Research progress of metal biomaterials with potential applications as cardiovascular stents and their surface treatment methods to improve biocompatibility.

Facing the growing issue of cardiovascular diseases, metallic materials with higher tensile strength and fatigue resistance play an important role in treating diseases. This review lists the advantages and drawbacks of commonly used medical metallic materials for vascular stents. To avoid post-procedural threats such as thrombosis and in-stent restenosis, surface treatments, and coating methods have been used to further improve the biocompatibility of these materials. Surface treatments including laser, plasma treatment, polishing, oxidization, and fluorination can improve biocompatibility by modifying the surface charges, surface morphology, and surface properties of the material. Coating methods based on polymer coatings, carbon-based coatings, and drug-functional coatings can regulate the surface properties, and also serve as an effective barrier to the interaction of metallic biomaterial surfaces with biomolecules, which can be used to improve corrosion resistance and stability, as well as improve their biocompatibility. Biocompatibility serves as the most fundamental property of cardiovascular stents, and maintaining the excellent and stable biocompatibility of cardiovascular stent surfaces is a current research bottleneck. Few reviews have been published on metallic biomaterials as cardiovascular stents and their surface treatments. For the purpose of advancing research on cardiovascular stents, common metal biomaterials, surface treatment methods, and coating methods to improve biocompatibility and comprehensive properties of the materials are described in this review. Finally, we suggest future directions for stent development, including continuously improving the durability and stability of permanent stents, accelerating the development of biodegradable stents, and strengthening feedback to improve the safety and reliability of cardiovascular stents.

Identification of a Pentasaccharide Lead Compound with High Affinity to the SARS-CoV-2 Spike Protein via In Silico Screening.

The spike (S) protein on the surface of the SARS-CoV-2 virus is critical to mediate fusion with the host cell membrane through interaction with angiotensin-converting enzyme 2 (ACE2). Additionally, heparan sulfate (HS) on the host cell surface acts as an attachment factor to facilitate the binding of the S receptor binding domain (RBD) to the ACE2 receptor. Aiming at interfering with the HS-RBD interaction to protect against SARS-CoV-2 infection, we have established a pentasaccharide library composed of 14,112 compounds covering the possible sulfate substitutions on the three sugar units (GlcA, IdoA, and GlcN) of HS. The library was used for virtual screening against RBD domains of SARS-CoV-2. Molecular modeling was carried out to evaluate the potential antiviral properties of the top-hit pentasaccharide focusing on the interactive regions around the interface of RBD-HS-ACE2. The lead pentasaccharide with the highest affinity for RBD was analyzed via drug-likeness calculations, showing better predicted druggable profiles than those currently reported for RBD-binding HS mimetics. The results provide significant information for the development of HS-mimetics as anti-SARS-CoV-2 agents.

Accurate Determination of Trace Water in Organic Solution by Quantitative Nuclear Magnetic Resonance.

A new method accompanied by a derived equation for accurate determination of trace water was developed by using quantitative 1H nuclear magnetic resonance (qNMR) spectroscopy. Given that the response for each chemically distinct moiety is uniformly proportional to the number of the corresponding resonant nuclei within the analyte, it is practicable to directly quantify the water content via its proton number using qNMR. In this study, three water standards with known water contents (e.g., 10.02, 1.006, and 0.103 mg/g), which were accurately determined by a well-established Coulometric Karl Fischer (CKF) titration method, were measured by using the developed qNMR method. An excellent agreement between the results from these two methods was obtained. Then, the water content of Sudan I was determined by high-field NMR (HF-NMR) spectroscopy, and the water contents of acetone and bioethanol were measured by low-field NMR (LF-NMR) spectroscopy. These results were compared with the water content measured by the CKF method to confirm the applicability of the established qNMR method. The developed method can eliminate the influences of environmental humidity and background water in the solvent; subsequently, the results calculated by the derived equation were comparable to the nominal values. Under the optimal conditions, the limit of quantitation of this method was as low as 6.7 µg. The recommended sample sizes for practical samples with various water contents (e.g., 10.02, 1.006, and 0.103 mg/g) were determined to be 5, 50, and 60 mg, respectively, which are much smaller than those required for the CKF method. The new method has a static and stable process without any side reactions, and the traceability to the SI unit can be directly achieved through the NMR internal standard. This method overcomes the limitations of the CKF method, especially for measuring methanol-insoluble substances.

Interacting polymer-modification enzymes in heparan sulfate biosynthesis.

Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcAß1-4GlcNSO3α1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotransferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluorescence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a functional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O-sulfated glucosamine residues. The inability to achieve simultaneous 2-O- and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme interactions in heparan sulfate biosynthesis.

In-depth characterization of 1,4-butanediol diglycidyl ether substituted hyaluronic acid hydrogels.

BDDE substituted HA hydrogels remain the most commonly used HA product in the biomedical field. The physical and biochemical properties of the hydrogels are dependent on the degree of modification and substitution patterns/positions, thus, characterizing their fine structure is of great importance for quality assurance. In this study, we developed novel LC-MS methods for accurate determination of MoD as well as in-depth characterization of the linkage network. Fragments resulted from enzymatic depolymerization were resolved by a porous graphitic carbon column followed by online tandem-MS for determining the modification site/residue. With high-resolution separation, two types of previously unknown structures were detected in the cross-linked fragments of 2-B-2 and 4-B-2. Based on the feature of resistance to NaBH4 reduction, these structures contain a GlcNAc residue modified at OH1. This special sugar unit likely derived from reducing end of the native polysaccharide could be a signature to discriminate subtle batch to batch variations.

Heparin mimetics as potential intervention for COVID-19 and their bio-manufacturing.

The COVID-19 pandemic has caused severe health problems worldwide and unprecedented decimation of the global economy. Moreover, after more than 2 years, many populations are still under pressure of infection. Thus, a broader perspective in developing antiviral strategies is still of great importance. Inspired by the observed multiple benefits of heparin in the treatment of thrombosis, the potential of low molecular weight heparin (LMWH) for the treatment of COVID-19 have been explored. Clinical applications found that LMWH decreased the level of inflammatory cytokines in COVID-19 patients, accordingly reducing lethality. Furthermore, several in vitro studies have demonstrated the important roles of heparan sulfate in SARS-CoV-2 infection and the inhibitory effects of heparin and heparin mimetics in viral infection. These clinical observations and designed studies argue for the potential to develop heparin mimetics as anti-SARS-CoV-2 drug candidates. In this review, we summarize the properties of heparin as an anticoagulant and the pharmaceutical possibilities for the treatment of virus infection, focusing on the perspectives of developing heparin mimetics via chemical synthesis, chemoenzymatic synthesis, and bioengineered production by microbial cell factories. The ultimate goal is to pave the eminent need for exploring novel compounds to treat coronavirus infection-caused diseases.

Antiviral Disaccharide Lead Compounds against SARS-CoV-2 through Computer-Aided High-Throughput Screen.

SARS-CoV-2 infects human epithelial cells through specific interaction with angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate proteoglycans act as the attachment factor to promote the binding of viral spike protein receptor binding domain (RBD) to ACE2 on host cells. Though the rapid development of vaccines has contributed significantly to preventing severe disease, mutated SARS-CoV-2 strains, especially the SARS-CoV-2 Omicron variant, show increased affinity of RBD binding to ACE2, leading to immune escape. Thus, there is still an unmet need for new antiviral drugs. In this study, we constructed pharmacophore models based on the spike RBD of SARS-CoV-2 and SARS-CoV-2 Omicron variant and performed virtual screen for best-hit compounds from our disaccharide library. Screening of 96 disaccharide structures identified two disaccharides that displayed higher binding affinity to RBD in comparison to reported small molecule antiviral drugs. Further, screening PharmMapper demonstrated interactions of the disaccharides with a number of inflammatory cytokines, suggesting a potential for disaccharides with multiple-protein targets.

A Cluster Sequencing Strategy To Determine the Consensus Affinity Domains in Heparin for Its Binding to Specific Proteins.

Glycosaminoglycans (GAGs) have high negative charge and are biologically and pharmaceutically important because their high charge promotes a strong interaction with many proteins. Due to the inherent heterogeneity of GAGs, multiple oligosaccharides, containing certain common domains, often can interact with clusters of basic amino acid residues on a target protein. The specificity of many GAG-protein interactions remains undiscovered since there is insufficient structural information on the interacting GAGs. Herein, we establish a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity GAG oligosaccharides, leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein. As a proof of concept, antithrombin III-binding oligosaccharides were examined, resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence. Repeating this approach, a new pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-γ (IFNγ). Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel GAG-based therapeutics.

Heparanase Expression Propagates Liver Damage in CCL4-Induced Mouse Model.

Heparanase is elevated in various pathological conditions, primarily cancer and inflammation. To investigate the significance and involvement of heparanase in liver fibrosis, we compared the susceptibility of wild-type (WT) and heparanase-overexpressing transgenic (Hpa-tg) mice to carbon tetrachloride (CCL4)-induced fibrosis. In comparison with WT mice, Hpa-tg mice displayed a severe degree of tissue damage and fibrosis, including higher necrotic tendency and intensified expression of smooth muscle actin. While damage to the WT liver started to recover after the acute phase, damage to the Hpa-tg liver was persistent. Recovery was attributed, in part, to heparanase-stimulated autophagic activity in response to CCL4, leading to increased apoptosis and necrosis. The total number of stellate cells was significantly higher in the Hpa-tg than the WT liver, likely contributing to the increased amounts of lipid droplets and smooth muscle actin. Our results support the notion that heparanase enhances inflammatory responses, and hence may serve as a target for the treatment of liver damage and fibrosis.

SI-traceable purity assignment of volatile material ethylbenzene by quantitative nuclear magnetic resonance spectroscopy.

In this study, a quantitative nuclear magnetic resonance (qNMR) method was developed to assign the SI-traceable purity of ethylbenzene, a volatile material, which is a colorless flammable liquid hydrocarbon at room temperature. An ethanol certified reference material having a similar boiling point was used as an internal standard to avoid measurement error arising from the volatilization of ethylbenzene. The reference value of the ethylbenzene study material was obtained by the mass balance method by subtracting all the impurities including water, inorganic impurities, and structurally related impurities (e.g. acetophenone, benzene, isobutylbenzene, sec-butylbenzene, methylcyclohexane), which is regarded as the traditional approach for purity assignment for organic compounds. The results of qNMR showed that the purity of the ethylbenzene study material was 998.6 ± 3.8 mg/g at a 95% confidence interval, which was consistent with the reference value of 998.9 ± 1.3 mg/g.

Elucidating the Binding Mode between Heparin and Inflammatory Cytokines by Molecular Modeling.

Heparan sulfate (HS) interacts with a broad spectrum of inflammatory cytokines, thereby modulating their biological activities. It is believed that there is a structural-functional correlation between each protein and sugar sequences in the HS polysaccharides, however, the information in this regard is limited. In this study, we compared the binding of four inflammatory cytokines (CCL8, IL-1beta, IL-2 and IL-6) to immobilized heparin by an SPR analysis. To define the molecular base of the binding, we used a heparin pentasaccharide as representative structure to dock into the 3D-molecular structure of the cytokines. The results show a discrepancy in KD values obtained by SPR analysis and theoretical calculation, pointing to the importance to apply more than one method when describing affinity between proteins and HS. By cluster analysis of the complex formed between the pentasaccharide and cytokines, we have identified several groups in heparin forming strong hydrogen bonds with all four cytokines, which is a significant finding. This molecular and conformational information should be valuable for rational design of HS/heparin-mimetics to interfere cytokine-HS interactions.

Re-expression of glucuronyl C5-epimerase in the mutant MEF cells increases heparan sulfate epimerization but has no influence on the Golgi localization and enzymatic activity of 2-O-sulfotransferase.

Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure-function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.

Coupling Liquid Chromatography and Tandem Mass Spectrometry to Electrophoresis for In-Depth Analysis of Glycosaminoglycan Drugs: Heparin and the Multicomponent Sulodexide.

Glycosaminoglycans (GAGs) contribute to the treatment of many human diseases, especially in the field of thrombosis, because of their anticoagulant activity. GAGs interrupt the coagulation process by interacting with multiple coagulation factors through defined sequences within their linear and negatively charged chains, which are not fully elucidated. Numerous methods have been developed to characterize the structure of pharmaceutical GAGs, including intravenously or subcutaneously administered heparin and orally administered sulodexide. However, most currently available methods only focus on the oligosaccharide portion or analyze the whole mixture because longer-chain polysaccharides are extremely difficult to resolve by chromatographic separation. We have established two novel electrophoresis-mass spectrometry methods to provide a panoramic view of the structures of pharmaceutical GAGs. In the first method, an in-gel digestion procedure was developed to recover GAGs from the polyacrylamide gels, while in the second method, a strong anion exchange ultrafiltration procedure was developed to extract multiple GAG species from the agarose gels. Both procedures are compatible with liquid chromatography-tandem mass spectrometry, and structural information, such as disaccharide composition and chain length, can be revealed for each GAG fraction. The applications of these two methods on analysis of two different GAG drugs, heparin and sulodexide, were demonstrated. The current study offers the first robust tool to directly elucidate the structure of larger GAG chains with more biological importance rather than obtaining a vague picture of all chains as a mixture, which is fundamental for better understanding the structure-activity relationship and quality control of the GAG drugs.

Oligosaccharides mapping of nitrous acid degraded heparin through UHPLC-HILIC/WAX-MS.

Building blocks characterization is a significant approach for understanding the molecular structure of heparin and its derivatives. Nitrous acid (HONO) depolymerization of heparin generates oligosaccharides that maintain the epimerization conformation on C5 of the uronic acids, reflecting the authentic structure of the parental chain. HONO treatment at pH 1.5 selectively cleaves the bond between N-sulfated glucosamine and hexuronic acid, resulting mainly disaccharides, as well as tetra-, tri-, and mono-saccharides. The tetrasaccharides are derived from the structure of N-acetylated domains while tri-, and mono-saccharides are derived from the reducing or the non-reducing end of the heparin chain. The resulted oligosaccharides were separated and analyzed using a UHPLC-HILIC/WAX-MS method. We succeeded in the identification of 19 tetrasaccharides, 19 trisaccharides and 4 monosaccharides species, majority of which is structurally characterized. By comparing the theoretical possibilities and actual occurrence of the well-characterized tetrasaccharides, we demonstrated that the biosynthesis of heparin is a systematic process.

Elucidating the Interactions Between Heparin/Heparan Sulfate and SARS-CoV-2-Related Proteins-An Important Strategy for Developing Novel Therapeutics for the COVID-19 Pandemic.

Owing to the high mortality and the spread rate, the infectious disease caused by SARS-CoV-2 has become a major threat to public health and social economy, leading to over 70 million infections and 1. 6 million deaths to date. Since there are currently no effective therapeutic or widely available vaccines, it is of urgent need to look for new strategies for the treatment of SARS-CoV-2 infection diseases. Binding of a viral protein onto cell surface heparan sulfate (HS) is generally the first step in a cascade of interaction that is required for viral entry and the initiation of infection. Meanwhile, interactions of selectins and cytokines (e.g., IL-6 and TNF-α) with HS expressed on endothelial cells are crucial in controlling the recruitment of immune cells during inflammation. Thus, structurally defined heparin/HS and their mimetics might serve as potential drugs by competing with cell surface HS for the prevention of viral adhesion and modulation of inflammatory reaction. In this review, we will elaborate coronavirus invasion mechanisms and summarize the latest advances in HS-protein interactions, especially proteins relevant to the process of coronavirus infection and subsequent inflammation. Experimental and computational techniques involved will be emphasized.

Selective binding of heparin oligosaccharides in a magnetic thermoresponsive molecularly imprinted polymer.

Heparin is a highly sulfated polysaccharide, applied in clinic for treatment of thrombotic diseases. The biological activity is closely related to its molecular structure e.g. compositions of disaccharides and oligosaccharides units. The classical method to isolate the oligosaccharides after depolymerization by heparinases or nitrous acid I s by size exclusion chromatography which is a time-consuming process. In this study, we explored the possibility for rapid separation of oligosaccharides using a novel polymer material. The magnetic thermoresponsive molecularly imprinted polymers (MIPs) were synthesized using heparin disaccharide as a template, AEM, NIPAAm, and AAm as functional monomer, and MBAA as crosslinker by surface radical polymerization in an aqueous media. Incubation of the MIP with hepairn oligosaccharides demonstrated specific binding to the template molecule. This binding to the targeted molecule was affected by reaction temperature with regard to binding capacity and specificity. The recognition specificity and selectivity can be modulated by varying the compositions of multi-functional monomers. The pseudo-second-order kinetic model and Langmuir isotherm model provide the best fit to the equilibrium adsorption of heparin disaccharides by MIPs. The results suggest that the new material can be used for rapid separation of di- and tetra-saccharides of heparin, which can also be adapted to the applications for isolation of oligosaccharides from other polysaccharides, e.g. heparan sulfate and chondoriting sulfate.

Characterization of epimerization and composition of heparin and dalteparin using a UHPLC-ESI-MS/MS method.

Heparin is a significant anticoagulant that has been used in clinic over decades. Although numerous efforts have been made to characterize the molecular structure of heparin and its derivatives for safety of the medicine, technical barriers still exist because of their structural complexity. In this study, we have established a method capable to evaluate both the epimerization and composition of heparin and dalteparin by a UHPLC-HILIC/WAX-MS/MS approach. Ten major disaccharide building blocks retaining the epimerization configuration of parental heparin chains were generated and well separated, 9 of which were unambiguously identified. Isomer identifications were achieved through high-resolution tandem mass spectrometry analysis with reference to elaborately prepared standards. The method was successfully applied for the sameness study of generic dalteparins in combination with an isotopic labelling procedure. We believe this robust method maybe adapted to quality control in pharmaceutical production of heparin and LMWHs.

Sequencing the oligosaccharide pool in the low molecular weight heparin dalteparin with offline HPLC and ESI-MS/MS.

Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI-MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC-MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization.

Slx5/Slx8 Promotes Replication Stress Tolerance by Facilitating Mitotic Progression.

Loss of minichromosome maintenance protein 10 (Mcm10) causes replication stress. We uncovered that S. cerevisiae mcm10-1 mutants rely on the E3 SUMO ligase Mms21 and the SUMO-targeted ubiquitin ligase complex Slx5/8 for survival. Using quantitative mass spectrometry, we identified changes in the SUMO proteome of mcm10-1 mutants and revealed candidates regulated by Slx5/8. Such candidates included subunits of the chromosome passenger complex (CPC), Bir1 and Sli15, known to facilitate spindle assembly checkpoint (SAC) activation. We show here that Slx5 counteracts SAC activation in mcm10-1 mutants under conditions of moderate replication stress. This coincides with the proteasomal degradation of sumoylated Bir1. Importantly, Slx5-dependent mitotic relief was triggered not only by Mcm10 deficiency but also by treatment with low doses of the alkylating drug methyl methanesulfonate. Based on these findings, we propose a model in which Slx5/8 allows for passage through mitosis when replication stress is tolerable.

Mapping ubiquitination sites of S. cerevisiae Mcm10.

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.