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Viruses Jan 2020The human OAS1 (hOAS1) gene produces multiple possible isoforms due to alternative splicing events and sequence variation among individuals, some of which affect...
The human OAS1 (hOAS1) gene produces multiple possible isoforms due to alternative splicing events and sequence variation among individuals, some of which affect splicing. The unique C-terminal sequences of the hOAS1 isoforms could differentially affect synthetase activity, protein stability, protein partner interactions and/or cellular localization. Recombinant p41, p42, p44, p46, p48, p49 and p52 hOAS1 isoform proteins expressed in bacteria were each able to synthesize trimer and higher order 2'-5' linked oligoadenylates in vitro in response to poly(I:C). The p42, p44, p46, p48 and p52 isoform proteins were each able to induce RNase-mediated rRNA cleavage in response to poly(I:C) when overexpressed in HEK293 cells. The expressed levels of the p42 and p46 isoform proteins were higher than those of the other isoforms, suggesting increased stability in mammalian cells. In a yeast two-hybrid screen, Fibrillin1 (FBN1) was identified as a binding partner for hOAS1 p42 isoform, and Supervillin (SVIL) as a binding partner for the p44 isoform. The p44-SVIL interaction was supported by co-immunoprecipitation data from mammalian cells. The data suggest that the unique C-terminal regions of hOAS1 isoforms may mediate the recruitment of different partners, alternative functional capacities and/or different cellular localization.
Topics: 2',5'-Oligoadenylate Synthetase; Alternative Splicing; Cloning, Molecular; Escherichia coli; Fibrillin-1; Gene Expression; HEK293 Cells; Humans; Membrane Proteins; Microfilament Proteins; Poly I-C; Polylysine; Protein Isoforms; Recombinant Proteins
PubMed: 32013110
DOI: 10.3390/v12020152 -
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 -
International Journal of Molecular... Oct 2021In this study, ε-polylysine and calcium phosphate precipitation (CPP) methods were employed to induce antibacterial effects and dentin tubule occlusion. Antibacterial...
In this study, ε-polylysine and calcium phosphate precipitation (CPP) methods were employed to induce antibacterial effects and dentin tubule occlusion. Antibacterial effects of ε-polylysine were evaluated with broth dilution assay against CPP solution from MCPM, DCPD, and TTCP was prepared. Four concentrations of ε-polylysine(ε-PL) solutions (0.125%, 0.25%, 0.5%, 1%) were prepared. Dentin discs were prepared from recently extracted human third molars. Dentin discs were incubated with (ATCC 33277) bacterial suspension (ca. 10 bacteria) containing Brain Heart Infusion medium supplemented with 0.1 g/mL Vitamin K, 0.5 mg/mL hemin, 0.4 g/mL L-cysteine in anaerobic jars (37 °C) for 7 days to allow for biofilm formation. -infected dentin specimens were randomly divided into four groups: CPP + 0.125% ε-PL, CPP + 0.25% ε-PL, CPP + 0.5% ε-PL, CPP + 1% ε-PL. On each dentin specimen, CPP solution was applied followed by polylysine solution with microbrush and immersed in artificial saliva. Precipitate formation, antibacterial effects, and occlusion of dentinal tubules were characterized in vitro over up to 72 h using scanning electron microscopy. ε-PL showed 34.97% to 61.19% growth inhibition levels against () after 24 h of incubation. On -infected dentin specimens, DCPD + 0.25% ε-PL, and DCPD + 0.5% ε-PL groups showed complete bacterial inhibition and 78.6% and 98.1% dentin tubule occlusion, respectively ( < 0.001). The longitudinal analysis on fractured dentin samples in DCPD and TTCP groups revealed deeply penetrated hydroxyapatite-like crystal formations in dentinal tubules after 72 h of incubation in artificial saliva.
Topics: Anti-Bacterial Agents; Calcium Phosphates; Dentin; Dentin Sensitivity; Humans; Microbial Sensitivity Tests; Polylysine; Spectrum Analysis; Surface Properties
PubMed: 34639022
DOI: 10.3390/ijms221910681 -
Biomaterials Advances Feb 2022The goal of this work is the design and the development of scaffolds based on maltodextrin (MD) to recover chronic lesions. MD was mixed with arginine/lysine/polylysine...
The goal of this work is the design and the development of scaffolds based on maltodextrin (MD) to recover chronic lesions. MD was mixed with arginine/lysine/polylysine and the electrospinning was successfully used to prepare scaffolds with uniform and continuous nanofibers having regular shape and smooth surface. A thermal treatment was applied to obtain insoluble scaffolds in aqueous environment, taking the advantage of amino acids-polysaccharide conjugates formed via Maillard-type reaction. The morphological analysis showed that the scaffolds had nanofibrous structures, and that the cross-linking by heating did not significantly change the nanofibers' dimensions and did not alter the system stability. Moreover, the heating process caused a reduction of free amino group and proportionally increased scaffold cross-linking degree. The scaffolds were elastic and resistant to break, and possessed negative zeta potential in physiological fluids. These were characterized by direct antioxidant properties and Fe chelation capability (indirect antioxidant properties). Moreover, the scaffolds were cytocompatible towards fibroblasts and monocytes-derived macrophages, and did not show any significant pro-inflammatory activity. Finally, those proved to accelerate the recovery of the burn/excisional wounds. Considering all the features, MD-poly/amino acids scaffolds could be considered as promising medical devices for the treatment of chronic wounds.
Topics: Amino Acids; Antioxidants; Polysaccharides; Tissue Engineering; Tissue Scaffolds
PubMed: 35527142
DOI: 10.1016/j.msec.2021.112593 -
Journal of Functional Biomaterials Dec 2023With the phase-out of amalgam and the increase in minimally invasive dentistry, there is a growing need for high-strength composite materials that can kill residual...
With the phase-out of amalgam and the increase in minimally invasive dentistry, there is a growing need for high-strength composite materials that can kill residual bacteria and promote tooth remineralization. This study quantifies how antibacterial polylysine (PLS) and re-mineralizing monocalcium phosphate monohydrate (MCPM) affect biofilms and the strength of dental composites. For antibacterial studies, the MCPM-PLS filler percentages were 0-0, 8-4, 12-6, and 16-8 wt% of the composite filler phase. Composite discs were immersed in 0.1% sucrose-supplemented broth containing (UA159) and incubated in an anaerobic chamber for 48 h. Surface biomass was determined by crystal violet (CV) staining. Growth medium pH was measured at 24 and 48 h. Biofilm bacterial viability (CFU), exo-polysaccharide (water-soluble glucan (WSG) and water-insoluble glucan (WIG)), and extracellular DNA (eDNA) were quantified. This was by serial dilution plate counting, phenol-sulfuric acid microassay, and fluorometry, respectively. The biaxial flexural strengths were determined after water immersion for 1 week, 1 month, and 1 year. The MCPM-PLS wt% were 8-4, 8-8, 16-4 and 16-8. The normalized biomass, WSG, and WIG showed a linear decline of 66%, 64%, and 55%, respectively, as the PLS level increased up to 8%. The surrounding media pH (4.6) was all similar. A decrease in bacterial numbers with the 12-6 formula and a significant reduction with 16-8 compared to the 0-0 formulation was observed. The eDNA concentrations in biofilms formed on 12-6 and 16-8 formulations were significantly less than the 0-0 control and 8-4 formulations. Doubling MCPM and PLS caused a 14 and 19% reduction in strength in 1 week, respectively. Average results were lower at 1 month and 1 year but affected less upon doubling MCPM and PLS levels. Moreover, a 4% PLS may help to reduce total biomass and glucan levels in biofilms on the above composites. Higher levels are required to reduce eDNA and provide bactericidal action, but these can decrease early strength.
PubMed: 38248680
DOI: 10.3390/jfb15010013 -
Advanced Science (Weinheim,... May 2024Non-antibiotic strategies are desperately needed to treat post-traumatic osteomyelitis (PTO) due to the emergence of superbugs, complex inflammatory microenvironments,...
Non-antibiotic strategies are desperately needed to treat post-traumatic osteomyelitis (PTO) due to the emergence of superbugs, complex inflammatory microenvironments, and greatly enriched biofilms. Previously, growing evidence indicated that quorum sensing (QS), a chemical communication signal among bacterial cells, can accelerate resistance under evolutionary pressure. This study aims to develop a medical dressing to treat PTO by inhibiting QS and regulating the inflammatory microenvironment, which includes severe oxidative stress and acid abscesses, through a reactive oxygen species (ROS)-responsive bond between N1- (4-borobenzoyl)-N3-(4-borobenzoyl)-the N1, the N1, N3, N3-tetramethylpropane-1,3-diamine (TSPBA) and polyvinyl alcohol (PVA), and the amino side chain of hyperbranched polylysine (HBPL). Physically enclosed QS inhibitors subsequently exerted the antibacterial effects. This hydrogel can scavenge hydrogen peroxide (HO), superoxide anion free radical (·O ), hydroxyl radicals (·OH) and 2,2-di(4-tert-octylphenyl)-1-picryl-hydrazyl (DPPH) to reduce oxidative stress and inhibit "bacteria-to-bacteria communication", thus clearing planktonic bacteria and biofilms, accelerating bacterial plasmolysis, reducing bacterial virulence and interfering with membrane transport. After in vivo treatment with hydrogel, nearly all bacteria are eliminated, inflammation is effectively inhibited, and osteogenesis and bone repair are promoted to facilitate recovery from PTO. The work demonstrates the clinical translational potential of the hydrogel in the treatment of drug-resistant bacteria induced PTO.
Topics: Quorum Sensing; Hydrogels; Osteomyelitis; Animals; Mice; Disease Models, Animal; Anti-Bacterial Agents; Oxidative Stress; Biofilms; Reactive Oxygen Species; Rats; Male
PubMed: 38482752
DOI: 10.1002/advs.202307969 -
Journal of Dairy Science May 2022ε-Polylysine (ε-PL) is a natural preservative of antimicrobial peptides with broad spectrum and high antibacterial properties. The electrostatic complex delivery...
ε-Polylysine (ε-PL) is a natural preservative of antimicrobial peptides with broad spectrum and high antibacterial properties. The electrostatic complex delivery system formed by ε-PL and whey protein can be used to maintain the stability of ε-PL and solve the problem of limited application of protein-based food. This work aimed to study the interaction between ε-PL and whey protein by multiple characterization methods. The spectroscopy results showed static quenching type and new stretching of C=O for ε-PL-whey protein complexes. Microstructure studies showed that the combination of ε-PL and whey protein made the structure of the complexes become rough and dense. The interaction between ε-PL and whey protein could improve the stability of the complexes system during storage. Additionally, the interaction affected critical gel temperatures and gel texture properties of complexes with change of whey protein concentration, mass ratio of ε-PL to whey protein, pH value in alkaline solutions, and ion concentration. Overall, this study confirmed the interaction between ε-PL and whey protein, and it will provide a reference for the application of ε-PL in protein food matrix.
Topics: Animals; Anti-Bacterial Agents; Polylysine; Rheology; Static Electricity; Whey Proteins
PubMed: 35282919
DOI: 10.3168/jds.2021-21219 -
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 -
Bioengineering & Translational Medicine May 2024Joint diseases, such as osteoarthritis, often require delivery of drugs to chondrocytes residing within the cartilage. However, intra-articular delivery of drugs to...
Joint diseases, such as osteoarthritis, often require delivery of drugs to chondrocytes residing within the cartilage. However, intra-articular delivery of drugs to cartilage remains a challenge due to their rapid clearance within the joint. This problem is further exacerbated by the dense and negatively charged cartilage extracellular matrix (ECM). Cationic nanocarriers that form reversible electrostatic interactions with the anionic ECM can be an effective approach to overcome the electrostatic barrier presented by cartilage tissue. For an effective therapeutic outcome, the nanocarriers need to penetrate, accumulate, and be retained within the cartilage tissue. Nanocarriers that adhere quickly to cartilage tissue after intra-articular administration, transport through cartilage, and remain within its full thickness are crucial to the therapeutic outcome. To this end, we used ring-opening polymerization to synthesize branched poly(l-lysine) (BPL) cationic nanocarriers with varying numbers of poly(lysine) branches, surface charge, and functional groups, while maintaining similar hydrodynamic diameters. Our results show that the multivalent BPL molecules, including those that are highly branched (i.e., generation two), can readily adhere and transport through the full thickness of cartilage, healthy and degenerated, with prolonged intra-cartilage retention. Intra-articular injection of the BPL molecules in mouse knee joint explants and rat knee joints showed their localization and retention. In summary, this study describes an approach to design nanocarriers with varying charge and abundant functional groups while maintaining similar hydrodynamic diameters to aid the delivery of macromolecules to negatively charged tissues.
PubMed: 38818117
DOI: 10.1002/btm2.10612 -
Journal of Nanobiotechnology May 2024Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid,...
Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired.
Topics: RNA, Ribosomal; Nucleic Acid Conformation; Nanostructures; Humans; RNA Folding; DNA; Polylysine; Polyethylene Glycols
PubMed: 38698435
DOI: 10.1186/s12951-024-02489-2