Heparinase III with High Activity and Stability: Heterologous Expression, Biochemical Characterization, and Application in Depolymerization of Heparin.

A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg-1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like →4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 → 4)ΔUA/ΔUA2S[1 → and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.

Detection of glycocalyx degradation in real time: A conceptual model of thromboelastography.

BACKGROUND: The endothelial glycocalyx is a critical component of the vascular barrier; its disruption after shock states may contribute to coagulopathy in a variety of conditions. Measurement of glycocalyx components in plasma have been used to index glycocalyx degradation but are not available as a point of care test. Heparanoids, such as heparan sulfate, may affect coagulation which may be detected by either thromboelastography or activated clotting time. METHODS: Endothelial glycocalyx components syndecan-1 and heparan sulfate were added to blood samples at clinically relevant concentrations. Thromboelastography values included clot reaction time, clot amplification and fibrinogen values, and maximum clot strength (maximum amplitude, platelets). The heparinase thromboelastography cartridge was used to detect a heparin-like effect. The activated clotting time test was performed subsequently using the heparan sulfate blood samples to compare a standard coagulation test with thromboelastography clot reaction times. RESULTS: Both thromboelastography clot reaction time (with comparison to heparinase) and activated clotting time were useful to detect effects of coagulation. Thromboelastography also detected platelet and fibrinogen abnormalities at higher heparan sulfate concentrations. Studies using thromboelastography or even activated clotting time may be useful to detect glycocalyx degradation after shock states and may guide clinical decision making. CONCLUSION: Thromboelastography and or activated clotting time may be useful to detect glycocalyx degradation as a point of care test in patients in the acute setting. Additionally, these assays may detect previous undisclosed coagulopathy due to glycocalyx degradation.

Identification and characterization of a novel heparinase PCHepII from marine bacterium Puteibacter caeruleilacunae.

Heparin (HP) and heparan sulfate (HS) are multifunctional polysaccharides widely used in clinical therapy. Heparinases (Hepases) are enzymes that specifically catalyse HP and HS degradation, and they are valuable tools for studying the structure and function of these polysaccharides and for preparing low molecular weight heparins. In this study, by searching the NCBI database, a novel enzyme named PCHepII was discovered in the genome of the marine bacterium Puteibacter caeruleilacuae. Heterologously expressed PCHepII in Escherichia coli (BL21) has high expression levels and good solubility, active in sodium phosphate buffer (pH 7.0) at 20°C. PCHepII exhibits an enzyme activity of 254 mU/mg towards HP and shows weak degradation capacity for HS. More importantly, PCHepII prefers to catalyse the high-sulfated regions of HP and HS rather than the low-sulfated regions. Although PCHepII functions primarily as an endolytic Hepase, it mainly generates disaccharide products during the degradation of HP substrates over time. Investigations reveal that PCHepII exhibits a preference for catalysing the degradation of small substrates, especially HP tetrasaccharides. The catalytic sites of PCHepII include the residues His199, Tyr254, and His403, which play crucial roles in the catalytic process. The study and characterization of PCHepII can potentially benefit research and applications involving HP/HS, making it a promising enzyme.

The pro-healing effects of heparan sulfate and growth factors are enhanced by the heparinase enzyme: New association for skin wound healing treatment.

Effective treatment strategies for skin wound repair are the focus of numerous studies. New pharmacological approaches appear necessary to guarantee a correct and healthy tissue regeneration. For these reasons, we purposed to investigate the effects of the combination between heparan sulfate and growth factors further adding the heparinase enzyme. Interestingly, for the first time, we have found that this whole association retains a marked pro-healing activity when topically administered to the wound. In detail, this combination significantly enhances the motility and activation of the main cell populations involved in tissue regeneration (keratinocytes, fibroblasts and endothelial cells), compared with single agents administered without heparinase. Notably, using an experimental C57BL/6 mouse model of skin wounding, we observed that the topical treatment of skin lesions with heparan sulfate + growth factors + heparinase promotes the highest closure of wounds compared to each substance mixed with the other ones in all the possible combinations. Eosin/hematoxylin staining of skin biopsies revealed that treatment with the whole combination allows the formation of a well-structured matrix with numerous new vessels. Confocal analyses for vimentin, FAP1α, CK10 and CD31 have highlighted the presence of activated fibroblasts, differentiated keratinocytes and endothelial cells at the closed region of wounds. Our results encourage defining this combined treatment as a new and appealing therapy expedient in skin wound healing, as it is able to activate cell components and promote a dynamic lesions closure.

Digital-like Enzyme Inhibition Mechanism-Based Strategy for the Digital Sensing of Heparin-Specific Biomarkers.

A new microbead (MB)-based digital flow cytometric sensing system is proposed for the sensitive detection of heparin-specific biomarkers, including heparin-binding protein (HBP) and heparinase. This strategy takes advantage of the inherent space-confined enzymatic behavior of T4 polynucleotide kinase phosphatase (T4 PNKP) around a single MB and the heparin's digital-like inhibitory effect on T4 PNKP. By integrating with an on-bead terminal deoxynucleotidyl transferase (TdT)-catalyzed fluorescence signal amplification technology, the concentration of HBP and heparinase can be digitally determined by the number of fluorescence-positive/-negative MBs which can be easily counted by flow cytometry. This is not only the first test to expand the application scenario of T4 PNKP to the digital detection of different biomarkers but also pioneers a new direction for fabricating digital biosensing platforms based on the enzyme inhibition mechanism.

A turn-on fluorescence sensor for detection of heparinase with heparin templated aggregation of tetracationic porphyrin derivative.

Heparinase is the only mammalian endoglycosidase that breaks down the commonly used blood-anticoagulant heparin into therapeutically relevant low-molecular-weight-heparin. Importantly, heparinase has been considered a malignant disease diagnostic marker. Thus, it is essential to develop detection scheme for heparinase. However, optical methods for heparinase determination are limited. In the present work, we report a turn-on fluorescence sensor for detection of heparinase that utilizes heparin-templated aggregation of a tetra-cationic porphyrin derivative, TMPyP4+, as a sensing framework. Heparinase cleaves the glycosidic linkage between hexosamine and uronic acid in the structure of heparin to destroy its polyelectrolytic nature that originally causes the aggregation of TMPyP4+. Thus, heparinase leads to dissociation of TMPyP4+ aggregates and generates an optical signal. This system leads to a sensitive and selective response towards heparinase with a Limit of Detection (LOD) of 0.3 pmol/L. Further, the same system is demonstrated to sense a trace amount of Oversulfated Chondrootin Sulphate (OSCS) in heparin, which is a heparin adulterant, by utilizing the fact that OSCS serves as an inhibitor for heparinase activity, which leads to reverse modulation in the photo-physical features of the monomer/aggregate equilibrium of the TMPyP4+-heparin-heparinase system. The sensing mechanism has been thoroughly demonstrated by ground-state absorption, steady-state emission, and time-resolved emission measurements. The selectivity of the sensor was tested using lysozyme, α-amylase, pepsin, trypsin, lipase, and glucose oxidase in the heparinase selectivity study and the method is also validated using another method reported in the literature. The study provides a new approach for the development of optical methods for the detection of heparinase and oversulfated chondroitin sulfate, which is currently limited.

The unremarkable alveolar epithelial glycocalyx: a thorium dioxide-based electron microscopic comparison after heparinase or pneumolysin treatment.

Recent investigations analyzed in depth the biochemical and biophysical properties of the endothelial glycocalyx. In comparison, this complex cell-covering structure is largely understudied in alveolar epithelial cells. To better characterize the alveolar glycocalyx ultrastructure, unaffected versus injured human lung tissue explants and mouse lungs were analyzed by transmission electron microscopy. Lung tissue was treated with either heparinase (HEP), known to shed glycocalyx components, or pneumolysin (PLY), the exotoxin of Streptococcus pneumoniae not investigated for structural glycocalyx effects so far. Cationic colloidal thorium dioxide (cThO2) particles were used for glycocalyx glycosaminoglycan visualization. The level of cThO2 particles orthogonal to apical cell membranes (≙ stained glycosaminoglycan height) of alveolar epithelial type I (AEI) and type II (AEII) cells was stereologically measured. In addition, cThO2 particle density was studied by dual-axis electron tomography (≙ stained glycosaminoglycan density in three dimensions). For untreated samples, the average cThO2 particle level was ≈ 18 nm for human AEI, ≈ 17 nm for mouse AEI, ≈ 44 nm for human AEII and ≈ 35 nm for mouse AEII. Both treatments, HEP and PLY, resulted in a significant reduction of cThO2 particle levels on human and mouse AEI and AEII. Moreover, a HEP- and PLY-associated reduction in cThO2 particle density was observed. The present study provides quantitative data on the differential glycocalyx distribution on AEI and AEII based on cThO2 and demonstrates alveolar glycocalyx shedding in response to HEP or PLY resulting in a structural reduction in both glycosaminoglycan height and density. Future studies should elucidate the underlying alveolar epithelial cell type-specific distribution of glycocalyx subcomponents for better functional understanding.

Combination of engineering the substrate and Ca2+ binding domains of heparinase I to improve the catalytic activity.

Heparinase I (EC 4.2.2.7), is an enzyme that cleaves heparin, showing great potential for eco-friendly production of low molecular weight heparin (LMWH). However, owing to its poor catalytic activity and thermal stability, the industrial application of heparinase I has been severely hindered. To improve the catalytic activity, we proposed to engineer both the substrate and Ca2+ binding domains of heparinase I. Several heparinases I from different organisms were selected for multiple sequence alignment and molecular docking to screen the key residues in the binding domain. Nine single-point mutations were selected to enhance the catalytic activity of heparinase I. Among them, T250D was the most highly active one, whereas mutations around Ca2+ binding domain yielded two active mutants. Mutant D152S/R244K/T250D with significantly increased catalytic activity was obtained by combined mutation. The catalytic efficiency of the mutant was 118,875.8 min-1·µM-1, which was improved 5.26 times. Molecular modeling revealed that the improved activity and stability of the mutants were probably attributed to the formation of new hydrogen bonds. The highly active mutant had great potential applications in industry and the strategy could be used to improve the performance of other enzymes.

HighlightsImproved catalytic activity of heparinase I by engineering the binding domains of substrate and Ca²⁺.The mutant D152S/R244K/T250D showed the highest catalytic performance.The increased hydrogen bonds attribute to the increased activity.

Interferon-ß alleviates sepsis by SIRT1-mediated blockage of endothelial glycocalyx shedding.

Sepsis is a life-threatening multi-organ dysfunction with high mortality caused by the body's improper response to microbial infection. No new effective therapy has emerged that can adequately treat patients with sepsis. We previously demonstrated that interferon-ß (IFN-ß) protects against sepsis via sirtuin 1-(SIRT1)-mediated immunosuppression. Another study also reported its significant protective effect against acute respiratory distress syndrome, a complication of severe sepsis, in human patients. However, the IFN-ß effect cannot solely be explained by SIRT1-mediated immunosuppression, since sepsis induces immunosuppression in patients. Here, we show that IFN-ß, in combination with nicotinamide riboside (NR), alleviates sepsis by blocking endothelial damage via SIRT1 activation. IFN-ß plus NR protected against cecal ligation puncture-(CLP)-induced sepsis in wild-type mice, but not in endothelial cell-specific Sirt1 knockout (EC-Sirt1 KO) mice. IFN-ß upregulated SIRT1 protein expression in endothelial cells in a protein synthesisindependent manner. IFN-ß plus NR reduced the CLP-induced increase in in vivo endothelial permeability in wild-type, but not EC-Sirt1 KO mice. IFN-ß plus NR suppressed lipopolysaccharide-induced up-regulation of heparinase 1, but the effect was abolished by Sirt1 knockdown in endothelial cells. Our results suggest that IFN-ß plus NR protects against endothelial damage during sepsis via activation of the SIRT1/heparinase 1 pathway. [BMB Reports 2023; 56(5): 314-319].

Heparan Sulfates Regulate Axonal Excitability and Context Generalization through Ca2+/Calmodulin-Dependent Protein Kinase II.

Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of ankyrin G at the axon initial segments in the CA1 region of the hippocampus ex vivo, impaired context discrimination in vivo, and increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in vitro. Here, we show that in vivo delivery of heparinase 1 in the CA1 region of the hippocampus elevated autophosphorylation of CaMKII 24 h after injection in mice. Patch clamp recording in CA1 neurons revealed no significant heparinase effects on the amplitude or frequency of miniature excitatory and inhibitory postsynaptic currents, while the threshold for action potential generation was increased and fewer spikes were generated in response to current injection. Delivery of heparinase on the next day after contextual fear conditioning induced context overgeneralization 24 h after injection. Co-administration of heparinase with the CaMKII inhibitor (autocamtide-2-related inhibitory peptide) rescued neuronal excitability and expression of ankyrin G at the axon initial segment. It also restored context discrimination, suggesting the key role of CaMKII in neuronal signaling downstream of heparan sulfate proteoglycans and highlighting a link between impaired CA1 pyramidal cell excitability and context generalization during recall of contextual memories.

[Advances in heparin structural analysis by chromatography technologies].

Heparin （Hp） is the most widely used anticoagulant drug in the clinics， with an annual global output of over 10 billion dollars. Hp， a member of the glycosaminoglycans （GAGs）， is prepared from porcine intestinal mucosa via extraction， separation， and purification. Hp is a linear polysaccharide with repeating disaccharide units. Low-molecular-weight heparins （LMWHs） are depolymerized from Hp via chemical or enzymatic degradation. Compared with Hp， LMWHs exhibit less bleeding side effect， milder immunogenicity， and higher bioavailability when injected subcutaneously. In general， Hps， including LMWHs， are high complex drugs with large molecular weights （MWs）， inhomogeneous MW distributions， and structural heterogeneity， including different degrees and locations of sulfonation， and unique residues generated from different production processes. Thus， developing efficient analytical methods to elucidate the structures of Hps and characterize or quantitate their properties is extremely challenging. Unfortunately， this problem limits their quality control， production optimization， clinical safety monitoring， and new applications. Research has constantly sought to elucidate the complicated structures of Hp drugs. Among the structural analysis and quality control methods of Hp currently available， chromatographic methods are the most widely studied and used. However， no literature thoroughly summarizes the specific applications of chromatographic methods in the structural analysis， manufacturing process， and quality control of Hp drugs. This paper systematically organizes and describes recent research progresses of the chromatographic methods used to analyze Hp drugs， including the identification and composition of monosaccharides， disaccharides， oligosaccharides， and polysaccharides. The applications， innovations， and limitations of these chromatographic methods are also summarized in this review. The insights obtained in this study will help production and quality control personnel， as well as drug researchers， obtain a deeper understanding of the complex structures of Hp drugs. This paper also provides a comprehensive reference for the structural analysis and quality control of Hps， proposes ideas for the development of new quality control methods， and lays a strong foundation for the in-depth structural elucidation of Hp drugs.

Enzymatic Sequencing of Heparin Oligosaccharides Using Exolyase.

Heparin/heparan sulfate (HP/HS) is a class of acidic polysaccharides with many potential medical applications, especially HP, and its derivatives, low molecular weight heparins (LMWHs), have been widely used as anticoagulants to treat thrombosis for decades. However, the complex structure endows HP/HS a variety of biological functions and hinders the structural and functional studies of HP/HS. Heparinases derived from bacteria are useful tools for the structural studies of HP/HS as well as the preparation of LMWHs. The enzymatic method for the structural analysis of HP/HS chains is easy to operate, requires less samples, and is low cost. Here, we describe an enzymatic approach to investigate the primary sequences of the HP/HS oligosaccharides using a recently discovered exotype heparinase.

Wasp-stung rat model translationally expresses the coagulopathy manifestations of human wasp patients.

Two over 80 wasp stings male victims appeared severe abnormal coagulation were consecutively examined by thromboelastography (TEG) guided with heparinase during hospitalization. However, the cause of coagulopathy remains unsolved. Rats were applied to establish a wasp-stung animal model highly resembled the manifestations of wasp-stung patients. According body surface area conversion, Sprague-Dawley rats were stung based on wasp sting numbers (0, 4, 8, 12 stings; n = 6 each) with various exposure times (0, 1, 3, 6 h) to determine the simulation of coagulopathy. The blood R, K values, and angle degree of wasp-stung rats were measured by TEG. The TEG profiles of stung rats were found to be concomitant with that of wasp-stung patients. Data showed that the endogenous heparinization of rats was time-dependent. Compared to the TEG profile of eight stings given rat, the coagulation time of 2 mm clot formation at 3 h (R value) was longer than that at 0 h. The coagulation time was prolonged with increasing sting numbers when compared to the various stings at 1, 3, and 6 h exposed. Interestingly, there was observed the peak coagulation at 3 h of eight stings. The Ck-standard and Ck-heparinase at 3 h after 8 stings given were R: 9.6-4.4 min; K: 3.8-1.8 min; angle degree: 49.8-68.0, respectively. The original data of R, K values and angle degree in two wasp-stung victims were 11.7-13.6 min, 4.3-5.5 min, and 41.2-32.8° in CK-standard, respectively; whereas those of the CK-heparinase groups were 5.6-6.7 min, 2.4-2.5 min, and 59.5-58.8°, correspondingly. Conclusively, this massive wasp-stung animal model can be applied to the investigations of pathogenesis and provides a clinical strategy or guideline for clinical intervention.

Importance of Heparan Sulfate Proteoglycans in Pancreatic Islets and ß-Cells.

ß-cells in the islets of Langerhans of the pancreas secrete insulin in response to the glucose concentration in the blood. When these pancreatic ß-cells are damaged, diabetes develops through glucose intolerance caused by insufficient insulin secretion. High molecular weight polysaccharides, such as heparin and heparan sulfate (HS) proteoglycans, and HS-degrading enzymes, such as heparinase, participate in the protection, maintenance, and enhancement of the functions of pancreatic islets and ß-cells, and the demand for studies on glycobiology within the field of diabetes research has increased. This review introduces the roles of complex glycoconjugates containing high molecular weight polysaccharides and their degrading enzymes in pancreatic islets and ß-cells, including those obtained in studies conducted by us earlier. In addition, from the perspective of glycobiology, this study proposes the possibility of application to diabetes medicine.

Enzymatic synthesis of low molecular weight heparins from N-sulfo heparosan depolymerized by heparanase or heparin lyase.

Low-molecular-weight heparin (LMWH) is prepared from the controlled chemical or enzymatic depolymerization of animal sourced heparins. It has been widely used as an anticoagulant. Concerns about the shortcomings of animal-derived heparin and the contamination of supply chain demand biochemical approaches for synthesizing LMWH. In the present study, two LMWHs were enzymatically synthesized from low molecular weight N-sulfated heparosan (LMW-NSH) cleaved by recombinant hydrolase, endo-ß-glucuronidase, (HepBp) or heparin lyase III (HepIII), followed by subsequent sulfotransferase modifications. Structural characterization shows that LMWH chains prepared using HepBp had a saturated uronic acid residue at their reducing ends, while chains of LMWH prepared using HepIII had an unsaturated uronic acid residue at their non-reducing end. Both LMWHs had anti-factor Xa and anti-factor IIa activities comparable to enoxaparin. This approach demonstrates that the hydrolase, HepBp, can be used to prepare a new type of LMWH that has no unsaturated uronic acid at its non-reducing end.

Expression and characterization of heparinase II with MBP tag from a novel strain, Raoultella NX-TZ-3-15.

The enzymes are biological macromolecules that biocatalyze certain biochemical reactions without undergoing any modification or degradation at the end of the reaction. In this work, we constructed a recombinant novel Raoultella sp. NX-TZ-3-15 strain that produces heparinase with a maltose binding tag to enhance its production and activity. Additionally, MBP-heparinase was purified and its enzymatic capabilities are investigated to determine its industrial application. Moreover, the recombinant plasmid encoding the MBP-heparinase fusion protein was effectively generated and purified to a high purity. According to SDS-PAGE analysis, the MBP-heparinase has a molecular weight of around 70 kDa and the majority of it being soluble with a maximum activity of 5386 U/L. It has also been noted that the three ions of Ca2 + , Co2 + , and Mg2 + can have an effect on heparinase activities, with Mg2 + being the most noticeable, increasing by about 85%, while Cu2 + , Fe2 + , Zn2 + having an inhibitory effect on heparinase activities. Further investigations on the mechanistic action, structural features, and genomes of Raoultella sp. NX-TZ-3-15 heparinase synthesis are required for industrial-scale manufacturing.

Sequencing analysis of heparin reducing terminals with orthogonal chromatographic approaches.

Heparin is a linear sulfated polysaccharide with a complex structure. It is important to figure out the sequences at the terminals of the sugar chains, as it will help us understand the heparin structure deeper and control its quality properly. The tetrasaccharide linkage region (LR) could be a tag to help us find out heparin terminals after digestion by different combinations of heparinases. In this work, orthogonal chromatographic approaches including SAX, SEC-MS and 2D-LC-MS were applied to qualitatively and quantitatively analyze the heparinase released LR-terminals. The disaccharides next to LR are those ones with low or non-sulfation, UA-GlcNAc and UA-GlcNAc6S, and then they are extended with the highly sulfated disaccharides, IdoA2S-GlcNS and IdoA2S-GlcNS6S. It is suggested that the sulfo transferases did not work at the sugar residues next to LR terminal, especially the 2-O-sulfo and N-sulfo transferases, which could be affected by steric hindrance from LR, when heparin is biosynthesized. This conclusion will be theoretical fundamental to help us understand heparin's structure deeper. The methods provided in this work could be potential ways to control heparin's quality and monitor the production processes of heparin properly.

Cloning and Expression of Heparinase Gene from a Novel Strain Raoultella NX-TZ-3-15.

Heparin is a class of highly sulfated, acidic, linear, and complex polysaccharide that belongs to the heparin/heparan sulfate (HS) glycosaminoglycans family. Enzymatic depolymerization of heparin by heparinases is a promising strategy for the production of ultra-low molecular weight heparins (ULMWHs) as anticoagulants. In the present study, a novel heparinase-producing strain Raoultella NX-TZ-3-15 was isolated and identified from soil samples. Herein, the heparinase gene MBP-H1 was cloned to the pBENT vector to enable expression in Escherichia coli. The optimized conditions made the activity of recombinant heparinase reach the highest level (2140 U/L). The overexpressed MBP-H1 was purified by affinity chromatography and a purity of more than 90% was obtained. The condition for biocatalysis was also optimized and three metal ions Ca2+, Co2+, and Mg2+ were utilized to activate the reaction. In addition, the kinetics regarding the new fusion heparinase was also determined with a Vm value of 11.29 µmol/min and a Km value of 31.2 µmol/L. In short, due to excellent Km and Vmax, the recombinant enzyme has great potential to be used in the clinic in medicine and industrial production of low or ultra-low molecule weight heparin.

Single-Cell Sequencing Analysis and Multiple Machine Learning Methods Identified G0S2 and HPSE as Novel Biomarkers for Abdominal Aortic Aneurysm.

Identifying biomarkers for abdominal aortic aneurysms (AAA) is key to understanding their pathogenesis, developing novel targeted therapeutics, and possibly improving patients outcomes and risk of rupture. Here, we identified AAA biomarkers from public databases using single-cell RNA-sequencing, weighted co-expression network (WGCNA), and differential expression analyses. Additionally, we used the multiple machine learning methods to identify biomarkers that differentiated large AAA from small AAA. Biomarkers were validated using GEO datasets. CIBERSORT was used to assess immune cell infiltration into AAA tissues and investigate the relationship between biomarkers and infiltrating immune cells. Therefore, 288 differentially expressed genes (DEGs) were screened for AAA and normal samples. The identified DEGs were mostly related to inflammatory responses, lipids, and atherosclerosis. For the large and small AAA samples, 17 DEGs, mostly related to necroptosis, were screened. As biomarkers for AAA, G0/G1 switch 2 (G0S2) (Area under the curve [AUC] = 0.861, 0.875, and 0.911, in GSE57691, GSE47472, and GSE7284, respectively) and for large AAA, heparinase (HPSE) (AUC = 0.669 and 0.754, in GSE57691 and GSE98278, respectively) were identified and further verified by qRT-PCR. Immune cell infiltration analysis revealed that the AAA process may be mediated by T follicular helper (Tfh) cells and the large AAA process may also be mediated by Tfh cells, M1, and M2 macrophages. Additionally, G0S2 expression was associated with neutrophils, activated and resting mast cells, M0 and M1 macrophages, regulatory T cells (Tregs), resting dendritic cells, and resting CD4 memory T cells. Moreover, HPSE expression was associated with M0 and M1 macrophages, activated and resting mast cells, Tregs, and resting CD4 memory T cells. Additional, G0S2 may be an effective diagnostic biomarker for AAA, whereas HPSE may be used to confer risk of rupture in large AAAs. Immune cells play a role in the onset and progression of AAA, which may improve its diagnosis and treatment.

Production, characteristics and applications of microbial heparinases.

Heparinases are enzymes that selectively cleave heparin and heparan sulfate chains, via cleavage of the glycosidic linkage between hexosamines and uronic acids, producing disaccharide and oligosaccharide products. While heparin is well known as an anti-coagulant drug, heparin and heparan sulfate are also involved in biological processes such as inflammation, cancer and angiogenesis and viral and bacterial infections and are of growing interest for their therapeutic potential. Recently, potential roles of heparin and heparan sulfate in relation to COVID-19 infection have been highlighted. The ability of heparinases to selectively cleave heparin chains has been exploited industrially to produce low molecular weight heparin, which has replaced heparin in several clinical applications. Other applications of heparinases include heparin and heparan sulfate structural analysis, neutralisation of heparin in blood and removal of the inhibitory effect of heparin on various enzymes. Heparinases are known to inhibit neovascularization and heparinase III is of interest for treating cancer and inhibiting tumour cell growth. Heparinase activity, first isolated from Pedobacter heparinus, has since been reported from several other microorganisms. Significant progress has been made in the production, characterisation and improvement of microbial heparinases in response to application demands in terms of heparinase yield and purity, which is likely to extend their usefulness in various applications. This review focuses on recent developments in the identification, characterisation and improvement of microbial heparinases and their established and emerging industrial, clinical and therapeutic applications.