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ACS Macro Letters Oct 2023The conjugation of a fluorophore and a variety of cell-penetrating peptides onto a RAFT agent allowed for the synthesis of polymers of defined sizes with quantifiable...
The conjugation of a fluorophore and a variety of cell-penetrating peptides onto a RAFT agent allowed for the synthesis of polymers of defined sizes with quantifiable cell-uptake. Each peptide-RAFT agent was used to polymerize acrylamide, acrylate, and styrene monomers to form high or low molecular weight polymers (here 50 or 7.5 kDa) with the peptide having no influence on the RAFT agent's control. The incorporation of a single fluorophore per polymer chain allowed cellular analysis of the uptake of the size-specific peptide-polymers via flow cytometry and confocal microscopy. The cell-penetrating peptides had a direct effect on the efficiency of polymer uptake for both high and low molecular weight polymers, demonstrating the versatility of the strategy. These "all-in-one", synthetically accessible RAFT agents allow highly controlled preparation of synthetic peptide-polymer conjugates and subsequent quantification of their delivery into cells.
Topics: Polymers; Cell-Penetrating Peptides; Lysine; Acrylamide; Styrene
PubMed: 37695265
DOI: 10.1021/acsmacrolett.3c00460 -
Poultry Science Oct 2023DNA vaccination has great potential to treat or prevent avian influenza pandemics, but the technique may be limited by low immunogenicity and gene delivery in clinical...
DNA vaccination has great potential to treat or prevent avian influenza pandemics, but the technique may be limited by low immunogenicity and gene delivery in clinical testing. Here, to improve the immune efficacy of DNA vaccines against avian influenza, we prepared and tested the immunogenicity of 4 recombinant DNA vaccines containing 2 or 3 AIV antigens. The results revealed that chickens and mice immunized with plasmid DNA containing 3 antigens (HA gene from H9N2, and NA and HA genes from H5N1) exhibited a robust immune response than chickens and mice immunized with plasmid DNAs containing 2 antigenic genes. Subsequently, this study used pβH9N1SH5 as a model antigen to study the effect of dendritic polylysine (DGL) nanoparticles as a gene delivery system and adjuvant on antigen-specific immunity in mice models. At a ratio of 1:3 DGL/pβH9N1SH5 (w/w), the pβH9N1SH5/DGL NPs showed excellent physical and chemical properties, induced higher levels of HI antibodies, and larger CD3+/CD4+ T lymphocyte and CD3+/CD8+ T lymphocyte population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2 compared with the naked pβH9N1SH5. Therefore, multiantigen gene expression methods using DGL as a delivery system may have broad application prospects in gene therapy.
Topics: Animals; Mice; Influenza in Birds; Vaccines, DNA; Chickens; Influenza Vaccines; Influenza A Virus, H5N1 Subtype; Influenza A Virus, H9N2 Subtype; Nanoparticles; Immunity; Antibodies, Viral
PubMed: 37542938
DOI: 10.1016/j.psj.2023.102925 -
Molecules (Basel, Switzerland) Jul 2023The aim of the present study was to develop an injectable hydrogel (HG) formulation of fuzapladib sodium (FZP), an animal drug for acute pancreatitis (AP), with the use...
The aim of the present study was to develop an injectable hydrogel (HG) formulation of fuzapladib sodium (FZP), an animal drug for acute pancreatitis (AP), with the use of polyethyleneoxide (PEO) and polylysine (pLys), a cationic polymer. A mixture of pLys and FZP was added to PEO to prepare an HG formulation, and the formulation was optimized by release test and viscosity measurements. Circular dichroism (CD) and infrared absorption (IR) spectral analyses were applied to clarify the intermolecular interactions between FZP and pLys. The pharmacokinetic behavior of FZP was evaluated after a subcutaneous administration of FZP samples (2.0 mg-FZP/kg) to rats. Although the immediate release of FZP was observed for the HG formulation, the addition of pLys at a 20-fold amount of FZP or higher led to the sustained release of FZP. Considering release behavior, the concentration of pLys was optimized as 100-fold that of FZP in the HG formulation. CD and IR spectroscopic analyses of FZP and/or pLys demonstrated an intermolecular interaction between FZP and pLys, as evidenced by the slight spectral transition. After a subcutaneous administration of HG formulation containing pLys to rats, compared with FZP alone, significant differences were observed in the pharmacokinetic behavior with a decrease of from 2.3 to 0.9 mg/mL and slower elimination kinetics. HG formulation using pLys might be a viable dosage option for FZP for the treatment of AP in animals.
Topics: Rats; Animals; Polylysine; Hydrogels; Delayed-Action Preparations; Lymphocyte Function-Associated Antigen-1; Acute Disease; Pancreatitis; Leukocytes
PubMed: 37513199
DOI: 10.3390/molecules28145325 -
International Journal of Molecular... Jul 2023The outer membrane of Gram-negative bacteria contains a variety of pore-forming structures collectively referred to as porins. Some of these are voltage dependent, but...
The outer membrane of Gram-negative bacteria contains a variety of pore-forming structures collectively referred to as porins. Some of these are voltage dependent, but weakly so, closing at high voltages. Triplin, a novel bacterial pore-former, is a three-pore structure, highly voltage dependent, with a complex gating process. The three pores close sequentially: pore 1 at positive potentials, 2 at negative and 3 at positive. A positive domain containing 14 positive charges (the voltage sensor) translocates through the membrane during the closing process, and the translocation is proposed to take place by the domain entering the pore and thus blocking it, resulting in the closed conformation. This mechanism of pore closure is supported by kinetic measurements that show that in the closing process the voltage sensor travels through most of the transmembrane voltage before reaching the energy barrier. Voltage-dependent blockage of the pores by polyarginine, but not by a 500-fold higher concentrations of polylysine, is consistent with the model of pore closure, with the sensor consisting mainly of arginine residues, and with the presence, in each pore, of a complementary surface that serves as a binding site for the sensor.
Topics: Humans; Ion Channel Gating; Porins; Thiourea; Translocation, Genetic
PubMed: 37511231
DOI: 10.3390/ijms241411473 -
Biosensors Jul 2023A highly sensitive unlabeled electrochemical aptasensor based on hydroxylated black phosphorus/poly-L-lysine (hBP/PLL) composite is introduced herein for the detection...
A highly sensitive unlabeled electrochemical aptasensor based on hydroxylated black phosphorus/poly-L-lysine (hBP/PLL) composite is introduced herein for the detection of malathion. Poly-L-lysine (PLL) with adhesion and coating properties adhere to the surface of the nanosheets by noncovalent interactions with underlying hydroxylated black phosphorus nanosheets (hBP) to produce the hBP/PLL composite. The as-synthesized hBP/PLL composite bonded to Au nanoparticles (Au NPs) firmly by assembling and using them as a substrate for the aptamer with high specificity as a probe to fabricate the sensor. Under optimal conditions, the linear range of the electrochemical aptasensor was 0.1 pM~1 μM, and the detection limit was 2.805 fM. The electrochemical aptasensor has great selectivity, a low detection limit, and anti-interference, which has potential application prospects in the field of rapid trace detection of pesticide residues.
Topics: Metal Nanoparticles; Malathion; Polylysine; Electrochemical Techniques; Gold; Phosphorus; Aptamers, Nucleotide; Limit of Detection; Biosensing Techniques
PubMed: 37504133
DOI: 10.3390/bios13070735 -
Journal of the Mechanical Behavior of... Sep 2023The aim was to quantify effects of polylysine (PLS, 2 or 5 wt%) and monocalcium phosphate (MCP, 4 or 8 wt%) on properties of dental composites.
OBJECTIVES
The aim was to quantify effects of polylysine (PLS, 2 or 5 wt%) and monocalcium phosphate (MCP, 4 or 8 wt%) on properties of dental composites.
METHODS
Light-activated, lower surface polymerisation kinetics versus sample depth (1-4 mm) of 4 formulations were quantified using ATR-FTIR. Water sorption and solubility (at 1 week) were assessed following ISO/4049. PLS release (over 1 month) and biaxial flexural strength (over 6 months) of fully-cured, water-immersed, 1 mm thick discs were determined. Surface mineral precipitation, following immersion in simulated body fluid (SBF), was assessed by SEM. Z250 was used as a conventional composite comparator.
RESULTS
With 40s light exposure, increasing depth (from 1 to 4 mm) led to enhanced delay before polymerisation (from 3 to 17s) and decreased final conversion (72-66%) irrespective of PLS and MCP level. Increasing PLS and MCP raised solubility (4-13 μg/mm). Water sorption (between 32 and 55 μg/mm) and final PLS release (8-13% of disc content) were raised primarily by increasing PLS. Higher PLS also reduced strength. Strength reached minimum values (69-94 MPa) at 3 months. Surface mineral deposition was enhanced by increased MCP. For Z250, polymerisation delays (3-6s) and final conversions (55-54%) at 1-4 mm depth, solubility (0 μg/mm), water sorption (16 μg/mm) and strength (180 MPa) were all significantly different.
CONCLUSION
Delay time increased whilst final conversion decreased with thicker samples. Higher PLS enhances its percentage release, but lower level is required to keep water sorption, solubility and mechanical properties within ISO 4049 recommendations. Doubling MCP raises solubility and enhances minerals reprecipitation with minimal mechanical property compromise.
Topics: Polylysine; Composite Resins; Materials Testing; Solubility; Phosphates; Water; Surface Properties; Dental Materials
PubMed: 37499523
DOI: 10.1016/j.jmbbm.2023.106039 -
Microbiology (Reading, England) Jul 2023Over the past decades, antibiotic resistance has become a major clinical problem, and searching for new therapeutic strategies seems to be necessary. Using novel natural...
Over the past decades, antibiotic resistance has become a major clinical problem, and searching for new therapeutic strategies seems to be necessary. Using novel natural compounds, antimicrobial peptides, and bacteriophages is the most promising solution. In this study, various cationic metabolite-producer bacteria were isolated from different soil samples. Two isolates were identified as HS4 (accession number: MW791428) and HS5 (accession number: MW791430) based on biochemical characteristics and phylogenetic analysis using 16S rRNA gene sequences. The cationic compound in the fermentation broth was precipitated and purified with sodium tetraphenylborate salt. The purified cationic peptide was confirmed to be epsilon-poly-l-lysine by structural and molecular analysis using High-Performance Liquid Chromatography, Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, and Fourier-transform infrared spectroscopy. The antibacterial activity of epsilon-poly-l-lysine was evaluated against ATCC 25923, ATCC 25922, ATCC 29212, ATCC 13880, and ATCC 13883 by microdilution method. Furthermore, the antibacterial effects of purified epsilon-poly-l-lysine in combination with two long non-contractile tail bacteriophages against vancomycin-resistant and colistin-resistant were investigated. The results indicated great antibacterial activity of epsilon-poly-l-lysine which was produced by two novel bacteria. The epsilon-poly-l-lysine as a potent cationic antimicrobial peptide is demonstrated to possess great antimicrobial activity against pathogenic and also antibiotic-resistant bacteria.
Topics: Polylysine; Stenotrophomonas maltophilia; Paenibacillus polymyxa; RNA, Ribosomal, 16S; Phylogeny; Anti-Bacterial Agents; Bacteria; Antimicrobial Cationic Peptides; Microbial Sensitivity Tests
PubMed: 37477972
DOI: 10.1099/mic.0.001363 -
Foods (Basel, Switzerland) Jul 2023In the present study, electrospun nanofiber mats were fabricated by mixing different ratios (96:4, 95:5, 94:6, 93:7, and 92:8) of Persian gum (PG) and poly (ethylene...
Fabrication of ɛ-Polylysine-Loaded Electrospun Nanofiber Mats from Persian Gum-Poly (Ethylene Oxide) and Evaluation of Their Physicochemical and Antimicrobial Properties.
In the present study, electrospun nanofiber mats were fabricated by mixing different ratios (96:4, 95:5, 94:6, 93:7, and 92:8) of Persian gum (PG) and poly (ethylene oxide) (PEO). The SEM micrographs revealed that the nanofibers obtained from 93% PG and 7% PEO were bead-free and uniform. Therefore, it was selected as the optimized ratio of PG:PEO for the development of antimicrobial nanofibers loaded with ɛ-Polylysine (ɛ-PL). All of the spinning solutions showed pseudoplastic behavior and the viscosity decreased by increasing the shear rate. Additionally, the apparent viscosity, G', and G″ of the spinning solutions increased as a function of PEO concentration, and the incorporation of ɛ-PL did not affect these parameters. The electrical conductivity of the solutions decreased when increasing the PEO ratio and with the incorporation of ɛ-PL. The X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectra showed the compatibility of polymers. The antimicrobial activity of nanofibers against () and () was investigated, and the samples loaded with ɛ-PL demonstrated stronger antimicrobial activity against
PubMed: 37444326
DOI: 10.3390/foods12132588 -
Journal of Biomedical Optics Aug 2023Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster...
SIGNIFICANCE
Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use.
AIM
We prove that absolute FRET efficiency can be measured on a simple setup, an order of magnitude more cost-effective than a standard FRET microscopy setup, by applying it to vinculin tension sensors (VinTS) at the focal adhesions of live CHO-K1 cells.
APPROACH
Our setup located at Université Paris-Saclay acquires donor and acceptor fluorescence in parallel on two low-cost CMOS cameras and uses two LEDs for rapid switching of the excitation wavelength at a reduced cost. The calibration required to extract FRET efficiency was achieved using a single construct (TSMod). FRET efficiencies were measured for VinTS and the tail-less control VinTL, lacking the actin-binding domain of vinculin. Measurements were confirmed on the same cell type using a more standard intensity-based setup located at Rutgers University.
RESULTS
The average FRET efficiency of VinTS () over more than 10,000 focal adhesions is significantly lower () than that of VinTL (), our control that is insensitive to force, in agreement with the force exerted on vinculin at focal adhesions. Attachment of the CHO-K1 cells on fibronectin decreases FRET efficiency, thus increasing the force, compared with poly-lysine. FRET efficiency for the VinTL control is consistent with all measurements currently available in the literature, confirming the validity of our measurements and hence of our simpler setup.
CONCLUSIONS
Force measurements, resolved spatially inside a cell, can be achieved using FRET-based tension sensors with a cost effective intensity-based setup. This will facilitate combining FRET with techniques for applying controlled forces such as optical tweezers.
Topics: Humans; Fluorescence Resonance Energy Transfer; Focal Adhesions; Vinculin; Cost-Benefit Analysis; Mechanical Phenomena
PubMed: 37441563
DOI: 10.1117/1.JBO.28.8.082808 -
Frontiers in Cellular Neuroscience 2023Glass coverslips are used as a substrate since Harrison's initial nerve cell culture experiments in 1910. In 1974, the first study of brain cells seeded onto polylysine...
INTRODUCTION
Glass coverslips are used as a substrate since Harrison's initial nerve cell culture experiments in 1910. In 1974, the first study of brain cells seeded onto polylysine (PL) coated substrate was published. Usually, neurons adhere quickly to PL coating. However, maintaining cortical neurons in culture on PL coating for a prolonged time is challenging.
METHODS
A collaborative study between chemical engineers and neurobiologists was conducted to find a simple method to enhance neuronal maturation on poly-D-lysine (PDL). In this work, a simple protocol to coat PDL efficiently on coverslips is presented, characterized, and compared to a conventional adsorption method. We studied the adhesion and maturation of primary cortical neurons with various morphological and functional approaches, including phase contrast microscopy, immunocytochemistry, scanning electron microscopy, patch clamp recordings, and calcium imaging.
RESULTS
We observed that several parameters of neuronal maturation are influenced by the substrate: neurons develop more dense and extended networks and synaptic activity is enhanced, when seeded on covalently bound PDL compared to adsorbed PDL.
DISCUSSION
Hence, we established reproducible and optimal conditions enhancing maturation of primary cortical neurons . Our method allows higher reliability and yield of results and could also be profitable for laboratories using PL with other cell types.
PubMed: 37416506
DOI: 10.3389/fncel.2023.1212097