ChemBioChem

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1e56ffborg.highwire.dtl.DTLVardef@1ac8b5org.highwire.dtl.DTLVardef@6f23c1org.highwire.dtl.DTLVardef@945c54_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell-free expression systems (CFES) are increasingly used alongside conventional biotechnological approaches to accelerate early-stage prototyping and are particularly valuable in point-of-use settings. However, their broader adoption remains limited by time- and cost-intensive preparation, as well as stringent cryogenic storage requirements. To address this, several studies have explored lyophilization with protective additives to generate stable, solid-state CFES. These approaches had to balance the protection gained with a loss of activity due to the additives. In this study, we present a CFES that contains a tardigrade-derived Cytosolic-Abundant Heat-Soluble (CAHS) protein to protect the biosynthetic machinery in lysates from damages during drying. We show that the CAHS protein, without any other additives, preserves protein synthesis activity during low-cost room temperature desiccation, while unprotected lysates are affected in mRNA synthesis kinetics and translation yields. The diversity of tardigrade-derived protective proteins is a treasure trove for cell-free synthetic biology, in particular for making CFES more accessible and portable. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/715078v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@8ecc2eorg.highwire.dtl.DTLVardef@ff0432org.highwire.dtl.DTLVardef@6c940eorg.highwire.dtl.DTLVardef@6c5390_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

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.

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.

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

The importance of human aldehyde oxidase (hAOX1) has increased over the last decades due to its involvement in drug metabolism. Inhibition studies concerning hAOX1 are extensive and a common reducing agent, dithiothreitol (DTT), was recently found to inactivate the enzyme. However, in previous crystallographic studies of hAOX1, DTT was found to be essential for crystallization. To surpass this concern another reducing agent used in crystallization trials. Using tris(2-carboxyethyl)phosphine (TCEP), a sulphur-free reducing agent, it was possible to obtain well-ordered crystals from hAOX1 wild type and variant, hAOX1_6A, which diffracted beyond 2.3 [A]. Instead of the typical star-shaped crystals of hAOX1, at pH 4.7, plates are obtained in the orthorhombic space group (P22121) with two molecules in the asymmetric unit. Activity assays with the enzyme incubated with both reducing agents show that contrary to DTT, TCEP does not lead to irreversible inactivation of the enzyme. The replacement of DTT with TCEP in crystallization of hAOX1 provides a strategy to circumvent enzyme inactivation during crystallographic studies, allowing future applications of new assays, such as time-resolved crystallography.

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.

Protein self-assembly enables precise nanoscale organization but rarely translates into macroscopic materials that retain functionality beyond aqueous environments. Here, we report that a bacterial microcompartment (BMC) trimer fused with SpyTag (T1-SpyTag), when expressed as a standalone component, undergoes rapid and spontaneous self-assembly into macroscopically visible fibers and layered sheets. These structures span from the nanoscale to the millimeter scale, forming robust three-dimensional protein materials that remain structurally intact and functionally accessible in both solution and dried states. Unlike previously reported SpyTag-enabled BMC systems that function primarily as passive cargo-loading modules, T1-SpyTag macromolecular structures exhibit emergent material behavior, including chemical robustness under denaturing conditions, while preserving covalent reactivity toward SpyCatcher-fused cargos. The multilayered architecture enables tunable surface display, access to ultrathin, processable protein films, and surface renewability through layer-by-layer removal and regeneration. This work demonstrates how a minimal genetic modification of a native protein building block can drive the formation of functional, macroscopic protein materials, thus expanding the design space of BMC-derived assemblies for biointerfaces, catalysis, and sustainable protein materials engineering.

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

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.

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. Here, we use site-directed mutagenesis to study the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Mutations at this position change the biochemical properties (KM for CO, product inhibition constant, catalytic bias...), and increase the resistance of the enzyme to the inhibitor O2, showing that isoleucine 563 indeed lines the gas channel. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far. We show that the size of the introduced amino acids is less important than their flexibility - along with the size of the cavity formed near the active site in the channel. We also conclude that O2 access to the active site cannot be slowed down without also affecting CO diffusion. This tradeoff will have to be considered in further attempts to use site-directed mutagenesis to make CODHs more O2 tolerant.

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

Rapid and highly selective sensing of ultra-low concentration protein biomarkers remains a critical challenge important for early disease diagnosis and monitoring. Here, we use conical SiO2 nanopore-based biosensing for the rapid detection of heart-type fatty acid binding protein (H-FABP). Antibodies were covalently immobilized on the nanopore surface through siloxane chemistry. The functionalized asymmetric nanopores generate a characteristic rectifying current-voltage response, which shows a distinct shift upon binding to the target protein due to partial neutralization of the negatively charged pore surface. The sensor exhibits excellent sensitivity in the attomolar to nanomolar concentration range with a detection limit (LOD) of [~]0.4 aM. Furthermore, the platform exhibits high selectivity, distinguishing H-FABP from non-target proteins (HSA and Hb) at concentrations six orders of magnitude higher. We also demonstrate that nanopores can be regenerated using sodium hypochloride and O2 plasma treatment, enabling repeated functionalization and reuse.

We discovered that palmatine (PAL), a well-known natural product, was inducing the fluorogenic fixation of live cells upon visible light irradiation. This ultrafast phenomenon proceeded under high spatiotemporal control down to single cells (SC), with persistence of well-preserved fixed-labeled cells. Cell "optofixing" was mediated by PAL interaction with nuclear and mitochondrial DNA, yielding reactive oxygen species (ROS) mainly singlet oxygen (1O2), lipid peroxidation (LPO) and LPO-derived fixing aldehydes. We found that other DNA dyes including conventional trackers were also capable of optofixing cells, furnishing a consistent methodology (fluorophore-mediated optofixation, FLUMO) across the visible spectrum. Our results pave the way for the functional ablation and labeling of target cell populations using small fluorophores, with applications in SC, organoid and whole organism biology.

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

Microneedle (MN) patches have emerged as a highly efficient platform for localized drug delivery, showing great promise in cancer therapy due to their ability to enable precise drug administration. However, conventional MN systems are limited by the low drug-loading capacity of their tips and primarily rely on biologically inert, non-therapeutic matrices for structural support, which restricts further gains in antitumor efficacy. Herein, we present a strategy turning toxicity into therapy by constructing palladium nanoparticle-loaded polyvinyl alcohol/polyethyleneimine (PVA/PEI@Pd) hydrogel microneedles (PPPd-MNs), which exploit the intrinsic cytotoxicity of PEI for synergistic melanoma therapy. The PPPd-MNs efficiently catalyze the deprotection of a doxorubicin prodrug (P-DOX), enabling in situ generation of active doxorubicin (DOX). Notably, the PEI matrix serves a dual function: acting as a robust ligand to stabilize Pd catalysts and functioning as a therapeutic agent that disrupts cancer cell membranes. Both in vitro and in vivo experiments demonstrate that the combination of Pd-mediated bioorthogonal activation of DOX and PEI-induced membrane damage achieves a remarkable synergistic therapeutic outcome in a murine melanoma model, resulting in a tumor inhibition rate of up to 98%. This work repurposes the inherent cytotoxicity of the carrier material as an active therapeutic component, offering a novel paradigm for the design of high-performance bioorthogonal catalytic systems.

Mitochondrial potassium channels, located in the inner mitochondrial membrane, play a crucial role in the cells life/death phenomenon. Activation of mitochondrial potassium channels by potassium channel openers may protect cells against ischemia-reperfusion injury. It is known that mitochondrial large conductance calcium-activated potassium channels interact with various mitochondrial proteins, including enzymes of the respiratory chain. Numerous studies indicate that the mitochondria, especially cytochrome c oxidase, play a crucial role as a chromatophore in the cellular response to red and near-infrared light. In this study, we employ the patch-clamp technique and single-channel recordings to investigate the regulation of glioblastoma mitochondrial large conductance calcium-activated potassium channel activity by infrared light. Specifically, we examined the effects of wavelengths 620 nm, 680 nm, 760 nm, and 820 nm in a redox-controlled environment. Our findings suggest that illuminating the inner mitochondrial membrane with these wavelengths may activate mitochondrial large conductance calcium-activated potassium channels. These results offer new insights into the regulation of mitochondrial potassium channels by cytochrome c oxidase, which may lead to the development of non-pharmacological interventions with potential cytoprotective benefits.