Self-assembling amyloid-like nanostructures from SARS-CoV-2 S1, S2, RBD and N recombinant proteins.

Self-assembling nanoparticles (saNP) and nanofibers were found in the recombinant coronavirus SARS-CoV-2 S1, S2, RBD and N proteins purified by affinity chromatography using Ni Sepharose. Scanning electron (SEM), atomic force (AFM) microscopy on mica or graphite surface and in liquid as well as dynamic light scattering (DLS) revealed nanostructures of various sizes. AFM in liquid cell without drying on the surface showed mean height of S1 saNP 80.03 nm, polydispersity index (PDI) 0.006; for S2 saNP mean height 93.32 nm, PDI = 0.008; for N saNP mean height 16.71 nm, PDI = 0.99; for RBD saNP mean height 16.25 nm, PDI = 0.55. Ratios between the height and radius of each saNP in the range 0.1-0.5 suggested solid protein NP but not vesicles with internal empty spaces. The solid but not empty structures of the protein saNP were also confirmed by STEM after treatment of saNP with the standard contrasting agent uranyl acetate. The saNP remained stable after multiple freeze-thaw cycles in water and hyperosmotic solutions for 2 years at -20 °C. Receptor-mediated penetration of the SARS-CoV-2 S1 and RBD saNP in the African green mokey kidney Vero cells with the specific receptors for ß-coronavirus reproduction was more efficient compared to unspecific endocytosis into MDCK cells without the specific receptors. Amyloid-like structures were revealed in the SARS-CoV-2 S1, S2, RBD and N saNP by means of their interaction with Thioflavin T and Congo Red dyes. Taken together, spontaneous formation of the amyloid-like self-assembling nanostructures due to the internal affinity of the SARS-CoV-2 virion proteins might induce proteinopathy in patients, including conformational neurodegenerative diseases, change stability of vaccines and diagnostic systems.

Environmental enrichment reverses proulcerogenic action of social isolation on the gastric mucosa and positively influences pain sensitivity and work capacity.

The aim of the study was to investigate the effects of rat housing conditions-standard conditions, social isolation, environmental enrichment-and the subsequent reversal of these conditions on the vulnerability of the gastric mucosa to ulcerogenic stimuli, somatic pain sensitivity, and treadmill work capacity. Rats, aged 30 days, were placed in standard conditions (SC), social isolation (Is), and environmental enrichment (EE) for 4 weeks. Then half of each group underwent a reversal of housing conditions: SC rats were moved to Is, Is rats were placed in EE, EE rats were moved to Is, for 2 weeks. The other half served as a control with no change in their initial housing. Two weeks after the reversal, vulnerability of the gastric mucosa to ulcerogenic action of indomethacin (IM, 35 mg/kg, sc), somatic pain sensitivity (hot plate test), and work capacity (measured by the running distance on a treadmill) were assessed in control and reversed groups. Social isolation induced a proulcerogenic effect, increasing IM-induced gastric erosions, which was effectively reversed when rats were transferred to an environmental enrichment. Conversely, transferring rats from an environmental enrichment to social isolation exacerbated ulcerogenic action of IM. Somatic pain sensitivity and treadmill work capacity were also influenced by housing conditions, with environmental enrichment showing positive effects. The present findings show that social isolation of rats induces a proulcerogenic effect. Environmental enrichment reverses proulcerogenic action of social isolation on the gastric mucosa and increases resilience to pain stimuli and treadmill work capacity.

Influence of pre-treatment methods on quality indicators and mineral composition of plant milk from different sources of raw materials.

BACKGROUND: Pre-treatment of plant materials is essential in producing plant-based products and can affect their various organoleptic and physicochemical characteristics. This work aimed to study the effect of pre-treatment of vegetable raw materials, namely ultrasonic processing and freezing of raw materials under various low-temperature conditions, to obtain multiple types of vegetable milk and determine their characteristics. RESULTS: It is shown that by applying a certain kind of pre-treatment of vegetable raw materials it is possible to adjust organoleptic parameters and the content of solids, protein, fat, carbohydrates, fiber and mineral composition of various types of vegetable milk from soy, rice, oats, wheat, peas, buckwheat, pumpkin seeds and lentils. Ultrasound pre-treatment allows increasing of polyphenol content by an average of 15-20% for all types of plant milk, except for lentil milk. The results showed that ultrasound treatment for 3 min had the most significant effect on the overall acceptability for lentils, pumpkin, rice and pea milk. Pre-freezing at a temperature regime of -17 and -85 °C contributed to an increase in Fe, K, Zn, Ca, Mg, Si and P by an average of 30-100%, depending on the plant material. CONCLUSION: Pre-treatment of vegetable raw materials, including freezing and ultrasonic treatment, can positively affect the macro- and micronutrient composition of plant milk. However, the effect may vary depending on the type of raw material and processing conditions. © 2023 Society of Chemical Industry.

Biodistribution of Fluorescent Albumin Nanoparticles among Organs of Laboratory Animals after Intranasal and Peroral Administration.

Natural, environmental and engineered nanoparticles (NP) penetrate into cells by endocytosis and induce innate immunity. The behaviour of the nanomaterials both in vitro and in vivo should be assessed. Our goal was to study protein NP stability in biological fluids and distribution in organs of animals after intranasal and oral administration. Bovine serum albumin (BSA) was labelled with the fluorescent dye RhoB and NP were fabricated by nanoprecipitation. The fluorescent protein NPwere administered intranasally and orally in laboratory-outbred mice ICR and rabbits. RhoB-BSA NP distribution in organs was detected using spectrofluorometry and fluorescent microscopy. Innate immunity was evaluated using reverse transcription with random hexanucleotide primer and subsequent real-time PCR with specific fluorescent hydrolysis probes. The labelled BSA NP were shown to remain stable in blood sera and nasopharyngeal swabs for 5 days at +37 °C. In vivo the maximal accumulation was found in the brain in 2 days posttreatment without prevalent accumulation in olfactory bulbs. For the intestine, heart and liver, the BSA NP accumulation was similar in 1 and 2 days, whereas for kidney samples even decreased after 1 day. Both intranasal and peroral administration of RhoB-BSA NP did not induce innate immunity. Thus, after intranasal or oral instillation RhoB-BSA NP were found mainly in the brain and intestine without interferon gene expression.

Reduction of transient carnosine radicals depends on ß-alanyl amino group charge.

Reduction of transient carnosine (ß-alanyl-L-histidine) radicals by L-tryptophan, N-acetyl tryptophan, and the Trp-Gly peptide in neutral and basic aqueous solutions was studied using the technique of time-resolved chemically induced dynamic nuclear polarization (TR CIDNP). Carnosine radicals were generated in the photoinduced reaction with triplet excited 3,3',4,4'-tetracarboxy benzophenone. In this reaction, carnosine radicals with their radical center at the histidine residue are formed. Modeling of CIDNP kinetic data allowed for the determination of pH-dependent rate constants of the reduction reaction. It was shown that the protonation state of the amino group of the non-reacting ß-alanine residue of the carnosine radical affects the rate constant of the reduction reaction. The results were compared to those obtained previously for the reduction of histidine and N-acetyl histidine free radicals and to newly obtained results for the reduction of radicals derived from Gly-His, a homologue of carnosine. Clear differences were demonstrated.

Deep Eutectic Solvents for Biotechnology Applications.

Deep eutectic solvents (DESs) are an alternative to traditional organic solvents and ionic liquids and meet the requirements of "green" chemistry. They are easy to prepare using low-cost constituents, are non-toxic and biodegradable. The review analyzes literature on the use of DES in various fields of biotechnology, provides data on the types of DESs, methods for their preparation, and properties. The main areas of using DESs in biotechnology include extraction of physiologically active substances from natural resources, pretreatment of lignocellulosic biomass to improve enzymatic hydrolysis of cellulose, production of bioplastics, as well as a reaction medium for biocatalytic reactions. The aim of this review is to summarize available information on the use of new solvents for biotechnological purposes.

Stable Enzymatic Nanoparticles from Nucleases, Proteases, Lipase and Antioxidant Proteins with Substrate-Binding and Catalytic Properties.

Limited membrane permeability and biodegradation hamper the intracellular delivery of the free natural or recombinant enzymes necessary for compensatory therapy. Nanoparticles (NP) provide relative protein stability and unspecific endocytosis-mediated cellular uptake. Our objective was the fabrication of NP from 7 biomedicine-relevant enzymes, including DNase I, RNase A, trypsin, chymotrypsin, catalase, horseradish peroxidase (HRP) and lipase, the analysis of their conformation stability and enzymatic activity as well as possible toxicity for eukaryotic cells. The enzymes were dissolved in fluoroalcohol and mixed with 40% ethanol as an anti-solvent with subsequent alcohol evaporation at high temperature and low pressure. The shapes and sizes of NP were determined by scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Enzyme conformations in solutions and in NP were compared using circular dichroism (CD) spectroscopy. The activity of the enzymes was assayed with specific substrates. The cytotoxicity of the enzymatic NP (ENP) was studied by microscopic observations and by using an MTT test. Water-insoluble ENP of different shapes and sizes in a range 50-300 nm consisting of 7 enzymes remained stable for 1 year at +4 °C without any cross-linking. CD spectroscopy of the ENP permitted us to reveal changes in proportions of α-helixes, ß-turns and random coils in comparison with fresh enzyme solutions in water. Despite the minor conformation changes of the proteins in the ENP, the enzymes retained their substrate-binding and catalytic properties. Among the studied bioactive ENP, only DNase NP were highly toxic for 3 cell lines with granulation in 1 day posttreatment, whereas other NP were less toxic (if any). Taken together, the enzymes in the stable ENP retained their catalytic activity and might be used for intracellular delivery.

Peculiar Properties of Template-Assisted Aniline Polymerization in a Buffer Solution Using Laccase and a Laccase-Mediator System as Compared with Chemical Polymerization.

The conventional chemical polymerization of aniline has been described in multiple publications, while enzymatic polymerization has been poorly explored. A comparative study of the template-assisted enzymatic and chemical polymerization of aniline in a buffer solution of sodium dodecylbenzenesulfonate micelles was performed for the first time. The high-redox potential laccase from the fungus Trametes hirsuta was used as a catalyst and air oxygen served as an oxidant. Potentiometric and spectral methods have shown that oligomeric/polymeric products of the enzymatic polymerization of aniline are synthesized in the conducting emeraldine salt form immediately after the reaction is initiated by the enzyme. The use of the laccase-mediator system enabled a higher rate of enzymatic polymerization and a higher yield of final products. Potassium octocyanomolybdate (IV) served as a redox mediator. The products of the enzymatic polymerization of aniline were studied by the ATR-FTIR, MALDI-TOF and atomic force microscopy methods. The chemical oxidative polymerization of aniline under the same conditions resulted in forming a non-conducting dark brown product.

Marcus Cross-Relationship Probed by Time-Resolved CIDNP.

The time-resolved CIDNP method can provide information about degenerate exchange reactions (DEEs) involving short-lived radicals. In the temperature range from 8 to 65 °C, the DEE reactions of the guanosine-5'-monophosphate anion GMP(-H)- with the neutral radical GMP(-H)•, of the N-acetyl tyrosine anion N-AcTyrO- with a neutral radical N-AcTyrO•, and of the tyrosine anion TyrO- with a neutral radical TyrO• were studied. In all the studied cases, the radicals were formed in the reaction of quenching triplet 2,2'-dipyridyl. The reorganization energies were obtained from Arrhenius plots. The rate constant of the reductive electron transfer reaction in the pair GMP(-H)•/TyrO- was determined at T = 25 °C. Rate constants of the GMP(-H)• radical reduction reactions with TyrO- and N-AcTyrO- anions calculated by the Marcus cross-relation differ from the experimental ones by two orders of magnitude. The rate constants of several other electron transfer reactions involving GMP(-H)-/GMP(-H)•, N-AcTyrO-/N-AcTyrO•, and TyrO-/TyrO• pairs calculated by cross-relation agree well with the experimental values. The rate of nuclear paramagnetic relaxation was found for the 3,5 and ß-protons of TyrO• and N-AcTyrO•, the 8-proton of GMP(-H)•, and the 3,4-protons of DPH• at each temperature. In all cases, the dependences of the rate of nuclear paramagnetic relaxation on temperature are described by the Arrhenius dependence.

Features of DNA-Montmorillonite Binding Visualized by Atomic Force Microscopy.

In the present work, complexes of DNA with nano-clay montmorillonite (Mt) were investigated by means of atomic force microscopy (AFM) under various conditions. In contrast to the integral methods of analysis of the sorption of DNA on clay, AFM allowed us to study this process at the molecular level in detail. DNA molecules in the deionized water were shown to form a 2D fiber network weakly bound to both Mt and mica. The binding sites are mostly along Mt edges. The addition of Mg2+ cations led to the separation of DNA fibers into separate molecules, which bound mainly to the edge joints of the Mt particles according to our reactivity estimations. After the incubation of DNA with Mg2+, the DNA fibers were capable of wrapping around the Mt particles and were weakly bound to the Mt edge surfaces. The reversible sorption of nucleic acids onto the Mt surface allows it to be used for both RNA and DNA isolation for further reverse transcription and polymerase chain reaction (PCR). Our results show that the strongest binding sites for DNA are the edge joints of Mt particles.

DETERMINATION OF SOMATIC, NEUROLOGICAL AND PSYCHO-EMOTIONAL MANIFESTATIONS OF THE ACUTE AND POSTCOVID PERIOD IN PATIENTS WITH A MILD COURSE OF COVID-19 IN WARTIME.

OBJECTIVE: The aim: To analyze the typical symptom complex at the stage of COVID-19 acute phase in the systemic relationship with somatic, psychosomatic, and neurological manifestations. PATIENTS AND METHODS: Materials and methods: The collection of primary material was performed by clinical-anamnestic method, laboratory, and sociological examination of patients treated out patiently. Summarizing of the results was performed according to the analysis of 100 completed cases of COVID-19 in patients aged 35-45 years (50 men and 50 women) who had no concomitant chronic pathology, and patients did not receive any vaccine dose before the disease (acute COVID-19) and during the next follow-up period (6 months). RESULTS: Results: The data of the analysis allowed us to make a grounded conclusion about the syndromic heterogeneity of COVID 19 in a standardized patients group with a mild course. CONCLUSION: Conclusions: the highest number of symptoms in the postcovid period by frequency, polymorphism, and life quality impact was found in the group of patients with subjectively tolerate acute COVID-19 most easily. Patients whose acute episode meets the mild criteria have pronounced neurological and psychoemotional manifestations during the postcovid period.

Reproducibility of global LV function and dyssynchrony parameters derived from phase analysis of gated myocardial perfusion SPECT: A multicenter comparison with core laboratory setting.

BACKGROUND: Gated myocardial perfusion scintigraphy (GMPS) phase analysis is an important tool to investigate the physiology of left ventricular (LV) dyssynchrony. We aimed to test the performance of GMPS LV function and phase analysis in different clinical settings and on a diverse population. METHODS: This is a post hoc analysis of a prospective, non-randomized, multinational, multicenter cohort study. Clinical evaluation and GMPS prior to cardiac resynchronization therapy (CRT)(baseline) and 6-month post CRT (follow-up) were done. LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), LV ejection fraction (LVEF), LV phase standard deviation (LVPSD), and percentage of left ventricle non-viable (PLVNV) were obtained by 10 centers and compared to the core lab. RESULTS: 276 GMPS studies had all data available from individual sites and from core lab. There were no statistically significant differences between all variables except for LVPSD. When subjects with no mechanical dyssynchrony were excluded, LVPSD difference became non-significant. LVESV, LVEF, LVPSD and PLVNV had strong correlation in site against core lab comparison. Bland-Altman plots demonstrated good agreement. CONCLUSIONS: The presented correlation and agreement of LV function and dyssynchrony analysis over different sites with a diverse sample corroborate the strength of GMPS in the management of heart failure in clinical practice.

Clinical and gated SPECT MPI parameters associated with super-response to cardiac resynchronization therapy.

PURPOSE: We sought to evaluate the behavior of cardiac mechanical synchrony as measured by phase SD (PSD) derived from gated MPI SPECT (gSPECT) in patients with super-response after CRT and to evaluate the clinical and imaging characteristics associated with super-response. METHODS: 158 subjects were evaluated with gSPECT before and 6 months after CRT. Patients with an improvement of LVEF > 15% and NYHA class I/II or reduction in LV end-systolic volume > 30% and NYHA class I/II were labeled as super-responders (SR). RESULTS: 34 patients were classified as super-responders (22%) and had lower PSD (32° ± 17°) at 6 months after CRT compared to responders (45° ± 24°) and non-responders 46° ± 28° (P = .02 for both comparisons). Regression analysis identified predictors independently associated with super-response to CRT: absence of previous history of CAD (odds ratio 18.7; P = .002), absence of diabetes mellitus (odds ratio 13; P = .03), and history of hypertension (odds ratio .2; P = .01). CONCLUSION: LV dyssynchrony after CRT implantation, but not at baseline, was significantly better among super-responders compared to non-super-responders. The absence of diabetes, absence of CAD, and history of hypertension were independently associated with super-response after CRT.

Unravelling structures of radicals of kynurenic acid formed in the photoinduced reactions with tryptophan and N-acetyl tyrosine.

Kynurenic acid (KNA) in the triplet state reacts with tryptophan (Trp) at neutral pH via proton-coupled electron transfer (PCET), which includes the stepwise transition of both electron and proton from Trp to triplet KNA. In the case of tyrosine (Tyr), the quenching reaction is H-transfer, a simultaneous transfer of electron and proton. In this work, we used the time-resolved chemically induced dynamic nuclear polarization (TR CIDNP) method to unveil the sites of H/H+ transfer within KNA. For this purpose, we obtained the values of 1H hyperfine coupling constants (HFCCs) and g-factors for different tautomeric forms of KNA radicals by the DFT method, then calculated CIDNP intensities using these g-factors and HFCCs according to the Adrian model. The calculated CIDNP intensities for different protons were correlated with their CIDNP intensities in the geminate spectra detected in the photoreactions of KNA with Trp, N-acetyl Trp, and N-acetyl Tyr. Best-fit proportionality relationships between calculated and experimental CIDNP intensities have shown that the KNA anion radical is present in two of the three possible tautomeric forms, which result from the H/H+ movement to the carbonyl oxygen of keto- and oxo-quinolinate forms of KNA, without any visible contribution of the H/H+ transfer to the nitrogen of the enol form. For 4-hydroxyquinoline (4HQN), being the chromophoric core of KNA and exhibiting the same PCET and H-transfer reactions with Trp and Tyr, a single possible tautomeric form of its radical has been revealed as H/H+ transfer to the carbonyl oxygen of the keto-form.

Randomization for the susceptibility effect of an infectious disease intervention.

Randomized trials of infectious disease interventions, such as vaccines, often focus on groups of connected or potentially interacting individuals. When the pathogen of interest is transmissible between study subjects, interference may occur: individual infection outcomes may depend on treatments received by others. Epidemiologists have defined the primary parameter of interest-called the "susceptibility effect"-as a contrast in infection risk under treatment versus no treatment, while holding exposure to infectiousness constant. A related quantity-the "direct effect"-is defined as an unconditional contrast between the infection risk under treatment versus no treatment. The purpose of this paper is to show that under a widely recommended randomization design, the direct effect may fail to recover the sign of the true susceptibility effect of the intervention in a randomized trial when outcomes are contagious. The analytical approach uses structural features of infectious disease transmission to define the susceptibility effect. A new probabilistic coupling argument reveals stochastic dominance relations between potential infection outcomes under different treatment allocations. The results suggest that estimating the direct effect under randomization may provide misleading conclusions about the effect of an intervention-such as a vaccine-when outcomes are contagious. Investigators who estimate the direct effect may wrongly conclude an intervention that protects treated individuals from infection is harmful, or that a harmful treatment is beneficial.

Effects of extensive mobilization and tension anastomosis in anorectal reconstruction (experimental study).

PURPOSE: Anorectoplasty and pull-through procedure can be performed with extensive mobilization or tension anastomosis, which can compromise bowel blood perfusion. We aimed to analyze the hypoxia biomarker values and histopathological findings in both conditions to correlate the occurrence of anal stenosis and defecation disorders in experimental models. METHODS: We created anorectal reconstruction models with impaired vascularization of the anorectum (group I) and tension anastomosis (group II) in rats. A third group of animals underwent sham operation (group III) and another as controls (group IV). Hypoxia biomarker values were assessed in all groups. The histopathological changes on the postoperative days 3 and 35, anal stenosis and defecation disorders on day 35 were compared. RESULTS: Hypoxia biomarker values confirmed postoperative ischemia in groups I-III compared to control. Group I and II rats had a similarly pronounced ischemia with histopathologic changes in the anorectum on the postoperative day 3 and accompanied by severe fibrosis on day 35. Compared to the sham operation, both groups showed defecation disorders with significant anal stenoses. CONCLUSION: Extensive rectal mobilization to about the same extent as tension anastomosis has a major impact on postoperative rectal ischemia, resulting in severe fibrotic changes in the anorectum and defecation disorders in the long term.

UAV and IoT-Based Systems for the Monitoring of Industrial Facilities Using Digital Twins: Methodology, Reliability Models, and Application.

This paper suggests a methodology (conception and principles) for building two-mode monitoring systems (SMs) for industrial facilities and their adjacent territories based on the application of unmanned aerial vehicle (UAV), Internet of Things (IoT), and digital twin (DT) technologies, and a set of SM reliability models considering the parameters of the channels and components. The concept of building a reliable and resilient SM is proposed. For this purpose, the von Neumann paradigm for the synthesis of reliable systems from unreliable components is developed. For complex SMs of industrial facilities, the concept covers the application of various types of redundancy (structural, version, time, and space) for basic components-sensors, means of communication, processing, and presentation-in the form of DTs for decision support systems. The research results include: the methodology for the building and general structures of UAV-, IoT-, and DT-based SMs in industrial facilities as multi-level systems; reliability models for SMs considering the applied technologies and operation modes (normal and emergency); and industrial cases of SMs for manufacture and nuclear power plants. The results obtained are the basis for further development of the theory and for practical applications of SMs in industrial facilities within the framework of the implementation and improvement of Industry 4.0 principles.

Characterization of Infants' General Movements Using a Commercial RGB-Depth Sensor and a Deep Neural Network Tracking Processing Tool: An Exploratory Study.

Cerebral palsy, the most common childhood neuromotor disorder, is often diagnosed through visual assessment of general movements (GM) in infancy. This skill requires extensive training and is thus difficult to implement on a large scale. Automated analysis of GM performed using low-cost instrumentation in the home may be used to estimate quantitative metrics predictive of movement disorders. This study explored if infants' GM may be successfully evaluated in a familiar environment by processing the 3D trajectories of points of interest (PoI) obtained from recordings of a single commercial RGB-D sensor. The RGB videos were processed using an open-source markerless motion tracking method which allowed the estimation of the 2D trajectories of the selected PoI and a purposely developed method which allowed the reconstruction of their 3D trajectories making use of the data recorded with the depth sensor. Eight infants' GM were recorded in the home at 3, 4, and 5 months of age. Eight GM metrics proposed in the literature in addition to a novel metric were estimated from the PoI trajectories at each timepoint. A pediatric neurologist and physiatrist provided an overall clinical evaluation from infants' video. Subsequently, a comparison between metrics and clinical evaluation was performed. The results demonstrated that GM metrics may be meaningfully estimated and potentially used for early identification of movement disorders.

Betaine-Based Deep Eutectic Solvent as a New Media for Laccase-Catalyzed Template-Guided Polymerization/Copolymerization of Aniline and 3-Aminobenzoic Acid.

Deep eutectic solvents (DESs) can compensate for some of the major drawbacks of traditional organic solvents and ionic liquids and meet all requirements of green chemistry. However, the potential of their use as a medium for biocatalytic reactions has not been adequately studied. In this work we used the DES betaine-glycerol with a molar ratio of 1:2 as co-solvent for enzymatic template-guided polymerization/copolymerization of aniline (ANI) and 3-aminobenzoic acid (3ABA). The laccase from the basidial fungus Trametes hirsuta and air oxygen served as catalyst and oxidant, respectively. Sodium polystyrene sulfonate (PSS) was used as template. Interpolyelectrolyte complexes of homopolymers polyaniline (PANI) and poly(3-aminobenzoic acid) (P3ABA) and copolymer poly(aniline-co-3-aminobenzoic acid) (P(ANI-3ABA)) were prepared and their physico-chemical properties were studied by UV-Vis and FTIR spectroscopy and cyclic voltammetry. According to the results obtained by atomic force microscopy, PANI/PSS had a granular shape, P(ANI-3ABA)/PSS had a spherical shape and P3ABA/PSS had a spindle-like shape. The copolymer showed a greater antimicrobial activity against Escherichia coli and Staphylcocus aureus as compared with the homopolymers. The minimal inhibitory concentration of the P(ANI-3ABA)/PSS against the gram-positive bacterium S. aureus was 0.125 mg mL-1.

Activation of Neutrophils by Mucin-Vaterite Microparticles.

Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite-mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of -1 ± 1 mV and -7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL-1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1ß, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1-10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin-vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption.