Enhancing wound healing with zinc and silver nanocomposites synthesized with ß-lactoglobulin: antimicrobial properties, collagen deposition, and systemic effects in a C57BL/6J mouse model.

This study explores the potential of zinc and silver nanocomposites, synthesized with ß-lactoglobulin, a whey protein, in promoting wound healing, using the C57BL/6J mouse model. Our research is distinct in its dual focus: assessing the antimicrobial efficacy of these nanocomposites and their impact on wound healing processes. The antimicrobial properties were investigated through minimum inhibitory concentration (MIC) assessments and colony-forming unit (CFU) tests, providing insights into their effectiveness against wound-associated microorganisms. Notably, the formulation's effective antibacterial concentration did not exhibit toxicity to mouse fibroblasts. A key aspect of our methodology involved the use of a stereoscopic microscope for detailed monitoring of the wound closure process. Additionally, the distribution and potential systemic effects of the zinc and silver ions were analyzed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). This analysis was crucial in evaluating metal ion absorption through the wound site and estimating any toxic effects on the body. Our findings are particularly significant in the field of regenerative medicine. Transmission electron microscopy (TEM) revealed that the tested nanocomposites notably enhanced collagen deposition, a vital component in the wound healing process. Furthermore, a reduction in glycogen levels in hepatocytes was observed following treatment with these metal-protein dressings. This novel finding warrants further investigation. Overall, our findings highlight the diverse roles of zinc and silver nanocomposites in wound healing. This study not only contributes to our understanding of metal-protein complexes in tissue regeneration but also opens new avenues for research into the delivery mechanisms of such treatments for hard-to-heal wounds.

Antifungal Properties of Bio-AgNPs against D. pinodes and F. avenaceum Infection of Pea (Pisum sativum L.) Seedlings.

Ascochyta blight and Fusarium root rot are the most serious fungal diseases of pea, caused by D. pinodes and F. avenaceum, respectively. Due to the lack of fully resistant cultivars, we proposed the use of biologically synthesized silver nanoparticles (bio-AgNPs) as a novel protecting agent. In this study, we evaluated the antifungal properties and effectiveness of bio-AgNPs, in in vitro (poisoned food technique; resazurin assay) and in vivo (seedlings infection) experiments, against D. pinodes and F. avenaceum. Moreover, the effects of diseases on changes in the seedlings' metabolic profiles were analyzed. The MIC for spores of both fungi was 125 mg/L, and bio-AgNPs at 200 mg/L most effectively inhibited the mycelium growth of D. pinodes and F. avenaceum (by 45 and 26%, respectively, measured on the 14th day of incubation). The treatment of seedlings with bio-AgNPs or fungicides before inoculation prevented the development of infection. Bio-AgNPs at concentrations of 200 mg/L for D. pinodes and 100 mg/L for F. avenaceum effectively inhibited infections' spread. The comparison of changes in polar metabolites' profiles revealed disturbances in carbon and nitrogen metabolism in pea seedlings by both pathogenic fungi. The involvement of bio-AgNPs in the mobilization of plant metabolism in response to fungal infection is also discussed.

Anthrax in one health in Southern and Southeastern Europe - the effect of climate change?

Anthrax is a serious infection caused by Bacillus anthracis. The anthracis spores are highly resistant and can persist in the environment for several decades. Therefore, anthrax is considered a global health threat affecting wildlife, livestock, and the general public. The resistance mechanism is influenced not only by the environment or the ecological niche but also by virulence factors. In the last 10 years the Southern and Southeastern Europe have been confronted with this threat. Recently, there have been 8 human anthrax cases reported in Croatia (2022), and 4 cases in Romania (2023). Moreover, this incident and the COVID situation could be a starting point to encourage researchers to raise the alarm. On the other hand, climate change is causing glaciers to melt and land to thaw, and many wetlands and swampy areas are being drained. It should not be forgotten that epidemiological and epizootic threats significantly affect the country's economic development. The Covid-19 epidemic best illustrates these threats.

Immobilized enzyme microreactors for analysis of tryptic peptides in ß-casein and ß-lactoglobulin.

In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: ß-casein (ßCN) and ß-lactoglobulin (ßLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (µ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The µ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the µ-IMER method demonstrated superior sequence coverage for oxidized molecules in ßCN (33 ± 1.5%) and ßLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for ßCN; 49 ± 2% for ßLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for ßCN; 60 ± 4% for ßLG) and µ-IMER (41 ± 3% for ßCN; 80 ± 5% for ßLG). Additionally, phosphorylations were identified. For ßCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With µ-IMER and µ-IMER-ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For ßLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and µ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and µ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of µ-IMER systems for enhanced sequence coverage and post-translational modification detection.

Pyrolized Diatomaceous Biomass Doped with Epitaxially Growing Hybrid Ag/TiO2 Nanoparticles: Synthesis, Characterisation and Antibacterial Application.

In the pursuit of innovative solutions for modern technologies, particularly in the design and production of new micro/nanostructured materials, microorganisms acting as "natural microtechnologists" can serve as a valuable source of inspiration. This research focuses on harnessing the capabilities of unicellular algae (diatoms) to synthesize hybrid composites composed of AgNPs/TiO2NPs/pyrolyzed diatomaceous biomass (AgNPs/TiO2NPs/DBP). The composites were consistently fabricated through metabolic (biosynthesis) doping of diatom cells with titanium, pyrolysis of the doped diatomaceous biomass, and chemical doping of the pyrolyzed biomass with silver. To characterize the synthesized composites, their elemental and mineral composition, structure, morphology, and photoluminescent properties were analysed using techniques such as X-ray diffraction, scanning and transmission electron microscopy, and fluorescence spectroscopy. The study revealed the epitaxial growth of Ag/TiO2 nanoparticles on the surface of pyrolyzed diatom cells. The antimicrobial potential of the synthesized composites was evaluated using the minimum inhibitory concentration (MIC) method against prevalent drug-resistant microorganisms, including Staphylococcus aureus, Klebsiella pneumonia, and Escherichia coli, both from laboratory cultures and clinical isolates.

Exogenously Applied Cyclitols and Biosynthesized Silver Nanoparticles Affect the Soluble Carbohydrate Profiles of Wheat (Triticum aestivum L.) Seedling.

Cyclitols, such as myo-inositol and its isomers and methyl derivatives (i.e., d-chiro-inositol and d-pinitol (3-O-methyl-chiro-inositol)), are classified as osmolytes and osmoprotectants and are significantly involved in plant responses to abiotic stresses, such as drought, salinity and cold. Moreover, d-pinitol demonstrates a synergistic effect with glutathione (GSH), increasing its antioxidant properties. However, the role of cyclitols in plant protection against stresses caused by metal nanoparticles is not yet known. Therefore, the present study examined the effects of myo-inositol, d-chiro-inositol and d-pinitol on wheat germination, seedling growth and changes in the profile of soluble carbohydrates in response to biologically synthesized silver nanoparticles ((Bio)Ag NPs). It was found that cyclitols were absorbed by germinating grains and transported within the growing seedlings but this process was disrupted by (Bio)Ag NPs. Cyclitols applied alone induced sucrose and 1-kestose accumulation in seedlings slightly, while (Bio)Ag NP doubled the concentrations of both sugars. This coincided with a decrease in monosaccharides; i.e., fructose and glucose. Cyclitols and (Bio)Ag NPs present in the endosperm resulted in reductions in monosaccharides, maltose and maltotriose, with no effect on sucrose and 1-kestose. Similar changes occurred in seedlings developing from primed grains. Cyclitols that accumulated in grain and seedlings during grain priming with d-pinitol and glutathione did not prevent the phytotoxic effects of (Bio)Ag NPs.

Characterization of the salivary microbiome before and after antibiotic therapy via separation technique.

In the present research, the MALDI-TOF MS technique was applied as a tool to rapidly identify the salivary microbiome. In this fact, it has been monitored the changes occurred in molecular profiles under different antibiotic therapy. Significant changes in the composition of the salivary microbiota were noticed not only in relation to the non antibiotic (non-AT) and antibiotic treatment (AT) groups, but also to the used media, the antibiotic therapy and co-existed microbiota. Each antibiotic generates specific changes in molecular profiles. The highest number of bacterial species was isolated in the universal culture medium (72%) followed by the selective medium (48% and 38%). In the case of non-AT patients, the prevalence of Streptococcus salivarius (25%), Streptococcus vestibularis (19%), Streptococcus oralis (13%), and Staphylococcus aureus (6%) was identified while in the case of AT, Streptococcus salivarius (11%), Streptococcus parasanguinis (11%), Staphylococcus epidermidis (12%), Enterococcus faecalis (9%), Staphylococcus hominis (8%), and Candida albicans (6%) were identified. Notable to specified that the Candida albicans was noticed only in AT samples, indicating a negative impact on the antibiotic therapy. The accuracy of the MALDI-TOF MS technique was performed by the 16S rRNA gene sequencing analysis-as a reference method. Conclusively, such an approach highlighted in the present study can help in developing the methods enabling a faster diagnosis of disease changes at the cellular level before clinical changes occur. Once the MALDI tool allows for the distinguishing of the microbiota of non-AT and AT, it may enable to monitor the diseases treatment and develop a treatment regimen for individual patients in relation to each antibiotic. KEY POINTS: The salivary microbiota of antibiotic-treated patients was more bacteria variety MALDI-TOF MS is a promising tool for recording of reproducible molecular profiles Our data can allow to monitor the treatment of bacterial diseases for patients.

Comprehensive study upon physicochemical properties of bio-ZnO NCs.

In this study, for the first time, the comparison of commercially available chemical ZnO NCs and bio-ZnO NCs produced extracellularly by two different probiotic isolates (Latilactobacillus curvatus MEVP1 [OM736187] and Limosilactobacillus fermentum MEVP2 [OM736188]) were performed. All types of ZnO formulations were characterized by comprehensive interdisciplinary approach including various instrumental techniques in order to obtain nanocomposites with suitable properties for further applications, i.e. biomedical. Based on the X- ray diffraction analysis results, all tested nanoparticles exhibited the wurtzite structure with an average crystalline size distribution of 21.1 nm (CHEM_ZnO NCs), 13.2 nm (1C_ZnO NCs) and 12.9 nm (4a_ZnO NCs). The microscopy approach with use of broad range of detectors (SE, BF, HAADF) revealed the core-shell structure of bio-ZnO NCs, compared to the chemical one. The nanoparticles core of 1C and 4a_ZnO NCs are coated by the specific organic deposit coming from the metabolites produced by two probiotic strains, L. fermentum and L. curvatus. Vibrational infrared spectroscopy, photoluminescence (PL) and mass spectrometry (LDI-TOF-MS) have been used to monitor the ZnO NCs surface chemistry and allowed for better description of bio-NCs organic coating composition (amino acids residues). The characterized ZnO formulations were then assessed for their photocatalytic properties against methylene blue (MB). Both types of bio-ZnO NCs exhibited good photocatalytic activity, however, the effect of CHEM_ZnO NCs was more potent than bio-ZnO NCs. Finally, the colloidal stability of the tested nanoparticles were investigated based on the zeta potential (ZP) and hydrodynamic diameter measurements in dependence of the nanocomposites concentration and investigation time. During the biosynthesis of nano-ZnO, the increment of pH from 5.7 to around 8 were observed which suggested possible contribution of zinc aquacomplexes and carboxyl-rich compounds resulted in conversion of zinc tetrahydroxy ion complex to ZnO NCs. Overall results in present study suggest that used accessible source such us probiotic strains, L. fermentum and L. curvatus, for extracellular bio-ZnO NCs synthesis are of high interest. What is important, no significant differences between organic deposit (e.g. metabolites) produced by tested strains were noticed-both of them allowed to form the nanoparticles with natural origin coating. In comparison to chemical ZnO NCs, those synthetized via microbiological route are promising material with further biological potential once have shown high stability during 7 days.

Synthesis and Antimicrobial Activity of 3D Micro-Nanostructured Diatom Biosilica Coated by Epitaxially Growing Ag-AgCl Hybrid Nanoparticles.

The 3D (three-dimensional) micro-nanostructured diatom biosilica obtained from cultivated diatoms was used as a support to immobilize epitaxially growing AgCl-Ag hybrid nanoparticles ((Ag-AgCl)NPs) for the synthesis of nanocomposites with antimicrobial properties. The prepared composites that contained epitaxially grown (Ag-AgCl)NPs were investigated in terms of their morphological and structural characteristics, elemental and mineral composition, crystalline forms, zeta potential, and photoluminescence properties using a variety of instrumental methods including SEM (scanning electron microscopy), TEM (transmission electron microscopy), EDX (energy-dispersive X-ray spectroscopy), XRD (X-ray powder diffraction), zeta-potential measurement, and photoluminescence spectroscopy. The content of (AgCl-Ag)NPs in the hybrid composites amounted to 4.6 mg/g and 8.4 mg/g with AgClNPs/AgNPs ratios as a percentage of 86/14 and 51/49, respectively. Hybrid nanoparticles were evenly dispersed with a dominant size of 5 to 25 nm in composite with an amount of 8.4 mg/g of silver. The average size of the nanoparticles was 7.5 nm; also, there were nanoparticles with a size of 1-2 nm and particles that were 20-40 nm. The synthesis of (Ag-AgCl)NPs and their potential mechanism were studied. The MIC (the minimum inhibitory concentration method) approach was used to investigate the antimicrobial activity against microorganisms Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus. The nanocomposites containing (Ag-AgCl)NPs and natural diatom biosilica showed resistance to bacterial strains from the American Type Cultures Collection and clinical isolates (diabetic foot infection and wound isolates).

Flow Cytometry - Sophisticated Tool for Basic Research or/and Routine Diagnosis; Impact of the Complementarity in Both Pre- as Well as Clinical Studies.

Flow cytometry is a sophisticated technology used widely in both basic research and as a routine tool in clinical diagnosis. The technology has progressed from single parameter detection in the 1970s and 1980s to high end multicolor analysis, with currently 30 parameters detected simultaneously, allowing the identification and purification of rare subpopulations of cells of interest. Flow cytometry continues to evolve and expand to facilitate the investigation of new diagnostic and therapeutic avenues. The present review gives an overview of basic theory and instrumentation, presents and compares the advantages and disadvantages of conventional, spectral and imaging flow cytometry as well as mass cytometry. Current methodologies and applications in both research, pre- and clinical settings are discussed, as well as potential limitations and future evolution. This finding encourages the reader to promote such relationship between basic science, diagnosis and multidisciplinary approach since the standard methods have limitations (e.g., in differentiating the cells after staining). Moreover, such path inspires future cytometry specialists develop new/alternative frontiers between pre- and clinical diagnosis and be more flexible in designing the study for both human as well as veterinary medicine.

Study on silver ions binding to ß-lactoglobulin.

The drug-resistant pathogen phenomenon, resulting in infections and deaths that are increasingly difficult to treat, requires research into searching new potential antimicrobial agents. The presented study is focused on the investigation of impact of silver ions (Ag+ ions) to ß-lactoglobulin (ßLG) structure and mechanism formation of silver-ß-lactoglobulin nanocomposites, that could find potential applications in medicine. To determine the physicochemical characteristics of silver ion binding, kinetics and isothermal models were used. The presence of functional groups involved in the binding process was investigated by spectroscopic methods (FTIR-ATR, Raman spectroscopy). The binding ability and nanocomplexes formation was determined by instrumental analyses (SEM, TEM, EDX). Based on the obtained results, the binding of Ag+ ions to ßLG were heterogeneous in nature consisting of three main steps: rapid sorption of Ag+ ions on the ßLG surface, intramolecular diffusion of Ag+ ions, and chemical equilibrium. Microscopic studies showed a change in the surface morphology of ßLG and the appearance of silver nanoparticles. Spectroscopic studies indicated that acidic (Glu-, Asp-) and Lys, Tyr, Met amino groups play a key role in the formation of the AgßLG nanocomplex. Finally, molecular dynamics (MD) and density functional theory (DFT) calculated studies as a comparative and complementary method have proven contribution of respective amino acids in the binding process.

Synthesis, characterization of silver/kaolinite nanocomposite and studying its antibacterial activity.

In the present study, silver/kaolinite nanocomposites were synthesized by impregnation in a silver nitrate solution. Silver nanoparticles are deposited onto the surface of the kaolinite by a simple wet reduction of a silver precursor using hydrogen peroxide as a reducing agent. Elemental, mineral composition, structure and morphology of natural kaolinite and synthesized nanocomposites are characterized by X-ray diffractometry, FT-IR spectroscopy, photoluminescence (PL), zeta potential, scanning electron microscopy, transmission electron microscopy and thermogravimetric analysis. The antibacterial activity of AgNPs/kaolinite nanocomposites to Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae, Escherichia coli strains was studied by the minimum inhibitory concentration method. The obtained AgNPs/kaolinite nanocomposite was shown to have antimicrobial potential.

Viability study of clinical bacterial strains by capillary electrophoresis and flow cytometry approaches.

One of the challenges medicine faces is the constantly growing resistance of pathogens to various classes of antibiotics. In this study, we investigated the use of capillary electrophoresis (CE) to characterize and assess the physiological states of three clinical bacterial strains-methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), and Escherichia coli extended-spectrum ß-lactamases (ESßL)-exposed to different antibiotics. All chosen bacteria are the leading causes of healthcare-associated and hospital-acquired invasive infections in adults. In the first part of the research, it was determined the optimal incubation time of the tested strains with antibiotics, represented as an optimal time of 24 h. In the second part, we have compared two approaches: flow cytometry (FC) as a standard method and CE as a proposed alternative approach. The viability of clinical strains treated with different class antibiotics calculated in CE measurements was strongly correlated (>0.83 for MSSA, >0.92 for ESßL and MRSA) with the viability obtained on the basis of FC measurements. As a result, CE has a chance to become a modern diagnostic method used in clinical practice. The CE cutoff was found to be 50%; above this value, the strain shows resistance to the action of the antibiotic.

Lacticaseibacillus paracasei as a Modulator of Fatty Acid Compositions and Vitamin D3 in Cream.

Butter is an important source of essential fatty acids, lipid-soluble vitamins, and antioxidants in the diet. However, this study showed that the presence of the Lacticaseibacillus paracasei strain has a great influence on the fatty acid profile as well as provitamin D3 and vitamin D3 content in the cream-the raw material from which the butter is obtained. The addition of this lactic acid bacteria enriches the cream in 9-hexadecenoic acid, oleic acid, octadeca-9,12-dienoic acid, and conjugated linoleic acid, which exhibit antimutagenic and anticarcinogenic properties. Moreover, a higher level of monounsaturated fatty acids can extend the shelf life of butter in the future. In the present work, we observed that the presence of lactic acid bacteria contributed to an increase in the level of provitamin D after 6 h of incubation and an increase in the levels of vitamin D3 after 24 and 48 h. Fatty acid profiles and the content of vitamins were largely dependent on the presence of light and mixing, which are probably associated with the status of lipid peroxidation.

In Vivo Efficacy of Wound Healing under External (Bio)AgNCs Treatment: Localization Case Study in Liver and Blood Tissue.

The present study reports on the in vivo application of (Bio)silver nanocomposite formulations (LBPC-AgNCs) on wound healing. Additionally, the present study emphasizes the limited uptake of silver by liver and blood tissues as well as the high viability of PBMCs following external LBPC-AgNCs treatment. The wound closure was monitored via stereoscopic microscope, a localization case study in liver and blood tissue was carried out by (Inductively Coupled Plasma-Mass Spectrometers (ICP/MS), and peripheral blood mononuclear cells (PMBC) viability was determined via flow cytometry technique. The silver formulation was applied externally on the site of the wound infection for a period of ten days. At the beginning of the experiment, a moderate decrease in body weight and atypical behavior was observed. However, during the last period of the experiment, no abnormal mouse behaviors were noticed. The wound-healing process took place in a gradual manner, presenting the regeneration effect at around 30% from the fourth day. From the seventh day, the wounds treated with the silver formulation showed 80% of the wound healing potential. The viability of PBMCs was found to be 97%, whereas the concentrations of silver in the liver and blood samples were determined to be 0.022 µg/g and 9.3 µg/g, respectively. Furthermore, the present report becomes a pilot study in transferring from in vitro to in vivo scale (e.g., medical field application) once LBPC-AgNCs have demonstrated a unique wound healing potential as well as a non-toxic effect on the liver and blood.