Biological Activity of Poly(1,3-propanediol citrate) Films and Nonwovens: Mechanical, Thermal, Antimicrobial, and Cytotoxicity Studies.

Polymers are of great interest for medical and cosmeceutical applications. The current trend is to combine materials of natural and synthetic origin in order to obtain products with appropriate mechanical strength and good biocompatibility, additionally biodegradable and bioresorbable. Citric acid, being an important metabolite, is an interesting substance for the synthesis of materials for biomedical applications. Due to the high functionality of the molecule, it is commonly used in biomaterials chemistry as a crosslinking agent. Among citric acid-based biopolyesters, poly(1,8-octanediol citrate) is the best known. It shows application potential in soft tissue engineering. This work focuses on a much less studied polyester, poly(1,3-propanediol citrate). Porous and non-porous materials based on the synthesized polyesters are prepared and characterized, including mechanical, thermal, and surface properties, morphology, and degradation. The main focus is on assessing the biocompatibility and antimicrobial properties of the materials.

Polytrimethylenimines: Highly Potent Antibacterial Agents with Activity and Toxicity Modulated by the Polymer Molecular Weight.

Cationic polymers have been extensively investigated as a potential replacement for traditional antibiotics. Here, we examined the effect of molecular weight (MW) on the antimicrobial, cytotoxic, and hemolytic activity of linear polytrimethylenimine (L-PTMI). The results indicate that the biological activity of the polymer sharply increases as MW increases. Thanks to a different position of the antibacterial activity and toxicity thresholds, tuning the MW of PTMI allows one to achieve a therapeutic window between antimicrobial activity and toxicity concentrations. L-PTMI presents significantly higher antimicrobial activity against model microorganisms than linear polyethylenimine (L-PEI) when polymers with a similar number of repeating units are compared. For the derivatives of L-PTMI and L-PEI, obtained through N-monomethylation and partial N,N-dimethylation of linear polyamines, the antimicrobial activity and toxicity were both reduced; however, resulting selectivity indices were higher. Selected materials were tested against clinical isolates of pathogens from the ESKAPE group and Mycobacteria, revealing good antibacterial properties of L-PTMI against antibiotic-resistant strains of Gram-positive and Gram-negative bacteria but limited antibacterial properties against Mycobacteria.

Valorisation of Whey Permeate in Sequential Bioprocesses towards Value-Added Products-Optimisation of Biphasic and Classical Batch Cultures of Kluyveromyces marxianus.

Whey permeate is categorised as hazardous wastewater for aquatic environments, mainly due to its high lactose content. Therefore, it must be valorised before being released into the environment. One pathway for whey permeate management is its use in biotechnological processes. Herein, we present roads for whey permeate valorisation with the K. marxianus WUT240 strain. The established technology is based on two bioprocesses. During first, 2.5 g/L 2-phenylethanol and fermented plant oils enriched with different flavourings are obtained after 48 h biphasic cultures at 30 °C. The second process leads to a maximum of 75 g ethanol/L (YP/S = 0.53 g/g) after 96 h at 30 °C. Moreover, established whey permeate valorisation pathways reduced its biochemical oxygen demand and chemical oxygen demand values by 12- to 3-fold, respectively. Together, the present study reports a complete, effective, and environmentally friendly whey permeate management strategy while simultaneously enabling the acquisition of valuable compounds with substantial application potential.

Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos-e.g., proteins, RNAs, and lipids-that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system.

Development of a Continuous System for 2-Phenylethanol Bioproduction by Yeast on Whey Permeate-Based Medium.

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L-1 h-1.

Kinetics of neomycin release from polylactide spheres and its antimicrobial activity.

BACKGROUND: Neomycin is a natural aminoglycoside antibiotic produced by actinomycete Streptomyces fradiae. It exerts bacteriostatic and bactericidal activity against Gram-negative bacteria, certain Gram-positive bacteria and Mycobacterium tuberculosis. Neomycin inhibits the biosynthesis of bacterial proteins by impairing their life functions, leading to death of cells. OBJECTIVES: To examine the effect of molecular weight of polylactide (PLA), the applied stabilizer as well as mixing speed used in the encapsulation process on the size of obtained spheres. Examination of the kinetics of neomycin release from the obtained PLA spheres and determination of the antimicrobial activity of the neomycin-containing spheres against selected strains of bacteria, yeast and fungi have also been necessary. MATERIAL AND METHODS: Polylactide (Mn 3000-40,000 g/mol) was obtained in-house. Other materials used in the study were as follows: L-lactic acid (PLLA; Mn 66,500 g/mol and 86,000 g/mol), polyvinyl alcohol (PVA) as a stabilizer of emulsion (Mw 30,000 g/mol, 130,000 g/mol; degree of hydrolysis 88%) as well as dichloromethane, p.a. and dimethyl sulfoxide (DMSO), p.a. as solvents. Distilled water was obtained in-house. Neomycin sulfate was used for encapsulation; phosphate (pH 7.2) and acetate (pH 4.5) buffers were used for the examination of the active pharmaceutical ingredient (API) dissolution profile. Antimicrobial activity was tested using commercial cell lines and the following media: Mueller-Hinton agar (MHA), Mueller-Hinton broth (MHB), yeast extract peptone dextrose (YPD), and potato dextrose agar (PDA). RESULTS: Neomycin-containing PLA spheres were obtained using an emulsion method. The average molecular weight of PLA, the average molecular weight of PVA and mixing speed on the size of obtained spheres were investigated. Furthermore, the profile of API dissolution from the spheres and antimicrobial activity of neomycin-containing spheres against certain strains of bacteria, yeast and fungi were determined. CONCLUSIONS: We demonstrated that efficient encapsulation of neomycin requires spheres of a <200 mm diameter.

Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity.

With the appearance of the antifungal resistance, novel antifungal agents need to be identified. In this context new 2,5-disubstituted tetrazole derivatives containing benzothiazole, benzoxazole or phenylsulfonyl moiety were synthesized by N-alkylation of aryltetrazole with 2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole or 2-[(3-chloropropyl)sulfanyl]-1,3-benzoxazole and Michael-type addition of aryltetrazole to phenyl vinyl sulfone. The chemical structures of the synthesized compounds were confirmed by means of 1H NMR, 13C NMR, IR and HRMS spectral data. The compounds were tested against the moulds: Fusarium sambucinum, Fusarium oxysporum, Colletotrichum coccodes, Aspergillus niger, and the yeast Candida albicans. The results showed that among the moulds only C. coccodes was significantly sensitive to all the structures examined. All the tetrazole derivatives acted at the same level against C. albicans and demonstrated a high cell growth inhibition (97-99%) at the concentrations ranging from 16 to 0.0313µg/mL. The mode of action of 2-({3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5c) and 2-({3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5d) was established by verifying fungal growth in the presence of osmotic protector-sorbitol. The effect of compound 5c or 5d combined with Fluconazole was determined using the checkerboard method. The calculated fractional inhibitory concentration index (FIC) indicated antagonism (FIC >1). Additionally, survival experiments with lepidopteran Galleria mellonella treated with compounds 5c and 5d were performed and demonstrated the lack of toxicity of these compounds.

Fructose bisphosphate aldolase is involved in the control of RNA polymerase III-directed transcription.

Yeast Fba1 (fructose 1,6-bisphosphate aldolase) is a glycolytic enzyme essential for viability. The overproduction of Fba1 enables overcoming of a severe growth defect caused by a missense mutation rpc128-1007 in a gene encoding the C128 protein, the second largest subunit of the RNA polymerase III complex. The suppression of the growth phenotype by Fba1 is accompanied by enhanced de novo tRNA transcription in rpc128-1007 cells. We inactivated residues critical for the catalytic activity of Fba1. Overproduction of inactive aldolase still suppressed the rpc128-1007 phenotype, indicating that the function of this glycolytic enzyme in RNA polymerase III transcription is independent of its catalytic activity. Yeast Fba1 was determined to interact with the RNA polymerase III complex by coimmunoprecipitation. Additionally, a role of aldolase in control of tRNA transcription was confirmed by ChIP experiments. The results indicate a novel direct relationship between RNA polymerase III transcription and aldolase.

Enzymatic hydrolysis of esters containing a tetrazole ring.

The lipase-catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4-(5-aryl-2H-tetrazol-2yl)butan-2-ol and 1-(5-aryl-2H-tetrazol-2yl)-propan-2-ol and their acetates with the highest optical purities (ee = 95%-99%) and excellent enantioselectivity (E>100). Some of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mixtures of 4-[5-(4-chlorophenyl)-2H-tetrazol-2-yl)butan-2-ol as well as pure enantiomers of this compound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes, and A. niger.

Unlocking the Potential: PEGylation and Molecular Weight Reduction of Ionenes for Enhanced Antifungal Activity and Biocompatibility.

Numerous synthetic polymers, imitating natural antimicrobial peptides, have demonstrated potent antimicrobial activity, positioning them as potential candidates for new antimicrobial drugs. However, the high activity of these molecules often comes at the cost of elevated toxicity against eukaryotic organisms. In this study, a series of cationic ionenes with varying molecular weights to assess the influence of polymer chain length on ionene activity is investigated. To enhance polymer antimicrobial activity and limit toxicity a PEG side chain is introduced into the repeating unit. The resulting molecules consistently exhibited high activity against three model organisms: E. coli, S. aureus and C. albicans. The incorporation of side PEG chain improves antifungal properties and biocompatibility, regardless of molecular weight. The most important finding of this work is that the reduction of polymer molecular mass led to increased antifungal activity and reduced cytotoxicity against HMF and MRC-5 cell lines simultaneously. As a result, the best-performing molecules reported herein displayed minimal inhibitory concentrations (MIC) as low as 2 and 0.0625 µg mL1 for C. albicans and C. tropicalis respectively, demonstrating exceptional selectivity. It is plausible that some of described herein molecules can serve as potential lead candidates for new antifungal drugs.

Are all yeast biosurfactants really capable of lowering surface tension below 30 mN/m ?

The study discusses pitfalls in attempts to determine reliable surface tension values for the culture media and their extracts for two biosurfactant-producing yeast strains: Rhodotorula graminis and Rhodotorula babjevae. The values obtained from an Axisymmetric Drop Shape Analysis (ADSA) tensiometer showed systematically more and more shallow dynamic surface tension decays, suggesting a deterioration of their surface activity. The rate of this apparent surface activity loss was shown to depend on the sample history, with slower changes observed in vigorously shaken samples. On the other hand, the force-based Wilhelmy plate method provided apparently stable surface tension values of the order of 30 mN/m, in accordance with numerous previous literature reports on similar yeast biosurfactants. Both observations can be justified by the presence of an oil emulsified by biosurfactants produced by the yeast. We show that the odd (apparent) surface tension results are in fact the measurement artifacts resulting from slow demulsification and subsequent oil-spreading assisted by the yeast biosurfactants. The apparent surface tension reduction is thus indeed caused by the presence of biosurfactants, but its value does not represent their real adsorption in a thermodynamic sense. Consequently, the often reported in the literature very low surface tension values for the yeast culture media, of the order of 30 ± 5 mN/m, should be treated with caution, especially if the emulsion stabilized with the biosurfactant had not been fully destabilized prior to the measurement.

In vitro immune evaluation of adenoviral vector-based platform for infectious diseases.

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

Prokaryotic ParA-ParB-parS system links bacterial chromosome segregation with the cell cycle.

While the essential role of episomal par loci in plasmid DNA partitioning has long been appreciated, the function of chromosomally encoded par loci is less clear. The chromosomal parA-parB genes are conserved throughout the bacterial kingdom and encode proteins homologous to those of the plasmidic Type I active partitioning systems. The third conserved element, the centromere-like sequence called parS, occurs in several copies in the chromosome. Recent studies show that the ParA-ParB-parS system is a key player of a mitosis-like process ensuring proper intracellular localization of certain chromosomal regions such as oriC domain and their active and directed segregation. Moreover, the chromosomal par systems link chromosome segregation with initiation of DNA replication and the cell cycle.

Influence of PEG Subunit on the Biological Activity of Ionenes: Synthesis of Novel Polycations, Antimicrobial and Toxicity Studies.

An alarming increase of antibiotic resistance among pathogens creates an urgent need to develop new antimicrobial agents. Many reported polycations show high antimicrobial activity along with low hemolytic activity. Unfortunately, most of those molecules remain highly cytotoxic against various mammalian cells. In this work, a systematic study on the impact of triethylene glycol monomethyl ether side groups (short polyethylene glycol (PEG) analog) on antimicrobial, hemolytic, and cytotoxic properties of novel amphiphilic ionenes is presented. A detailed description of synthesis, leading to well-defined alternating polymers, which differ in structural elements responsible for hydrophilicity (PEG) and hydrophobicity (alkyl chain), is presented. Obtained results show that the PEG moiety and fine-tuned hydrophilic-lipophilic balance of ionenes synergistically lead to low cytotoxic, low hemolytic molecules with high activity against S. aureus, including methicillin-resistant strains (MRSA). Additionally, the results of mechanistic studies on bacterial cells and fluorescently labeled liposomes are also discussed.

Main-chain flexibility and hydrophobicity of ionenes strongly impact their antimicrobial activity: an extended study on drug resistance strains and Mycobacterium.

The spread of antibiotic-resistant pathogens and the resurgence of tuberculosis disease are major motivations to search for novel antimicrobial agents. Some promising candidates in this respect are cationic polymers, also known as synthetic mimics of antimicrobial peptides (SMAMPs), which act through the membrane-lytic mechanism. Development of resistance toward SMAMPs is less likely than toward currently employed antibiotics; however, further studies are needed to better understand their structure-activity relationship. The main objective of this work is to understand the cross-influence of hydrophobicity, main-chain flexibility, and the topology of ionenes (polycations containing a cationic moiety within the main-chain) on activity. To fulfill this goal, a library of ionenes was developed and compared with previously investigated molecules. The obtained compounds display promising activity against the model microorganisms and drug-resistance clinical isolates, including Mycobacterium tuberculosis. The killing efficiency was also investigated, and results confirm a strong effect of hydrophobicity, revealing higher activity for molecules possessing the flexible linker within the polymer main-chain.

Influence of lipid bilayer composition on the activity of antimicrobial quaternary ammonium ionenes, the interplay of intrinsic lipid curvature and polymer hydrophobicity, the role of cardiolipin.

Incorporation of hydrophobic component into amphiphilic polycations structure is frequently accompanied by an increase of antimicrobial activity. There is, however, a group of relatively hydrophilic polycations containing quaternary ammonium moieties along mainchain, ionenes, which also display strong antimicrobial and limited hemolytic properties. In this work, an influence of a hydrophobic side group length on antimicrobial mechanism of action is investigated in a series of novel amphiphilic ionenes. High antimicrobial activity was found by determination of minimum inhibitory concentration (MIC) and minimum bactericidal, and fungicidal concentration (MBC and MFC) in both growth media and a buffer. Biocompatibility was estimated by hemolytic and mammalian cells viability assays. Mechanistic studies were performed using large unilamellar vesicles (LUVs) with different lipid composition, as simplified models of cell membranes. The investigated ionenes are potent and selective antimicrobial molecules displaying a decrease of antimicrobial activity correlated with increase of hydrophobicity. Studies using LUVs revealed that the cardiolipin is an essential component responsible for the lipid bilayer permeabilization by investigated ionens. In contrast to relatively hydrophilic ionenes, more hydrophobic polymers showed an ability to stabilize membranes composed of lipids with negative spontaneous curvature in a certain range of polymer to lipid ratio. The results substantially contribute to the understanding of antimicrobial activity of the investigated class of polymers.

Antimicrobial films of poly(2-aminoethyl methacrylate) and its copolymers doped with TiO2 and CaCO3.

Polymeric coatings with positive surface charge offer potential antimicrobial activity, which they owe to a simple electrostatic attraction with negatively charged bacterial walls and membranes. We describe synthesis and characterization of poly(2-aminoethyl methacrylate) and its copolymers with methyl methacrylate and butyl acrylate, as potential binders for antimicrobial solvent-cast paints. TiO2 and CaCO3 mineral particles were employed as model pigments/fillers, as they are used in most real-life paint formulations. Electrokinetic (ζ) potential and antimicrobial activity of thin films made of the (co)polymers in the absence and presence of TiO2 and CaCO3 nanopowders were assessed using streaming current measurements and microbial growth inhibition tests, respectively. Independently of the structure of the monomers used for the synthesis, the films showed positive ζ-potential values (up to +95 mV) in the pH range 3.5-8.0. The presence of mineral particles at 50% dry weight of the films did not affect significantly the ζ(pH) curves. The films made of the mixed dispersions remained positively charged and inhibited growth of both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, as well as yeast (C. albicans). The mixed polymeric-mineral films described in this study seem to be promising potential candidates for designing antimicrobial coatings aimed to prevent spreading of bacterial infections.

Hydrophilic Quaternary Ammonium Ionenes-Is There an Influence of Backbone Flexibility and Topology on Antibacterial Properties?

The antimicrobial properties of polycations are strongly affected by the structural features such as the backbone flexibility and topology (isomerism) through the polymer ability to attain proper conformation in interaction with the cell membrane. In this paper, a synthesis and biocidal properties evaluation of ionenes characterized by different backbone topology (isomerism) and flexibility are presented. The findings reveal influence of variation in topology on activity against different microorganisms, and general positive effect of improved flexibility. Furthermore, one of the obtained ionenes displays degradable properties in near physiological environment (phosphate-buffered saline pH 7.4, 37 °C). The degradation proceeds via Hofmann elimination reaction and the products are not of acidic character. For the first time a new class of degradable ionenes with a high antimicrobial potential is presented.

Carotenoid-producing yeasts: Identification and Characteristics of Environmental Isolates with a Valuable Extracellular Enzymatic Activity.

Sixteen cold-adapted reddish-pigmented yeast strains were obtained from environmental samples. According to the PCR-based detection of classical yeast markers combined with phylogenetic studies, the yeasts belong mainly to the genera Rhodotorula, Sporobolomyces and Cystobasidium, all within the subphylum Pucciniomycotina. All strains produced carotenoids within a 0.25-10.33 mg/L range under non-optimized conditions. Noteworthily, among them, representatives of the Cystobasidium genus were found; of particular value are the strains C. laryngis and C. psychroaquaticum, poorly described in the literature to date. Interestingly, carotenoid production with representatives of Cystobasidium was improved 1.8- to 10-fold at reduced temperature. As expected, most of the isolated yeasts biosynthesized extracellular lipases, but within them also one proteolytic and four cellulolytic strains were revealed. We succeeded in isolating strain Cystofilobasidium macerans WUT145 with extraordinarily high cellulolytic activity at 22°C (66.23 ± 0.15 µmol/mg protein·min) that is described here for the first time. Consequently, a set of yeasts capable of producing both carotenoids and extracellular enzymes was identified. Taking into account those abilities, the strains might be applicable for a development of carotenoids production on an agro-industrial waste, e.g., lignocellulose.

Amphiphilic Polymethyloxazoline-Polyethyleneimine Copolymers: Interaction with Lipid Bilayer and Antibacterial Properties.

Polycations, mimicking activity of antibacterial peptides, belong to an important class of molecules investigated as a support or as an alternative to antibiotics. In this work, studies of modified linear amphiphilic statistical polymethyloxazoline (PMOX) and polyethyleneimine copolymers (PMOX_PEI) series are presented. Variation of PEI content in the structure results in controllable changes of polymeric aggregates zeta potential. The structure with the highest positive charge shows the best antimicrobial activity, well visible in tests against model Gram-positive and Gram-negative bacteria, fungi, and mycobacterium strains. The polymer toxicity is evaluated with MTT and hemolysis assay as a reference. Quartz crystal microbalance (QCM-D) is used to investigate interaction between polycations and a model lipid membrane. Polymer activity correlates well with molecular structure, showing that amphiphilic component is altering polymer behavior in contact with the lipid bilayer.