Predicting outcomes of the acute phase of COVID-19. High sensitive prognostic model, based on the results of the international registry "analysis of chronic non-infectious diseases dynamics after COVID-19 infection in adult patients" (ACTIV).

The aim of this study is to investigate the course of the acute period of COVID-19 and devise a prognostic scale for patients hospitalized. Materials and methods: The ACTIV registry encompassed both male and female patients aged 18 years and above, who were diagnosed with COVID-19 and subsequently hospitalized. Between June 2020 and March 2021, a total of 9364 patients were enrolled across 26 medical centers in seven countries. Data collected during the patients' hospital stay were subjected to multivariate analysis within the R computational environment. A predictive mathematical model, utilizing the "Random Forest" machine learning algorithm, was established to assess the risk of reaching the endpoint (defined as in-hospital death from any cause). This model was constructed using a training subsample (70% of patients), and subsequently tested using a control subsample (30% of patients). Results: Out of the 9364 hospitalized COVID-19 patients, 545 (5.8%) died. Multivariate analysis resulted in the selection of eleven variables for the final model: minimum oxygen saturation, glomerular filtration rate, age, hemoglobin level, lymphocyte percentage, white blood cell count, platelet count, aspartate aminotransferase, glucose, heart rate, and respiratory rate. Receiver operating characteristic analysis yielded an area under the curve of 89.2%, a sensitivity of 86.2%, and a specificity of 76.0%. Utilizing the final model, a predictive equation and nomogram (termed the ACTIV scale) were devised for estimating in-hospital mortality amongst COVID-19 patients. Conclusion: The ACTIV scale provides a valuable tool for practicing clinicians to predict the risk of in-hospital death in patients hospitalized with COVID-19.

Extremely High Soil Copper Content, yet Low Phytotoxicity: A Unique Case of Monometallic Soil Pollution at Kargaly, Russia.

Ecotoxicological studies on soil metal toxicity often rely on artificially contaminated soils. A major difficulty in using soils contaminated by anthropogenic activities (e.g., mining and agriculture) is the presence of multiple metals, which can make it impossible to distinguish the toxic effects of a particular metal. Therefore, sites with monometallic pollution have great potential for ecotoxicological research. One such site is an agricultural field in Kargaly, Orenburg region, Russia, where copper mining and smelting activities were carried out during the 18th-20th centuries. Samples of Mollisols (chernozems) were collected in the studied field. At several sampling points there were copper ore rocks on the surface, containing malachite (CuCO3 · Cu(OH)2 ). The soil samples had a high copper content, up to approximately 10 g kg-1 , compared with 75 mg kg-1 in the background soil. Importantly, the content of other elements in all soil samples was similar to that in the background soil, highlighting the uniqueness of the monometallic contamination in the study area. Despite the extremely high total copper content, exchangeable copper was relatively low, with a maximum of approximately 0.5 mg kg-1 . We performed a short-term (21-day) ecotoxicity assessment using perennial ryegrass as an indicator of copper toxicity. Contrary to expectations, plant growth was not affected by the high copper content in the studied soils. The low copper phytotoxicity may be explained by the low solubility of malachite. However, future long-term experiments may be warranted to determine copper toxicity thresholds for plants under field conditions. The site discovered in the present study could potentially acquire the same significance as the Danish Hygum site for the study of monometallic soil contamination. Environ Toxicol Chem 2023;42:707-713. © 2023 SETAC.

Stimuli-Responsive Polypeptide Nanoparticles for Enhanced DNA Delivery.

The development of non-viral delivery systems for effective gene therapy is one of the current challenges in modern biomedicinal chemistry. In this paper, the synthesis of pH- and redox-responsive amphiphilic polypeptides for intracellular DNA delivery is reported and discussed. Two series of polypeptides consisting of L-lysine, L-phenylalanine, L-histidine, and L-cysteine as well as the same amino acids with L-glutamic acid were synthesized by a combination of copolymerization of N-carboxyanhydrides of α-amino acids and post-polymerization modification of the resulting copolymers. The presence of histidine provided pH-sensitive properties under weakly acidic conditions specific to endosomal pH. In turn, the presence of cysteine allowed for the formation of redox-responsive disulfide bonds, which stabilized the self-assembled nanoparticles in the extracellular environment but could degrade inside the cell. The formation of intraparticle disulfide bonds resulted in their compactization from 200-250 to 55-100 nm. Empty and pDNA-loaded cross-linked nanoparticles showed enhanced stability in various media compared to non-crosslinked nanoparticles. At the same time, the addition of glutathione promoted particle degradation and nucleic acid release. The delivery systems were able to retain their size and surface charge at polypeptide/pDNA ratios of 10 or higher. GFP expression in HEK 293 was induced by the delivery of pEGFP-N3 with the developed polypeptide nanoparticles. The maximal transfection efficacy (70%) was observed when the polypeptide/pDNA ratio was 100.

Polymer Particles Bearing Recombinant LEL CD81 as Trapping Systems for Hepatitis C Virus.

Hepatitis C is one of the most common social diseases in the world. The improvements in both the early diagnostics of the hepatitis C and the treatment of acute viremia caused by hepatitis C virus are undoubtedly an urgent task. In present work, we offered the micro- and nanotraps for the capturing of HCV. As a capturing moiety, we designed and synthesized in E. coli a fusion protein consisting of large extracellular loop of CD81 receptor and streptavidin as spacing part. The obtained protein has been immobilized on the surface of PLA-based micro- and nanoparticles. The developed trapping systems were characterized in terms of their physico-chemical properties. In order to illustrate the ability of developed micro- and nanotraps to bind HCV, E2 core protein of HCV was synthesized as a fusion protein with GFP. Interaction of E2 protein and hepatitis C virus-mimicking particles with the developed trapping systems were testified by several methods.

An electrochemical biosensor for direct detection of hepatitis C virus.

This paper is aimed at the development of a biosensor for direct detection of Hepatitis C virus (HCV) surface antigen: envelope protein (E2). A recombinant LEL fragment of biological cell receptor CD81 and two short synthetic peptides imitating the fragment of LEL sequence of CD81 (linear and loop-like peptides) capable of specific binding to E2 were tested as molecular recognition elements of the biosensor. For this purpose the selected ligands were immobilized to the surface of a screen-printed electrode utilized as an electrochemical sensor platform. The immobilization parameters such as the ligand concentration and the immobilization time were carefully optimized for each ligand. Differential pulse voltammetry used to evaluate quantitatively binding of E2 to the ligands revealed their similar binding affinity towards E2. Thus, the linear peptide was selected as a less expensive and easily prepared ligand for the HCV biosensor preparation. The resulting HCV biosensor demonstrated selectivity towards E2 in the presence of interfering protein, conalbumin. Moreover, it was found that the prepared biosensor effectively detected E2 bound to hepatitis C virus-mimetic particles (HC VMPs) at LOD value of 2.1∙10-5 mg/mL both in 0.01 M PBS solution (pH 7.4) and in simulated blood plasma.

Slow Many-Body Delocalization beyond One Dimension.

We study the delocalization dynamics of interacting disordered hard-core bosons for quasi-1D and 2D geometries, with system sizes and timescales comparable to state-of-the-art experiments. The results are strikingly similar to the 1D case, with slow, subdiffusive dynamics featuring power-law decay. From the freezing of this decay we infer the critical disorder W_{c}(L,d) as a function of length L and width d. In the quasi-1D case W_{c} has a finite large-L limit at fixed d, which increases strongly with d. In the 2D case W_{c}(L,L) grows with L. The results are consistent with the avalanche picture of the many-body localization transition.

Towards the Development of a 3-D Biochip for the Detection of Hepatitis C Virus.

The early diagnostics of hepatitis C virus (HCV) infections is currently one of the most highly demanded medical tasks. This study is devoted to the development of biochips (microarrays) that can be applied for the detection of HCV. The analytical platforms of suggested devices were based on macroporous poly(glycidyl methacrylate-co-di(ethylene glycol) dimethacrylate) monolithic material. The biochips were obtained by the covalent immobilization of specific probes spotted onto the surface of macroporous monolithic platforms. Using the developed biochips, different variants of bioassay were investigated. This study was carried out using hepatitis C virus-mimetic particles (VMPs) representing polymer nanoparticles with a size close to HCV and bearing surface virus antigen (E2 protein). At the first step, the main parameters of bioassay were optimized. Additionally, the dissociation constants were calculated for the pairs "ligand-receptor" and "antigen-antibody" formed at the surface of biochips. As a result of this study, the analysis of VMPs in model buffer solution and human blood plasma was carried out in a format of direct and "sandwich" approaches. It was found that bioassay efficacy appeared to be similar for both the model medium and real biological fluid. Finally, limit of detection (LOD), limit of quantification (LOQ), spot-to-spot and biochip-to-biochip reproducibility for the developed systems were evaluated.

Effect of Double Substitution in Cationic Chitosan Derivatives on DNA Transfection Efficiency.

Recently, much effort has been expended on the development of non-viral gene delivery systems based on polyplexes of nucleic acids with various cationic polymers. Natural polysaccharide derivatives are promising carriers due to their low toxicity. In this work, chitosan was chemically modified by a reaction with 4-formyl-n,n,n-trimethylanilinium iodide and pyridoxal hydrochloride and subsequent reduction of the imine bond with NaBH4. This reaction yielded three novel derivatives, n-[4-(n',n',n'-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (Pyr-CS), and n-[4-(n',n',n''-trimethylammonium)benzyl]-n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (PyrTMAB-CS). Their structures and degrees of substitution were established by 1H NMR spectroscopy as DS1 = 0.22 for TMAB-CS, DS2 = 0.28 for Pyr-CS, and DS1 = 0.21, DS2 = 0.22 for PyrTMAB-CS. Dynamic light scattering measurements revealed that the new polymers formed stable polyplexes with plasmid DNA encoding the green fluorescent protein (pEGFP-N3) and that the particles had the smallest size (110-165 nm) when the polymer:DNA mass ratio was higher than 5:1. Transfection experiments carried out in the HEK293 cell line using the polymer:DNA polyplexes demonstrated that Pyr-CS was a rather poor transfection agent at polymer:DNA mass ratios less than 10:1, but it was still more effective than the TMAB-CS and PyrTMAB-CS derivatives that contained a quaternary ammonium group. By contrast, TMAB-CS and PyrTMAB-CS were substantially more effective than Pyr-CS at higher polymer:DNA mass ratios and showed a maximum efficiency at 200:1 (50%-70% transfected cells). Overall, the results show the possibility of combining substituent effects in a single carrier, thereby increasing its efficacy.

Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus.

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.

Enhanced Delivery of 4-Thioureidoiminomethylpyridinium Perchlorate in Tuberculosis Models with IgG Functionalized Poly(Lactic Acid)-Based Particles.

The compound 4-thioureidoiminomethylpyridinium perchlorate (perchlozone©) is a novel anti-tuberculosis drug that is active in multiple drug resistance cases, but the compound is hepatotoxic. To decrease the systemic load and to achieve targeting, we encapsulated the drug into poly(lactic acid)-based micro- (1100 nm) and nanoparticles (170 nm) that were modified with single-chain camel immunoglobulin G (IgG) for targeting. Both micro- and nanoparticles formed stable suspensions in saline solution at particle concentrations of 10⁻50 mg/mL. The formulations were injected intraperitoneally and intravenously into the mice with experimental tuberculosis. The survival of control animals was compared to that of mice which were treated with daily oral drug solution, single intraperitoneal administration of drug-loaded particles, and those treated both intravenously and intraperitoneally by drug-loaded particles modified with polyclonal camel IgGs. The distribution of particles in the organs of mice was analyzed with immunofluorescence and liquid chromatography/mass spectrometry. Morphological changes related to tuberculosis and drug toxicity were registered. Phagocytic macrophages internalized particles and transported them to the foci of tuberculosis in inner organs. Nanoparticle-based drug formulations, especially those with IgG, resulted in better survival and lower degree of lung manifestations than the other modes of treatment.

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

The persistence of high concentrations of beta-2-microglobulin (ß2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of ß2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length ß2M (ß2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6ß2m and ΔN10ß2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of ß2M amyloid fibrils with affinity ~104 M-1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with ß2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-ß2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10ß2m fibrils from other ß2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between ß2m and ΔN6ß2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the ß2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit ß2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

Novel Pathway for Efficient Covalent Modification of Polyester Materials of Different Design to Prepare Biomimetic Surfaces.

To form modern materials with biomimic surfaces, the novel pathway for surface functionalization with specific ligands of well-known and widely used polyester-based rigid media was developed and optimized. Two types of material bases, namely, poly(lactic acid) and poly(ε-caprolactone), as well as two types of material design, e.g., supermacroporous matrices and nanoparticles (NPs), were modified via covalent attachment of preliminary oxidized polyvinylsaccharide poly(2-deoxy-N-methacryloylamido-d-glucose) (PMAG). This polymer, being highly biocompatible and bioinspired, was used to enhance hydrophilicity of the polymer surface and to provide the elevated concentration of reactive groups required for covalent binding of bioligands of choice. The specialties of the interaction of PMAG and its preliminary formed bioconjugates with a chemically activated polyester surface were studied and thoroughly discussed. The supermacroporous materials modified with cell adhesion motifs and Arg-Gly-Asp-containing peptide (RGD-peptide) were tested in the experiments on bone tissue engineering. In turn, the NPs were modified with bioligands ("self-peptide" or camel antibodies) to control their phagocytosis that can be important, for example, for the preparation of drug delivery systems.

N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride: Synthesis, interaction with DNA and evaluation of transfection efficiency.

А novel cationic chitosan derivative, N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), with different degrees of substitution (DS) was synthesized by a chemoselective interaction of 4-formyl-N,N,N-trimethylanilinium iodide with chitosan amino groups using a reductive amination method. Several factors (pH, reactant ratio, reaction time, and chitosan structure) were studied for their effects on the DS of the resulting TMAB-CS. The obtained derivatives were characterized by 1H NMR and FTIR spectroscopy. Turbidimetric titration showed enhanced solubility over a wide pH range even for low-substituted TMAB-CS. TMAB-CS provided strong DS-dependent binding of plasmid DNA. Dynamic light scattering measurements revealed the formation of stable polyplexes with hydrodynamic diameters of 200-300nm and ζ-potential of 20-30mV. TMAB-CS with relatively low DS (25%) demonstrated more pronounced transfection efficiency (up to 2000 cell/cm2) of plasmid DNA into the HEK293 cell line promoted by free TMAB-CS. The positive effects of lower DS can be related to a better polyplex dissociation within the cell. The cytotoxicity of TMAB-CS was comparable to that of the initial chitosan at concentrations up to 300ng/µL, even at high DS.

Fabrication and mechanical properties of composite based on ß-chitin and polyacrylic acid.

Squid ß-chitin has been exfoliated in aqueous acrylic acid (AA), after which a composite film of chitin microfibrils in polyacrylic acid (PAA) has been prepared by in situ polymerization of the AA. The segregated chitin fibrils in the composite are 4-6nm in diameter, with an aspect ratio >250. After drying cast films of the composites containing 1, 2 and 3% (w/w) chitin at 140°C for four hours, there was a dramatic resistance to swelling in water, in that the dried films showed only small changes in shape and properties after four hours immersed in water.The most profound impact of the reinforcement on the mechanical properties is observed at high relative humidity (RH), when the PAA is in the rubbery state. At 97.5% RH and room temperature, the elastic moduli of the composites with 1, 2 and 3% (w/w) chitin were 150, 230 and 2100MPa respectively, compared to 65MPa for pure PAA. The main contribution to the filler-reinforcing effect is the high aspect ratio of fibrils and non-covalent interactions, but the stability in water suggests the presence of chemical bonding between the PAA and chitin.

Structure of ß-chitin from Berryteuthis magister and its transformation during whisker preparation and polymerization filling.

Models for the structures of the ß-chitin-protein complex of native and deproteinized squid pen (Berryteuthis magister) based on SAXS and WAXS data are proposed. Chitin fibrils of 25 Å in diameter and persistence length of 1200 Å are immersed in protein matrix. Average distance between fibrils is 42 Å. Deproteinization of the squid pen led to disappearance of the lateral fibril order stabilized by the protein matrix of the native sample. Swelling in water and acrylic acid resulted in an increase in the chitin 010 D-spacing to 14 and 18 Å, respectively. A preparation routine for individual chitin nanofibers of few microns in length and with diameter of 40-60 Å has been developed. During exfoliation of the chitin in acrylic acid the degree of acetylation does not change. Chitin-based nanocomposites can be prepared by polymerization of acrylic acid in swelled deproteinized samples which takes place mainly in the interfibrillar space of ß-chitin mainly.