The construction of nano-drug carriers based on deoxyribonucleic acid (DNA) has demonstrated significant therapeutic potential. Similarly, supramolecular therapeutic systems utilizing host-guest interactions have emerged as promising in nanomedicine. Building upon these approaches, we designed a size-controllable, multi-responsive supramolecular DNA nanogel (SDN) based on host-guest recognition for dual-drug co-delivery in cancer combination therapy. The nanogel incorporates doxorubicin (DOX, a chemotherapeutic agent) and methylene blue (MB, a photosensitizer). The assembly of SDN is driven by cucurbit[8]uril (CB[8]), which selectively binds two MB molecules-one from each of two Y-shaped DNA building blocks-forming a 1 : 2 host-guest complex that crosslinks the structures into a nanogel network. Meanwhile, the double-stranded DNA scaffold efficiently encapsulates DOX via intercalation, enabling SDN@DOX to co-deliver both drugs in a precisely controlled ratio. Notably, MB's photodynamic activity is initially suppressed upon CB[8] binding. However, upon cellular uptake, SDN@DOX responds to overexpressed spermine or specific peptide sequences in the tumor microenvironment, triggering MB release and restoring its photodynamic function. Concurrently, DNase I-mediated DNA degradation liberates DOX, enabling synergistic chemo-photodynamic therapy (PDT). In vitro studies confirmed that SDN@DOX enhances reactive oxygen species (ROS) generation in cancer cells and achieves superior therapeutic efficacy through combined PDT and chemotherapy. This stimuli-responsive, dual-drug delivery system offers a potentially robust and controllable platform for precision cancer treatment.

Synergistic effects of plasma-activated water and potassium chloride on the structure, function, and digestibility of low-sodium chicken myofibrillar protein.

Peptides are important tools in biological, medical, and pharmaceutical research. Solid-phase peptide synthesis (SPPS) is the primary method for their preparation in high yields and purity. However, SPPS often encounters difficulties due to the occurrence of side reactions, which can generate by-products that are difficult to remove. Side reactions can occur under both basic and acidic conditions, resulting in reduced yields, costly purification processes, and sometimes potential inaccessibility of the target peptides. By-products include the formation of aspartimide, glutarimide, and pyroglutamic acid. The problem of unwanted intramolecular cyclizations is well known and unavoidable, in some cases minimized, as it depends on the intrinsic thermodynamics of the final structures. In this context, we report a systematic analysis of the side reactions that occur during SPPS in peptides containing the Glu-Asp-Tyr motif. This study is the first to investigate the formation of three different by-products within the same peptide sequence and to provide valuable insights into the experimental conditions that favor one reaction over the others. The study aims to identify the optimal experimental conditions to mitigate these by-products' formation. Our results highlight that the steric and conformational effects of the growing protected peptide chain play a role that is often overlooked or misinterpreted.

Cereals: a source of allergenic proteins and their characterization by proteomics.

Synthesis of a hemicyanine-based near-infrared fluorescent probe for the diagnosis of pulmonary fibrosis and acute lung injury.

Herein we report the development of a novel linker platform for tumour-responsive antibody-drug conjugates. A series of functionalised homo- and heterobivalent BisFab conjugates have been synthesised, comprising an MMP-cleavable peptide sequence which facilitates selective monomerisation in the tumour microenvironment of the BisFab into two smaller cytotoxic species. The platform was then expanded to produce bispecific BisFab conjugates.

Human inflammatory caspases (caspase-1, -4, and -5) are key players in the innate immune response. These enzymes have been shown to cleave proinflammatory substrates, implicating them in many inflammatory disease states. Their activity is frequently assessed using in vitro fluorogenic assays, with all three human inflammatory caspases preferring the same WEHD tetrapeptide. The study examines the specificity of these enzymes C-terminal to the cleaved aspartate residue with Förster resonance energy transfer peptide-based assays using 7-methoxycoumaryl alanine [A(MCA)] as the donor and lysine-conjugated dabsyl [K(Dab)] as the quencher. The P4-P1 peptide sequences A(MCA)EHD, A(MCA)VAD, and A(MCA)QPD are varied on the C-terminal (prime) side of the peptide. Historically, caspase-4 and caspase-5 have been grouped together in their reactivity. Herein, caspase-5 only appreciably cleaves the A(MCA)EHDGK(Dab) peptide, whereas caspase-4 displays broader reactivity. All base sequences react more considerably with caspase-4 when a glycine is included C-terminal to Asp. The specificity of caspase-1 at this position varies based on the P3-P1 sequence of the peptide. These results highlight the interconnectedness of the prime and nonprime side amino acid sequences and the different behavior of each enzyme, which can be useful in understanding these potential drug targets.

Uncovering the pivotal roles of two pairs of maize CLE genes in regulating leaf senescence and conferring drought tolerance through genome-wide analysis.

Systematic gait analysis in alpha-synuclein transgenic line 62 mice using the CatWalk.

Physical condensation of oligopeptides provides a reductionist platform for probing the underwater adhesion of marine adhesive proteins. The link between peptide sequence and adhesion remains unclear at the molecular level. Using cationic peptide isomers and anionic H4SiW12O40 (SiW) clusters, we explored how sequence isomerism governs the adhesion of the peptide/SiW condensates via regulating the layout of two lysine residues within the peptide isomers. On stainless steel (SS), one-end-on isomers with lysine residues at one terminal exhibited adhesion strengths of 43.1-54.2 kPa; end-side-on isomers with lysine residues at both terminals and midsite yielded adhesion of 64.1-67.8 kPa; while double-end-on isomers with lysine residues at both termini achieved adhesion strength of 74.4-87.1 kPa. Furthermore, introducing one glutamic acid residue enables protonation-/deprotonation-driven charge redistribution and adhesion regulation. This work establishes a design principle, whereby the adhesion of peptide condensates can be controlled by optimizing binding site arrangements in peptide sequences.

ABSTRACT Candida species are major contributors to nosocomial infections, with biofilm formation being a critical virulence factor that enables persistence in clinical settings and resistance to antifungal therapies. Central to biofilm development is the adhesion of fungal cells, a process mediated by surface proteins such as Als5p in Candida albicans. The amyloid-forming peptide sequence within Als5p (322SNGIVIVATTRTV334) has been implicated in mediating adhesion and biofilm formation; however, its role in shaping the biofilm architecture has not been fully elucidated. In this study, we demonstrated that the addition of Als5pFP promoted biomass accumulation in C. albicans biofilms under laboratory conditions, including complex media and at temperatures compatible with clinical biofilm assays. Using advanced image analysis of microscopy images, we show that the Als5p peptide induces a distinct morphological effect on biofilms: a shape-edging of microcolony structures, characterized by the concentration of fungal cells into denser aggregates and the reduction of cells in intermediate spaces. These observations suggest a potential role of amyloid-like fibrils formed by the Als5p peptide in influencing the spatial organization of C. albicans biofilms. This discovery presents a novel aspect on how these fibrils affect the biofilm architecture extending beyond previous studies, which primarily focused on biomass accumulation. Our findings contribute to the understanding of the architectural development of C. albicans biofilms and provide a foundation for future research aimed at targeting the amyloid structures within fungal biofilms. Furthermore, the results may support the design of biofilm-targeting antifungal agents and development of biosensors for monitoring amyloid formation during infection.

Casanovo is a state-of-the-art deep learning model for de novo peptide sequencing from mass spectrometry and proteomics data. Here, we report on a series of enhancements to Casanovo, aimed at improving the interpretability of the scores assigned to predicted peptides, generalizing the software for use in database searches, speeding up training and prediction runtimes, and providing workflows and visualization tools to facilitate adoption of Casanovo and interpretation of its results. Our goal is to make Casanovo accurate and easy to use for applications such as metaproteomics, antibody sequencing, immunopeptidomics, and the discovery of novel peptide sequences in standard proteomics analyses. Casanovo is available as open source at https://github.com/Noble-Lab/casanovo.

Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80% across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved >60% sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows.

Combination cancer therapies offer to minimize toxicity and alleviate patient burden, but a key challenge is independent control over the release of multiple therapeutics, especially in systems with chemotherapeutic drugs of similar size and structure. Due to MMP upregulation in cancer microenvironments, matrix metalloproteinase (MMP)-degradable linkers are often exploited for targeted release; however, their short substrates exhibit overlap with multiple proteases, which confounds kinetic control. Here, a library of MMP-responsive "peptomer" drug linkers was developed to control release on fast and slow timescales. Peptomers are hybrid molecules of peptides and non-natural peptoids (N-substituted glycines), which hinder proteolytic susceptibility. Systematic variation of peptoid substitutions within a pan-MMP-cleavable peptide sequence yielded distinctdegradation kinetics to multiple MMPs. Two chemotherapeutics, doxorubicin and geldanamycin, were conjugated to peptomer linkers and incorporated into polyethylene glycol (PEG)-based hydrogels for sequential delivery. Fluorogenic and mass spectrometry-based assays demonstrated decoupled release of each drug in response to MMP-2 and MMP-9. In vitro studies using MDA-MB-231 and A549 cells showed that cell death rates correlated with the order of drug release. These findings highlight peptomers as modular, biocompatible linkers capable of kinetic control over multiple therapeutic agents, providing a versatile platform for improving the precision of combination drug delivery systems.

Therapeutic peptides have become an important direction in drug discovery because of their high targeting and low side effects, and are used to treat many diseases. Peptides are short-chain molecules formed by connecting amino acids through peptide bonds and play key roles in the body. The stability and production costs of peptides are challenges that need to be overcome for their pharmaceutical applications. Researchers have improved the accuracy of therapeutic peptide sequence function predictions by constructing and integrating peptide features from different sources. However, accurately predicting multi-functional therapeutic peptides is challenging due to the limitations of handcrafted feature properties, which are unable to capture the full complexity of biological systems. In this study, we introduce a novel method TPpred-CMvL for the prediction of multi-functional therapeutic peptide (MTP) based on a contrastive multi-view learning model. This framework directly integrates semantic information pretraining TAPE from protein large language model and evolutionary information. Subsequently, TPpred-CMvL leverages contrastive multi-view learning to comprehensively capture representations of peptide sequences, thereby enhancing the prediction accuracy of MTPs. We utilized adaptive synthetic sampling and focal loss to address the classification imbalance arising from the long-tailed distribution. The experimental results demonstrate that the proposed method outperforms existing related approaches and exhibits the most advanced performance. We developed a contrast multi-view learning model TPpred-CMvL utilizing sequential semantic information TAPE and evolutionary information PSSM. Compared with existing related methods, this method achieved state-of-the-art performance. Finally, a web server has been established and is accessible at http://bliulab.net/TPpred-CMvL .

Nonviral protein vehicles for RNA delivery hold significant promises for various biotechnological applications due to their scalability, ease of engineering, and high biocompatibility. Directed evolution is a powerful approach to enhance protein activity, yet few nonviral protein vehicles have been evolved due to the challenges in linking genotype and phenotype. Here, we report engineering and directed evolution of a neo-nucleocapsid enabling receptor-specific RNA delivery. We applied the DogTag/DogCatcher protein ligation system to an artificial nucleocapsid and conducted directed evolution to obtain nucleocapsid displaying proteins. To create designer neo-nucleocapsids, we lasso-grafted a pharmacophore sequence of a pseudo-cyclic peptide binding to the hepatocyte growth factor receptor MET into the loop of DogCatcher. After the cellular-based directed evolution, endocytic uptake of neo-nucleocapsids was improved whereas cytosolic release and subsequent protein expression from the delivered RNA were not detected. This experimental approach provides a versatile system for constructing protein cages delivering various cargos to specific cells.

Osteoarthritis (OA) affects a large population worldwide, causing chronic pain, functional decline, and increased personal and societal medical costs. A major challenge in developing disease-modifying OA drugs (DMOADs) is the inefficient delivery to diseased chondrocytes. Here we synthesize a viral glycoprotein-mimicking peptide (CMP) containing a type II collagen-adhesive motif and a matrix metalloproteinase-13-activated cell-penetrating peptide sequence. The CMP peptide was conjugated to small-sized micelles loaded with the model drug IOX4, enabling the micelles to adhere to cartilage and chondrocyte surfaces through collagen binding and achieve proteinase-induced selective uptake by diseased chondrocytes. In an OA mouse model, our micelles demonstrated prolonged joint retention and exhibited a higher uptake by diseased chondrocytes compared with unmodified micelles and normal chondrocytes, respectively. In both OA mice and a clinically relevant OA sheep model, our system maintained metabolic homeostasis in cartilage, attenuating OA pathological changes and improving symptoms without causing additional toxicity. These findings suggest that our nanoformulation is a promising DMOAD candidate and provides an efficient delivery strategy for other potential DMOADs targeting intracellular sites of diseased chondrocytes.

Uptake of proteins and ampholytic solutes into polyelectrolyte brushes underlies some biological processes and also applications in sensing or biomedicine. Especially uptake on the "wrong" side of the isoelectric point (pI) remains puzzling, with charge regulation and solute patchiness proposed as possible mechanisms. Using a hierarchy of approximations, coarse-grained molecular simulations, self-consistent mean-field, and a simple phenomenological model, we investigated the uptake of model ampholytic solutes into polyanionic brushes across varying pH, salt concentrations, pKa values, and peptide sequences. In a narrow pH range on the wrong side of pI, charge regulation enables uptake of the ampholytes by inducing charge inversion so that they become positively charged in the brush despite being negatively charged in the bulk. This charge inversion can be calculated from the pH difference between the brush and the bulk, which is related to the Donnan potential. It is strongest for ampholytes with small differences between acidic and basic pKa values and decreases with increasing salt. Our phenomenological model reproduces the universal effect of charge regulation promoting ampholyte uptake into brushes but fails to be quantitative. The mean field model is close to explicit simulations for alternating sequences, but fails to describe the effect of charge patchiness, which is only captured by explicit simulations. Thus, our phenomenological framework offers a practical rule of thumb for estimating uptake from experimentally accessible parameters without sophisticated calculations. Deviations from this rule of thumb for complex ampholytes, such as proteins or peptides with patterned charge sequences, are captured only by explicit simulations.

Efficient and safe Messenger RNA (mRNA) delivery remains a central challenge in nucleic acid therapeutics. While lipid nanoparticles dominate clinically, their biosafety concerns and poor endosomal escape hinder broader application. Phase-separating peptides (PSPs) offer a biocompatible alternative, but often lack structural stability and precise control over intracellular release. In this study, HBpep-SS4 was developed as a chemically defined coacervate system with intrinsic redox-responsiveness encoded by tandem cysteines in its peptide sequence. This minimalist, single-component design eliminates the need for postsynthetic modifications or protein conjugations, simplifying the synthesis process and reducing potential toxicity. HBpep-SS4 forms stable coacervates capable of encapsulating >95% mRNA and retains responsiveness to glutathione, enabling cytosolic RNA release. It delivers a broad spectrum of RNA cargos─including linear, circular, and self-amplifying RNAs (∼9700 nt)─and achieves high transfection efficiency across multiple cell lines. Functionally, it supports genome editing via SpCas9 mRNA/sgRNA delivery, reaching 86.0% EGFP disruption and 72.5% editing at the HBB locus. Mechanistic studies reveal that HBpep-SS4 enters cells via phagocytosis and bypasses endosomal trafficking, disassembling in reductive environments without toxic byproducts. Building on these findings, HBpep-SS4 was designed with primary sequence-encoded environmental responsiveness, enabling integration of structure, function, and redox sensitivity within a single peptide-based system. Embedding functional reactivity into the peptide backbone supports glutathione-triggered disassembly, which may contribute to improved safety, manufacturing scalability, and potential applicability in RNA-based delivery platforms.

Bacteria-induced on-membrane assembly of oligopeptides showed the potential for balancing their persistent conflict between antibacterial activity and biocompatibility. However, molecular determinants linking the peptide sequence, assembly behavior, antibacterial efficacy, and biocompatibility remain insufficiently understood. Here, we investigated a series of heptapeptides, (RX)3R (X = A, I, F), and demonstrated the significant influence of hydrophobic residues on bacteria-induced assembly, antibacterial performance, cytotoxicity, and hemolytic activity. Negatively charged liposomes or bacterial membranes could induce the assembly of (RI)3R and (RF)3R into nanofibers, which in turn exhibited enhanced antibacterial efficacy against Gram-negative Escherichia coli with minimum inhibitory concentrations (MICs) of 60 μM but poor antibacterial activity on Gram-positive Staphylococcus aureus (MICs: 400-500 μM). Importantly, both peptides demonstrated excellent biocompatibility, with cytotoxicity thresholds of 600 μM (RI)3R and 2000 μM (RF)3R and hemolysis thresholds up to 2000 and 4000 μM, respectively. These findings provide valuable insights for the rational design of antibacterial oligopeptides.