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Cane molasses, a sugar-rich agro-industrial byproduct, was used to enhance the production of ε-poly-l-lysine (ε-PL) in Streptomyces albulus PD-1 as a cost-effective carbon source. The sucrose metabolism pathway was engineered by heterologously expressing sucrose-6-phosphate hydrolase from Escherichia coli W. The optimization of various promoters identified the SP44 promoter, increasing the total sugar utilization rate by 2.76-fold compared with the ermEp* promoter. Additionally, adaptive laboratory evolution improved the total sugar utilization rate. The evolved strain achieved an ε-PL titer of 2.65 ± 0.15 g/L in flask experiments, increasing the ε-PL titer by 7.16-fold compared with the unevolved strain. Comparative transcriptomic analyses revealed that the enhanced tolerance of the evolved strain to high concentrations of cane molasses was primarily due to modifications in the sucrose metabolism pathways, microbial metabolism in heavy metals and phenols, and the amino acids transport and metabolic pathways. These changes enabled more efficient ε-PL production. During fed-batch fermentation in a 5-L fermentor using a concentration of 50 g/L cane molasses, the ε-PL titer reached 36.88 ± 0.62 g/L, and dry cell weight was 41.1 ± 1.0 g/L. This study illustrates that cane molasses is an economical carbon source for producing ε-PL on an industrial scale.

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine consisting of 25-35 l-α-lysine residues (ε-PαL 25-35 ) and ε-poly-l-β-lysine consisting of l-β-lysine residues (ε-PβL 4-13 ), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5-14 l-α-lysine residues (ε-PαL 5-14 ) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL 5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL 5-14 -FAM, with HeLa cells. Unlike ε-PαL 25-35 -FAM, ε-PαL 5-14 -FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 μM) was required for the internalization events. ε-PαL 5-14 has a chain length almost equal to that of the membrane permeable ε-PβL 4-13 , which can enter cells at low concentrations. Considering that the basicity of the β-amino group is higher than that of α-amino acid at physiological pH, ε-PβL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PβL would be more advantageous for cellular internalization of cargo proteins than ε-PαL 25-35 ., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

ε-Poly-l-lysine (ε-PL), a natural food preservative with various advantages, is primarily produced by Streptomyces. It has attracted considerable attentions for the outstanding antibacterial activity, safety, heat stability, water solubility and other remarkable properties. In this study, a food-grade recombinant Bacillus subtilis was constructed for the biocatalysis of ε-PL. Firstly, the d-alanine racemase gene (alrA) was deleted from the genome of Bacillus subtilis 168 to construct an auxotrophic B. subtilis 168 (alrA - ). Based on the shuttle plasmid pMA5, a food-grade plasmid pMA5a was constructed by replacing the genes of kanamycin resistance (Kan r ) and ampicillin resistance (Amp r ) with alrA and the gene encoding α-peptide of β-galactosidase (lacZα), respectively. Subsequently, codon-optimized ε-PL synthase gene (pls) and P-pls were ligated into pMA5a and transformed in E. coli DH5α and expressed in B. subtilis 168 (alrA - ). Finally, the whole-cell biocatalysis conditions for ε-PL production by B. subtilis 168 (alrA - )/pMA5a-pls were optimized, and the optimal conditions were 30°C, pH 4, l-lysine concentration of 0.6 g/L, bacterial concentration of 15 % (w/v) and a catalytic time of 7 h. The ε-PL production reached a maximum of 0.33 ± 0.03 g/L. The product was verified to be ε-PL by HPLC and tricine-SDS-PAGE. The information obtained in this study shows critical reference for the food-grade heterologous expression of ε-PL., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants., (© 2024. The Author(s).)

Safe and eco-friendly preservatives are crucial to preventing food spoilage and illnesses, as foodborne diseases caused by pathogens result in approximately 600 million cases of illness and 420,000 deaths annually. ε-Poly-L-lysine (ε-PL) is a novel food preservative widely used in many countries. However, its commercial application has been hindered by high costs and low production. In this study, ε-PL's biosynthetic capacity was enhanced in Streptomyces albulus WG608 through metabolic engineering guided by multi-omics techniques. Based on transcriptome and metabolome data, differentially expressed genes (fold change >2 or <0.5; p < 0.05) and differentially expressed metabolites (fold change >1.2 or <0.8) were separately subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The integrative analysis of transcriptome, metabolome, and overexpression revealed the essential roles of isocitrate lyase, succinate dehydrogenase, flavoprotein subunit, diaminopimelate dehydrogenase, polyphosphate kinase, and polyP:AMP phosphotransferase in ε-PL biosynthesis. Subsequently, a strain with enhanced ATP supply, L-lysine supply, and ε-PL synthetase expression was constructed to improve its production. Finally, the resulting strain, S. albulus WME10, achieved an ε-PL production rate of 77.16 g/L in a 5 L bioreactor, which is the highest reported ε-PL production to date. These results suggest that the integrative analysis of the transcriptome and metabolome can facilitate the identification of key pathways and genetic elements affecting ε-PL synthesis, guiding further metabolic engineering and thus significantly enhancing ε-PL production. The method presented in this study could be applicable to other valuable natural antibacterial agents.

Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro . Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo . Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: The aim of this study was to construct crosslinked polylysine-hyaluronic acid microspheres (pl-HAM) ladened with gingival mesenchymal stem cells (GMSCs) and explore its biologic behavior in soft tissue regeneration., Methods: The effects of the crosslinked pl-HAM on the biocompatibility and the recruitment of L-929 cells and GMSCs were detected in vitro. Moreover, the regeneration of subcutaneous collagen tissue, angiogenesis and the endogenous stem cells recruitment were investigated in vivo. We also detected the cell developing capability of pl-HAMs., Results: The crosslinked pl-HAMs appeared to be completely spherical-shaped particles and had good biocompatibility. L-929 cells and GMSCs grew around the pl-HAMs and increased gradually. Cell migration experiments showed that pl-HAMs combined with GMSCs could promote the migration of vascular endothelial cells significantly. Meanwhile, the green fluorescent protein-GMSCs in the pl-HAM group still remain in the soft tissue regeneration area 2 weeks after surgery. The results of in vivo studies showed that denser collagen deposition and more angiogenesis-related indicator CD31 expression in the pl-HAMs+ GMSCs + GeL group compared with the pl-HAMs + GeL group. Immunofluorescence showed that CD44, CD90, CD73 co-staining positive cells surrounded the microspheres in both pl-HAMs + GeL group and pl-HAM + GMSCs + GeL group., Conclusions: The crosslinked pl-HAM ladened with GMSCs system could provide a suitable microenvironment for collagen tissue regeneration, angiogenesis and endogenous stem cells recruitment, which may be an alternative to autogenous soft tissue grafts for minimally invasive treatments for periodontal soft tissue defects in the future., (© 2023 American Academy of Periodontology.)

Streptomyces albulus NBRC14147 produces a homopoly(amino acid), ε-poly-L-lysine (ε-PL). Due to its antibiotic activity, thermostability, biodegradability, and non-toxicity to humans, ε-PL is used as a food preservative. In this study, homology searches of diaminopimelate (DAP) pathway genes (dapB and dapE), in an S. albulus genome database, were shown to encode predicted enzymes using dapB or dapE in Escherichia coli strain complementation assays. We observed that dapB and dapE transcriptional levels were weak during ε-PL production stages. Therefore, we strengthened this expression using an ermE constitutive promoter. Engineered strains generated faster growth and ε-PL production rates when compared with the control strain. Moreover, maximum ε-PL yields in S. albulus, where dapB was constitutively expressed, were approximately 14% higher when compared with the control strain. These findings showed that enhanced lysine biosynthetic gene expression generated faster and higher ε-PL production levels., (© 2023. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

The purpose of the study was to investigate the mechanism of inactivation of Serratia liquefaciens by different treatments, namely corona discharge plasma (CDP), ε-polylysine (ε-PL), and corona discharge plasma combined with ε-polylysine (CDP plus ε-PL). The results showed that the combined treatment of CDP and ε-PL exhibited significant antibacterial effects. The total number of colonies of S. liquefaciens dropped by 0.49 log CFU/mL following 4 min of CDP treatment, 4MIC ε-PL treatment for 6 h alone decreased the amounts of colonies by 2.11 log CFU/mL, and 6 h of treatment with 4MIC ε-PL after the bacterium was treated with CDP could decrease the number of colonies by 6.77 log CFU/mL. Scanning electron microscopy images showed that the combined treatment of CDP and ε-PL caused the most serious damage to the cell morphology. Electrical conductivity, nucleic acid, and PI staining indicated that the combined treatment dramatically enhanced the permeability of the cell membrane. In addition, the combined treatment led to a significant decrease in SOD and POD enzyme activities in S. liquefaciens, which prevented energy metabolism. Finally, the determination of free and intracellular ε-PL concentrations confirmed that the treatment of CDP could cause the bacteria to bind more ε-PL and exert more significant bacterial inhibition. Therefore, CDP and ε-PL had a synergistic effect in the inhibition of S. liquefaciens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest None., (Published by Elsevier B.V.)

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance, and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases., (© 2021 S. Karger AG, Basel.)

The effects of ε-polylysine (ε-PL) at different concentrations (0.005 %, 0.010 %, 0.020 %, and 0.030 %) on the structure and gelling behavior of pork myofibrillar protein (MP) under oxidative stress were explored. The incorporation of ε-PL significantly restrained oxidation-induced sulfhydryl and solubility losses (up to 9.72 % and 41.9 %, respectively) as well as protein crosslinking and aggregation. Compared with the oxidized control, ε-PL at low concentrations (0.005 % - 0.020 %) promoted further unfolding and destabilization of MP, while 0.030 % ε-PL led to refolding of MP and enhanced its thermal stability. The ε-PL-induced physicochemical changes favored the formation of a finer and more homogeneous three-dimensional network structure, therefore obviously enhancing the strength and water-holding capacity (WHC) of thermally induced oxidized MP gels, with the ε-PL at 0.020 % showed the greatest enhancement. This work revealed for the first time that ε-PL can significantly ameliorate the oxidation stability and gel-forming ability of meat proteins., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

ε-poly-L-lysine (ε-PL) is the main secondary metabolite of Streptomyces albulus, and it is widely used in the food industry. Polylysine synthetase (Pls) is the last enzyme in the ε-PL biosynthetic pathway. Our previous study revealed that Pls overexpressed in S. albulus CICC11022 result in the efficient production of ε-PL. In this study, a Pls gene knockout strain was initially constructed. Then, genomic, transcriptomic and metabolomic approaches were integrated to study the effects of the high expression and knockout of Pls on the gene expression and metabolite synthesis of S. albulus. The high expression of Pls resulted in 598 significantly differentially expressed genes (DEGs) and 425 known differential metabolites, whereas the inactivation of Pls resulted in 868 significant DEGs and 374 known differential metabolites. The expressions of 8 and 35 genes were negatively and positively associated with the Pls expression, respectively. Subsequently, the influence mechanism of the high expression and inactivation of Pls on the ε-PL biosynthetic pathway was elucidated. Twelve metabolites with 30% decreased yield in the high-expression strain of Pls but 30% increased production in the Pls knockout strain were identified. These results demonstrate the influence of Pls on the metabolism of S. albulus. The present work can provide the theoretical basis for improving the production capacity of ε-PL by means of metabolic engineering or developing bioactive substances derived from S. albulus., (© 2022. The Author(s).)

ε-Poly-l-lysine (ε-PL) is a wide-spectrum antimicrobial agent, while its biosynthesis-inducing signals are rarely reported. This study found that Botrytis cinerea extracts could act as a microbial call to induce a physiological modification of Streptomyces albulus for ε-PL efficient biosynthesis and thereby resulted in ε-PL production (34.2 g/liter) 1.34-fold higher than control. The elicitors could be primary isolated by ethanol and butanol extraction, which resulted in more vibrant, aggregate and stronger mycelia. The elicitor-derived physiological changes focused on three aspects: ε-PL synthase, energy metabolism, and lysine biosynthesis. After elicitor addition, upregulated sigma factor hrdD and improved transcription and expression of pls directly contributed to the high ε-PL productivity; upregulated genes in tricarboxylic acid (TCA) cycle and energy metabolism promoted activities of citrate synthase and the electron transport system; in addition, pool enlargements of ATP, ADP, and NADH guaranteed the ATP provision for ε-PL assembly. Lysine biosynthesis was also increased based on enhancements of gene transcription, key enzyme activities, and intracellular metabolite pools related to carbon source utilization, the Embden-Meyerhof pathway (EMP), the diaminopimelic acid pathway (DAP), and the replenishment pathway. Interestingly, the elicitors stimulated the gene transcription for the quorum-sensing system and resulted in upregulation of genes for other antibiotic production. These results indicated that the Botrytis cinerea could produce inducing signals to change the Streptomyces mycelial physiology and accelerate the ε-PL biosynthesis. IMPORTANCE This work identified the role of microbial elicitors on ε-PL production and disclosed the underlying mechanism through analysis of gene transcription, key enzyme activities, and intracellular metabolite pools, including transcriptome and metabolome analysis. It was the first report for the inducing effects of the "microbial call" to Streptomyces albulus and ε-PL biosynthesis, and these elicitors could be potentially obtained from decayed fruits infected by Botrytis cinerea; hence, this may be a way of turning a biohazard into bioproduct wealth. This study provided a reference for application of microbial signals in secondary metabolite production, which is of theoretical and practical significance in industrial antibiotic production.

The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been vaguely explained. Our studies revealed that on both, Candida albicans and Saccharomyces cerevisiae, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were 250 μg·mL -1 ; EPL produced a K + and Ca 2+ efflux, and at higher concentrations also an efflux of material absorbing at 260 nm, small peptides, and phosphate is produced, along with the inhibition of fermentation and extracellular acidification and respiration. Moreover, growth was inhibited, reactive oxygen species (ROS) production increased, and cell viability decreased. The polycation also produced plasma membrane potential hyperpolarization. The effects were dependent both on the cell quantity and polycation concentration, as well as the media used. The plasma membrane disruption was confirmed by TEM and PI staining., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

Ferrocene-based polymers as redox mediators are considered versatile and important in the study of glucose biosensors. Poly-L-lysine (PLL), as a cationic polymer, possesses good properties including biocompatibility, biodegradation and water solubility. In this work, PLL was modified with ferrocene carboxylate in a very simple way by activating the carboxyl group of Fc, which reacted with the amino groups of the polymer. The resulting product was analysed by FTIR. Performance as a redox mediator (Fc-PLL) with the enzyme glucose oxidase was tested by cyclic voltammetry and showed an increase in the oxidation current in the presence of glucose in PBS pH 7.4. Additionally, performance as a biosensor was evaluated by amperometry and gave a linear range of 0-10 mM, a limit of detection of 23 μM, a sensitivity of 6.55 μA/cm 2 mM and high selectivity. To evaluate the charged regions of Fc-PLL/GOx on the electrode surface, analysis by scanning electrochemical microscopy showed remarkable activity. The Fc-PLL redox polymer as a glucose biosensor has been well accepted as this kind of material, and the results showed remarkable activity as an electron transfer mediator between the redox polymer and the GOx enzyme., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cryopreservation of human erythrocytes via 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% via 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.

Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNA Lys or Val-tRNA Lys reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Epsilon-poly-L-lysine (ε-PL) is an unusual biopolymer composed of L-lysine produced by several microorganisms, especially by the genus Streptomyces. Due to its excellent antimicrobial activity, good water solubility, high safety, and biodegradable nature, ε-PL with a GRAS status has been widely used in food and pharmaceutical industries. In the past years, studies have focused on the biotechnological production of ɛ-PL, the biosynthetic mechanism of microbial ɛ-PL, and its application. To provide new perspectives from recent advances, the review introduced the methods for the isolation of ɛ-PL producing strains and the biosynthetic mechanism of microbial ɛ-PL. We summarized the strategies for the improvement of ɛ-PL producing strains, including physical and chemical mutagenesis, ribosome engineering and gene engineering, and compared the different metabolic regulation strategies for improving ɛ-PL production, including medium optimization, nutrient supply, pH control, and dissolved oxygen control. Then, the downstream purification methods of ɛ-PL and its recent applications in food and medicine industries were introduced. Finally, we also proposed the potential challenges and the perspectives for the production of ε-PL., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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 Sn ). First, we identified a novel AdpA homolog named AdpA Sa 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 Sn 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 Sn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpA Sn 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 Sn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74&#95;2409), were identified, suggesting that AdpA Sn 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 Sn 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 Sn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces., (© 2022. The Author(s).)

ε-poly-l-lysine (ε-PL) synthetase (Pls) is a membrane protein that possesses both adenylation and thiolation domains, characteristic of non-ribosomal peptide synthetases (NRPSs). Pls catalyzes the polymerization of l-Lys molecules in a highly specific manner within proteinogenic amino acids. However, this enzyme accepts certain l-Lys analogs which contain small substituent groups at the middle position of the side chain. From the crystal structures of the adenylation domain from NRPSs, the amino acid residues involved in substrate binding can be assumed; however, the precise interactions for better understanding the Pls recognition of l-Lys and its analogs have not yet been fully elucidated. Here, we determined the crystal structure of the adenylation domain of Pls in complex with the intermediate lysyl adenylate at 2.3 Å resolution. This is the first structure determination of the l-Lys activating adenylation domain. The crystal structure reveals that the shape of the substrate-binding pocket determines the specific recognition of l-Lys and its analogs and the electrostatic and hydrogen-bonding interactions further strengthen substrate binding. This study helps us understand the ε-PL synthesis mechanism and contributes to improving our knowledge of the molecular mechanism of NRPS adenylation domains towards their successful application in bioengineering., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

After an injury, soft tissue structures in the body undergo a natural healing process through specific phases of healing. Adhesions occur as abnormal attachments between tissues and organs through the formation of blood vessels and/or fibrinous adhesions during the regenerative repair process. In this study, we developed an adhesion-preventing membrane with an improved physical protection function by modifying the surface of chondrocyte-derived extracellular matrices (CECM) with anti-adhesion function. We attempted to change the negative charge of the CECM surface to neutral using poly-L-lysine (PLL) and investigated whether it blocked fibroblast adhesion to it and showed an improved anti-adhesion effect in animal models of tissue adhesion. The surface of the membrane was modified with PLL coating (PLL 10), which neutralized the surface charge. We confirmed that the surface characteristics except for the potential difference were maintained after the modification and tested cell attachment in vitro. Adhesion inhibition was identified in a peritoneal adhesion animal model at 1 week and in a subcutaneous adhesion model for 4 weeks. Neutralized CECM (N-CECM) suppressed fibroblast and endothelial cell adhesion in vitro and inhibited abdominal adhesions in vivo. The CECM appeared to actively inhibit the infiltration of endothelial cells into the injured site, thereby suppressing adhesion formation, which differed from conventional adhesion barriers in the mode of action. Furthermore, the N-CECM remained intact without degradation for more than 4 weeks in vivo and exerted anti-adhesion effects for a long time. This study demonstrated that PLL10 surface modification rendered a neutral charge to the polymer on the extracellular matrix surface, thereby inhibiting cell and tissue adhesion. Furthermore, this study suggests a means to modify extracellular matrix surfaces to meet the specific requirements of the target tissue in preventing post-surgical adhesions., (© 2021 John Wiley & Sons Ltd.)

Abstract: This study was conducted to determine the sterilization effect of a combination of high pressure thermal sterilization (HPTS) and ε-polylysine (ε-PL) on Bacillus subtilis spores. The spores were treated with HPTS (550 MPa at 25, 65, and 75°C) and ε-PL at 0.1 and 0.3%. HPTS and ε-PL synergistically decreased the number of surviving spores and increased the release of the intracellular components in the spore suspension, with the maximal effects from treatment with 550 MPa at 75°C plus 0.3% ε-PL. Maximum fluidity and permeability of the cell inner membrane were observed with 550 MPa at 75°C plus 0.3% ε-PL. Changes in membrane lipids were detected from 3,000 to 2,800 cm-1 by Fourier transform infrared spectroscopy. The results provide new insights into the mechanism by which HPTS and ε-PL synergistically sterilize B. subtilis spores., (Copyright ©, International Association for Food Protection.)

Currently, glycerol is a conventional cryoprotectant of human red blood cells (hRBCs), but the time-consuming thawing and deglycerolization processes are essential before transfusion. Much of the research up to now has been conducted on the delivery of impermeable trehalose to hRBCs at 37 °C, but the cryoprotective effect of trehalose and deterioration of cells still remain challenging. Encouraged by the interaction of hydrophobic or cationic groups on cell membranes and osmotic stabilization, herein, we propose a novel cryopreservation system to facilitate trehalose entry into hRBCs at 4 °C and pH 7.4. High intracellular trehalose contents and cryosurvival of hRBCs were achieved with small function variations via the assistance of self-assembled nanoparticles of alkylated ε-poly(L-lysine) (ε-PL) along with poly(vinyl pyrrolidone) (PVP). The effect of amphipathic alkylated ε-PL with various alkyl chains and grafting ratios on membrane perturbation with protection of PVP was systematically investigated. Overall, by the combination of alkylated ε-PL and PVP, the intracellular trehalose could be enhanced to 109.7 ± 6.1 mM and subsequently hRBC cryosurvival reached 91.7 ± 5.5%, significantly higher than those containing trehalose only, 11.9 ± 1.1 mM and 50.0 ± 2.1%, respectively. It was observed that the biocompatible trehalose-loading system could benefit glycerol-free cryopreservation of hRBCs and also provide a feasible way for impermeable biomacromolecule delivery.

Diabetic ulcers, a difficult problem faced by clinicians, are strongly associated with an increase in cellular senescence. Few empirical studies have focused on exploring a targeted strategy to cure diabetic wounds by eliminating senescent fibroblasts (SFs) and reducing side effects. In this study, poly-l-lysine/sodium alginate (PLS) is modified with talabostat (PT100) and encapsulates a PARP1 plasmid (PARP1@PLS-PT100) for delivery to target the dipeptidyl peptidase 4 (DPP4) receptor and eliminate SFs. PARP1@PLS-PT100 releases encapsulated plasmids, displaying high selectivity for SFs over normal fibroblasts by targeting the DPP4 receptor, decreasing senescence-associated secretory phenotypes (SASPs), and stimulating the secretion of anti-inflammatory factors. Furthermore, the increased apoptosis of SFs and the disappearance of cellular senescence alleviates SASPs, accelerates re-epithelialization and collagen deposition, and significantly induces macrophage M2 polarization, which mediates tissue repair and the inflammatory response. This innovative strategy has revealed the previously undefined role of PARP1@PLS-PT100 in promoting diabetic wound healing, suggesting its therapeutic potential in refractory wound repair., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6 K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The delayed healing of infected post-operative wounds has turned into a worldwide medical problem. In the clinical treatment, effective bacterial clearance and promoted wound healing were considered as two crucial aspects. However, the effect of current dressings with antibacterial activity was limited due to the declined efficacy against antibiotic-resistant bacteria, and poor mechanical property during skin extension and compression movement. In this project, a lyotropic liquid crystal (LLC)-based bacteria-resistant and self-healing spray dressing loaded with ε-polylysine (PLL) was designed. Owing to the unique antibacterial mechanism, PLL was expected to kill antibiotic-resistant bacteria efficiently, even the "superbug" methicillin-resistant Staphylococcus aureus ( MRSA ). The cubic cells of LLC were applied to encapsulate PLL to improve its stability and induce a sustained release, further realizing a long-term antibacterial effect. Meanwhile, the LLC precursor (LLCP) could extend to the irregular edges of the wound, and spontaneously transited to a cubic phase gel once exposed to physiological fluid. This 3D structure was also endowed with mechanically responsive viscoelasticity that formed a robust and flexible defense for wounds. An excellent antibacterial activity with more than 99% MRSA killed in 3 h was demonstrated by a killing kinetics study. The long-term effect was also proved by measuring the bacteriostatic circle test within 48 h. In addition, the unique sol-gel phase transition behavior and superior self-healing capacity of PLL-LLCP was verified with the rheological study and self-recoverable conformal deformation test in vivo . In the infected post-operative wound model, satisfactory bacterial clearance and prominent wound healing promotion were realized by PLL-LLCP, with the survival of the bacteria at lower than 0.1% and the wound closure at higher than 90%. Thus, PLL-LLCP was believed to be an excellent candidate for the therapy of infected post-operative wounds.

The C-type lectin receptor DC-SIGN mediates interactions with envelope glycoproteins of many viruses such as SARS-CoV-2, ebola, and HIV and contributes to virus internalization and dissemination. In the context of the recent SARS-CoV-2 pandemic, involvement of DC-SIGN has been linked to severe cases of COVID-19. Inhibition of the interaction between DC-SIGN and viral glycoproteins has the potential to generate broad spectrum antiviral agents. Here, we demonstrate that mannose-functionalized poly-l-lysine glycoconjugates efficiently inhibit the attachment of viral glycoproteins to DC-SIGN-presenting cells with picomolar affinity. Treatment of these cells leads to prolonged receptor internalization and inhibition of virus binding for up to 6 h. Furthermore, the polymers are fully bio-compatible and readily cleared by target cells. The thermodynamic analysis of the multivalent interactions reveals enhanced enthalpy-driven affinities and promising perspectives for the future development of multivalent therapeutics., (© 2021 Wiley-VCH GmbH.)

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., Competing Interests: Competing interests: AMS is Chairman, CEO, Scientific Director and cofounder of Oncovir., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

ε-Poly-l-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special nonribosomal peptide synthetase (NRPS)-like enzyme called Pls. In this study, we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-l-lysine. A comprehensive sequence analysis suggested that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-l-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces , also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-l-lysine may naturally exist in cheese and on our skin, and ε-poly-l-lysine producers are not limited to filamentous actinobacteria., (Copyright © 2021 Jiang et al.)

The removal of excessive blood lead ions (Pb2+) is very important to human health, but current effective removal technology is still lacking because of the complex existence state of Pb2+ in blood, which can be attributed to the fact that most of the blood Pb2+ is combined with haemoglobin (Hb) located in red blood cells (RBCs). Here, a new type of magnetic mesoporous silica/ε-polylysine nanomotor-based remover (MMS/P NR) with abundant chelation sites was designed, synthesized and used to remove Pb2+ from blood. The magnetic core can make the nanocomposites become nanomotors with autonomous movement under an external variable magnetic field, which can effectively improve the contact probability between the MMS/P NRs and Pb2+-contaminated Hb in RBCs. The amino rich ε-polylysine (ε-PL) was used as the co-template of mesoporous silica. Mesoporous channels can provide a confinement effect for Pb2+-contaminated Hb to stabilize the captured blood Pb2+. The movement behavior of the MMS/P NRs in and out of RBCs and the capture mechanism of Pb2+ in the blood were studied. The results indicate that the MMS/P NRs we propose have good blood compatibility, low cytotoxicity, magnetic properties, autonomous movement ability and recyclability under the condition of an external magnetic field. Moreover, compared with the experimental conditions without an external variable magnetic field (0.01485 mg g-1), the MMS/P NRs show a higher blood Pb2+ removal ability under the condition of an external variable magnetic field (0.05525 mg g-1). The design strategy of this remover based on nanomotor technology has great potential in the future medical treatment of heavy metal poisoning.

An effective antibacterial nanofiber film was prepared through the incorporation of ε-polylysine (ε-PL) into gelatin/chitosan-based polymers. All nanofiber films had uniformly disordered fibrous structure with good diameter distribution. The weight ratio of the gelatin/chitosan/ε-PL (G/C/P) influenced the solution property and nanofiber morphology. The addition of ε-PL can decrease the viscosity and increase the conductivity of solutions, which lead to a decrease in the diameter of nanofibers. The three polymers of gelatin, chitosan, and ε-PL were interacted by hydrogen bonding, and the crystallinity of nanofiber films was decreased by the electrospinning process. The addition of ε-PL can enhance the thermal stability, and decrease the water vapor permeability and oxygen permeability of the films, and ε-PL did not easily release from the nanofiber films. The G/C/P (6:1:0.125) nanofiber film was more effective to control six foodborne pathogens than the G/C nanofiber films by destroying the bacterial cell membranes. The result indicated that the gelatin/chitosan/ε-PL nanofiber films can be used as a food-packaging material to reduce the risk of foodborne pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Thylakoid membranes of photosynthetic plant chloroplasts are involved in a wide range of energy producing pathways. Their stacking can be employed in order to provide increased surface area for biocatalytic purposes. Here we probe the aggregate formation of higher plants' thylakoids using low-molecular poly-l-lysine as an electrostatic polymer linker in low ionic strength media. Microelectrophoresis, actinic light scattering, millisecond-delayed fluorescence and free radical production of thylakoid membranes are measured and analyzed in the presence of the cationic polypeptide to track its influence on the surface electrical properties, the electron-transport processes and the proton gradient accumulation across membranes. Enhanced proton gradient in polylysine-treated thylakoids is obtained upon illumination due to alterations of the proton intake across the membrane resulting from the non-specific electrostatic interactions of the cationic polypeptide with thylakoids. We report lower rates of lipid peroxidation in polylysine-treated thylakoids measured both in the dark and under illumination in salt-free medium. The gained insight on the effect of polycations on photosynthetic membranes may be used in future developments of thylakoid-based approaches for energy transfer applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Multilayered coated liposomes were prepared using the layer-by-layer (LbL) technique in an effort to improve their stability in biological media. The formulation strategy was based on the alternate deposition of two biocompatible and biodegradable polyelectrolytes - poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) - on negatively charged small unilamellar vesicles (SUVs). Some parameters of the formulation process were optimized such as the polyelectrolyte concentration and the purification procedure. This optimized procedure has allowed the development of very homogeneous formulations of liposomes coated with up to 6 layers of polymers (so-called layersomes). The coating was characterized by dynamic light scattering (DLS), zeta potential measurements and Förster resonance energy transfer (FRET) between two fluorescently labeled polyelectrolytes. Studies on the stability of the formulations at 4 °C in a buffered solution have shown that most structures are stable over 1 month without impacting their encapsulation capacity. In addition, fluorophore release experiments have demonstrated a better resistance of the layersomes in the presence of a non-ionic detergent (Triton™ X-100) as well as in the presence of phospholipase A 2 and human plasma. In conclusion, new multilayered liposomes have been developed to increase the stability of conventional liposomes in biological environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: The aim of this study was to design naproxen sodium (NS)-containing, biomimetic, porous poly(lactide- co -glycolide) (PLGA) scaffolds for regeneration of damaged corneal epithelium. Methods: NS-incorporated PLGA scaffolds were prepared using the emulsion freeze-drying method and then coated with collagen or poly-l-lysine. Porosity measurements of the scaffolds were performed by the gas adsorption/desorption method and the scaffolds demonstrated highly porous, open-cellular pore structures with pore sizes from 150 to 200 μm. Results: The drug loading efficiency of scaffolds was found to be higher than 84%, and about 90%-98% of NS was released at the end of 7 days with a fast drug release rate at the initial period of time and then in a slow and sustained manner. The corneal epithelial cells were isolated from New Zealand white rabbits. The obtained cells were seeded onto scaffolds and continued to increase during the time period of the study, indicating that the scaffolds might promote corneal epithelial cell proliferation without causing toxic effects for at least 10 days. Conclusions: The NS-loaded PLGA scaffolds exhibited a combination of controlled drug release and biomimetic properties that might be attractive for use in treatment of corneal damage both for controlled release and biomedical applications.

Multistage delivery systems with size reduction capacity have been proposed as a powerful strategy for improving tissue drug penetration. Here we developed a simple and fast supramolecular approach to construct size-shrinkable polyamine-salt aggregates by ionic cross-linking of biodegradable poly-L-lysine dendrigraft with tripolyphosphate anion. The use of a peptide dendrimer as a nanobuilding block (∼7 nm in diameter) allows the formation of supraparticles (SPs) with well-defined dimensions (∼200 nm in diameter), narrow size distribution and great capacity to encapsulate different molecules, including chemotherapeutic agents as Curcumin and Doxorubicin. When exposed to slightly acidic environments, the crosslinked matrix is instantaneously disassembled to free dendrimer units. Subsequently, model cargo molecules entrapped in the dendrimer architecture can be released by the action of trypsin enzyme through peptide biodegradation. Therefore, these SPs with proved sequential pH and enzyme-responsiveness could be exploited as nanocarriers in multistage drug delivery systems., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ras proteins must localize to the plasma membrane (PM) for biological function. The membrane anchor of the K-Ras4B isoform comprises a farnesylated and methylated C-terminal cysteine together with an adjacent hexa-lysine polybasic domain (PBD). Traditionally, polybasic sequences have been thought to interact electrostatically with negatively charged membranes showing no specificity for anionic lipid head groups. By contrast we recently showed that the K-Ras membrane anchor actually exhibits a very high degree of specificity for phosphatidylserine (PtdSer). The selectivity for PtdSer is determined by a combinatorial code comprising the PBD sequence plus the prenyl anchor. Lipid binding specificity is therefore altered by PBD point mutations that in turn modulate signaling output. For example, mutating Lys177 or Lys178 to glutamine switches K-Ras4B lipid affinity from PtdSer to phosphoinositol 4,5-bisphosphate (PIP 2 ). Changing the lipid anchor from farnesyl to geranylgeranyl or the PBD lysines to arginines also changes lipid binding specificity. All-atom molecular dynamics simulations reveal the structural basis for these K-Ras anchor lipid-binding preferences. Here we examine the PM interactions of a series of geranylgeranylated PBD mutants and provide further evidence that the precise PBD sequence and prenyl lipid determines lipid sorting specificity of the K-Ras anchor and hence biological function.

The disruption of the cellular pathways of protein biosynthesis through the mechanism of RNA interference has been recognized as a tool of great diagnostic and therapeutic significance. However, in order to fully exploit the potential of this phenomenon, efficient and safe carriers capable of overcoming extra- and intracellular barriers and delivering siRNA to the target cells are needed. Recently, attention has focused on the possibility of the application of multifunctional nanoparticles, dendrimers, as potential delivery devices for siRNA. The aim of the present work was to evaluate the formation of dendriplexes using novel poly(lysine) dendrimers (containing lysine and arginine or histidine residues in their structure), and to verify the hypothesis that the use of these polymers may allow an efficient method of siRNA transfer into the cells in vitro to be obtained. The fluorescence polarization studies, as well as zeta potential and hydrodynamic diameter measurements were used to characterize the dendrimer:siRNA complexes. The cytotoxicity of dendrimers and dendriplexes was evaluated with the resazurin-based assay. Using the flow cytometry technique, the efficiency of siRNA transport to the myeloid cells was determined. This approach allowed us to determine the properties and optimal molar ratios of dendrimer:siRNA complexes, as well as to demonstrate that poly(lysine) dendrimers may serve as efficient carriers of genetic material, being much more effective than the commercially available transfection agent Lipofectamine 2000. This outcome provides the basis for further research on the application of poly(lysine) dendrimers as carriers for nucleic acids in the field of gene therapy.

RGD peptides play a pivotal role in growing and diverse areas of biological research, ranging from in vitro experiments probing fundamental molecular mechanisms of cell adhesion to more applied in vivo strategies in medical imaging and cancer therapeutics. To better understand the outcomes of RGD-based approaches, we quantified the degree to which cyclic RGD (cRGD) activity is blocked by nonspecific binding of commonly used medium constituents. First, we show that recombinant α V β 3 integrins can be used as a highly sensitive cell-free sensor to quantitatively and reliably characterize the activity of cRGD-functionalized surfaces via surface plasmon resonance (SPR). Next, SPR experiments were utilized to measure the extent of blocking of cRGD-functionalized surfaces by the commonly used agents BSA, PLL- g -PEG, and fetal calf serum (FCS)-supplemented media, using recombinant α V β 3 integrin as a probe for cRGD binding activity in the presence of blocking agents. All three additives were highly efficient blockers of cRGD activity, as exemplified by cell culture media containing 1% FCS which reduced the cRGD activity by 33-fold. We then developed a strategy to combat these deleterious effects by employing the recombinant integrins as a protective cap. We show that the unblocked cRGD activity can be preserved in the presence of PLL- g -PEG by employing the α V β 3 integrin as a removable protective cap, both in cell-free and in vitro experiments. In vitro studies with MDA-MB-231 cells cultured atop cRGD-functionalized surfaces found that cell adhesion and migration prevented by PLL- g -PEG were restored when this protective cap approach was used.

Trypsin-responsive near-infrared fluorescent (NIRF) and magnetic resonance (MR) dual-imaging composite nanoparticle/polypeptide coacervate nanoprobes with tunable sizes, have been constructed herein via electrostatic interaction-induced self-assembly. Considering the requirements of in vivo metabolism on nanoparticle size, three coacervate nanoprobes with diameters of around 100, 200, and 300 nm were fabricated with a polydispersity of around 0.2. These coacervate nanoprobes consist of Fe 3 O 4 magnetic nanoparticles surface-decorated with poly acrylic acid and Cy5.5-modified poly-l-lysine (PLL- g -Cy5.5) serving as MR imaging and trypsin-responsive substrate/NIRF agents, respectively. The notable fluorescence signal from PLL- g -Cy5.5 is self-quenched due to the short distances between the fluorescent Cy5.5 molecules after construction of the coacervate nanoprobes. Remarkably, coacervate nanoprobes with a diameter of around 100 nm are selectively disintegrated into fragmented segments upon the hydrolysis of PLL by trypsin, resulting in an 18-fold amplification of the NIRF intensity in comparison with the self-assembled coacervate nanoprobes in the quenched state. Moreover, the MR imaging enhancement is also related to the disintegration of the coacervate nanoprobes. Cellular experiments and in vivo studies demonstrate that the coacervate nanoprobes exhibit remarkable trypsin-sensitive NIRF and MR dual-imaging capabilities and thus have excellent potential to serve as dual-imaging nanoprobes for the efficient mapping of malignant tumors in which trypsin is often overexpressed. In consideration of their excellent capability to enrich charged molecules, the coacervate nanoprobes provide a conceptually novel and promising platform toward in vivo trypsin mapping and controlled delivery of targeted payloads.

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg 2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.

Phenothiazines are very effective antipsychotic drugs, which also have anticancer and antimicrobial activities. Despite being used in human treatment, the molecular mechanism of the biological actions of these molecules is not yet understood in detail. The role of the interactions between phenothiazines and proteins or lipid membranes has been much discussed. Herein, fourier-transform infrared (FTIR) spectroscopic studies were used to investigate the effect of three phenothiazines: fluphenazine (FPh); chlorpromazine (ChP); and propionylpromazine (PP) on the structures of a positively charged poly-l-lysine (PLL) peptide, a negatively charged dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) membrane, and on the mutual interactions between electrostatically associated PLL molecules and DPPC/DPPG membranes. Phenothiazine-induced alterations in the secondary structure of PLL, the conformational state (trans/gauche) of the hydrocarbon lipid chains, and the hydration of the DPPC/DPPG membrane interface were studied on the basis of amide I' vibrations, antisymmetric and symmetric stretching vibrations of the CH 2 groups of the lipid hydrocarbon chains (ν s CH 2 ), and stretching vibrations of the lipid C=O groups (νC = O), respectively. It was shown that in the presence of negatively charged DPPC/DPPG membranes, the phenothiazines were able to modify the secondary structure of charged PLL molecules. Additionally, the effect of PLL on the structure of DPPC/DPPG membranes was also altered by the presence of the phenothiazine molecules., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Clusters of positively-charged basic amino acid residues, particularly lysine, are known to promote the interaction of many peripheral membrane proteins with the cytoplasmic surface of the plasma membrane via electrostatic interactions. In this work, cholesterol's effects on the interaction between lysine residues and membranes have been studied. Using poly-l-lysine (PLL) and vesicles as models to mimic the interaction between lysine-rich protein domains and the plasma membrane, light scattering measurements indicated cholesterol enhanced the electrostatic interaction through indirectly affecting the negatively charged phospholipid dioleoylphosphatidylserine, DOPS. Addition of PLL to lipid vesicles containing DOPS showed an initial increase in static light scattering (SLS), attributed to binding of PLL to the vesicle surface, followed by a slower continuously declining SLS signal, which, from comparison with fluorescent dye leakage studies could be attributed to vesicle lysis. Although electrostatic interactions between PLL and the membrane were not necessary for penetration to occur, cholesterol promoted membrane disruption of negatively charged vesicles, possibly by increasing the electrostatic interactions between PLL and the membrane. In contrast, cholesterol lowered the susceptibility of uncharged vesicles (formed using dioleoylphosphatidylcholine, DOPC) to PLL penetration. This can be explained by the absence of electrostatic interactions and cholesterol's known ability to increase membrane thickness and mechanical strength. Thus, the ability of cationic peptides to penetrate membranes including cholesterol is likely to depend on the membrane's PS:PC ratio., (Copyright © 2019 Elsevier B.V. All rights reserved.)

ε-Poly-l-lysine (ε-PL) consists of 25-35 lysine residues which are linked by an isopeptide bond formed by dehydration condensation of α-carboxyl and ε-amino groups and has good antibacterial activity and broad-spectrum inhibition range. However, there is no clear conclusion about the structure and antibacterial mechanism of ε-PL in aqueous solution. Herein, a high purity of ε-PL was prepared using Amberlite IRC-50 ion-exchange resin. Membrane filtration and dynamic light scattering were used to study the variations of ε-PL aggregation in aqueous solution with pH value. The conformational changes and antibacterial activities of ε-PL and carbamoylated ε-PL in different water environments were studied with circular dichroism (CD) and inhibition zone. The structural changes during the spray-drying process were determined by Fourier transform infrared spectroscopy. The results indicated that the side chain amino charge played a decisive role in the ε-PL conformation and aggregation. ε-PL exhibited the properties of a β-sheet during spray drying from acidic liquids to solids. The cation enhanced the antibacterial activity of ε-PL but did not play a key role. Instead, the backbone of ε-PL might determine the mechanism of ε-PL antibacterial.

Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome's decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.

Programmable behavior combined with tailored stiffness and tunable biomechanical response are key requirements for developing successful materials. However, these properties are still an elusive goal for protein-based biomaterials. Here, we use protein-polymer interactions to manipulate the stiffness of protein-based hydrogels made from bovine serum albumin (BSA) by using polyelectrolytes such as polyethyleneimine (PEI) and poly-L-lysine (PLL) at various concentrations. This approach confers protein-hydrogels with tunable wide-range stiffness, from ~10-64 kPa, without affecting the protein mechanics and nanostructure. We use the 6-fold increase in stiffness induced by PEI to program BSA hydrogels in various shapes. By utilizing the characteristic protein unfolding we can induce reversible shape-memory behavior of these composite materials using chemical denaturing solutions. The approach demonstrated here, based on protein engineering and polymer reinforcing, may enable the development and investigation of smart biomaterials and extend protein hydrogel capabilities beyond their conventional applications.

The combination of high mechanical properties, antibacterial activity and a green synthesis of the polyvinyl alcohol (PVA) based films remains challenging. This study presents a ternary system of PVA films containing bacterial cellulose (BC) and epsilon-polylysine (ε-PL) by a green solution casting method. The prepared composite films showed more than 99% antibacterial properties against both Staphylococcus aureus and Escherichia coli bacteria. Moreover, the films were collected after a single use and were reused twice, which still exhibited strong antibacterial activity. The films showed thermal stability and higher mechanical properties as compared to pure PVA films. In addition, the cytotoxicity of the films was evaluated by MTT assay against NIH 3T3 mouse fibroblast cells. The results showed no toxicity of the films towards tested cells. We believe that these antibacterial films may find applications in active food packaging and biomedical fields., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.

DNA condensed agents can improve the transfection efficiency of the cationic liposome delivery system. However, various condensed agents have distinct transfection efficiency and cellular cytotoxicity. The object of this study was to screen the optimal agents with the high transfection efficiency and low cytotoxicity from four polymer compressive materials, polyethylenimine (PEI), chitosan, poly-l-lysine (PLL), and spermidine. DNA was precompressed with these four agents and then combined to cationic liposomes. Subsequently, the entrapment and transfection efficiency of the obtained complexes were investigated. Finally, the particle sizes, cytotoxicity, and endocytosis fashion of these copolymers (Lipo-PEI, Lipo-chitosan, Lipo-PLL, and Lipo-spermidine) were examined. It was found that these four copolymers had significantly lower cytotoxicity and higher transfection efficiency (45.5%, 42.4%, 36.8%, and 47.4%, respectively) than those in the control groups. The transfection efficiency of Lipo-PEI and Lipo-spermidine copolymers were better than the other two copolymers. In 293T cells, nystatin significantly inhibited the transfection efficiency of Lipo-PEI-DNA and Lipo-spermidine-DNA (51.88% and 46.05%, respectively), which suggest that the endocytosis pathway of Lipo-spermidine and Lipo-PEI copolymers was probably caveolin dependent. Our study indicated that these dual-degradable copolymers especially liposome-spermidine copolymer could be used as the potential biocompatible gene delivery carriers.