Heparin Binding Research Articles

Time-course remodeling and pathology intervention of α-synuclein amyloid fibril by heparin and heparin-like oligosaccharides.

Amyloid fibrils represent a pathological state of protein polymer that is closely associated with various neurodegenerative diseases. Polysaccharides have a prominent role in recognizing amyloid fibrils and mediating their pathogenicity. However, the mechanism underlying the amyloid-polysaccharide interaction remains elusive. We also do not know its impact on the structure and pathology of formed fibrils. Here, we used cryo-electron microscopy to analyze the atomic structures of mature α-synuclein (α-syn) fibrils upon binding with polymeric heparin and heparin-like oligosaccharides. The fibril structure, including the helical twist and conformation of α-syn, changed over time upon the binding of heparin but not oligosaccharides. The sulfation pattern and numbers of saccharide units are important for the binding. Similarly, negatively charged biopolymers typically interact with amyloid fibrils, including tau and various α-syn polymorphs, leading to alterations in their conformation. Moreover, we show that heparin-like oligosaccharides can not only block neuronal uptake and propagation of formed α-syn fibrils but also inhibit α-syn fibrillation. This work demonstrates a distinctive activity of heparin and biopolymers in remodeling amyloid fibrils and suggests the pharmaceutical potential of heparin-like oligosaccharides.

Synthesis, Biological Evaluation and Reversal of Sulfonated Di- and Triblock Copolymers as Novel Parenteral Anticoagulants.

Despite targeting different coagulation cascade sites, all Food and Drug Administration-approved anticoagulants present an elevated risk of bleeding, including potentially life-threatening intracranial hemorrhage. Existing studies have not thoroughly investigated the efficacy and safety of sulfonate polymers in animal models and fully elucidate the precise mechanisms by which these polymers act. The activity and safety of sulfonated di- and triblock copolymers containing poly(sodium styrenesulfonate) (PSSS), poly(sodium 2-acrylamido-2-methylpropanesulfonate) (PAMPS), poly(ethylene glycol) (PEG), poly(sodium methacrylate) (PMAAS), poly(acrylic acid) (PAA), and poly(sodium 11-acrylamidoundecanoate) (PAaU) blocks are synthesized and assessed. PSSS-based copolymers exhibit greater anticoagulant activity than PAMPS-based ones. Their activity is mainly affected by the total concentration of sulfonate groups and molecular weight. PEG-containing copolymers demonstrate a better safety profile than PAA-containing ones. The selected copolymer PEG47-PSSS32 exhibits potent anticoagulant activity in rodents after subcutaneous and intravenous administration. Heparin Binding Copolymer (HBC) completely reverses the anticoagulant activity of polymer in rat and human plasma. No interaction with platelets is observed. Selected copolymer targets mainly factor XII and fibrinogen, and to a lesser extent factors X, IX, VIII, and II, suggesting potential application in blood-contacting biomaterials for anticoagulation purposes. Further studies are needed to explore its therapeutic applications fully.

Refinement and optimisation of Neisseria gonorrhoeae NHBA and MetQ vaccine candidates

Neisseria gonorrhoeae has a significant impact on reproductive health with an estimated 82 million new cases of infection per year worldwide. Due to the ongoing emergence of multidrug-resistant N. gonorrhoeae strains, the high number of asymptomatic cases, and the risk of disease sequelae, the development of a gonococcal vaccine is urgently needed. We have previously described two potential gonococcal vaccine antigens, cNHBA (C-terminal fragment of the Neisseria Heparin Binding Antigen) and MetQ (methionine-binding protein). This study aimed to optimise these antigens for improved immune responses and to facilitate vaccine production, by investigating cNHBA fusions with the full-length MetQ protein or N-terminal and C-terminal MetQ fragments (Met1 and Met2, respectively) adjuvanted with aluminium hydroxide. The cNHBA and MetQ fragments and fusion antigens were all immunogenic in mice, generating a predominantly IgG1 response. Antibodies mediated bacterial killing via both serum bactericidal activity (SBA) and opsonophagocytic activity (OPA), and reduced adherence to cervical and urethral epithelial cells. Among the antigen fusions tested, MetQ-cNHBA and cNHBA-Met2 generated the highest SBA, OPA and adherence blocking titres and are proposed as promising optimised antigens for N. gonorrhoeae vaccine development.

Predicting gene signature in breast cancer patients with multiple machine learning models

AimsThe aim of this study was to predict gene signatures in breast cancer patients using multiple machine learning models.MethodsIn this study, we first collated and merged the datasets GSE54002 and GSE22820, obtaining a gene expression matrix comprising 16,820 genes (including 593 breast cancer (BC) samples and 26 normal control (NC) samples). Subsequently, we performed enrichment analyses using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Disease Ontology (DO).ResultsWe identified 177 differentially expressed genes (DEGs), including 40 up-regulated and 137 down-regulated genes, through differential expression analysis. The GO enrichment results indicated that these genes are primarily involved in extracellular matrix organization, positive regulation of nervous system development, collagen-containing extracellular matrix, heparin binding, glycosaminoglycan binding, and Wnt protein binding, among others. KEGG enrichment analysis revealed that the DEGs were primarily associated with pathways such as focal adhesion, the PI3K–Akt signaling pathway, and human papillomavirus infection. DO enrichment analysis showed that the DEGs play a significant role in regulating diseases such as intestinal disorders, nephritis, and dermatitis. Further, through LASSO regression analysis and SVM-RFE algorithm analysis, we identified 9 key feature DEGs (CF-DEGs): ANGPTL7, TSHZ2, SDPR, CLCA4, PAMR1, MME, CXCL2, ADAMTS5, and KIT. Additionally, ROC curve analysis demonstrated that these CF-DEGs serve as a reliable diagnostic index. Finally, using the CIBERSORT algorithm, we analyzed the infiltration of immune cells and the associations between CF-DEGs and immune cell infiltration across all samples.ConclusionsOur findings provide new insights into the molecular functions and metabolic pathways involved in breast cancer, potentially aiding in the discovery of new diagnostic and immunotherapeutic biomarkers.

Characterisation and Immunogenicity of Neisseria cinerea outer membrane vesicles displaying NadA, NHBA and fHbp from Neisseria meningitidis serogroup B.

More affordable and effective vaccines against bacterial meningitis caused by Neisseria meningitidis serogroup B are still required for global prevention. We have previously shown that modified outer membrane vesicles (mOMVs) from commensal Neisseria cinerea can be used as a platform to induce immune responses against meningococcal antigens. The aim of the present study was to use a combination of two genetically engineered mOMVs to express multiple antigens from N. meningitidis known to be involved in protective immunity to meningococcal meningitis (different variants of factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisseria Adhesin A (NadA)). Antigen expression in the mOMVs was confirmed by Western blotting; detoxification of the lipooligosaccharide (LOS) was confirmed by measuring human Toll-like receptor 4 (hTLR4) activation using in vitro cell assays. Mice immunised with a combination of two mOMVs expressing fHbp, NHBA and NadA produced antibodies to all the antigens. Furthermore, serum bactericidal activity (SBA) was induced by the immunisation, with mOMVs expressing NadA displaying high SBA titres against a nadA+ MenB strain. The work highlights the potential of mOMVs from N. cinerea to induce functional immune responses against multiple antigens involved in the protective immune response to meningococcal disease.

Fgf17: A regulator of the mid/hind brain boundary in mammals

The Fibroblast growth factor (FGFs) family consists of at least 22 members that exert their function by binding and activating fibroblast growth factor receptors (FGFRs). The Fgf8/FgfD subfamily member, Fgf17, is located on human chromosome 8p21.3 and mouse chromosome 14 D2. In humans, FGF17 can be alternatively spliced to produce two isoforms (FGF17a and b) whereas three isoforms are present in mice (Fgf17a, b, and c), however, only Fgf17a and Fgf17b produce functional proteins. Fgf17 is a secreted protein with a cleavable N-terminal signal peptide and contains two binding domains, namely a conserved core region and a heparin binding site. Fgf17 mRNA is expressed in a wide range of different tissues during development, including the rostral patterning centre, midbrain-hindbrain boundary, tailbud mesoderm, olfactory placode, mammary glands, and smooth muscle precursors of major arteries. Given its broad expression pattern during development, it is surprising that adult Fgf17−/− mice displayed a rather mild phenotype; such that mutants only exhibited morphological changes in the frontal cortex and mid/hind brain boundary and changes in certain social behaviours. In humans, FGF17 mutations are implicated in several diseases, including Congenital Hypogonadotropic Hypogonadism and Kallmann Syndrome. FGF17 mutations contribute to CHH/KS in 1.1% of affected individuals, often presenting in conjunction with mutations in other FGF pathway genes like FGFR1 and FLRT3. FGF17 mutations were also identified in patients diagnosed with Dandy-Walker malformation and Pituitary Stalk Interruption Syndrome, however, it remains unclear how FGF17 is implicated in these diseases. Altered FGF17 expression has been observed in several cancers, including prostate cancer, hematopoietic cancers (acute myeloid leukemia and acute lymphoblastic leukemia), glioblastomas, perineural invasion in cervical cancer, and renal cell carcinomas. Furthermore, FGF17 has demonstrated neuroprotective effects, particularly during ischemic stroke, and has been shown to improve cognitive function in ageing mice.

Reprint of: Fibroblast Growth Factor 6

Fibroblast Growth Factor 6 (FGF6), also referred to as HST2 or HBGF6, is a member of the Fibroblast Growth Factor (FGF), the Heparin Binding Growth Factor (HBGF) and the Heparin Binding Secretory Transforming Gene (HST) families. The genomic and protein structure of FGF6 is highly conserved among varied species, as is its expression in muscle and muscle progenitor cells. Like other members of the FGF family, FGF6 regulates cell proliferation, differentiation, and migration. Specifically, it plays key roles in myogenesis and muscular regeneration, angiogenesis, along with iron transport and lipid metabolism. Similar to others from the FGF family, FGF6 also possesses oncogenic transforming activity, and as such is implicated in a variety of cancers.

Structural Insights into Endostatin-Heparan Sulfate Interactions Using Modeling Approaches.

Glycosaminoglycans (GAGs) play a key role in a variety of biological processes in the extracellular matrix (ECM) via interactions with their protein targets. Due to their high flexibility, periodicity and electrostatics-driven interactions, GAG-containing complexes are very challenging to characterize both experimentally and in silico. In this study, we, for the first time, systematically analyzed the interactions of endostatin, a proteolytic fragment of collagen XVIII known to be anti-angiogenic and anti-tumoral, with heparin (HP) and representative heparan sulfate (HS) oligosaccharides of various lengths, sequences and sulfation patterns. We first used conventional molecular docking and a docking approach based on a repulsive scaling-replica exchange molecular dynamics technique, as well as unbiased molecular dynamic simulations, to obtain dynamically stable GAG binding poses. Then, the corresponding free energies of binding were calculated and the amino acid residues that contribute the most to GAG binding were identified. We also investigated the potential influence of Zn2+ on endostatin-HP complexes using computational approaches. These data provide new atomistic details of the molecular mechanism of HP's binding to endostatin, which will contribute to a better understanding of its interplay with proteoglycans at the cell surface and in the extracellular matrix.

Identification of small molecule antagonists of sonic hedgehog/heparin binding with activity in hedgehog functional assays

Sonic hedgehog (Shh) is a morphogen with important roles in embryonic development and in the development of a number of cancers. Its activity is modulated by interactions with binding partners and co-receptors including heparin and heparin sulfate proteoglycans (HSPG). To identify antagonists of Shh/heparin binding, a diverse collection of 34,560 chemicals was screened in single point 384-well format. We identified and confirmed twenty six novel small molecule antagonists with diverse structures including four scaffolds that gave rise to multiple hits. Nineteen of the confirmed hits blocked binding of the N-terminal fragment of Shh (ShhN) to heparin with IC50 values < 50 μM. In the Shh-responsive C3H10T1/2 cell model, four of the compounds demonstrated the ability to block ShhN-induced alkaline phosphatase activity. To demonstrate a direct and selective effect on ShhN ligand mediated activity, two of the compounds were able to block induction of Gli1 mRNA, a primary downstream marker for Shh signaling activity, in Shh-mediated but not Smoothened agonist (SAG)-mediated C3H10T1/2 cells. Direct binding of the two compounds to ShhN was confirmed by thermal shift assay and molecular docking simulations, with both compounds docking with the N-terminal heparin binding domain of Shh. Overall, our findings indicate that small molecule compounds that block ShhN binding to heparin and act to inhibit Shh mediated activity in vitro can be identified. We propose that the interaction between Shh and HSPGs provides a novel target for identifying small molecules that bind Shh, potentially leading to novel tool compounds to probe Shh ligand function.

Intrinsically Disordered Proteins on Au Surfaces

Intrinsically disordered protein Tau, is a neuronal protein and one of the biomarkers of neurodegenerative diseases. Tau protein undergoes conformational changes, post-translational modifications, and aggregation, and is a viable drug target. We reported on using electrochemical impedance spectroscopy and cyclic voltammetry to study various aspects of tau protein biochemistry on Au surfaces (1-2). By immobilizing the tau protein onto the Au surface, the interactions with other proteins, ligands, and enzymes were investigated. Data show that the electrochemical methods are viable biophysical tools that provide new biochemical information which is not always possible with traditional biochemical methods. References A) Carlin, N., Martic-Milne, S. (2018). Anti-tau antibodies based electrochemical sensor for detection of tau protein. J. Electrochem. Soc.165: G3018-G3025. B) Jahshan, A., Esteves, J.O.V., Martic-Milne, S. (2016). Evaluation of ferritin and transferrin binding to tau protein. J. Inorg. Biochem. 162: 127-134. Trzeciakiewicz, H., Esteves, J.O.V., Carlin, N., Martic, S. (2015). Electrochemistry of heparin binding to tau protein on Au surfaces. Electrochim. Acta. 162: 24-30.

Alteration of Neuropilin-1 and Heparan Sulfate Interaction Impairs Murine B16 Tumor Growth.

Neuropilin-1 acts as a coreceptor with vascular endothelial growth factor receptors to facilitate binding of its ligand, vascular endothelial growth factor. Neuropilin-1 also binds to heparan sulfate, but the functional significance of this interaction has not been established. A combinatorial library screening using heparin oligosaccharides followed by molecular dynamics simulations of a heparin tetradecasaccharide suggested a highly conserved binding site composed of amino acid residues extending across the b1 and b2 domains of murine neuropilin-1. Mutagenesis studies established the importance of arginine513 and lysine514 for binding of heparin to a recombinant form of Nrp1 composed of the a1, a2, b1, and b2 domains. Recombinant Nrp1 protein bearing R513A,K514A mutations showed a significant loss of heparin-binding, heparin-induced dimerization, and heparin-dependent thermal stabilization. Isothermal calorimetry experiments suggested a 1:2 complex of heparin tetradecasaccharide:Nrp1. To study the impact of altered heparin binding in vivo, a mutant allele of Nrp1 bearing the R513A,K514A mutations was created in mice (Nrp1D) and crossbred to Nrp1+/- mice to examine the impact of altered heparan sulfate binding. Analysis of tumor formation showed variable effects on tumor growth in Nrp1D/D mice, resulting in a frank reduction in tumor growth in Nrp1D/- mice. Expression of mutant Nrp1D protein was normal in tissues, suggesting that the reduction in tumor growth was due to the altered binding of heparin/heparan sulfate to neuropilin-1. These findings suggest that the interaction of neuropilin-1 with heparan sulfate modulates its stability and its role in tumor formation and growth.

Engineered intrinsically fluorescent galectin-8 variants with altered valency, ligand recognition and biological activity

Galectin-8 is a small soluble lectin with two carbohydrate recognition domains (CRDs). N- and C-terminal CRDs of Gal-8 differ in their specificity for glycan ligands. Here, we wanted to find out whether oligomerization of individual CRDs of galectin-8 affects its biological activity. Using green fluorescent protein polygons (GFPp) as an oligomerization scaffold, we generated intrinsically fluorescent CRDs with altered valency. We show that oligomers of C-CRD are characterized by significant cell surface affinity. Furthermore, the multivalency of the resulting variants has an impact on cellular activities such as cell signaling, heparin binding and proliferation. Our data indicates that tunable valence is a useful tool for modifying the biological activity of CRDs of galectins.

Exploitation of porphyrin-based titanium-rich porous organic polymers for targeted phosphopeptide enrichment from the serum of colorectal cancer individuals.

Aporphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol),highselectivity (β-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143mg·g-1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC-MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC.

Changes in the chikungunya virus E1 glycoprotein domain II and hinge influence E2 conformation, infectivity, and virus-receptor interactions.

In a previous study to understand how the chikungunya virus (CHIKV) E1 glycoprotein β-strand c functions, we identified several attenuating variants at E1 residue V80 and the emergence of second-site mutations in the fusion loop (E1-M88L) and hinge region (E1-N20Y) with the V80 variants in vivo. The emergence of these mutations led us to question how changes in E1 may contribute to CHIKV infection at the molecular level. Here, we use molecular dynamics to understand how changes in the E1 glycoprotein may influence the CHIKV glycoprotein E1-E2 complex. We found that E1 domain II variants lead to E2 conformational changes, allowing us to hypothesize that emerging variants E1-M88L and E1-N20Y could also change E2 conformation and function. We characterized CHIKV E1-M88L and E1-N20Y in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. We found that CHIKV E1-N20Y enhanced infectivity in mosquito cells, while the CHIKV E1-M88L variant enhanced infectivity in both BHK-21 and C6/36 cells and led to changes in viral cholesterol-dependence. Moreover, we found that E1-M88L and E1-N20Y changed E2 conformation, heparin binding, and interactions with the receptor Mxra8. Interestingly, the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, yet was attenuated in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type-dependent entry. Taken together, these studies show that key residues in the CHIKV E1 domain II and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry.IMPORTANCEArboviruses are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo to address these gaps in our knowledge. Here, we show that changes in the CHIKV E1 domain II and hinge alter E2 conformations leading to changes in virus-receptor and -glycosaminoglycan interactions and cell-specific infection. These results highlight that adaptive changes in E1 can have a major effect on virus attachment and entry, furthering our knowledge of how alphaviruses infect mammals and insects.

Unlocking precision gene therapy: harnessing AAV tropism with nanobody swapping at capsid hotspots.

Adeno-associated virus (AAV) has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2/5-fold wall and 5-fold pore of the AAV capsid that are amenable to insertion of larger protein domains, including nanobodies. Here, we demonstrate that these hotspots facilitate AAV tropism switching through simple nanobody replacement without extensive optimization in both VP1 and VP2. Our data suggest that engineering VP2 is the preferred path for maintaining both virus production yield and infectivity. We demonstrate highly specific targeting of human cancer cells expressing fibroblast activating protein (FAP). Furthermore, we found that the combination of FAP nanobody insertion plus ablation of the heparin binding domain can reduce off-target infection to a minimum, while maintaining a strong infection of FAP receptor-positive cells. Taken together, our study shows that nanobody swapping at multiple capsid locations is a viable strategy for nanobody-directed cell-specific AAV targeting.

Determination of molecular pathways and gene ontology of genes associated with Raynaud’s phenomenon

Abstract Objectives Raynaud’s phenomenon (RF) is a disease that causes discoloration of the fingers. The purpose of this study is to identify the molecular pathways in which genes related to RP illness are involved, as well as uncover the biological processes and molecular functions connected with those genes via the use of gene ontology (GO) analysis. Methods Genes associated with RP in the MalaCards Human Diseases database were detected. Twenty genes obtained from the MalaCards Human Diseases database were included in the study for gene ontology analysis via the STRING database. Accordingly, possible interactions between 20 genes were determined through STRING and network enrichment was performed. Results A significant enrichment by gene ontology enrichment analysis was detected in a subset of genes involved in biological processes including cellular response to luteinizing hormone stimulus, negative regulation of fibrinolysis, negative regulation of smooth muscle cell apoptotic process, plasminogen activation, cellular response to follicle-stimulating hormone stimulus. The assay for molecular function determined enrichment of a subset of genes in chemoattractant activity, growth factor activity, heparin binding, sulfur compound binding, growth factor receptor binding. Through the use of KEGG pathways, we were able to identify many molecular processes that contribute to RP, including the AGE-RAGE signaling pathway in diabetic complications, complement and coagulation cascades, fluid shear stress, atherosclerosis. Conclusions Some individuals may have a genetic predisposition to the onset of Raynaud’s phenomenon. Our data showed that it is associated with genes involved in vascular damage and fibrosis, especially in RP. Therefore, we can include RP disease in the group of vascular diseases.

Ti4+ functionalized zirconium metal-organic frameworks with polymer brushes for specific identification of phosphopeptides in human serum and skimmed milk.

In the present study, click chemistry and Schiff base reactions were simultaneously applied to prepare polymer brush (PEG)-functionalized MOF materials (UiO-66-NH2) and immobilized with Ti4+ (MOF-Brush-THBA-Ti4+) for phosphopeptide analysis. The material has a detection limit of 0.5 fmol, a selectivity of 2000:1, and a loading capacity of 133 mg/g for phosphopeptides. It also demonstrated great repeatability (10 cycles) and recovery rate (96.7 ± 1.4%). During the analysis of bio-samples, 4 specific phosphopeptides were identified in endogenous breast cancer serum, while 11 phosphopeptides were identified in skimmed milk. Moreover, 47 phosphopeptides correlated with 29 phosphorylated proteins were selectively identified from normal control serum, and 66 phosphopeptides correlated with 26 phosphorylated proteins were identified from breast cancer serum. Further analysis of gene ontology (GO) revealed that the detected phosphorylated proteins associated with breast cancer included positive regulation of receptor-mediated endocytosis, proteolysis, extracellular exosome, heparin binding, and chaperone binding. These findings suggest that these associated pathways might contribute to the etiology of breast cancer. Overall, this application exhibits enormous potential in the identification of phosphorylated peptides within bio-samples.

Molecular Mechanisms of the Impaired Heparin Pentasaccharide Interactions in 10 Antithrombin Heparin Binding Site Mutants Revealed by Enhanced Sampling Molecular Dynamics.

Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments.

MMP-2/GSH-responsive nanobomb for tumor chemo-immunotherapy with hierarchical delivery and TAMs re-education by regulating hbegf-activated ERK/MAPK signal pathway

The low targeting of immunity regulators & chemotherapy drugs and immune tolerance in the tumor microenvironment significantly hindered the chemo-immunotherapy effect. In this work, an MMP-2/GSH-responsive nanobomb loading Resiquimod (R848) and prodrug Cisplatin (Pt) with hierarchical drug delivery and tumor associated macrophages (TAMs) polarization traits was designed for improved chemo-immunotherapy. The Pt-carrying dendrimers penetrate the depth of tumor tissues under MMP-2 and activate the cytotoxicity of Pt prodrug in response to GSH, causing effective tumor damage and enhancing immunogenicity. Meanwhile, R848 is effectively uptake by TAMs, which were re-educated and then greatly relieved immune resistance. In vitro and in vivo results collectively revealed that the nanosystem could not only overcome the barrier of tumor penetration and the heterogeneity action sites of drugs but also re-educate TAMs and suppress immune evasion via activating extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signal pathway regulated by heparin binding EGF like growth factor (Hbegf) gene. The gene silencing or knockout in vitro and in vivo demonstrated that Hbegf could regulate the re-education of TAMs and the progression of tumors.

Heparin binding protein as a reliable prognostic biomarker for severity of sepsis in the intensive care unit

ABSTRACT Background It is still challenging to identify people who are at risk for developing sepsis quickly and early. Heparin-binding protein (HBP) has been demonstrated a promising data that can be used as predictive qualities in identifying organ failure. Methods This prospective observational investigation of 55 adult patients who have been proven to have sepsis, and were hospitalized into the intensive care unit. By carrying out HBP, procalcitonin (PROCAL), C-reactive protein (CRP), serum lactate, SOFA score on admission and after 72 hours and detecting 28-day mortality. Results Despite PROCAL and HBP were higher in survival than non-survival patients at day 0 (1010.32 ± 341.72 vs 770.21 ± 327.97, p = 0.0112) (16.73 ± 7.19 vs 13.19 ± 7.26, p = 0.077) respectively, It was significantly lower in survival than non-survival at day 3 (542.09 ± 191.98 vs 995.00 ± 333.74, p =<0.0001) (9.03 ± 2.92 vs 16.67 ± 7.55,p =<0.0001) respectively. Our main marker HBP decreased significantly from day 0 to day 3 for survival patients with paired difference −7.69 ± 6.78 with p value < 0.0001, while it is increased with non-significant value for non-survival patients with paired difference 3.48 ± 9.45 with p value 0.084. ROC analysis for mortality showed for HBP that AUC at day 0 was 0.323 (p=0.025). At cut-off value of>15.5ng/ml, sensitivity was 29.2%, specificity was 64.5%, while at day 3 was 0.831 (p=0.000). At cut-off value of>9.5ng/ml, sensitivity was 83.33%, specificity was 77.42%. Conclusion HPB showed a strong prognostic marker of mortality in ICU septic patients at day 3 more than day 0 with important value and trend.