Mistletoe lectin dissociates into catalytic and binding subunits before translocation across the membrane to the cytoplasm

AbstractHybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A-chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome-inactivating proteins. Anti-MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin

Immobilization of Antioxidant Enzyme Catalase on Porous Hybrid Microparticles of Vaterite with Mucin

Catalase is one of the crucial antioxidant enzymes with diverse applications in textile, food industries, wastewater treatment, cosmetics, and pharmaceutics, which, however, is highly sensitive to environmental challenges. Resisting the loss of activity and prolongation of formulation storage can be achieved via the catalase entrapment into insoluble carriers. Affordable and degradable vaterite is proposed as amicable material for catalase immobilization. To improve the carrier properties of the vaterite, it was co‐precipitated with mucin from the pig's stomach producing ca 5 μm hybrid mucin/vaterite microparticles. Catalase is impregnated into the crystals by means of adsorption without chemical modifications. The presence of mucin matrix partially hinders catalase penetration into the crystals and reduces the adsorption capacity (for 0.1 mg mL−1 catalase, ca 2.3 vs ca 1.5 mg g−1 for pristine and hybrid microparticles, respectively) but significantly promotes the protection of antioxidant activity upon storage and under the action of temperature, organic solvent (acetonitrile), and proteolytic enzyme (trypsin). Hybrid microcrystals are pH‐sensitive and better retain the enzyme at pH 3–5 due to catalase‐mucin complexation. Immobilized catalase can be used for 5–6 consecutive cycles until it loses catalytic activity. Altogether, these findings indicate promises of hybrid mucin/vaterite microparticles for immobilization of antioxidant enzymes

Thermal effects of carbonated hydroxyapatite modified by glycine and albumin

In this work calcium phosphate powders were obtained by precipitation method from simulated solutions of synovial fluid containing glycine and albumin. X-ray diffraction and IR spectroscopy determined that all samples are single-phase and are presented by carbonate containing hydroxyapatite (CHA). The thermograms of solid phases of CHA were obtained and analyzed; five stages of transformation in the temperature range of 25-1000°C were marked. It is shown that in this temperature range dehydration, decarboxylation and thermal degradation of amino acid and protein connected to the surface of solid phase occur. The tendency of temperature lowering of the decomposition of powders synthesized from a medium containing organic substances was determined. Results demonstrate a direct dependence between the concentration of the amino acid in a model solution and its content in the solid phase

Novel Biodegradable Polymeric Microparticles Facilitate Scarless Wound Healing by Promoting Re-epithelialization and Inhibiting Fibrosis

Despite decades of research, the goal of achieving scarless wound healing remains elusive. One of the approaches, treatment with polymeric microcarriers, was shown to promote tissue regeneration in various in vitro models of wound healing. The in vivo effects of such an approach are attributed to transferred cells with polymeric microparticles functioning merely as inert scaffolds. We aimed to establish a bioactive biopolymer carrier that would promote would healing and inhibit scar formation in the murine model of deep skin wounds. Here we characterize two candidate types of microparticles based on fibroin/gelatin or spidroin and show that both types increase re-epithelialization rate and inhibit scar formation during skin wound healing. Interestingly, the effects of these microparticles on inflammatory gene expression and cytokine production by macrophages, fibroblasts, and keratinocytes are distinct. Both types of microparticles, as well as their soluble derivatives, fibroin and spidroin, significantly reduced the expression of profibrotic factors Fgf2 and Ctgf in mouse embryonic fibroblasts. However, only fibroin/gelatin microparticles induced transient inflammatory gene expression and cytokine production leading to an influx of inflammatory Ly6C+ myeloid cells to the injection site. The ability of microparticle carriers of equal proregenerative potential to induce inflammatory response may allow their subsequent adaptation to treatment of wounds with different bioburden and fibrotic content

Fibroblasts upregulate expression of adhesion molecules and promote lymphocyte retention in 3D fibroin/gelatin scaffolds

Bioengineered scaffolds are crucial components in artificial tissue construction. In general, these scaffolds provide inert three-dimensional (3D) surfaces supporting cell growth. However, some scaffolds can affect the phenotype of cultured cells, especially, adherent stromal cells, such as fibroblasts. Here we report on unique properties of 3D fibroin/gelatin materials, which may rapidly induce expression of adhesion molecules, such as ICAM-1 and VCAM-1, in cultured primary murine embryonic fibroblasts (MEFs). In contrast, two-dimensional (2D) fibroin/gelatin films did not show significant effects on gene expression profiles in fibroblasts as compared to 3D culture conditions. Interestingly, TNF expression was induced in MEFs cultured in 3D fibroin/gelatin scaffolds, while genetic or pharmacological TNF ablation resulted in diminished ICAM-1 and VCAM-1 expression by these cells. Using selective MAPK inhibitors, we uncovered critical contribution of JNK to 3D-induced upregulation of these adhesion molecules. Moreover, we observed ICAM-1/VCAM-1-dependent adhesion of lymphocytes to fibroblasts cultured in 3D fibroin/gelatin scaffolds, but not on 2D fibroin/gelatin films, suggesting functional reprogramming in stromal cells, when exposed to 3D environment. Finally, we observed significant infiltration of lymphocytes into 3D fibroin/gelatin, but not into collagen scaffolds in vivo upon subcapsular kidney implantation in mice. Together our data highlight the important features of fibroin/gelatin scaffolds, when they are produced as 3D sponges rather than 2D films, which should be considered when using these materials for tissue engineering

Probing Red Blood Cell Membrane Microviscosity Using Fluorescence Anisotropy Decay Curves of the Lipophilic Dye PKH26

Red blood cell (RBC) aggregation and deformation are governed by the molecular processes occurring on the membrane. Since several social important diseases are accompanied by alterations in RBC aggregation and deformability, it is important to develop a diagnostic parameter of RBC membrane structural integrity and stability. In this work, we propose membrane microviscosity assessed by time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe as a diagnostic parameter. We measured the fluorescence decay curves of the PKH26 probe in the RBC membrane to establish the optimal parameters of the developed fluorescence assay. We observed a complex biphasic profile of the fluorescence anisotropy decay characterized by two correlation times corresponding to the rotational diffusion of free PKH26, and membrane-bounded molecules of the probe. The developed assay allowed us to estimate membrane microviscosity ηm in the range of 100–500 cP depending on the temperature, which paves the way for assessing RBC membrane properties in clinical applications as predictors of blood microrheological abnormalities

CAR-tropic extracellular vesicles carry tumor-associated antigens and modulate CAR T cell functionality

Abstract Tumor-derived extracellular vesicles (EVs) are active contributors in metastasis and immunosuppression in tumor microenvironment. At least some of the EVs carry tumor surface molecules such as tumor-associated antigens (TAAs) and/or checkpoint inhibitors, and potentially could interact with T cells or CAR T cells. Upon contact with T cells, EVs could alter their phenotype and functions by triggering signaling through TCR or CAR reprogramming them to escape immune response. We hypothesize that EVs that possess TAA on the surface will probably interact with CAR T cells which can recognize and bind corresponding TAA. This interaction between EVs and CAR T cells may change the outcome of CAR T-based cancer immunotherapy since it should affect CAR T cells. Also, EVs could serve as adjuvants and antigenic components of antitumor vaccines. Herein, we isolated EVs from B cell precursor leukemia cell line (pre-B ALL) Nalm-6 and demonstrated that recognition and binding of CD19+EVs with CD19-CAR T cells strongly depends on the presence of CD19 antigen. CD19+EVs induce secretion of pro-inflammatory cytokines (IL-2 and IFN-y) and upregulated transcription of activation-related genes (IFNG, IFNGR1, FASLG, IL2) in CD19-CAR T cells. Tumor necrosis factor receptor superfamily (TNFRSF4 and TNFRSF9) and T-cell exhaustion markers (CTLA4, LAG3, TIM3 and PDCD1LG2) were also upregulated in CD19-CAR T cells after incubation with CD19+EVs. Long-term cultivation of CD19+ or PD-L1+EVs with CD19-CAR T cells led to increased terminal differentiation and functional exhaustion according to elevated expression of PD-1, TIGIT, CD57. In summary, our results suggest that chronic exposure of CD19-CAR T cells to CD19+EVs mediates activation and systemic exhaustion in antigen-specific manner, and this negative effect is accompanied by the impaired cytotoxic activity in vitro

Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell’s lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells

Combined Impact of Magnetic Force and Spaceflight Conditions on Escherichia coli Physiology

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows