Selective Clearance of Circulating Histones Based on Dodecapeptide-Grafted Copolymer Material for Sepsis Blood Purification.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Research indicates that circulating histones, as pathogenic factors, may represent a therapeutic target for sepsis. However, effectively clearing circulating histones poses a challenge due to their structural similarity to normal blood proteins, their low abundance in the bloodstream, and serious interference from other blood biomacromolecules. Here we design a dodecapeptide-based functional polymer that can selectively adsorb circulating histones from the blood. The peptide, named P1 (HNHHQLALVESY), was discovered through phage display screening and demonstrated a strong affinity for circulating histones while exhibiting negligible affinities for common proteins in the blood, such as human serum albumin (HSA), immunoglobulin G (IgG), and transferrin (TRF). Furthermore, the P1 peptide was incorporated into a functional polymer design, poly(PEGMA-co-P1), which was immobilized onto a silica gel surface through reversible addition-fragmentation chain transfer polymerization. The resulting material was characterized using solid nuclear magnetic resonance, thermogravimetric analysis, and X-ray photoelectron spectroscopy. This material demonstrated the ability to selectively and efficiently capture circulating histones from both model solutions and whole blood samples while also exhibiting satisfactory blood compatibility, good antifouling properties, and resistance to interference. Satisfactory binding affinity and efficient capture capacity toward histone were also observed for the other screened peptide P2 (QMSMDLFGSNFV)-grafted polymer, validating phage display as a reliable ligand screening strategy. These findings present an approach for the specific clearance of circulating histones and hold promise for future clinical applications in blood purification toward sepsis.

Precise Capture and Dynamic Release of Circulating Liver Cancer Cells with Dual-Histidine-Based Cell Imprinted Hydrogels.

Circulating tumor cells (CTCs) detection presents significant advantages in diagnosing liver cancer due to its noninvasiveness, real-time monitoring, and dynamic tracking. However, the clinical application of CTCs-based diagnosis is largely limited by the challenges of capturing low-abundance CTCs within a complex blood environment while ensuring them alive. Here, an ultrastrong ligand, l-histidine-l-histidine (HH), specifically targeting sialylated glycans on the surface of CTCs, is designed. Furthermore, HH is integrated into a cell-imprinted polymer, constructing a hydrogel with precise CTCs imprinting, high elasticity, satisfactory blood compatibility, and robust anti-interference capacities. These features endow the hydrogel with excellent capture efficiency (>95%) for CTCs in peripheral blood, as well as the ability to release CTCs controllably and alive. Clinical tests substantiate the accurate differentiation between liver cancer, cirrhosis, and healthy groups using this method. The remarkable diagnostic accuracy (94%), lossless release of CTCs, material reversibility, and cost-effectiveness ($6.68 per sample) make the HH-based hydrogel a potentially revolutionary technology for liver cancer diagnosis and single-cell analysis.

A highly sensitive nanochannel device for the detection of SUMO1 peptides.

SUMOylation is an important and highly dynamic post-translational modification (PTM) process of protein, and its disequilibrium may cause various diseases, such as cancers and neurodegenerative disorders. SUMO proteins must be accurately detected to understand disease states and develop effective drugs. Reliable antibodies against SUMO2/3 are commercially available; however, efficient detectors are yet to be developed for SUMO1, which has only 50% homology with SUMO2 and SUMO3. Here, using phage display technology, we identified two cyclic peptide (CP) sequences that could specifically bind to the terminal dodecapeptide sequence of SUMO1. Then we combined the CPs and polyethylene terephthalate conical nanochannel films to fabricate a nanochannel device highly sensitive towards the SUMO1 terminal peptide and protein; sensitivity was achieved by ensuring marked variations in both transmembrane ionic current and Faraday current. The satisfactory SUMO1-sensing ability of this device makes it a promising tool for the time-point monitoring of the SENP1 enzyme-catalyzed de-SUMOylation reaction and cellular imaging. This study not only solves the challenge of SUMO1 precise recognition that could promote SUMO1 proteomics analysis, but also demonstrates the good potential of the nanochannel device in monitoring of enzymes and discovery of effective drugs.

Identifying Umami Peptides Specific to the T1R1/T1R3 Receptor via Phage Display.

Umami peptides are small molecular weight oligopeptides that play a role in umami taste attributes. However, the identification of umami peptides is easily limited by environmental conditions, and the abundant source and high chromatographic separation efficiency remain difficult. Herein, we report a robust strategy based on a phage random linear heptapeptide library that targets the T1R1-Venus flytrap domain (T1R1-VFT). Two candidate peptides (MTLERPW and MNLHLSF) were readily identified with high affinity for T1R1-VFT binding (KD of MW-7 and MF-7 were 790 and 630 nM, respectively). The two peptides exhibited umami taste and significantly enhanced the umami intensity when added to the monosodium glutamate solution. Overall, this strategy shows that umami peptides could be developed via phage display technology for the first time. The phage display platform has a promising application to discover other taste peptides with affinity for taste receptors of interest and has more room for improvement in the future.

Order-order assembly transition-driven polyamines detection based on iron-sulfur complexes.

Innovative modes of response can greatly push forward chemical sensing processes and subsequently improve sensing performance. Classical chemical sensing modes seldom involve the transition of a delicate molecular assembly during the response. Here, we display a sensing mode for polyamine detection based on an order-order transition of iron-sulfur complexes upon their assembly. Strong validation proves that the unique order-order transition of the assemblies is the driving force of the response, in which the polyamine captures the metal ion of the iron-sulfur complex, leading it to decompose into a metal-polyamine product, accompanied by an order-order transition of the assemblies. This mechanism makes the detection process more intuitive and selective, and remarkably improves the detection efficiency, achieving excellent polyamines specificity, second-level response, convenient visual detection, and good recyclability of the sensing system. Furthermore, this paper also provides opportunities for the further application of the iron-sulfur platform in environment-related fields.

H2O2 and Phosphorylated Peptide Dual-Responsive Nanochannel Device.

Oxidation and protein phosphorylation are critical mechanisms involved in regulating various cellular activities. Increasing research has suggested that oxidative stress could affect the activities of specific kinases or phosphatases, leading to alterations in the phosphorylation status of certain proteins. Ultimately, these alterations can affect cellular signaling pathways and gene expression patterns. However, the relationship between oxidation and protein phosphorylation remains complex and not yet fully understood. Therefore, the development of effective sensors capable of detecting both oxidation and protein phosphorylation simultaneously presents an ongoing challenge. To address this need, we introduce a proof-of-concept nanochannel device that is dual-responsive to both H2O2 and phosphorylated peptide (PP). Specifically, we design a peptide GGGCEG(GPGGA)4CEGRRRR, which contains an H2O2-sensitive unit CEG, an elastic peptide fragment (GPGGA)4, and a phosphorylation site recognition fragment RRRR. When the peptides are immobilized on the inner walls of conical nanochannels in a polyethylene terephthalate membrane, this peptide-modified nanochannel device exhibits a sensitive response to both H2O2 and PPs. The peptide chains undergo a random coil-to-α-helix transition in response to H2O2, which leads to a close-to-open transition of the nanochannel, accompanied with a remarkable increase in the transmembrane ionic current. In contrast, binding of the peptides with PPs shields the positive charge of the RRRR fragments, causing a decrease of the transmembrane ionic current. These unique features enable the sensitive detection of reactive oxygen species released by 3T3-L1 cells stimulated by platelet-derived growth factor (PDGF) as well as PDGF-induced change in the PP level. Real-time kinase activity monitoring further confirms the device's potential utility for kinase inhibitor screening.

Specific Clearance of Lipopolysaccharide from Blood Based on Peptide Bottlebrush Polymer for Sepsis Therapy.

Lipopolysaccharide (LPS) is the primary bacterial toxin that is vital to the pathogenesis and progression of sepsis associated with extremely high morbidity and mortality worldwide. However, specific clearance of LPS from circulating blood is highly challenging because of the structural complexity and its variation between/within bacterial species. Herein, a robust strategy based on phage display screening and hemocompatible peptide bottlebrush polymer design for specific clearance of targeted LPS from circulating blood is proposed. Using LPS extracted from Escherichia coli as an example, a novel peptide (HWKAVNWLKPWT) with high affinity (KD < 1.0 nм), specificity, and neutralization activity (95.9 ± 0.1%) against the targeted LPS is discovered via iterative affinity selection coupled with endotoxin detoxification screening. A hemocompatible bottlebrush polymer bearing the short peptide [poly(PEGMEA-co-PEP-1)] exhibits high LPS selectivity to reduce circulating LPS level from 2.63 ± 0.01 to 0.78 ± 0.05 EU mL-1 in sepsis rabbits via extracorporeal hemoperfusion (LPS clearance ratio > 70%), reversing the LPS-induced leukocytopenia and multiple organ damages significantly. This work provides a universal paradigm for developing a highly selective hemoadsorbent library fully covering the LPS family, which is promising to create a new era of precision medicine in sepsis therapy.

Sensitive and specific detection of saccharide species based on fluorescence: update from 2016.

Increasing evidence supports the critical role of saccharides in various pathophysiological steps of tumor progression, where they regulate tumor proliferation, invasion, hematogenic metastasis, and angiogenesis. The identification and recognition of these saccharides provide a solid foundation for the development of targeted drug preparations, which are however not fully understood due to their complex and similar structures. In order to achieve fluorescence sensing of saccharides, extensive research has been conducted to design molecular probes and nanoparticles made of different materials. This paper aims to provide in-depth discussion of three main topics that cover the current status of the carbohydrate sensing based on the fluorescence sensing mechanism, including a phenylboronic acid-based sensing platform, non-boronic acid entities, as well as an enzyme-based sensing platform. It also highlights efforts made to understand the recognition mechanisms and improve the sensing properties of these systems. Finally, we present the challenge of achieving high selectivity and sensitivity recognition of saccharides, and suggest possible future avenues for exploration.

Phage display derived peptides for Alzheimer's disease therapy and diagnosis.

Alzheimer's disease (AD) is an incurable and fatal progressive neurodegenerative disorder associated with memory and cognition impairment. AD is one of the top medical care concerns across the world with a projected economic burden of $2 trillion by 2030. To date, however, there remains no effective disease-modifying therapy available. It is more important than ever to reveal novel therapeutic approaches. Peptide-based biotherapeutics has been a great potential strategy attributed to their distinct and superior biochemical characteristics, such as reproducible chemical synthesis and modification, rapid cell and tissue permeability, and fast blood clearance. Phage display, one of today's most powerful platforms, allows selection and identification of suitable peptide drug candidates with high affinities and specificity toward target, demonstrating the potential to overcome challenges and limitations in AD diagnosis/treatment. We aim to provide the first comprehensive review to summarize the status in this research direction. The biological overview of phage display is described, including basic biology of the phage vectors and construction principle of phage library, biopanning procedure, mirror image phage display, and various binding affinity evaluation approaches. Further, the applications of phage display in AD therapy, targeted drug delivery, and early detection are presented. Finally, we discuss the current challenges and offer a future outlook for further advancing the potential application of phage display on AD and other neurodegenerative diseases.

Enrichment of IgG and HRP glycoprotein by dipeptide-based polymeric material.

Separation, purification, and identification of glycoproteins are essential for understanding their vital roles in biological and pathological processes. However, glycoproteins are difficult to be captured due to their low abundance, strong interference from non-glycosylated proteins. Here, we report a promising dipeptide-based saccharide recognition platform to selectively enrich two typical glycoproteins, named immunoglobin G (IgG) and horseradish peroxidase (HRP). Different from the conventional glycoprotein enrichment method based on boronic acid affinity or hydrophilic interaction with glycans, the present method was established based on affinity between Pro-Glu (PE) dipeptide and mannose, which is a key unit in the pentasaccharide core of the IgG and HRP glycans. The prepared PE homopolymer surface was proved to selectively bind IgG and HRP superior to that of bovine serum albumin (BSA). Benefiting from this feature, selective enrichment of IgG and HRP was achieved from a protein mixture containing 200-fold BSA interference by using polyPE@SiO2 under a dispersive solid-phase extraction (dSPE) mode. High adsorption capacity, controllable and selective adsorption behaviors, as well as satisfactory recovery demonstrated the high potential of the dipeptide-based polymeric material in IgG and HRP enrichment. This study might provide a new insight to solve the challenging problem of glycoprotein separation.

Discerning Tyrosine Phosphorylation from Multiple Phosphorylations Using a Nanofluidic Logic Platform.

Discerning tyrosine phosphorylation (pTyr) catalyzed by Tyr kinase is central to the revelation of oncogenic mechanisms and the development of targeted anticancer drugs. Despite some techniques, this goal remains challenging, especially when faced with the interference of multiple phosphorylation events, including serine (pSer) and threonine phosphorylation (pThr). We describe here a functional polymer-modified artificial ion nanochannel, which enables the sensitive and selective recognition of phosphotyrosine (pY) peptide by the distinct ionic current change. Such a recognition effect allows for the nanochannel to work in a complex protein digest condition. Further, the implementation of nanofluidic logic functions with the addition of Ca2+ dramatically improves the selectivity of the nanochannel to pY peptide and thus can discern pTyr by the Tyr kinase from pSer by the Ser/Thr kinase through simultaneously monitoring multisite phosphorylation at the same or different peptide substrates in one-pot. This logic sensing platform displays the potential in differentiating Tyr kinase and Ser/Thr kinase and assessing multi-kinase activities in multi-targeted drug design.

Self-assembly gel-based dynamic response system for specific recognition of N-acetylneuraminic acid.

Sialic acids located at the terminal end of glycans are densely attached to cell surfaces and play crucial and distinctive roles in a variety of physiological and pathological processes, such as neural development, cell-cell interactions, autoimmunity and cancers. However, due to the subtle structural differences of sialic acid species and the complicated composition of glycans, the precise recognition of sialylated glycans is difficult. Here, a fluorescent dynamic response system based on a pyrene-conjugated histidine (PyHis) supramolecular gel is proposed. Driven by π-π stacking and intermolecular hydrogen bonds, PyHis exhibits a strong self-assembly ability and forms stable gels. It is found that introduction of N-acetylneuraminic acid (a typical sialic acid) can prevent this self-assembly process, whereas other monosaccharides or sialic acid analogs have no significant effect on it. Interestingly, a sialylated glycan also has a remarkable inhibitory effect on the gel formation, which highlights the high selectivity of the gel dynamic response system. Analysis of the mechanism reveals that the sialic acid or sialylated glycan can interact closely with two PyHis molecules stacked together in the assemblies via hydrogen bonding interactions, thereby preventing the ordered accumulation of the gelators. It is worth noting that the high-efficiency sialic acid recognition effect is not observed at the single molecule level but at the supramolecular level, indicating the unique superiority of the supramolecular self-assembly system in biomolecular recognition and response. This work shows the promising prospects of using supramolecular gels in assembly engineering, regenerative medicine, tumour cell sorting and cancer diagnosis.

A methylation-inspired mesoporous coordination polymer for identification and removal of organic pollutants in aqueous solutions.

Qualitative analysis of contamination events and rapid removal of hazardous substances from water are in urgent need for a sustainable environment and human health. Porous coordination polymers (PCPs) bridged by organic ligands through metal nodes in an extendable and periodic manner have emerged as competitive candidates for the detection and removal of hazardous substances. However, the majority of them suffer from high production costs, poor structural stability and environmental problems, which has become a bottleneck for commercial translation. Here, we report a class of phenylalanine-based metal-biomolecule complexes and discuss the impact of subtle sequence variations in modular ligands on their assembly behaviors and structural properties. The phenomenon in which the bioligands dominate the structure formation and surface wettability has been revealed. A Cu(ii)-aspartame coordination polymer, Cu(mDF), with satisfactory chemical stability was selected for removal of organic pollutants in aqueous solution. The mesoporous structure, surface charge and high specific surface area (233.71 m2 g-1, Dmean = 5.65 nm) promote its rapid equilibrium of anionic adsorption within 10 min. In addition, Cu(mDF) possessing an adsorption-induced color-shifting feature provides an ideal platform for organic pollutant detection. Furthermore, Cu(mDF) with biocompatibility and low cost fabrication exhibits antimicrobial properties against C. albicans, E. coli and S. aureus, and may be a potential purifier in wastewater treatment.

Highly Efficient Separation of Methylated Peptides Utilizing Selective Complexation between Lysine and 18-Crown-6.

Protein methylation is one of the most common and important post-translational modifications, and it plays vital roles in epigenetic regulation, signal transduction, and chromatin metabolism. However, due to the diversity of methylation forms, slight difference between methylated sites and nonmodified ones, and ultralow abundance, it is extraordinarily challenging to capture and separate methylated peptides from biological samples. Here, we introduce a simple and highly efficient method to separate methylated and nonmethylated peptides using 18-crown-6 as a mobile phase additive in high-performance liquid chromatography. Selective complexation between lysine and 18-crown-6 remarkably increases the retention of the peptides on a C18 stationary phase, leading to an excellent baseline separation between the lysine methylated and nonmethylated peptides. A possible binding mechanism is verified by nuclear magnetic resonance titration, biolayer interferometry technology, and quantum chemistry calculation. Through establishment of a simple enrichment methodology, a good selectivity is achieved and four methylated peptides with greatly improved signal-to-noise (S/N) ratios are successfully separated from a complex peptide sample containing 10-fold bovine serum albumin tryptic digests. By selecting rLys N as an enzyme to digest histone, methylation information in the histone could be well identified based on our enrichment method. This study will open an avenue and provide a novel insight for selective enrichment of lysine methylated peptides in post-translational modification proteomics.

Functional Nanochannels for Sensing Tyrosine Phosphorylation.

Tyrosine phosphorylation (pTyr), much of which occurred on localized multiple sites, initiates cellular signaling, governs cellular functions, and its dysregulation is implicated in many diseases, especially cancers. pTyr-specific sensing is of great significance for understanding disease states and developing targeted anticancer drugs, however, it is very challenging due to the slight difference from serine (pSer) or threonine phosphorylation (pThr). Here we present polyethylenimine-g-phenylguanidine (PEI-PG)-modified nanochannels that can address the challenge. Rich guanidinium groups enabled PEI-PG to form multiple interactions with phosphorylated residues, especially pTyr residue, which triggered the conformational change of PEI-PG. By taking advantage of the "OFF-ON" change of the ion flux arising from the conformational shrinkage of the grafted PEI-PG, the nanochannels could distinguish phosphorylated peptide (PP) from nonmodified peptide, recognize PPs with pSer, pThr, or pTyr residue and PPs with different numbers of identical residues, and importantly could sense pTyr peptides in a biosample. Benefiting from the strong interaction between the guanidinium group and the pTyr side-chain, the specific sensing of pTyr peptide was achieved by performing a simple logic operation based on PEI-PG-modified nanochannels when Ca2+ was introduced as an interferent. The excellent pTyr sensing capacity makes the nanochannels available for real-time monitoring of the pTyr process by c-Abl kinase on a peptide substrate, even under complicated conditions, and the proof-of-concept study of monitoring the kinase activity demonstrates its potential in kinase inhibitor screening.

What Is Hidden Behind Schiff Base Hydrolysis? Dynamic Covalent Chemistry for the Precise Capture of Sialylated Glycans.

The aberrant expression of sialylated glycans (SGs) is closely associated with the occurrence, progression, and metastasis of various cancers, and sialylated glycoproteins have been widely used as clinical biomarkers for cancers. However, the identification and comprehensive analysis of SGs are exceptionally complex, which urgently need an innovative and effective method of capturing SGs from biosamples prior to MS analysis. Here, we report that a novel dynamic covalent chemistry strategy based on Schiff base hydrolysis can be applied to the precise capture of SGs. The prepared glucopyranoside-Schiff base-modified silica gel displays extraordinary enrichment selectivity (even at a ratio of 1:5000 with interference), high adsorption capacity (120 mg·g-1), and satisfying enrichment recovery (95.5%) toward sialylated glycopeptides, contributing to a highly specific, efficient, mild, and reversible SG capturing approach that can remarkably promote the development of glycoproteomics and sialic acid sensing devices and can be considerably promising in cancer biomarker discovery. Meanwhile, the facile hydrolysis characteristic of our Schiff base material completely subverts conventional knowledge of enrichment materials, the chemical stability of which is usually regarded as a prerequisite. Importantly, we find an exciting story hidden behind the Schiff base hydrolysis reaction, which demonstrates the unique advantage of dynamic covalent chemistry in glycoproteomics and biomolecule sensing.

cAMP sensitive nanochannels driven by conformational transition of a tripeptide-based smart polymer.

Inspired by biological nanochannels, a novel cyclic 3',5'-adenosine monophosphate (cAMP)-regulated artificial nanochannel based on a tripeptide Arg-Thr-Ala (RTA) design is developed. Highly specific binding between the tripeptide and cAMP triggers an obvious conformational transition of a smart polymer chain from a contracted state to a swollen one, which leads to a dynamic modulation of the gating behaviours of the nanochannels.

Molecular chirality mediated amyloid formation on phospholipid surfaces.

One of the neuropathological features of Alzheimer's disease (AD) is the misfolding of amyloid-ß to form amyloid aggregates, a process highly associated with biological membranes. However, how molecular chirality affects the amyloid formation on phospholipid surfaces has seldom been reported. Here, l- and d-aspartic acid-modified 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (l-/d-Asp-DPPE) is synthesized to construct chiral phospholipid bilayers. We discover that the l-Asp-DPPE liposomes slightly inhibit the Aß(1-40) nucleation process but cannot affect the oligomer elongation process. By contrast, the d-Asp-DPPE liposomes strongly inhibit both nucleation and elongation of the peptide. Notably, l- and d-Asp-DPPE liposomes not only have good biocompatibility but can also rescue Aß(1-40)-aggregation induced cytotoxicity with significant chiral discrimination, in which the cell viability is higher in the presence of d-Asp-DPPE liposomes. Mechanism analysis and molecular dynamics simulation clearly demonstrate that differential electrostatic interactions of Lys16 in Aß(1-40) with l- or d-Asp on the phospholipid contribute to the remarkable chiral discrimination. This study provides a deeper understanding of the crucial amyloidosis process from the perspective of the chiral interface and reveals that the convergence of d-amino acids with the liposomes might be a feasible route for AD prevention.

Biomimetic nanochannels for the discrimination of sialylated glycans via a tug-of-war between glycan binding and polymer shrinkage.

Sialylated glycans that are attached to cell surface mediate diverse cellular processes such as immune responses, pathogen binding, and cancer progression. Precise determination of sialylated glycans, particularly their linkage isomers that can trigger distinct biological events and are indicative of different cancer types, remains a challenge, due to their complicated composition and limited structural differences. Here, we present a biomimetic nanochannels system integrated with the responsive polymer polyethyleneimine-g-glucopyranoside (Glc-PEI) to solve this problem. By using a dramatic "OFF-ON" change in ion flux, the nanochannels system achieves specific recognition for N-acetylneuraminic acid (Neu5Ac, the predominant form of sialic acid) from various monosaccharides and sialic acid species. Importantly, different "OFF-ON" ratios of the conical nanochannels system allows the precise and sensitive discrimination of sialylated glycan linkage isomers, α2-3 and α2-6 linkage (the corresponding ion conductance increase ratios are 96.2% and 264%, respectively). Analyses revealed an unusual tug-of-war mechanism between polymer-glycan binding and polymer shrinkage. The low binding affinity of Glc-PEI for the α2-6-linked glycan caused considerable shrinkage of Glc-PEI layer, but the high affinity for the α2-3-linked glycan resulted in only a slight shrinkage. This competition mechanism provides a simple and versatile materials design principle for recognition or sensing systems that involve negatively charged target biomolecules. Furthermore, this work broadens the application of nanochannel systems in bioanalysis and biosensing, and opens a new route to glycan analysis that could help to uncover the mysterious and wonderful glycoworld.