadruplex unfolding factors such as UP1. This interaction leads to a marked downregulation of KRAS, c-MYC, c-KIT, and BCL2 expression. Suppression of these oncogenes profoundly affects pancreatic cancer cell fate, as evidenced by reduced expression of proliferation (Ki67) and anti-apoptotic (BCL2) markers. In addition, NLG4 treatment decreases inflammatory signaling mediated by NF-{kappa}B and inhibits major pro-proliferative kinase pathways, including ERK, AKT, and phosphorylated AKT. The therapeutic relevance of this decoy strategy is further supported by the observed potentiation of gemcitabine antitumor activity. Overall, these findings highlight NLG4 as a promising anticancer approach that simultaneously targets multiple oncogenic pathways through G-quadruplex-based decoy mechanisms, with translational potential for future pancreatic cancer treatment.

TEA domain (TEAD) proteins bind co-activator Yes-associated protein (YAP) to regulate the expression of target genes of the Hippo pathway. The TEAD*YAP protein-protein interaction is not druggable, but TEADs possess a unique and deep palmitate pocket with a highly conserved cysteine located outside the TEAD*YAP protein-protein interaction interface. Here, we screen a fragment library of acrylamide electrophiles and identify a fragment that forms an adduct with the conserved palmitate pocket cysteine and inhibits TEAD4 binding to YAP. Synthesis of a focused set of derivatives and time- and concentration-dependent studies with four TEADs provide reaction rates and binding constants. Co-crystal structures of fragments bound to TEAD2 and TEAD3 reveal reaction at the conserved palmitate pocket cysteine but also at another less conserved cysteine located in the palmitate pocket of TEAD2 closer to the TEAD*YAP interface. These fragments provide a starting point for the development of allosteric acrylamide small-molecule covalent TEAD*YAP inhibitors.

Pseudomonas aeruginosa is an adaptable organism, frequently found in chronic infections, and for which antimicrobial resistance is a growing concern. Therefore, there is an urgent need for alternative therapeutic strategies. Cationic antimicrobial peptides (AMPs) offer potent bactericidal activity but suffer from limited selectivity and potential host toxicity. To enhance species-specific targeting, we designed two prodrug variants of the AMP D-Bac8CLeu2,5 - EEEE-D-Bac8CLeu2,5 and ELEG-D-Bac8CLeu2,5 -- engineered for activation by the P. aeruginosa extracellular aminopeptidase PaAP. While both prodrug motifs effectively neutralized the positive charge of D-Bac8CLeu2,5 and prevented DNA-peptide complex formation, EEEE-D-Bac8CLeu2,5 showed negligible antimicrobial activity due to slow and incomplete activation. In contrast, ELEG-D-Bac8CLeu2,5 underwent rapid PaAP-mediated activation, restoring bactericidal activity in planktonic cultures and biofilms. PaAP contributed significantly to complete prodrug activation, particularly within biofilms, where the accumulation of partially activated intermediates correlated with biphasic killing kinetics. The prodrug showed reduced activity against other ESKAPEE pathogens, demonstrating selective activation by P. aeruginosa. Experiments selecting resistant bacteria revealed distinct mutations in lipopolysaccharide biosynthesis pathways for D-Bac8CLeu2,5 and the prodrug, with limited cross-resistance. These findings establish aminopeptidase-activated AMP prodrugs as a promising approach for species-selective antimicrobial therapy and highlight the feasibility of exploiting bacterial enzymes for controlled antimicrobial peptide activation. Table of contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/715093v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4a5505org.highwire.dtl.DTLVardef@13e578org.highwire.dtl.DTLVardef@3e3080org.highwire.dtl.DTLVardef@e24266_HPS_FORMAT_FIGEXP M_FIG C_FIG

Isoprenoid quinones are ubiquitous redox lipids that mediate electron transfer in various cellular processes across all domains of life. These molecules also serve as taxonomic and metabolic markers, facilitating the characterisation of microbial communities. However, their structural diversity and extreme hydrophobicity pose challenges for comprehensive detection and quantification in complex biological matrices. In this study, we present a semi-quantitative HPLC-MS/MS method that enables the sensitive analysis of the widest range of quinones reported to date. Using a 16-quinone standard mixture, we optimized separation within a 14-minute HPLC gradient and achieved femtomole-level sensitivity in targeted analyses. When applied to sewage sludges sampled weekly over three weeks, our method detected 57 distinct quinones, revealing stage-specific quinone profiles that reflect shifts in bacterial communities during wastewater treatment. This rapid and sensitive workflow provides a robust tool for accurate quinone profiling in complex samples, opening avenues for the discovery of novel quinones through untargeted approaches. By pushing the boundaries of quinone profiling, our method holds significant promise for advancing microbial ecology, environmental monitoring, and biotechnological applications. HighlightsO_LIuHPLC-Orbitrap method for the semi-quantitative profiling of isoprenoid quinones C_LIO_LIAnalysis of the widest range of isoprenoid quinones to date C_LIO_LIFemtomole-level sensitivity in just 14 minutes of chromatographic separation C_LIO_LIDetection of 57 quinones in complex wastewater sludge matrices C_LIO_LIMost comprehensive set of quinone standards including microbially-purified quinones C_LI

Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mechanisms. In our study, we employed two single-molecule methods, Forster Resonance Energy Transfer (smFRET) and high-resolution optical tweezers, to investigate the transcription of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) from initiation through to termination. We aim to provide a set of comprehensive biophysical tools to deepen our current understanding of MtbRNAP and its transcription factors. These experimental assays represent an important step towards unraveling the molecular dynamics and interactions that support transcription in Mycobacterium tuberculosis.

Flexible and biocompatible piezoelectric materials are crucial for next-generation wearable and bio-integrated electronics. In this work, we report a sustainable bio-composite film by incorporating lysozyme, a naturally abundant protein, into a polyvinyl alcohol matrix to achieve efficient electromechanical conversion. The composite exploits the intrinsic molecular dipoles of lysozyme, which are effectively stabilized and aligned within the polymer network. Under applied bending strain and vertical pressure, the film exhibits a pronounced piezoelectric response, as evidenced by time-dependent electrical measurements under forward and reverse bias conditions. The deformation of -helices and other helical structures within lysozyme induces dipole reorientation and charge separation, generating a measurable electrical output. In contrast, pure polyvinyl alcohol films show no detectable response, confirming the essential role of lysozyme in the observed piezoelectricity. Furthermore, the device enables real-time human motion sensing, highlighting its potential for flexible, eco-friendly, and biocompatible electronic applications.

Formate is emerging as an important molecule in carbon capture and utilization technologies. However, its low electron density makes formate less attractive for energy storage. Some hydrogenotrophic methanogens can reduce formate to methane, thereby upgrading it into an established energy carrier. The bottleneck in this process is that 75% of the carbon is lost as carbon dioxide, and achieving a complete formate-to-methane conversion requires co-feeding hydrogen. However, hydrogen-dependent genetic regulation of formate metabolism inhibits simultaneous formate and hydrogen utilization in hydrogenotrophic methanogens. Here, we compared the catalytic performance of the genetically modified strain Methanothermobacter thermautotrophicus {Delta}H (pFdh) with M. thermautotrophicus Z-245 by conducting continuous cultivation at different hydrogen concentrations. While M. thermautotrophicus Z-245 is a natural formatotroph, M. thermautotrophicus {Delta}H (pFdh) was engineered to enable formate utilization via episomal expression of a formate dehydrogenase-gene cassette. We found that M. thermautotrophicus {Delta}H (pFdh) can simultaneously utilize formate and hydrogen. It continuously consumed formate at {approx} 0.1 mM dissolved hydrogen, enabling a 75.6% formate-to-methane conversion efficiency. M. thermautotrophicus Z-245 showed a declining formate consumption at {approx} 0.016 mM and only reached a maximum stable efficiency of 36.3%. These results suggest that M. thermautotrophicus {Delta}H (pFdh) is largely insensitive to hydrogen-induced genetic regulation; however, it still faces redox-related metabolic limitations at dissolved hydrogen concentrations above 0.4 mM. Overall, the findings reveal a potential strategy to circumvent hydrogen-induced regulation of formate metabolism and identify M. thermautotrophicus {Delta}H (pFdh) as a promising biocatalyst for formate-to-methane conversion.

Tumour biomarkers such as CA125, CA15-3, CA19-9, AFP and CEA are routinely used in the oncology clinic to diagnose cancer, monitor response to therapy, and detect relapse. However, their quantification depends on immunoassay-based methods that are time-consuming, reagent-dependent, and poorly suited to resource-limited settings. Here, we present a machine learning-assisted ATR-FTIR spectroscopy approach for label-free tumour biomarker analysis to enable simple and rapid quantification at the bedside. Using principal component analysis (PCA), we first demonstrate that these five clinically relevant biomarkers are spectrally separable, with the protein-associated region (1200-1700 cm-1) providing the greatest discriminative information. We then develop partial least squares regression (PLSR) models to quantify CA125 in phosphate-buffered saline (R2 = 0.95) and in human serum across a clinically relevant concentration range, achieving reliable predictions at and above the clinical decision threshold of 35 U/mL. A semi-quantitative classification model further demonstrated robust identification of elevated CA125, with a macro-average sensitivity of 0.86 and specificity of 0.92. These results support ATR-FTIR spectroscopy as a rapid, reagent-free platform for cancer biomarker monitoring, with potential utility in resource-limited settings. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/714840v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1be9c03org.highwire.dtl.DTLVardef@f49e5eorg.highwire.dtl.DTLVardef@1c93e39org.highwire.dtl.DTLVardef@1141e6f_HPS_FORMAT_FIGEXP M_FIG C_FIG