Two-Year Study on the Intra-Individual Dynamics of Gut Microbiota and Short-Chain Fatty Acids Profiles in Healthy Adults.

While the gut microbiome has been intensively investigated for more than twenty years already, its role in various disorders remains to be unraveled. At the same time, questions about what changes in the gut microbiota can be considered as normal or pathological and whether communities are able to recover after exposure to negative factors (diseases, medications, environmental factors) are still unclear. Here, we describe changes in the gut microbiota composition and the content of short-chain fatty acids in adult healthy volunteers (n = 15) over a 24 month-period. Intraindividual variability in gut microbial composition was 40%, whereas the short chain fatty acids profile remained relatively stable (2-year variability 20%, inter-individual 26%). The changes tend to accumulate over time. Nevertheless, both short-term and long-term changes in the gut microbiome composition were significantly smaller within individuals than interindividual differences (two-year interindividual variability was 75%). Seasonal changes in gut microbiota were found more often in autumn and spring involving the content of minor representatives (less than 1.5% of the community in average) in the phyla Actinobacteriota, Firmicutes and Proteobacteria.

Antibacterial Activity of Various Morphologies of Films Based on Guanidine Derivatives of Pillar[5]arene: Influence of the Nature of One Substitute on Self-assembly.

The progress of the pillar[5]arene chemistry allowed us to set out a new concept on application of the supramolecular assemblies to create antimicrobial films with variable surface morphologies and biological activities. Antibacterial films were derived from the substituted pillar[5]arenes containing nine pharmacophoric guanidine fragments and one thioalkyl substituent. Changing the only thioalkyl fragment in the macrocycle structure made it possible to control the biological activity of the resulting antibacterial coating. Pretreatment of the surface with aqueous solution of the amphiphilic pillar[5]arenes reduced the biofilm thickness by 56 ± 10% of Gram-positive Staphylococcus aureus in the case of the pillar[5]arene containing a thiooctyl fragment and by 52 ± 7% for the biofilm of Gram-negative Klebsiella pneumoniae in the case of pillar[5]arene containing a thiooctadecyl fragment. Meanwhile, the cytotoxicity of the synthesized macrocycles was examined at a concentration of 50 µg/mL, which was significantly lower than that of bis-guanidine-based antimicrobial preparations.

The Antimicrobial Potential of the Hop (Humulus lupulus L.) Extract against Staphylococcus aureus and Oral Streptococci.

Plant extracts are in the focus of the pharmaceutical industry as potential antimicrobials for oral care due to their high antimicrobial activity coupled with low production costs and safety for eukaryotic cells. Here, we show that the extract from Hop (Humulus lupulus L.) exhibits antimicrobial activity against Staphylococcus aureus and Streptococci in both planktonic and biofilm-embedded forms. An extract was prepared by acetone extraction from hop infructescences, followed by purification and solubilization of the remaining fraction in ethanol. The effect of the extract on S. aureus (MSSA and MRSA) was comparable with the reference antibiotics (amikacin, ciprofloxacin, and ceftriaxone) and did not depend on the bacterial resistance to methicillin. The extract also demonstrated synergy with amikacin on six S. aureus clinical isolates, on four of six isolates with ciprofloxacin, and on three of six isolates with ceftriaxone. On various Streptococci, while demonstrating lower antimicrobial activity, an extract exhibited a considerable synergistic effect in combination with two of three of these antibiotics, decreasing their MIC up to 512-fold. Moreover, the extract was able to penetrate S. aureus and S. mutans biofilms, leading to almost complete bacterial death within them. The thin-layer chromatography and LC-MS of the extract revealed the presence of prenylated flavonoids (2',4',6',4-tetrahydroxy-3'-geranylchalcone) and acylphloroglucides (cohumulone, colupulone, humulone, and lupulone), apparently responsible for the observed antimicrobial activity and ability to increase the efficiency of antibiotics. Taken together, these data suggest an extract from H. lupulus as a promising antimicrobial agent for use both as a solely antiseptic and to potentiate conventional antimicrobials.

Effect of meso-substituents and medium properties on the photo- and pH-stability, penetration efficiency into bacterial and microscopic fungi cells of terpene-BODIPY conjugates.

In order to expand the arsenal of tools and areas for practical use of BODIPY dyes as bifunctional fluorescent theranostics, we studied the effect of the meso-substituents nature and medium properties on photo- and pH-stability, efficiency of singlet oxygen generation, and affinity to biostructures of terpene-BODIPY conjugates. The BODIPYs fused with myrtenol or thiotherpenoid via carboxylic acid residues exhibit high stability over a wide pH range and the presence of a bulky substituent at the meso-position of BODIPY conjugates increases their photostability two-fold compared to structurally related meso-unsubstituted analogues. Furthermore, the photodegradation rate of the conjugates directly depends on their ability to generate singlet oxygen and the course probability of the corresponding red-ox reactions involving reactive oxygen species. The conjugate of BODIPY with a thiotherpenoid demonstrated high ability to penetrate the membranes of filamentous and yeast-like fungi and bind to membrane of organelles in the fungal cell. At the same time, this compound also had a high ability to penetrate into biofilms of Staphylococcus aureus and Klebsiella pneumoniae and into bacterial cells within the matrix, which makes this compound promising for staining intracellular structures of eukaryotic cells and bacteria embedded into biofilms.

Unraveling the Mechanism of Platelet Aggregation Suppression by Thioterpenoids: Molecular Docking and In Vivo Antiaggregant Activity.

Natural monoterpenes and their derivatives are widely considered the effective ingredients for the design and production of novel biologically active compounds. In this study, by using the molecular docking technique, we examined the effects of two series of "sulfide-sulfoxide-sulfone" thioterpenoids containing different (e.g., bornane and pinane) monoterpene skeletons on the platelet's aggregation. Our data revealed that all the synthesized compounds exhibit inhibitory activities on platelet aggregation. For example, compound 1 effectively inhibited platelet activation and demonstrated direct binding with CD61 integrin, a well-known platelet GPIIb-IIIa receptor on platelets. We further examined the antiaggregant activity of the most active compound, 1, in vivo and compared its activity with that of acetylsalicylic acid and an oral GPIIb-IIIa blocker, orbofiban. We found that compound 1 demonstrates antiaggregant activity in rats when administered per os and its activity was comparable with that of acetylsalicylic acid and orbofiban. Moreover, similarly, tirofiban, a well-known GPIIb-IIIa blocker, compound 1, effectively decreased the expression of P-selectin to the values similar to those of the intact platelets. Collectively, here, we show, for the first time, the potent antiaggregant activity of compound 1 both in vitro and in vivo due to its ability to bind with the GPIIb-IIIa receptor on platelets.

Reversal in the drought stress response of the Scots pine forest ecosystem: Local soil water regime as a key to improving climate change resilience.

In a changing climate, forest ecosystems have become increasingly vulnerable to continuously exacerbating heat and associated drought conditions. Climate stress resilience is governed by a complex interplay of global, regional, and local factors, with hydrological conditions being among the key players. We studied a Scots pine (Pinus sylvestris L.) forest ecosystem located near the southern edge of the boreal ecotone, which is particularly subjected to frequent and prolonged droughts. By comparing the dendrochronological series of pines growing in apparently contrasting hydrological conditions ranging from the waterlogged peat bog area to the dry soil at the surrounding elevations, we investigated how the soil water regime affects the climate response and drought stress resilience of the forest ecosystem. We found that in the dry land area, a significant fraction of the trees were replaced after two major climate extremes: prolonged drought and extremely low winter temperatures. The latter has also been followed by a three- to ten-fold growth reduction of the trees that survived in the next year, whereas no similar effect has been observed in the peat bog area. Multi-scale detrended partial cross-correlation analysis (DPCCA) indicated that tree-ring width (TRW) was negatively correlated with spring and summer temperatures and positively correlated with the Palmer drought severity index (PDSI) for the same year. For the elevated dry land area, the above effect extends to interannual scales, indicating that prolonged heatwaves and associated droughts are among the factors that limit tree growth. In marked contrast, in the waterlogged peat bog area, a reversed tendency was observed, with prolonged dry periods as well as warmer springs and summers over several consecutive years, leading to increasing tree growth with a one- to three-year time lag. Altogether, our results indicate that the pessimal conditions of a warming climate could become favorable through the preservation of the soil water regime.

The Effect of Ficin Immobilized on Carboxymethyl Chitosan on Biofilms of Oral Pathogens.

In the last decade, Ficin, a proteolytic enzyme extracted from the latex sap of the wild fig tree, has been widely investigated as a promising tool for the treatment of microbial biofilms, wound healing, and oral care. Here we report the antibiofilm properties of the enzyme immobilized on soluble carboxymethyl chitosan (CMCh) and CMCh itself. Ficin was immobilized on CMCh with molecular weights of either 200, 350 or 600 kDa. Among them, the carrier with a molecular weight of 200 kDa bound the maximum amount of enzyme, binding up to 49% of the total protein compared to 19-32% of the total protein bound to other CMChs. Treatment with pure CMCh led to the destruction of biofilms formed by Streptococcus salivarius, Streptococcus gordonii, Streptococcus mutans, and Candida albicans, while no apparent effect on Staphylococcus aureus was observed. A soluble Ficin was less efficient in the destruction of the biofilms formed by Streptococcus sobrinus and S. gordonii. By contrast, treatment with CMCh200-immobilized Ficin led to a significant reduction of the biofilms of the primary colonizers S. gordonii and S. mutans. In model biofilms obtained by the inoculation of swabs from teeth of healthy volunteers, the destruction of the biofilm by both soluble and immobilized Ficin was observed, although the degree of the destruction varied between artificial plaque samples. Nevertheless, combined treatment of oral Streptococci biofilm by enzyme and chlorhexidine for 3 h led to a significant decrease in the viability of biofilm-embedded cells, compared to solely chlorhexidine application. This suggests that the use of either soluble or immobilized Ficin would allow decreasing the amount and/or concentration of the antiseptics required for oral care or improving the efficiency of oral cavity sanitization.

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

Terpene-Functionalized Fluoroquinolones as Potential Antimicrobials: Synthesis and Properties.

This study was the first to synthesize terpene-containing conjugates of fluoroquinolones, ciprofloxacin and norfloxacin, and to evaluate their antibacterial activity against gram-positive methicillin sensitive (MSSA) and methicillin resistant (MRSA) S. aureus, gram-negative P. aeruginosa as well as antifungal activity against C. albicans. The ability of obtained fluoroquinolones to inhibit S. aureus growth was found to depend upon the presence of a linker separating the bulky terpene and fluoroquinolone fragments, and this activity diminished with increasing its length. The highest activity against MSSA was demonstrated by ciprofloxacin derivatives with campholenic (MIC 1 µg/mL) and 2-(isobornan-2-yl-sulfanyl)acetyl (MIC 0.5 µg/mL) substituents. The compound with the last fragment showed high activity against MRSA (MIC 8 µg/mL). The terpene-functionalized norfloxacin derivatives generally proved to be less active than those containing ciprofloxacin fragment. Camphor-10-sulfonylamide derivative with the ciprofloxacin fragment was the only one of all compounds that showed high antifungal activity against C. albicans (8 µg/mL). The study presents data on docking fluoroquinolones to S. aureus DNA gyrase to explain the reasons for manifestation or disappearance of antibacterial activity. The cytotoxicity of fluoroquinolones that showed any antimicrobial activity was investigated against bovine primary lung cells, and they were found to be not toxic in most cases.

Synthesis and biological evaluation of fluoroquinolones containing a pyridoxine derivatives moiety.

We report herein the design, synthesis and biological evaluation of series of 7-substituted fluoroquinolones with pyridoxine derivatives. In vitro screening of antibacterial activity and toxicity of 39 synthesized fluoroquinolones defined compounds 7 and 28 as lead compounds for further investigations. On various clinical isolates lead compounds 7 and 28 exhibited antibacterial activity comparable with reference fluoroqinolones. Mutagenic effects haven't been observed for these compounds in SOS-chromotest. Compound 7 are non-toxic in vivo on mice (LD50 > 2000 mg/kg, oral) and rats (LD50 > 2000 mg/kg, oral). Compound 28 was more toxic (LD50 = 474 mg/kg, oral, mice). Moreover compound 7 showed greater in vivo efficacy compared to ciprofloxacin in a murine model of staphylococcal sepsis. Taken together the described active compound are promising candidate for preclinical trials.

Repression of Staphylococcus aureus and Escherichia coli by Lactiplantibacillus plantarum Strain AG10 in Drosophila melanogaster In Vivo Model.

Probiotic bacteria exhibiting antagonistic activities against pathogenic bacteria are widely considered as potential options for the prevention and treatment of various infectious diseases and represent potential substitutes of antibiotics. Here we show that the L. plantarum AG10 strain represses the growth of Staphylococcus aureus and Escherichia coli in vitro and diminishes their negative effects in vivo in a Drosophila melanogaster model of survival on embryonic (larvae) and pupa stages. In an agar drop diffusion test, L. plantarum AG10 exhibited antagonistic properties against Escherichia coli, Staphylococcus aureus, Serratia marcescens and Pseudomonas aeruginosa, and repressed the growth of E. coli and S. aureus during milk fermentation. In a Drosophila melanogaster model, L. plantarum AG10 alone did not provide any significant effect, either during the embryonic stage or during further development of the flies. Despite this, it was able to restore the viability of groups infected with either E. coli and S. aureus, almost to the level of non-treated control at all stages of development (larvae, pupa and adult). Moreover, in the presence of L. plantarum AG10, pathogens-induced mutation rates and recombination events reduced 1.5-2-fold. The genome of L. plantarum AG10 was sequenced and deposited at NCBI under the accession number PRJNA953814 and consists of annotated genome and raw sequence data. It consists of 109 contigs and is 3,479,919 bp in length with a GC content of 44.5%. The analysis of the genome has revealed considerably few putative virulence factors and three genes responsible for the biosynthesis of putative antimicrobial peptides, with one of them exhibiting a high probability of antimicrobial properties. Taken together, these data allow the suggestion that the L. plantarum AG10 strain is promising for use in both dairy production and probiotics as a preservative from foodborne infections.

Alterations in Antibiotic Susceptibility of Staphylococcus aureus and Klebsiella pneumoniae in Dual Species Biofilms.

In the last decades, it has been shown that biofilm-associated infections in most cases are caused by rather two or even more pathogens than by single microorganisms. Due to intermicrobial interactions in mixed communities, bacteria change their gene expression profile, in turn leading to alterations in the biofilm structure and properties, as well as susceptibility to antimicrobials. Here, we report the alterations of antimicrobials efficiency in mixed biofilms of Staphylococcus aureus-Klebsiella pneumoniae in comparison with mono-species biofilms of each counterpart and discuss possible mechanisms of these alterations. In cell clumps detached from dual-species biofilms, S. aureus became insensitive to vancomycin, ampicillin, and ceftazidime compared to solely S. aureus cell clumps. In turn, the increased efficiency of amikacin and ciprofloxacin against both bacteria could be observed, compared to mono-species biofilms of each counterpart. Scanning electron microscopy and confocal microscopy indicate the porous structure of the dual-species biofilm, and differential fluorescent staining revealed an increased number of polysaccharides in the matrix, in turn leading to more loose structure and thus apparently providing increased permeability of the dual-species biofilm to antimicrobials. The qRT-PCR showed that ica operon in S. aureus became repressed in mixed communities, and polysaccharides are produced mainly by K. pneumoniae. While the molecular trigger of these changes remains undiscovered, detailed knowledge of the alterations in antibiotic susceptibility to given drugs opens doors for treatment correction options for S. aureus-K. pneumoniae biofilm-associated infections.

The Novel Chiral 2(5H)-Furanone Sulfones Possessing Terpene Moiety: Synthesis and Biological Activity.

Over the past decades, 2(5H)-furanone derivatives have been extensively studied because of their promising ability to prevent the biofilm formation by various pathogenic bacteria. Here, we report the synthesis of a series of optically active sulfur-containing 2(5H)-furanone derivatives and characterize their biological activity. Novel thioethers were obtained by an interaction of stereochemically pure 5-(l)-menthyloxy- or 5-(l)-bornyloxy-2(5H)-furanones with aromatic thiols under basic conditions. Subsequent thioethers oxidation by an excess of hydrogen peroxide in acetic acid resulted in the formation of the corresponding chiral 2(5H)-furanone sulfones. The structure of synthesized compounds was confirmed by IR and NMR spectroscopy, HRMS, and single crystal X-ray diffraction. The leading compound, 26, possessing the sulfonyl group and l-borneol moiety, exhibited the prominent activity against Staphylococcus aureus and Bacillus subtilis with MICs of 8 µg/mL. Furthermore, at concentrations of 0.4-0.5 µg/mL, the sulfone 26 increased two-fold the efficacy of aminoglycosides gentamicin and amikacin against S. aureus. The treatment of the model-infected skin wound in the rat with a combination of gentamicin and sulfone 26 speeded up the bacterial decontamination and improved the healing of the wound. The presented results provide valuable new insights into the chemistry of 2(5H)-furanone derivatives and associated biological activities.

Brightfield vs Fluorescent Staining Dataset-A Test Bed Image Set for Machine Learning based Virtual Staining.

Differential fluorescent staining is an effective tool widely adopted for the visualization, segmentation and quantification of cells and cellular substructures as a part of standard microscopic imaging protocols. Incompatibility of staining agents with viable cells represents major and often inevitable limitations to its applicability in live experiments, requiring extraction of samples at different stages of experiment increasing laboratory costs. Accordingly, development of computerized image analysis methodology capable of segmentation and quantification of cells and cellular substructures from plain monochromatic images obtained by light microscopy without help of any physical markup techniques is of considerable interest. The enclosed set contains human colon adenocarcinoma Caco-2 cells microscopic images obtained under various imaging conditions with different viable vs non-viable cells fractions. Each field of view is provided in a three-fold representation, including phase-contrast microscopy and two differential fluorescent microscopy images with specific markup of viable and non-viable cells, respectively, produced using two different staining schemes, representing a prominent test bed for the validation of image analysis methods.

Video-based marker-free tracking and multi-scale analysis of mouse locomotor activity and behavioral aspects in an open field arena: A perspective approach to the quantification of complex gait disturbances associated with Alzheimer's disease.

Introduction: Complex gait disturbances represent one of the prominent manifestations of various neurophysiological conditions, including widespread neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Therefore, instrumental measurement techniques and automatic computerized analysis appears essential for the differential diagnostics, as well as for the assessment of treatment effectiveness from experimental animal models to clinical settings. Methods: Here we present a marker-free instrumental approach to the analysis of gait disturbances in animal models. Our approach is based on the analysis of video recordings obtained with a camera placed underneath an open field arena with transparent floor using the DeeperCut algorithm capable of online tracking of individual animal body parts, such as the snout, the paws and the tail. The extracted trajectories of animal body parts are next analyzed using an original computerized methodology that relies upon a generalized scalable model based on fractional Brownian motion with parameters identified by detrended partial cross-correlation analysis. Results: We have shown that in a mouse model representative movement patterns are characterized by two asymptotic regimes characterized by integrated 1/f noise at small scales and nearly random displacements at large scales separated by a single crossover. More detailed analysis of gait disturbances revealed that the detrended cross-correlations between the movements of the snout, paws and tail relative to the animal body midpoint exhibit statistically significant discrepancies in the Alzheimer's disease mouse model compared to the control group at scales around the location of the crossover. Discussion: We expect that the proposed approach, due to its universality, robustness and clear physical interpretation, is a promising direction for the design of applied analysis tools for the diagnostics of various gait disturbances and behavioral aspects in animal models. We further believe that the suggested mathematical models could be relevant as a complementary tool in clinical diagnostics of various neurophysiological conditions associated with movement disorders.

Docosahexaenoic acid-loaded chitosan/alginate membrane reduces biofilm formation by P. aeruginosa and promotes MSC-mediated burn wound healing.

Aims: Chitosan, like docosahexaenoic acid (DHA) and mesenchymal stem cells (MSCs), is used in medicine as a wound healing accelerator. Thus, in this study, chitosan-alginate (CA) membranes containing DHA and MSCs were produced, and their antibacterial and antibiofilm activities against burn infections caused by Pseudomonas aeruginosa were investigated.Methods: Physicochemical properties were assessed by SEM, Fourier transform infrared (FTIR), and X-ray diffraction (XRD). Porosity, cytocompatibility, and antibacterial and antibiofilm activities were evaluated both in vitro and in vivo. The viability and apoptosis of MSCs were studied using flow cytometry. Wound healing effects were analyzed based on histopathological features, the wound contraction rate (WCR) ratio, and bacterial clearance.Results: The CA membranes showed antibiofilm activity both in vivo and in vitro, accompanied by reduced lasI and rhlI expressions and pyocyanin production. The membranes were highly porous and biocompatible and showed favorable physicochemical properties. Docosahexaenoic acid incorporation to CA membranes improved their antibacterial and antibiofilm activities, as well as MSCs' viability by reducing crystallinity and increasing porosity (p = .008). Treatment with CA-DHA-MSC accelerated burn wound healing (with complete healing being observed after 14 days, WCR = 85%) and augmented antibacterial and antibiofilm activities in vivo compared to CA-DHA and CA-MSC. The CA-DHA-MSC group delivered a significantly higher WCR and lower inflammation than the CA-MSC group (p = .0001).Conclusion: In combination with DHA-loaded CA membranes, MSCs reduced the healing time of burn wounds, offering a viable option for designing effective wound dressings.

The Bovhyaluronidase Azoximer (Longidaza®) Disrupts Candida albicans and Candida albicans-Bacterial Mixed Biofilms and Increases the Efficacy of Antifungals.

Background and Objectives: Candida albicans causes various diseases ranging from superficial mycoses to life-threatening systemic infections often associated with biofilm formation, including mixed fungal−bacterial consortia. The biofilm matrix protects cells, making Candida extremely resistant to treatment. Here, we show that the bovhyaluronidase azoximer (Longidaza®) in vitro destroys the biofilm formed by either C. albicans alone or mixed with bacteria, this way decreasing the concentrations of antimicrobials required for the pathogen's eradication. Materials and Methods: Bovhyaluronidase azoximer, Longidaza® was obtained from NPO Petrovax Pharm Ltd., Moscow, Russia as lyophilized powder. The antifungal activity was assessed by microdilution assay and CFUs counting. Antibiofilm activity was evaluated via biofilms staining and scanning electron microscopy. Results: Thus, treatment with Longidaza® reduced the biofilm biomass of nine C. albicans clinical isolates by 30−60%, while mixed biofilms of C. albicans with various bacteria were destroyed by 30−40%. Furthermore, the concentration of fluconazole required to achieve a similar reduction of the residual respiratory activity of detached cell clumps of four C. albicans isolates has been reduced four-fold when combined with Longidaza®. While in the biofilm, two of four isolates became significantly more susceptible to fluconazole in combination with Longidaza®. Conclusion: Taken together, our data indicate that Longidaza® is capable of suppression of tissues and artificial surfaces biofouling by C. albicans biofilms, as well as facilitating drug penetration into the cell clumps, this way decreasing the effective MIC of antifungals.

Increasing the Efficacy of Treatment of Staphylococcus aureus-Candida albicans Mixed Infections with Myrtenol.

Infectious diseases caused by various nosocomial microorganisms affect worldwide both immunocompromised and relatively healthy persons. Bacteria and fungi have different tools to evade antimicrobials, such as hydrolysis damaging the drug, efflux systems, and the formation of biofilm that significantly complicates the treatment of the infection. Here, we show that myrtenol potentiates the antimicrobial and biofilm-preventing activity of conventional drugs against S. aureus and C. albicans mono- and dual-species cultures. In our study, the two optical isomers, (-)-myrtenol and (+)-myrtenol, have been tested as either antibacterials, antifungals, or enhancers of conventional drugs. (+)-Myrtenol demonstrated a synergistic effect with amikacin, fluconazole, and benzalkonium chloride on 64-81% of the clinical isolates of S. aureus and C. albicans, including MRSA and fluconazole-resistant fungi, while (-)-myrtenol increased the properties of amikacin and fluconazole to repress biofilm formation in half of the S. aureus and C. albicans isolates. Furthermore, myrtenol was able to potentiate benzalkonium chloride up to sixteen-fold against planktonic cells in an S. aureus-C. albicans mixed culture and repressed the adhesion of S. aureus. The mechanism of both (-)-myrtenol and (+)-myrtenol synergy with conventional drugs was apparently driven by membrane damage since the treatment with both terpenes led to a significant drop in membrane potential similar to the action of benzalkonium chloride. Thus, due to the low toxicity of myrtenol, it seems to be a promising agent to increase the efficiency of the treatment of infections caused by bacteria and be fungi of the genus Candida as well as mixed fungal-bacterial infections, including resistant strains.

Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features.

Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and N-(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively). In addition, all derivatives of chitosan studied in this work form hydrogen bonds with His158 located in the active site of bromelain (except N-(2-hydroxypropyl)-3-trimethylammonium chitosan), apparently explaining a significant decrease in the activity of biocatalysts. The N-(2-hydroxypropyl)-3-trimethylammonium chitosan displays only physical interactions with His158, thus possibly modulating the structure of the bromelain active site and leading to the hyperactivation of the enzyme, up to 208% of the total activity and 158% of the specific activity. The FTIR analysis revealed that interaction between N-(2-hydroxypropyl)-3-trimethylammonium chitosan and bromelain did not significantly change the enzyme structure. Perhaps this is due to the slowing down of aggregation and the autolysis processes during the complex formation of bromelain with a carrier, with a minimal modification of enzyme structure and its active site orientation.

Amine-Reactive BODIPY Dye: Spectral Properties and Application for Protein Labeling.

A boron-dipyrromethene (BODIPY) derivative reactive towards amino groups of proteins (NHS-Ph-BODIPY) was synthesized. Spectroscopic and photophysical properties of amine-reactive NHS-Ph-BODIPY and its non-reactive precursor (COOH-Ph-BODIPY) in a number of organic solvents were investigated. Both fluorescent dyes were characterized by green absorption (521-532 nm) and fluorescence (538-552 nm) and medium molar absorption coefficients (46,500-118,500 M-1·cm-1) and fluorescence quantum yields (0.32 - 0.73). Solvent polarizability and dipolarity were found to play a crucial role in solvent effects on COOH-Ph-BODIPY and NHS-Ph-BODIPY absorption and emission bands maxima. Quantum-chemical calculations were used to show why solvent polarizability and dipolarity are important as well as to understand how the nature of the substituent affects spectroscopic properties of the fluorescent dyes. NHS-Ph-BODIPY was used for fluorescent labeling of a number of proteins. Conjugation of NHS-Ph-BODIPY with bovine serum albumin (BSA) resulted in bathochromic shifts of absorption and emission bands and noticeable fluorescence quenching (about 1.5 times). It was demonstrated that the sensitivity of BSA detection with NHS-Ph-BODIPY was up to eight times higher than with Coomassie brilliant blue while the sensitivity of PII-like protein PotN (PotN) detection with NHS-Ph-BODIPY and Coomassie brilliant blue was almost the same. On the basis of the molecular docking results, the most probable binding sites of NHS-Ph-BODIPY in BSA and PotN and the corresponding binding free energies were estimated.