BACKGROUND:The global heparin supply relies heavily on porcine mucosa, prompting renewed interest in ruminant-derived alternatives. However, concerns regarding heparin-induced thrombocytopenia remain a major barrier to their clinical reintroduction. OBJECTIVES:To systematically evaluate the platelet factor 4 (PF4)-dependent immunogenic potential of porcine mucosal heparin, bovine lung heparin, and ovine mucosal heparin, as well as their low-molecular-weight heparin (LMWH) derivatives, and to delineate the structural features governing these interactions. METHODS:Size-exclusion chromatography and a competitive ELISA established in this study were used to monitor PF4-heparin complex stoichiometry and antibody recognition. Flow cytometry quantified functional heparin-induced platelet activation (CD62P/CD63 expression). PF4 affinity chromatography-hydrophilic interaction liquid chromatography-mass spectrometry and molecular docking characterized high-affinity glycan features and evaluated sulfation patterns in stabilizing PF4-heparin interactions. RESULTS:Under equipotent dosing conditions, PF4 complex formation, antibody recognition, and platelet activation followed the order bovine lung heparin > ovine mucosal heparin > porcine mucosal heparin. Differences among heparins were largely explained by molecular weight-dependent stoichiometry. In contrast, source-dependent differences among LMWHs persisted after potency normalization, with bovine LMWH forming significantly more PF4 complexes and triggering the most robust platelet activation. Affinity profiling identified degree of polymerization 6-degree of polymerization 8 (dp6-dp8) as minimal PF4-binding units and revealed that increased sulfation and reduced N-acetylation enhance PF4 affinity. Molecular docking confirmed that N-sulfation stabilizes PF4-heparin interactions. CONCLUSION:Heparins derived from different animal sources exhibit distinct PF4-binding properties and immunogenic profiles. These findings provide mechanistic insight into species-dependent heparin-induced thrombocytopenia risk-driven by stoichiometry in heparin and fine structural features in LMWH-and support diversification of the global heparin supply.

Heparan sulfate (HS), one of the mostly negatively charged biomacromolecules anchored on the membrane surface of nearly all mammal cells, plays critical regulatory roles through interacting with a variety of proteins. However, there is still no method capable to directly sequence the domain alterations of HS in pathological states. In the current study, the pathological alterations of HS were elucidated for the first time in APAP-induced acute liver injury by a deep learning-driven chemical derivatization-tandem mass spectrometry strategy, and the sequence changes up to octasaccharides within the bioactive domain "GlcA-GlcNS6S" were successfully decoded. GAG-Explorer, a software incorporated with a comprehensive deep learning model capable of predicting the fragmentation patterns of HS oligomers under actual MS/MS condition was developed to facilitate large-scale sequencing of natural HS structures. The HS alterations in the sequence aspect were elucidated thoroughly in APAP-induced acute liver injury, rather than their compositional changes, which is of great significance for the applications of HS-based therapeutic agents in the biomedical field.

Chondroitin sulfate (CS) plays critical regulatory roles in numerous biological processes, but definitive sequence of these bio-active polymers is still lacking, with only a few natural/synthetic CS oligomers had been welly sequenced, which hinders the further understanding of structure-function relationship of CS in physiological process. In our study, a uronic acid reduction-tandem mass spectrometry strategy was developed for large-scale online sequencing CS oligosaccharide. High-throughput and widely applicable HILIC-HCD-MS/MS was performed on sequencing saccharide chain for further application in glycomics. The accurate sulfation patterns of CS-A and CS-C disaccharide to tetradecasaccharide were firstly attributed with no significant loss of sulfate groups in HCD-MS/MS dissociation, including the locations of O/C units in CS-A chain and D units in CS-C chain, which enables us to decoding the CS sequence in various tissues, cells and their pathological change in diseases. Remarkably, by completely shielding the carboxyl groups and increasing the charge state of precursor ion, the sulfate loss during HCD-MS/MS dissociation was significantly suppressed, with no sulfate loss detected in the fragment-ions. Hence, the above strategy effectively addressed the critical challenge of decoding the information contained in functional CS motifs, which is of great significance for elucidating the changes in CS during disease progression and their applications in biomedical field.

Non-small cell lung cancer (NSCLC) is a highly prevalent and aggressive type of lung cancer, often associated with a poor prognosis. Cucurbitacin B, a natural tetracyclic triterpene, has demonstrated remarkable anticancer activity. In this study, we engineered a novel drug delivery system, Dex-APDMS@CP1@1@ Cucurbitacin B, which incorporates synthetically derived compound 1 to enhance the therapeutic efficacy of Cucurbitacin B. The system was comprehensively characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and nitrogen adsorption techniques. Results revealed excellent stability, a uniform particle size of approximately 500 nm, and a high drug-loading efficiency of 35.2 +/- 2.1 wt%. Additionally, the in vitro drug release study indicated that 88.6 +/- 3.5% of Cucurbitacin B was released within 12 hours, demonstrating a rapid release profile. The successful encapsulation of the drug was further confirmed by effective fluorescence quenching. In vitro experiments showed that the Dex-APDMS@CP1@1@Cucurbitacin B system significantly inhibited the proliferation of NSCLC cells, highlighting its great potential for targeted cancer therapy.

Parkinson's disease (PD) is a progressive neurodegenerative disorder with limited therapeutic options and suboptimal long-term efficacy. Although glycosaminoglycans such as chondroitin sulfate (CS) possess neuroprotective potential, their clinical application is hampered by poor oral bioavailability and limited target specificity. In this study, we first established a highly sensitive LC-MS/MS MRM assay to quantify CS in plasma. Using animal models, we demonstrated that co-administration with N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) significantly enhanced the oral bioavailability of CS compared to deoxycholic acid (DOCA) modification. Subsequently, we synthesized a structurally defined 6-O-desulfated chondroitin sulfate octasaccharide (CS-dp8-de6S) and evaluated its therapeutic efficacy in MPTP-induced PD mouse models via co-administration with SNAC. Treatment with CS-dp8-de6S/SNAC improved motor performance in the pole and hanging tests, partially restored striatal dopamine levels, and upregulated tyrosine hydroxylase expression in the substantia nigra, these effects were significantly greater than those observed with non-desulfated CS-dp8, demonstrating its enhanced specificity. Mechanistic studies using gel mobility shift assay and dual immunohistochemistry revealed that CS-dp8-de6S treatment effectively reduced pathological fibrinogen β-chain (FGB) deposition in brain tissues. These findings highlight the potential of structurally defined CS oligosaccharides as novel disease-modifying therapeutics for PD and provide a basis for the development of carbohydrate-based strategies targeting proteinopathies in neurodegenerative disorders.

The precise structure of glycosaminoglycans is critical for their bioactivity and the development of glycopharmaceuticals. Herein, cellular and animal experiments were conducted to assess the differences in the activities of heparin (HP) and heparan sulfate (HS) against liver cancer and drug-induced liver injury. Label-free quantitative proteomics, bioinformatics, biolayer interferometry, and immunohistochemical analyses were used to determine key proteins with differential expression. As a result, HP demonstrated superior antiliver cancer activity compared with HS, whereas HS exhibited strong potential in resisting acetaminophen-induced liver injury. DIRAS family GTPase 2 (DIRAS2) was identified as a key HS-binding protein that was strongly associated with cell proliferation, and its expression levels in cells and tissues showed opposite trends following HP and HS administration. HP significantly reduced the abundance of DIRAS2 in the tumor tissue, thereby inhibiting tumor cell proliferation, whereas HS promoted proliferation by increasing DIRAS2 expression. Cluster sequencing revealed that consecutive GlcNS6S-IdoA2S domains in HP and IdoA2S-GlcNS6S, GlcA-GlcNS6S, and IdoA-GlcNAc domains in HS were required for affinity binding within the decasaccharide region. Molecular docking suggested that differences in the binding modes of HP and HS chains to DIRAS2 underlie their functional diversity. These findings indicate that HP and HS oligosaccharides with well-defined structures may serve as potential therapeutic agents for liver-related diseases.

Heparin, a bio-molecule with the highest negative charge density, is pharmaceutically important to prevent SARS-CoV-2 infection due to its strong competitive binding to spike protein compared with cellular heparan sulfate, which was confirmed as a co-receptor for virus-host cell interaction. Hence, the refined structural characterization of heparin targeting viral protein-HS interaction was significant for developing antiviral pharmaceuticals. In our study, heparin oligomers (dp >= 4) were prepared using heparinase I. The affinity oligosaccharides binding to Omicron spike protein RBD were separated by affinity chromatography and size exclusion chromatography. HILIC-ESI-FTMS was used for chain mapping analysis. The basic building blocks were analyzed and the binding domain sequence was produced by Seq-GAG software and further measured by SAX chromatography. As results, heparin octasaccharide was found with significantly higher binding ability than hexasaccharide and tetrasaccharide, and the octasaccharide [Delta UA-GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2SGlcNS6S] with 12 sulfate groups showed high binding to RBD. The mechanism of this structurally well-defined octasaccharide binding to RBD was further investigated by molecular docking. The affinity energy of optimal pose was -6.8 kcal/mol and the basic amino acid residues in RBD sequence (Arg403, Arg452, Arg493 and His505) were identified as the major contribution factor to interacting with sulfate/carboxyl groups on saccharide chain. Our study demonstrated that heparin oligosaccharide with well-defined structure could be potentially developed as anti-SARS-CoV-2 drugs.

Diabetic nephropathy (DN) is one of the most important comorbidities for diabetic patients, which is the main factor leading to end-stage renal disease. Heparin analogs can delay the progression of DN, but the mechanism is not fully understood. In this study, we found that low molecular weight heparin therapy significantly upregulated some downstream proteins of the peroxisome proliferator-activated receptor (PPAR) signaling pathway by label-free quantification of the molecular weight heparin can protect the heparan sulfate of renal tubular epithelial cells from being degraded by heparanase that is highly expressed in a high-glucose environment, enhance the endocytic recruitment of fatty acid-binding protein 1, a coactivator of the PPAR pathway, and then regulate the activation level of intracellular PPAR. In addition, we have elucidated for the first time the molecular mechanism of heparan sulfate and fatty acid-binding protein 1 interaction. These findings provide new insights into understanding the role of heparin in the pathogenesis of DN and developing corresponding treatments.

A low-triggering potential and a narrow-potential window are anticipated to decrease the electrochemical interference and cross talk of electrochemiluminescence (ECL). Herein, by exploiting the low oxidative potential (0.82 V vs Ag/AgCl) of dihydrolipoic acid-capped sliver nanoclusters (DHLA-AgNCs), a coreactant ECL system of DHLA-AgNCs/hydrazine (N2H4) is proposed to achieve efficient and oxidative-reduction ECL with a low-triggering potential of 0.82 V (vs Ag/AgCl) and a narrow-potential window of 0.22 V. The low-triggering-potential and narrow-potential-window nature of ECL can be primarily preserved upon labeling DHLA-AgNCs to probe DNA and immobilizing DHLA-AgNCs onto the Au surface via sandwiched hybridization, which eventually enables a selective ECL strategy for the gene assay at +0.82 V. This gene assay strategy can sensitively determine the gene of human papillomavirus from 10 to 1000 pM with a low limit of detection of 5 pM (S/N = 3) and would open a way to improve the applied ECL bioassay.

Parkinson's disease (PD) is a neurodegenerative disorder influenced by various factors, including age, genetics, and the environment. Current treatments provide symptomatic relief without impeding disease progression. Previous studies have demonstrated the therapeutic potential of exogenous heparin and chondroitin sulfate in PD. However, their therapeutic mechanisms and structure-activity relationships remain poorly understood. In this study, low-molecular-weight heparin (L-HP) and chondroitin sulfate (L-CS) exhibited favorable therapeutic effects in a mouse model of PD. Proteomics revealed that L-HP attenuated mitochondrial dysfunction through its antioxidant properties, whereas L-CS suppressed neuroinflammation by inhibiting platelet activation. Two glycosaminoglycan (GAG)-binding proteins, manganese superoxide dismutase (MnSOD2) and fibrinogen beta chain (FGB), were identified as potential targets of L-HP and L-CS, and we investigated their structure-activity relationships. The IdoA2S-GlcNS6S/GlcNAc6S unit in HP bound to SOD2, whereas the GlcA-GalNAc4S and GlcA-GalNAc4S6S units in CS preferred FGB. Furthermore, N-S and 2-O-S in L-HP, and 4-O-S, 6-O-S, and -COOH in L-CS contributed significantly to the binding process. These findings provide new insights and evidence for the development and use of glycosaminoglycan-based therapeutics for PD.

Chemiluminescence (CL), especially commercialized CL immunoassay (CLIA), is normally performed within the eye-visible region of the spectrum by exploiting the electronic-transition-related emission of the molecule luminophore. Herein, dual-stabilizers-capped CdTe nanocrystals (NCs) is employed as a model of nanoparticulated luminophore to finely tune the CL color with superior color purity. Initialized by oxidizing the CdTe NCs with potassium periodate (KIO4), intermediates of the reactive oxygen species (ROS) tend to charge CdTe NCs in both series-connection and parallel-connection routes and dominate the charge-transfer CL of CdTe NCs. The CdTe NCs/KIO4 system can exhibit color-tunable CL with the maximum emission wavelength shifted from 694 nm to 801 nm, and the red-shift span is over 100 nm. Both PL and CL of each of the CdTe NCs are bandgap-engineered; the change in the NCs surface state via CL reaction enables CL of each of the CdTe NCs to be red-shifted for ∼20 nm to PL, while the change in the NCs surface state via labeling CdTe NCs to secondary-antibody (Ab2) enables CL of the CdTe NCs-Ab2 conjugates to be red-shifted for another ∼20 nm to bare CdTe NCs. The CL of CdTe753-Ab2/KIO4 is ∼791 nm, which can perform near-infrared CL immunoassay and semi-automatically determined procalcitonin (PCT) on commercialized in vitro diagnosis (IVD) instruments.

Ultrashort laser pulses can serve as fast probes to record instant events. The isolated attosecond pulses (IAPs) generated from high-order harmonic generation (HHG) have been shortened down to about 2 atomic units in time, empowering us to study quantum behaviors of electrons in atoms, molecules, and solids with unprecedented time resolution. Following the cutoff energy law of HHG, the shortest IAP reported so far is driven with short-wavelength infrared (SWIR) pulses, which require additional broadband frequency conversion techniques and raise the bar for attosecond researches. Here, we show that with few-cycle near-infrared (NIR) laser pulses, IAP with pulse duration of 51 ± 4 as is generated during 1-fs linear polarization gate formed by generalized double optical gating (GDOG) technique. The characterization is done with attosecond streak camera, and phase reconstruction is performed with quick phase retrieval by omega oscillation filtering (qPROOF). Furthermore, we show that the IAP generation favors certain carrier envelope phases (CEPs) in the narrow gate, i.e., IAP is only efficiently produced for certain CEPs, which eliminates the requirement of CEP stabilization. The demonstrated scheme for IAP generation in principle has much higher conversion efficiency than the long-wave driver scheme according to the wavelength scaling law of HHG. Our work suggests an alternative way to generate ultrashort IAPs by applying GDOG on few-cycle free-CEP NIR driving pulses, and is thereby of great importance to facilitate the development of attosecond science and technology.

Depression is a common neuropsychiatric disease which brings an increasing burden to all countries globally. Baicalin, a flavonoid extracted from the dried roots of Scutellaria, has been reported to exert anti-inflammatory, antioxidant, and neuroprotective effects in the treatment of depression. However, the potential biological mechanisms underlying its antidepressant effect are still unclear. In the present study, we conducted extensive research on the potential mechanisms of baicalin’s antidepressant effect using the methods of network pharmacology, including overlapped terms-based analysis, protein–protein interaction (PPI) network topology analysis, and enrichment analysis. Moreover, these results were further verified through molecular docking, weighted gene co-expression network analysis (WGCNA), differential gene expression analysis, and subsequent animal experiments. We identified forty-one genes as the targets of baicalin in the treatment of depression, among which AKT1, IL6, TP53, IL1B, and CASP3 have higher centrality in the more core position. Meanwhile, the roles of peripheral genes derived from direct potential targets were also observed. Our study suggested that biological processes, such as inflammatory reaction, apoptosis, and oxidative stress, may be involved in the therapeutic process of baicalin on depression. These mechanisms were validated at the level of structure, gene, protein, and signaling pathway in the present study. Taken together, these findings propose a new perspective on the potential mechanisms underlying baicalin’s antidepressant effect, and also provide a new basis and clarified perspective for its clinical application.

The low-triggering-potential and potential-selective electrochemiluminescence for a highly selective immunoassay by exploiting AgInS 2 /ZnS nanocrystals as a tag and N 2 H 4 as a coreactant.

The commercialized electrochemiluminescence (ECL) immunoassay is carried out by holding luminophore Ru(bpy)32+ at a given potential. Designing an electrochemiluminophore with a narrow triggering potential window is strongly anticipated to decrease the electrochemical cross-talk and improve the flux of the commercialized ECL immunoassay in a potential-resolved way. Herein, L-penicillamine-capped silver nanoclusters (LPA-AgNCs) are facilely synthesized and utilized as tags to perform the ECL immunoassay with a sole and narrow triggering potential window of 0.24 V by employing hydrazine (N2H4) as a coreactant. The maximum ECL emission of the LPA-AgNCs/N2H4 system is located ca. +1.27 V. Upon immobilizing LPA-AgNCs onto the electrode surface via forming a sandwich immunocomplex, the ECL of LPA-AgNCs/N2H4 can be utilized to sensitively and selectively determine human carcinoembryonic antigen from 0.5 to 1000 pg/mL with a low limit of detection of 0.1 pg/mL (S/N = 3). This work might open a way to screen electrochemiluminophores for the multiple ECL immunoassay in a potential-resolved way.

Coreactant-free electrochemiluminescence (ECL) is promising for removing the exogenous effects of coreactant and simplify the operation procedures and setups of commercialized ECL bioassays. Herein, an electrosterically involved strategy for achieving a low-triggering-potential (+0.21 V vs Ag/AgCl) and coreactant-free ECL from dual-stabilizer-capped CdTe nanocrystals (NCs) is proposed with mercaptopropionic acid (MPA) and hexametaphosphate (HMP) as the capping agents of luminophores. Upon employing the CdTe NCs as the ECL tag for the immunoassay, all the tags in the bioconjugates of the CdTe NCs and the secondary antibody (Ab2|CdTe) as well as in the final achieved sandwich-type immunocomplexes can exhibit efficient coreactant-free ECL with an electrosterically involved triggering potential nature. The bioconjugates of Ab2|CdTe with Ab2 no more than 30 kDa, such as the thyroid stimulating hormone (30 kDa) and the recombinant pro-gastrin releasing peptide (ProGRP, 14 kDa), merely exhibit coreactant-free ECL around +0.24 V, while bioconjugates of Ab2|CdTe with an Ab2 beyond 30 kDa only give off coreactant-free ECL around +0.82 V. Due to the further enhanced electrosteric effect in sandwich-type immunocomplexes, only the ECL immunosensor with ProGRP as the target can give off coreactant-free ECL around +0.24 V. The electrosterically involved and coreactant-free ECL of CdTe NCs is consequently utilized to sensitively and selectively determine the molecular protein ProGRP, which demonstrates a wide linearity range from 0.1 to 2000 pg/mL and a low limit of detection at 0.05 pg/mL (S/N = 3). This low-triggering-potential and coreactant-free combined ECL platform indicates that engineering the surface of CdTe NCs with a protein can improve the performance of ECL tags in a protein-weight-involved electrosterical way.

To determine the intrinsic effects of body elements on the electrochemiluminescence (ECL) of metal nanoclusters (NCs), herein, a valence-state engineering strategy is developed to adjust the NCs' ECL with bovine serum albumin (BSA)-stabilized AuNCs as a model, in which engineering the valence state of the Au body element, i.e., Au(0) and Au(I), is performed via successively reducing the precursor AuCl4- to Au(I) and Au(0) with BSA. The obtained BSA-AuNCs/N2H4 system leads to three anodic ECL processes at 0.37 (ECL-1), 0.85 (ECL-2), and 1.45 V (ECL-3). ECL-1 is generated from the BSA-Au(0) section of BSA-AuNCs in a surface-defect-involved route and is much stronger and red-shifted compared to ECL-2 and ECL-3, which are generated from the BSA-Au(I) section of BSA-AuNCs in the band-gap-engineered route. Each of the anodic ECL processes can be selectively generated and/or suppressed via adjusting the Au(I)/Au(0) ratio of BSA-AuNCs, tunable ECL generation route, and triggering potential, and the emission intensity and waveband of metal NCs are conveniently achieved in body-element-involved valence-state engineering.

A single-stabilizer-capped strategy is proposed for achieving highly efficient and surface-defect-involved electrochemiluminescence (ECL) from unary copper nanoclusters (NCs) via employing l-cysteine (Cys) as a capping agent of luminophore. The Cys-capped CuNCs (Cys-CuNCs) can be electrochemically injected with valence band (VB) holes and exhibit eye-touchable ECL processes around +0.95 and +1.15 V upon employing TPrA as a coreactant. Both accumulated ECL spectra and spooling ECL spectra demonstrated that the two ECL processes are of the same single waveband and spectrally identical to each other with the same maximum emission wavelength of 640 nm. Promisingly, ECL of the Cys-CuNCs/TPrA system is obviously red-shifted for ∼150 nm to PL of Cys-CuNCs, indicating that the bandgap-engineered routes for ECLs of Cys-CuNCs are completely blocked. The oxidative-reduction ECL process of the Cys-CuNCs/TPrA system is a kind of highly efficient, eye-visible, and single-color emission in surface-defect-involved route. The capping agent of Cys can enable the CuNCs/TPrA system with a stronger ECL than other thiol capping agents, so that Cys-CuNCs are utilized as ECL tags for sensitive and selective immunoassays, which exhibit a wide linear response range from 0.05 pg/mL to 0.5 ng/mL and a limit of detection of 0.01 pg/mL (S/N = 3) with carcinoembryonic antigen as the analyte. Moreover, both the luminophore Cys-CuNCs and conjugates Ab2-CuNCs can be safely stored in aqueous media without any protector, which is promising for the evolution and clinic application of metal NC ECL in the surface-defect-involved route.

Acid-base titration is the most basic experiment in analytical chemistry.The titration curve reflects the whole process of neutralization reaction and is very important to understand acid-base titration.Based on three basic hypotheses, the chemical equilibrium, electroneutrality principle and mass balance, we established mathematical model between ionization degree and pH as well as titration percentage by keeping one parameter unchanged and the other two changed at the initial point.In addition, we recovered the acid-base titration curve by performing mathematical analysis and derivatization of the function.Through the analysis of two initial conditions in mathematical formula and the effects on the function, the quantitative validation on interpretations was introduced for reactions of different concentrations and strength of acids in titrations described in traditional teaching materials.With theoretical expansion of titration curves, we proposed a possible method for weak acid titration.

Multiplexed gene assay for simultaneously detecting the multi-targets of nucleic acids is strongly anticipated for the accurate diseases diagnosis and prediction, and all commercial available gene assays for IVD are a kind of single-target assay. Herein, a dual-potential encoded and coreactant-free electrochemiluminescence (ECL) strategy is proposed for the multiplexed gene assay, which can be conveniently carried out by directly oxidizing the same luminescent tag of dual-stabilizers-capped CdTe nanocrystals (NCs). The CdTe NCs linked with sulfhydryl-RNA via Cd-S bond merely exhibits one ECL process around 0.32 V with a narrow triggeringpotential-window of 0.35 V, while CdTe NCs linked with amino-RNA via amide linkage solely gives off one ECL process around 0.82 V with a narrow triggering-potential-window of 0.30 V. Multiplexing ECL of both sulfhydryl-RNA-functionalized CdTe NCs and amino-RNA-functionalized CdTe NCs can be utilized to simultaneously detect the open reading frame 1ab (ORF1ab) and the nucleoprotein (N) genes without crosstalk, in which ECL of sulfhydryl-RNA-functionalized CdTe NCs can dynamically determine ORF1ab from 200 aM to 10 fM with a limit of detection (LOD) of 100 aM, while ECL of amino-RNA-functionalized CdTe NCs can linearly detect N gene from 5 fM to 1 pM with a LOD of 2 fM. Post-engineering CdTe NCs with RNA in a labeling-bond engineering way would provide a potential-selective and encoded ECL strategy for multiplexed gene assay with one luminophore.