Although biomedical devices have greatly evolved, none of the materials that have been used to date are able to meet all the hemocompatibility criteria. The rapid accumulation of proteins at the ...implant surface and the subsequent physiological response are the main causes of failure. Thus, the appropriate design of antithrombotic materials is of the utmost importance. In this work, we employed Carbothane® electrospun matrices (PCU) for lysine surface modification, using oligomers obtained from allyl glycidyl ether (AGE) reaction as spacers. This technique enables the binding of several lysine molecules per urethane linkage, which, along with the large surface-to-volume ratio of the electrospun membranes, leads to high ε-amino free lysine grafting (29 ± 2 nmol cm−2). The incorporation of AGE oligomers significantly reduced the nonspecific protein adsorption, while further modification with lysine led to a more pronounced decrease (25% for BSA, 35% for fibrinogen, and 30% for PNP proteins, with respect to PCU membranes). The lysine-modified matrices presented increased plasminogen adsorption capacity and in vitro clot lysis ability after incubation in pooled normal human plasma and tissue plasminogen activator, confirming the plasminogen adsorption selectivity and thus improving the hemocompatibility behavior of these matrices. Therefore, the obtained electrospun membranes are promising coatings for biomedical devices with fibrinolytic activity.In this work, we employed Carbothane® electrospun matrices (PCU) for lysine surface modification, using oligomers obtained from allyl glycidyl ether (AGE) reaction as spacers. The incorporation of AGE oligomers significantly reduced the nonspecific protein adsorption, while further modification with lysine led to a more pronounced decrease. The lysine-modified matrices presented increased plasminogen adsorption capacity and in vitro clot lysis ability, confirming the plasminogen adsorption selectivity and thus improving the hemocompatibility behavior of these matrices.
•StSBTc-3 activity is optimized using response surface methodology.•StSBTc-3 has fibrinogenolytic activity at physiological conditions.•StSBTc-3 has potential use in biomedical devices.•rsm package ...with R is a suitable tool to perform plant protease activity optimizations.
The aim of this study was to optimize in vitro conditions to enhance fibrinogenolytic activity of Solanum tuberosum subtilisin-like protease (StSBTc-3). The effects of StSTBc-3 concentration (0.2–5 μM), pH value (6–10) and temperature (35–50 °C) on fibrinogenolytic activity were studied through response surface methodology (RSM). We obtained a model that predicts the response accurately. The relationship between enzyme concentration and fibrinogenolytic activity was linear, while the main effect from pH and temperature on the response was quadratic. From the RSM generated model the optimum pH was 8 and the optimum temperature was 43 °C, while higher concentrations of enzyme produce higher activities.
Under optimum conditions there were no statistically significant differences between the experimental responses and the ones predicted from the model. This model also predicts the activity under physiological conditions. These results confirm that StSTBc-3 is a good candidate to be considered for therapeutic uses. The generated model will be useful for biotechnological purposes.
The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. ...The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bβ3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bβ3 preventing the interaction between α2bβ3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.
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•StSBTc-3 inhibits platelet aggregation.•Platelet aggregation inhibition by StSBTc-3 is agonist independent.•StSBTc-3 reduces the binding of fibrinogen onto platelets.•StSBTc-3 shows strong interaction with α2bβ3 integrin.
The use of green-soluble inhibitors in the corrosive medium as alternatives to traditional inhibitors has increased due to the toxicity of the commonly used substances. These novel substances are ...selected owing to their low cost, ease of application as well as maintenance, and low environmental risk. This work aims to evaluate ethanolic-water extracts from post-harvest soybean (Glycine max) by-products as corrosion inhibitors for mild steel in an aggressive medium of sodium chloride. Soybean extracts were obtained by percolation at a controlled flow rate and temperature. To evaluate the anticorrosive efficiency, polarization curves of AISI 1030 steel were carried out at 1 and 7 days of immersion with different concentrations of the soybean extract. Weight loss measurements were carried out alongside potentiodynamic measurements. The steel samples were analyzed by scanning electron microscopy. The soybean extracts obtained reduced the corrosion rate of the steel, showing an efficiency of 30% at day 1. The inhibition efficiency increased up to 80% after 7 days of incubation with 2000 ppm of the extract. A Langmuir adsorption model was fitted to weight loss measurements. K
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obtained from the model were characteristic of physisorption. The corrosion potential was shifted toward more negative values, classifying the ethanolic-water soybean extract as a cathodic inhibitor. The steel surface for the samples incubated with 2000 ppm of soybean extract was greatly improved showing significantly fewer agglomeration of corrosion products. Soybean leaves are a promising by-product useful to produce ethanolic-water extracts to be used as green corrosion inhibitors.
To identify new coagulant enzymes as rennet substitutes, the aim of this study was to determine and to characterise the milk-clotting activity (MCA) of two potato aspartic proteases (StAPs). Both ...enzymes exhibited MCA in a dose-dependent manner. Optimum MCA values were determined at pH 5 and 30 °C. The ability of StAPs to degrade casein subunits was also evaluated. β-Casein was preferentially hydrolysed by both StAPs, followed by αS-casein and, to a lesser extent, κ-casein. These results confirmed the suitability of StAPs for producing curd and the possibility of using these proteases in artisanal cheese production.
The plant-specific insert of Solanum tuberosum aspartic proteases (StAP-PSI) has high structural similarity with NK-lysin and granulysin, two saposin-like proteins (SAPLIPs) with antimicrobial ...activity. Recombinant StAP-PSI and some SAPLIPs show antimicrobial activity against pathogens that affect human and plants. In this work, we transformed Arabidopsis thaliana plants with StAP-PSI encoding sequence with its corresponding signal peptide under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Results obtained show that StAP-PSI significantly enhances Arabidopsis resistance against Botrytis cinerea infection. StAP-PSI is secreted into the leaf apoplast and acts directly against pathogens; thereby complementing plant innate immune responses. Data obtained from real-time PCR assays show that the constitutive expression of StAP-PSI induces the expression of genes that regulate jasmonic acid signalling pathway, such as PDF1.2, in response to infection due to necrotrophic pathogens. On the other hand, according to the data described for other antimicrobial peptides, the presence of the StAP-PSI protein in the apoplast of A. thaliana leaves is responsible for the expression of salicylic acid-associated genes, such as PR-1, irrespective of infection with B. cinerea. These results indicate that the increased resistance demonstrated by A. thaliana plants that constitutively express StAP-PSI owing to B. cinerea infection compared to the wild-type plants is a consequence of two factors, i.e., the antifungal activity of StAP-PSI and the overexpression of A. thaliana defense genes induced by the constitutive expression of StAP-PSI. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the use of pesticides.
We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the spreading of resistance of agriculturally important pathogens.
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•The constitutive expression of StAP-PSI induces defense genes in Arabidopsis thaliana.•The StAP-PSI domain exerts cytotoxic activity toward Botrytis cinerea.•The constitutive expression of StAP-PSI reduces B. cinerea infection in A. thaliana.•The constitutive expression of StAP-PSI increases growth in A. thaliana.
Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable success as an immunotherapy for hematological malignancies, and its potential for treating solid tumors is an active area of ...research. However, limited trafficking and mobility of T cells within the tumor microenvironment (TME) present challenges for CAR T cell therapy in solid tumors. To gain a better understanding of CAR T cell function in solid tumors, we subjected CD70-specific CAR T cells to a challenge by evaluating their immune trafficking and infiltration through a confined 3D microchannel network in a bio-conjugated liquid-like solid (LLS) medium. Our results demonstrated successful CAR T cell migration and anti-tumor activity against CD70-expressing glioblastoma and osteosarcoma tumors. Through comprehensive analysis of cytokines and chemokines, combined with in situ imaging, we elucidated that immune recruitment occurred via chemotaxis, and the effector-to-target ratio plays an important role in overall antitumor function. Furthermore, through single-cell collection and transcriptomic profiling, we identified differential gene expression among the immune subpopulations. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach. STATEMENT OF SIGNIFICANCE: The use of specialized immune cells named CAR T cells to combat cancers has demonstrated remarkable success against blood cancers. However, this success is not replicated in solid tumors, such as brain or bone cancers, mainly due to the physical barriers of these solid tumors. Currently, preclinical technologies do not allow for reliable evaluation of tumor-immune cell interactions. To better study these specialized CAR T cells, we have developed an innovative in vitro three-dimensional model that promises to dissect the interactions between tumors and CAR T cells at the single-cell level. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach.
Abstract
BACKGROUND
In recent years, emerging evidence indicates that tumor-associated myeloid cells (TAMCs) affect cancer progression. But the molecular mechanisms and signaling pathways involved in ...the TAMCs’ interaction with extracellular matrix (ECM) and tumor stroma are incompletely understood. Checkpoint inhibition therapy targets immune inhibitory receptors, such as CTLA-4 and PD-1, which have become a major weapon in fighting cancer. These antibodies demonstrate apparent advantages such as being easy to use, broad applicability, and durable clinical response. Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR1), also called CD305, a collagen-binding immunoreceptor tyrosine-based inhibition motifs (ITIM)-bearing inhibitory receptor, was identified to present on almost all immune cell populations and overexpressed in cancers of human patients. However, LAIR1’s role and regulation in solid cancers are poorly defined.
OBJECTIVE
To identify LAIR1 as a new class of immune checkpoints in cancers that impacts TAMCs-associated tumor immunity. Further functional investigation warrants understanding the crosstalk between LAIR1-related immune checkpoint blocking agent(s), immune micro-environment, and its underlying mechanisms for targeted therapy development.
METHODS
Murine GBM cell line KR158-Luciferase cell line was used to set up the mouse model. Tumor-bearing mice were administered the Anti-LAIR1 blockade and IgG, followed by IVIS imaging for tumor growth and survival were recorded. The presentation and phenotype of immune cell populations in tumors and spleens were measured through scRNA-Seq.
RESULTS
GBM is one of these tumors that overexpress LAIR1 based on the TCGA analysis. LAIR1 molecules express highly on macrophages, monocytes, DCs, and activated T cells, not on the naive T cells. We found that LAIR1 blockade enhanced survival in preclinical GBM models. LAIR1 blockade reduced the presentation and function of TAMCs in tumors. What's more, LAIR1 blockade provided a synergetic antitumor effect with CD70 CAR T cells.
CONCLUSION
This study suggests that we identified a novel immune checkpoint molecule, LAIR1, which can limit tumor progression.
Abstract
BACKGROUND
Our research study focuses on the recently discovered immune checkpoint known as Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR1), which is found to be overexpressed on ...tumor-associated myeloid cells (MAMC), including Tumor-associated macrophages (TAM) and Myeloid-derived suppressive cells (MDSC), in GBM and other solid cancers. The overexpression of LAIR1 in gliomas is associated with poor survival, which has prompted investigations into the potential of LAIR1 blockade (aLAIR1) as a therapeutic strategy. In preclinical mouse models, Objective: A) To investigate the mechanism of LAIR1, a new class of immune checkpoint. B) To develop strategies for blocking LAIR1 signaling as a means of cancer therapeutics, thereby enabling novel mono and combinatorial treatments for GBM.
METHODS
1) Conduct coculture experiments using human TAMs and autologous CAR T cells in a 3D microgel bio-printing system, which allows us to recreate TME using patient-derived immune cells and a heterogeneous glioma model. By monitoring this coculture system, we aim to gain insights into the inhibitory effects of TAMs on T cells mediated by LAIR1. 2) Utilize tumor-burden wild-type and LAIR1 knockout mice to compare the intratumoral immune cell infiltration between WT and KO mice. 3) Employ sc-RNA-seq to identify key molecules involved in the TAM-T cell interaction. 4) Perform pathway analysis to identify signaling pathways that are influenced by this interaction.
RESULTS
aLAIR1 shifted macrophage polarization from M1 to M2 and increased memory CD8+ T cells intratumorally. It reversed MDSC- or M2- macrophage-mediated T-cell suppression, resulting in enhanced antitumor response as a standalone treatment and when used in combination with CAR T cell therapy in PD-1-resistant tumor models.
CONCLUSIONS
LAIR1 is a newly identified innate immune checkpoint that is highly expressed on TAMC in solid tumors, contributing to tumor progression and drug resistance. Blocking the LAIR1 signaling pathway shows promise as an effective approach for cancer therapy.
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