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Microscopy Research and Technique Apr 2022Bacterial sample preparation is crucial for its observation by scanning electron microscopy (SEM). However, the current polylysine (PLL) method leads to bacterial...
Bacterial sample preparation is crucial for its observation by scanning electron microscopy (SEM). However, the current polylysine (PLL) method leads to bacterial morphological changes. To overcome this problem, we employed chitosan (CS) to coat coverslips to prepare bacteria for SEM and compared it with the PLL method. Coverslips coated with 0.025% (w/v) CS showed satisfactory bacterial binding ability. Within 30 min of binding time, the number of bacteria on CS-coated and PLL-coated coverslips exhibited no differences. Four bacteria strains were employed to compare the differences in SEM images between the two methods. Most of the bacteria showed irregular surface or sticky substances after settling on PLL-coated coverslips, while bacteria with clear surface texture were observed on CS-coated coverslips. Transmission electron microscopy (TEM) images showed deformed bacterial envelope on PLL-coated coverslips; meanwhile, similar intact envelope was observed from the bacteria on CS-coated coverslips and the bacteria without any treatment. The TEM results verified the morphological differences of SEM between the two methods. Except for morphology, the length of the rod-shaped bacteria was longer on CS-coated coverslips than that on PLL-coated coverslips, less shrinkage of the sample was observed, and CS could preserve the length of the rod-shaped bacteria better than PLL in its preparation for SEM. It is demonstrated that the low-cost CS could be utilized in bacterial preparation for SEM to acquire preferable images. Bacterial suspension with optical density at 600 nm of about 0.5, deposited on 0.025% CS-coated coverslips for 30 min, and followed by routine fixation, dehydration, and drying are optimal parameters.
Topics: Bacteria; Chitosan; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Polylysine
PubMed: 34851006
DOI: 10.1002/jemt.23992 -
Small (Weinheim An Der Bergstrasse,... Mar 2022Encapsulation of live cells in protective, semipermeable microcapsules is one of the kernel techniques for in vitro tissue regeneration, cell therapies, and...
Encapsulation of live cells in protective, semipermeable microcapsules is one of the kernel techniques for in vitro tissue regeneration, cell therapies, and pharmaceutical screening. Advanced fabrication techniques for cell encapsulation have been developed to meet different requirements. Existing cell encapsulation techniques place substantial constraints on the spatial patterning of live cells as well as on the compartmentalization of heterotypic cells. Alginate-Poly-L-lysine-alginate (APA) microcapsules that use sodium alginate as the polyanion and poly-L-lysine (PLL) as the polycation have been extensively employed for cell microencapsulation due to their excellent biocompatibility and biodegradability. This study proposes a novel method for developing programmable Janus APA microcapsules with variable shapes and sizes by using electrodeposition. By the versatile design of the microelectrode device, sequential electrodeposition is triggered to electro-address the cells at specific locations immobilized within a Janus APA microcapsule. The osteogenesis is evaluated by resembling cell compartmentalized and vascularized osteoblast-laden constructs. This technique allows precise spatial patterning of heterotypic cells inside the APA microcapsule, enabling the observation of cellular growth, interactions, and differentiation in a well-controlled chemical and mechanical microenvironment.
Topics: Alginates; Capsules; Electroplating; Polylysine
PubMed: 34921585
DOI: 10.1002/smll.202106363 -
Journal of Agricultural and Food... Jun 2011ε-Polylysine (ε-PL) is a food-grade cationic antimicrobial that is highly effective against a wide range of food pathogens and spoilage organisms. However, its...
ε-Polylysine (ε-PL) is a food-grade cationic antimicrobial that is highly effective against a wide range of food pathogens and spoilage organisms. However, its application within foods and beverages is currently limited because of its tendency to associate with anionic substances, thereby increasing product turbidity or forming sediments. In this study, we examined a potential means of overcoming these problems by forming electrostatic complexes between cationic ε-PL and anionic pectin. The nature of the complexes formed depended on the mass ratio of pectin to ε-PL (R(P-PL)), since this determined their electrical characteristics, aggregation stability, and antimicrobial efficacy. The electrical charge on the complexes went from positive to negative with increasing R(P-PL), with the point of zero charge being around R(P-PL) ∼ 8. Soluble complexes or stable colloidal dispersions were formed at low and high R(P-PL) levels, but insoluble complexes were formed at intermediate levels (i.e., 4 ≤ R(P-PL) ≤ 16). The complexes maintained good antimicrobial activity (minimum inhibitory concentration of ε-PL < 10 μg/mL) at R(P-PL) ≤ 20 against two acid resistant spoilage yeasts: Zygosaccharomyces bailli and Saccharomyces cerevisiae. Finally, we showed that certain ε-PL-pectin complexes (10 μg/mL ε-PL; R(P-PL) ≥ 2) could be incorporated into green tea beverages without adversely affecting their appearance or physical stability. This work has shown that the function of a cationic antimicrobial agent (ε-polylysine) can be improved by incorporating it within electrostatic complexes using a food-grade anionic biopolymer (pectin).
Topics: Anti-Infective Agents; Food Preservatives; Pectins; Polylysine; Static Electricity; Yeasts
PubMed: 21574554
DOI: 10.1021/jf201162g -
Biochimica Et Biophysica Acta Sep 1984This paper characterizes the complex formed in vitro between polylysine and heparin in the presence of heparin excess, and investigates the interaction of this complex...
This paper characterizes the complex formed in vitro between polylysine and heparin in the presence of heparin excess, and investigates the interaction of this complex with the surface of Chinese hamster ovary cells. It examines the kinetics of surface binding and cellular uptake of the complex and shows that both processes can be distinguished from those of free heparin and free polylysine. The view that these three ligands bind to different surface sites is further supported by the fact that their interaction with cells is influenced differently by cell detachment with trypsin, detachment with EGTA or exposure to acid pH. Membrane transport of the complex is a saturable process suggestive of receptor-mediated endocytosis. It is, however, less effective than would be expected on the basis of the binding kinetics. Only 40% of the complex bound at 0 degrees C is internalized during a 2 h reincubation period at 37 degrees C, suggesting some degree of uncoupling between binding and endocytosis. These data confirm prior results obtained with methotrexate-polylysine conjugates. We had shown that the addition of heparin to a medium containing a methotrexate-polylysine conjugate leads unexpectedly to a marked cellular uptake of drug conjugate, which is capable of killing cells that are otherwise resistant to free methotrexate (Shen, W.-C. and Ryser, H.J.-P. (1981) Proc. Natl. Acad. Sci. USA 78, 7589-7593). The polylysine X heparin complex is therefore of interest as a potential carrier for intracellular drug delivery through endocytosis.
Topics: Animals; Carbon Radioisotopes; Cell Line; Cell Membrane; Cell Survival; Cricetinae; Cricetulus; Female; Heparin; Kinetics; Ovary; Peptides; Polylysine; Receptors, Cell Surface; Tritium
PubMed: 6087917
DOI: 10.1016/0304-4165(84)90219-8 -
Methods in Molecular Biology (Clifton,... 2016Engineered PEG-cleavable catiomers based on poly-L-lysine have been developed as nonviral gene vectors, which have been found to be one of important methods to balance...
Engineered PEG-cleavable catiomers based on poly-L-lysine have been developed as nonviral gene vectors, which have been found to be one of important methods to balance "PEG dilemma." In this protocol, we aim at the standardization of the method and procedure of PEG-cleavable catiomers. Major steps including ring-opening polymerization (ROP) of ε-benzyloxycarbonyl-L-lysine N-carboxyanhydride (zLL-NCA) monomers to yield PEG-cleavable polylysine, examination on bio-stability and bio-efficacy of its gene complexes are described.
Topics: Biological Transport; Cell Line; Disulfides; Drug Carriers; Drug Stability; Humans; Polyethylene Glycols; Polylysine; Polymerization; RNA, Small Interfering
PubMed: 26472441
DOI: 10.1007/978-1-4939-3112-5_5 -
Science (New York, N.Y.) Oct 2003
Topics: Adsorption; Aluminum Silicates; Amino Acids; Catalysis; Chemical Phenomena; Chemistry, Physical; Clay; Genetic Code; Geologic Sediments; Hydrogen-Ion Concentration; Lipid Bilayers; Lipids; Micelles; Models, Theoretical; Oceans and Seas; Peptides; Polylysine; RNA; Surface Properties; Water
PubMed: 14576411
DOI: 10.1126/science.1091765 -
Cellular & Molecular Biology Letters 2004The construction of an efficient carrier for genetic material is a major research objective that needs to be achieved before gene therapy can become a viable...
The construction of an efficient carrier for genetic material is a major research objective that needs to be achieved before gene therapy can become a viable pharmacological approach. Artificial aggregates containing nucleic acids are one of the options for the systemic delivery of genetic information. The diversity of functions the aggregate is expected to fulfill necessitates its complex architecture. In order to obtain a complex supramolecular aggregate, formed from elements that are themselves complex molecules, appropriate procedures based on the detailed understanding of processes at the molecular level are required. In this study, we investigated how the various properties of cationic compounds affect nucleic acid condensation. The combination of two condensing agents, differing in their affinity towards water, when mixed with plasmids, resulted in aggregates which are resistant to enzymatic digestion and which form particles with well-defined size distributions. Such uniform and well-defined complexes may subsequently be further modified in order to obtain a fully functional genetic material carrier.
Topics: Gene Transfer Techniques; Genetic Therapy; Lipids; Plasmids; Polylysine
PubMed: 15048147
DOI: No ID Found -
Biomacromolecules Aug 2007As part of the search of novel degradable polymers, amphiphilic and cationic poly(epsilon-caprolactone)-g-poly(l-lysine) (PCL-g-PlL) copolymers have been synthesized...
As part of the search of novel degradable polymers, amphiphilic and cationic poly(epsilon-caprolactone)-g-poly(l-lysine) (PCL-g-PlL) copolymers have been synthesized following a grafting "onto" or a grafting "from" method both applied to a macropolycarbanionic PCL derivative. The first approach led to PCL-g-PZlL containing 36% of epsilon-caprolactone and 64% of N-epsilon-Z-l-lysine units, by reaction of activated poly(N-epsilon-Z-l-lysine) on the macropolycarbanion derived from PCL. The second route was based on the anionic ring opening polymerization of N-carboxyanhydride of N-epsilon-benzyloxycarbonyl-l-lysine initiated by the macropolycarbanion derived from PCL and led to a similar copolymer containing 45% of of epsilon-caprolactone and 55% of N-epsilon-Z-l-lysine units. After deprotection of the lysine units, PCL-g-PlL copolymers were obtained. These copolymers are water-soluble and form nanometric micelle-like objects with mean diameters between 60 and 500 nm in distilled water depending on the synthesis route.
Topics: Biocompatible Materials; Polyesters; Polylysine; Solubility; Water
PubMed: 17625909
DOI: 10.1021/bm700449c -
Journal of Materials Chemistry. B Jun 2022Cryopreservation of human erythrocytes suitable cryoprotectants is essential for transfusion during emergencies, but the conventional glycerolization method requires a...
Cryopreservation of human erythrocytes suitable cryoprotectants is essential for transfusion during emergencies, but the conventional glycerolization method requires a tedious thawing-deglycerolization process. Alternatively, trehalose, a nonreducing disaccharide, has gained much attention as a biocompatible cryoprotectant due to its nature in living organisms capable of surviving extreme cold and desiccation. In this work, cryopreservation of human erythrocytes was realized through high intracellular trehalose enhanced by benzyl alcohol at 4 °C with membrane stabilization of maltotriose-grafted ε-poly(L-lysine). Intracellular trehalose could reach 94.2 ± 12.1 mM with slight impacts on morphology and cell functions, and the post-storage cryosurvival of human erythrocytes could achieve 96.2 ± 3.4% membrane protection by the glycopeptide. It has been demonstrated that the functional glycopeptide performed as an extracellular cryoprotectant accompanied by high intracellular trehalose for synergistic cryopreservation of human erythrocytes in the biocompatible glycerol-free conditions. This two-step approach involving augmentation of intracellular trehalose at a hypothermic temperature and membrane stabilization of the functional glycopeptide could be an alternative way for human cell cryopreservation.
Topics: Cryopreservation; Cryoprotective Agents; Erythrocytes; Glycopeptides; Humans; Polylysine; Trehalose; Trisaccharides
PubMed: 35604178
DOI: 10.1039/d2tb00445c -
Microbial Cell Factories Apr 2022AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the...
BACKGROUND
AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces.
RESULTS
In this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpA). First, we identified a novel AdpA homolog named AdpA in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpA was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpA was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpA in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpA was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpA may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus.
CONCLUSIONS
Here, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpA promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpA may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.
Topics: Phylogeny; Polylysine; Secondary Metabolism; Streptomyces
PubMed: 35397580
DOI: 10.1186/s12934-022-01785-6