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Journal of Neuroscience Methods May 2023The blood-brain barrier (BBB) is a protective cellular anatomical layer with a dynamic micro-environment, tightly regulating the transport of materials across it. To...
The blood-brain barrier (BBB) is a protective cellular anatomical layer with a dynamic micro-environment, tightly regulating the transport of materials across it. To achieve in-vivo characteristics, an in-vitro BBB model requires the constituent cell types to be layered in an appropriate order. A cost-effective in-vitro BBB model is desired to facilitate central nervous system (CNS) drug penetration studies. Enhanced integrity of tight junctions observed during the in-vitro BBB establishment and post-experiment is essential in these models. We successfully developed an in-vitro BBB model mimicking the in-vivo cell composition and a distinct order of seeding primary human brain cells. Unlike other in-vitro BBB models, our work avoids the need for pre-coated plates for cell adhesion and provides better cell visualization during the procedure. We found that using bovine collagen-I coating, followed by bovine fibronectin coating and poly-L-lysine coating, yields better adhesion and layering of cells on the transwell membrane compared to earlier reported use of collagen and poly-L-lysine only. Our results indicated better cell visibility and imaging with the polyester transwell membrane as well as point to a higher and more stable Trans Endothelial Electrical Resistance values in this plate. In addition, we found that the addition of zinc induced higher claudin 5 expressions in neuronal cells. Dolutegravir, a drug used in the treatment of HIV, is known to appear in moderate concentrations in the CNS. Thus, dolutegravir was used to assess the functionality of the final model and cells. Using primary cells and an in-house coating strategy substantially reduces costs and provides superior imaging of cells and their tight junction protein expression. Our 4-cell-based BBB model is a suitable experimental model for the drug screening process.
Topics: Animals; Cattle; Humans; Blood-Brain Barrier; Cell Line; Polylysine; Endothelial Cells; Microscopy, Confocal
PubMed: 37116621
DOI: 10.1016/j.jneumeth.2023.109867 -
Journal of Extracellular Vesicles Nov 2021Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) possess a great therapeutical potential for osteoarthritis (OA) treatment. However, the steric and...
Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) possess a great therapeutical potential for osteoarthritis (OA) treatment. However, the steric and electrostatic hindrance of cartilage matrix leads to very limited distribution of MSC-sEVs in cartilage and low bioavailability of MSC-sEVs after intra-articular injection. To overcome this, a strategy to reverse the surface charge of MSC-sEVs by modifying the MSC-sEVs with a novel cationic amphiphilic macromolecule namely ε-polylysine-polyethylene-distearyl phosphatidylethanolamine (PPD) was developed in this study. Through incubation with 100 μg/ml PPD, positively charged MSC-sEVs (PPD-sEVs) were obtained, and the modification process showed nearly no disturbance to the integrity and contents of sEVs and exhibited good stability under the interference of anionic macromolecules. A more effective cellular uptake and homeostasis modulation ability of PPD-sEVs than unmodified MSC-sEVs to chondrocytes was demonstrated. More importantly, PPD-sEVs demonstrated significantly enhanced cartilage uptake, cartilage penetration, and joint retention capacity as compared to MSC-sEVs. Intra-articular injection of PPD-sEVs into a mouse OA model showed significantly improved bioavailability than MSC-sEVs, which resulted in enhanced therapeutic efficacy with reduced injection frequency. In general, this study provides a facile and effective strategy to improve the intra-articular bioavailability of MSC-sEVs and has a great potential to accelerate the clinical practice of MSC-sEVs based OA therapy.
Topics: Adolescent; Animals; Cartilage; Cell- and Tissue-Based Therapy; Cells, Cultured; Chondrocytes; Disease Models, Animal; Extracellular Vesicles; Humans; Induced Pluripotent Stem Cells; Injections, Intra-Articular; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Osteoarthritis; Phosphatidylethanolamines; Polyethylene Glycols; Polylysine; Swine; Treatment Outcome
PubMed: 34724347
DOI: 10.1002/jev2.12160 -
Bioorganic & Medicinal Chemistry Feb 2016Raloxifene, a selective estrogen receptor modulator (SERM), reduces fracture risk at least in part by improving the mechanical properties of bone in a cell- and estrogen...
Raloxifene, a selective estrogen receptor modulator (SERM), reduces fracture risk at least in part by improving the mechanical properties of bone in a cell- and estrogen receptor-independent manner. In this study, we determined that raloxifene directly interacts with the bone tissue. Through the use of multiple and complementary biophysical techniques including nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR), we show that raloxifene interacts specifically with the organic component or the organic/mineral composite, and not with hydroxyapatite. Structure-activity studies reveal that the basic side chain of raloxifene is an instrumental determinant in the interaction with bone. Thus, truncation of portions of the side chain reduces bone binding and also diminishes the increase in mechanical properties. Our results support a model wherein the piperidine interacts with bone matrix through electrostatic interactions with the piperidine nitrogen and through hydrophobic interactions (van der Waals) with the aliphatic groups in the side chain and the benzothiophene core. Furthermore, in silico prediction of the potential binding sites on the surface of collagen revealed the presence of a groove with sufficient space to accommodate raloxifene analogs. The hydroxyl groups on the benzothiophene nucleus, which are necessary for binding of SERMs to the estrogen receptor, are not required for binding to the bone surface, but mediate a more robust binding of the compound to the bone powder. In conclusion, we report herein a novel property of raloxifene analogs that allows them to interact with the bone tissue through potential contacts with the organic matrix and in particular collagen.
Topics: Animals; Bone Matrix; Collagen; Dogs; Durapatite; Femur; Hydrophobic and Hydrophilic Interactions; Male; Piperidines; Polylysine; Protein Binding; Raloxifene Hydrochloride; Receptors, Estrogen; Static Electricity; Structure-Activity Relationship; Thiophenes
PubMed: 26795112
DOI: 10.1016/j.bmc.2015.12.045 -
Microbial Cell Factories Mar 2023ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL...
BACKGROUND
ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered.
RESULTS
In this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2B from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2B for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains.
CONCLUSIONS
In this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites.
Topics: Polylysine; Fermentation; Adenosine Triphosphate; Regeneration
PubMed: 36918890
DOI: 10.1186/s12934-023-02057-7 -
Biochimica Et Biophysica Acta.... Jun 2018The Na,K-ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na and K electrochemical potential gradients across...
The Na,K-ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na and K electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state P and H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations.
Topics: Amino Acid Sequence; Animals; Cell Membrane; Conserved Sequence; Molecular Docking Simulation; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Polylysine; Protein Conformation; Sequence Alignment; Sequence Homology, Amino Acid; Sodium-Potassium-Exchanging ATPase; Spectrophotometry, Ultraviolet; Sus scrofa
PubMed: 29522705
DOI: 10.1016/j.bbamem.2018.03.002 -
Journal of Microbiology and... 2013We recently showed that polylysine, the polymer of lysines, retains anti-prion activity. Although the effectiveness of prion inhibition by polylysine was demonstrated...
We recently showed that polylysine, the polymer of lysines, retains anti-prion activity. Although the effectiveness of prion inhibition by polylysine was demonstrated with the regimen tolerated in mice, a determination of quantitative polylysine toxicity is necessary to precisely address the in vivo toxicity level of polylysine. In this communication, we report the results of body weight monitoring and hematologic tests performed in CD-1 mice that received two different tolerable dosages of polylysine for an either 5-day or 4-week period. We found that there was no significant alteration in overall serum chemistry, blood cell count, and body weight gain compared with controls. The only notable quantitative change with statistical significance was the decrease of platelet numbers in mice subchronically administered with polylysine. Our results suggest that polylysine is harmless in mice if administered for a short period, but administrations of polylysine in mice may require considerate attention for safety in future investigations as mice chronically receive tolerable doses of polylysine.
Topics: Animals; Blood Chemical Analysis; Body Weight; Mice; Platelet Count; Polylysine
PubMed: 23711515
DOI: 10.4014/jmb.1302.02055 -
Journal For Immunotherapy of Cancer Jun 2021While adoptive transfer of T-cells has been a major medical breakthrough for patients with B cell malignancies, the development of safe and effective T-cell-based...
BACKGROUND
While adoptive transfer of T-cells has been a major medical breakthrough for patients with B cell malignancies, the development of safe and effective T-cell-based immunotherapy for central nervous system (CNS) tumors, such as glioblastoma (GBM), still needs to overcome multiple challenges, including effective homing and persistence of T-cells. Based on previous observations that interleukin (IL)-17-producing T-cells can traffic to the CNS in autoimmune conditions, we evaluated CD8 T-cells that produce IL-17 and interferon-γ (IFN-γ) (Tc17-1) cells in a preclinical GBM model.
METHODS
We differentiated Pmel-1 CD8 T-cells into Tc17-1 cells and compared their phenotypic and functional characteristics with those of IFN-γ-producing CD8 T (Tc1) and IL-17-producing CD8 T (Tc17) cells. We also evaluated the therapeutic efficacy, persistence, and tumor-homing of Tc17-1 cells in comparison to Tc1 cells using a mouse GL261 glioma model.
RESULTS
In vitro, Tc17-1 cells demonstrated profiles of both Tc1 and Tc17 cells, including production of both IFN-γ and IL-17, although Tc17-1 cells demonstrated lesser degrees of antigen-specific cytotoxic activity compared with Tc1 cells. In mice-bearing intracranial GL261-Quad tumor and treated with temozolomide, Tc1 cells, but not Tc17-1, showed a significant prolongation of survival. However, when the T-cell transfer was combined with poly-ICLC and Pmel-1 peptide vaccine, both Tc1 and Tc17-1 cells exhibited significantly prolonged survival associated with upregulation of very late activation antigen-4 on Tc17-1 cells in vivo. Glioma cells that recurred following the therapy lost the susceptibility to Pmel-1-derived cytotoxic T-cells, indicating that immuno-editing was a mechanism of the acquired resistance.
CONCLUSIONS
Tc17-1 cells were equally effective as Tc1 cells when combined with poly-ICLC and peptide vaccine treatment.
Topics: CD8-Positive T-Lymphocytes; Carboxymethylcellulose Sodium; Glioma; Humans; Interferon-gamma; Interleukin-17; Poly I-C; Polylysine; Vaccines, Subunit
PubMed: 34193567
DOI: 10.1136/jitc-2021-002426 -
Biomaterials May 2016The ability to construct easily in vitro networks of primary neurons organized with imposed topologies is required for neural tissue engineering as well as for the...
The ability to construct easily in vitro networks of primary neurons organized with imposed topologies is required for neural tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. However, accumulating evidence suggests that the mechanical properties of the culture matrix can modulate important neuronal functions such as growth, extension, branching and activity. Here we designed robust and reproducible laminin-polylysine grid micropatterns on cell culture substrates that have similar biochemical properties but a 100-fold difference in Young's modulus to investigate the role of the matrix rigidity on the formation and activity of cortical neuronal networks. We found that cell bodies of primary cortical neurons gradually accumulate in circular islands, whereas axonal extensions spread on linear tracks to connect circular islands. Our findings indicate that migration of cortical neurons is enhanced on soft substrates, leading to a faster formation of neuronal networks. Furthermore, the pre-synaptic density was two times higher on stiff substrates and consistently the number of action potentials and miniature synaptic currents was enhanced on stiff substrates. Taken together, our results provide compelling evidence to indicate that matrix stiffness is a key parameter to modulate the growth dynamics, synaptic density and electrophysiological activity of cortical neuronal networks, thus providing useful information on scaffold design for neural tissue engineering.
Topics: Action Potentials; Animals; Biocompatible Materials; Cell Adhesion; Cell Culture Techniques; Cell Movement; Cells, Cultured; Cerebellar Cortex; Elastic Modulus; Laminin; Nerve Net; Neurons; Polylysine; Rats; Tissue Engineering
PubMed: 26946402
DOI: 10.1016/j.biomaterials.2016.02.041 -
European Journal of Histochemistry : EJH May 2017In a single transient middle cerebral artery occlusion model of stroke and using immunohistochemical techniques, the effects of a new therapeutic approach named Gemst (a...
In a single transient middle cerebral artery occlusion model of stroke and using immunohistochemical techniques, the effects of a new therapeutic approach named Gemst (a member of the Poly-L-Lysine innovative therapies) have been studied in the rat brain. The expression of inflammatory (CD45, CD11b), oxidative (NO-tryptophan, NO2-tyrosine) and indoleamine 2, 3-dioxygenase pathway (kynurenic acid, 3-hydroxy anthranilic acid) markers has been evaluated in early and late phases of stroke. For this purpose, we have developed eight highly specific monoclonal antibodies directed against some of these markers. In the early phase (3 and 5 days of the stroke, we observed no effect of Gemst treatment (7.5 mg/day, subcutaneously for 3, 5 days). In the late phase (21 days) of stroke and exclusively in the ipsilateral side of non-treated animals an overexpression of kynurenic acid, 3-hydroxy anthranilic acid, CD45, CD11b, GFAP and ionized calcium-binding adapter molecule 1 (IBA-1) was found. In treated animals, the overexpression of the four former markers was completely abolished whereas the overexpression of the two latter ones was decreased down to normal levels. Gemst reversed the pathological conditions of stroke to normal situations. Gemst exerts a multifunctional action: down-regulates the indoleamine 2, 3-dioxygenase pathway and abolishes brain infiltration, microglial activation and gliosis. Moreover, Gemst has no effect on the expression of doublecortin, a protein involved in neuronal migration. Gemst could be a new drug for the treatment of stroke since it reverses the pathological findings of stroke and normalizes brain tissue conditions following the ischemic insult.
Topics: Animals; Brain; Doublecortin Protein; Drug Combinations; Enzyme-Linked Immunosorbent Assay; Gene Expression; Immunohistochemistry; Male; Neuroprotective Agents; Polylysine; Rats; Stroke
PubMed: 28735520
DOI: 10.4081/ejh.2017.2790 -
Biomacromolecules Nov 2010Complexes of recombinant silk-polylysine molecules with ppTG1 peptide, a lysine-rich cell membrane destabilizing peptide to bind plasmid DNA (pDNA), are designed as...
Complexes of recombinant silk-polylysine molecules with ppTG1 peptide, a lysine-rich cell membrane destabilizing peptide to bind plasmid DNA (pDNA), are designed as less-cytotoxic and highly efficient gene carriers. The peptide destabilizes the cell membrane and promotes gene transfer. Our particular interest is in how ppTG1 enhances transfection efficiency of the silk-based delivery system into human cells. Genetically engineered silk proteins containing polylysine and the monomeric and dimeric ppTG1 sequences are synthesized in Escherichia coli , followed by transfection experiments. The pDNA complexes of silk-polylysine-ppTG1 dimer recombinant proteins prepared at an N/P 2 (the ratio of number of amines/phosphates from pDNA) shows the highest transfection efficiency into human embryonic kidney (HEK) cells, the level of which is comparable to the transfection reagent Lipofectamine 2000. The assemblies show a globular morphology with an average hydrodynamic diameter of 99 nm and almost no β-sheet structure. Additionally, the silk-based pDNA complexes demonstrate excellent DNase resistance as well as efficient release of the pDNA by enzymes that degrade silk proteins. Also, comparison with β-sheet induced silk-based pDNA complexes indicates that the β-sheet structure content of the silk sequence of the pDNA complexes controls the enzymatic degradation rate of the complexes and, hence, can regulate the release profile of genes from the complexes. The bioengineered silk-based gene delivery vehicles containing cell membrane destabilizing peptides are therefore concluded to have potential for a less-toxic and controlled-release gene delivery system.
Topics: Cell Membrane; Cell Survival; Cells, Cultured; DNA; HEK293 Cells; Humans; Peptides; Plasmids; Polylysine; Silk; Structure-Activity Relationship; Transfection
PubMed: 20942485
DOI: 10.1021/bm101055m