Accessing Properties of Molecular Compounds Involved in Cellular Metabolic Processes with Electron Paramagnetic Resonance, Raman Spectroscopy, and Differential Scanning Calorimetry.

In this work, we review some physical methods of macroscopic experiments, which have been recently argued to be promising for the acquisition of valuable characteristics of biomolecular structures and interactions. The methods we focused on are electron paramagnetic resonance spectroscopy, Raman spectroscopy, and differential scanning calorimetry. They were chosen since it can be shown that they are able to provide a mutually complementary picture of the composition of cellular envelopes (with special attention paid to mycobacteria), transitions between their molecular patterning, and the response to biologically active substances (reactive oxygen species and their antagonists-antioxidants-as considered in our case study).

Single-cell transcriptomics combined with proteomics of intrathecal IgG reveal transcriptional heterogeneity of oligoclonal IgG-secreting cells in multiple sclerosis.

The phenotypes of B lineage cells that produce oligoclonal IgG in multiple sclerosis have not been unequivocally determined. Here, we utilized single-cell RNA-seq data of intrathecal B lineage cells in combination with mass spectrometry of intrathecally synthesized IgG to identify its cellular source. We found that the intrathecally produced IgG matched a larger fraction of clonally expanded antibody-secreting cells compared to singletons. The IgG was traced back to two clonally related clusters of antibody-secreting cells, one comprising highly proliferating cells, and the other consisting of more differentiated cells expressing genes associated with immunoglobulin synthesis. These findings suggest some degree of heterogeneity among cells that produce oligoclonal IgG in multiple sclerosis.

Combination of Machine Learning and Analytical Correlations for Establishing Quantitative Compliance between the Trolox Equivalent Antioxidant Capacity Values Obtained via Electron Paramagnetic Resonance and Ultraviolet-Visible Spectroscopies.

Recent interest in the antioxidant capacity of foods and beverages is based on the established medical knowledge that antioxidants play an essential role in counteracting the damaging effects of free radicals, preventing human neurodegenerative diseases, cardiovascular disorders, and even cancer. At the same time, there is no "the method" that uniquely defines the antioxidant capacity of substances; moreover, the question of interrelation between results obtained by different experimental techniques is still open. In this work, we consider the trolox equivalent antioxidant capacity (TEAC) values obtained by electron paramagnetic resonance (EPR) spectroscopy and ultraviolet-visible (UV-vis) spectroscopy using the classic objects for such studies as an example: red, rosé, and white wine samples. Based on entirely different physical principles, these two methods give values that are not so simply interrelated; this creates a demand for machine learning as a suitable tool for revealing quantitative correspondence between them. The consideration consists of an approximate correlation-based analytical model for the key argument (i.e., TEACEPR) with subsequent adjustment by machine learning-based processing utilizing the CatBoost algorithm with the usage of auxiliary chemical data, such as the total phenolic content and color index, which cannot be accurately described by analytical expressions.

Stereotyped B-cell responses are linked to IgG constant region polymorphisms in multiple sclerosis.

Clonally related B cells infiltrate the brain, meninges, and cerebrospinal fluid of MS patients, but the mechanisms driving the B-cell response and shaping the immunoglobulin repertoires remain unclear. Here, we used single-cell full-length RNA-seq and BCR reconstruction to simultaneously assess the phenotypes, isotypes, constant region polymorphisms, and the paired heavy- and light-chain repertoires in intrathecal B cells. We detected extensive clonal connections between the memory B cell and antibody-secreting cell (ASC) compartments and observed clonally related cells of different isotypes including IgM/IgG1, IgG1/IgA1, IgG1/IgG2, and IgM/IgA1. There was a strong dominance of the G1m1 allotype constant region polymorphisms in ASCs, but not in memory B cells. Tightly linked to the G1m1 allotype, we found a preferential pairing of the immunoglobulin heavy-chain variable (IGHV)4 gene family with the κ variable (IGKV)1 gene family. The IGHV4-39 gene was most used and showed the highest frequency of pairing with IGKV1-5 and IGKV1(D)-33. These results link IgG constant region polymorphisms to stereotyped B-cell responses in MS and indicate that the intrathecal B-cell response in these patients could be directed against structurally similar epitopes.

Transition Metals (Cr3+) and Lanthanides (Eu3+) in Inorganic Glasses with Extremely Different Glass-Formers B2O3 and GeO2.

Glasses containing two different network-forming components and doped with optically active ions exhibit interesting properties. In this work, glass systems based on germanium dioxide and boron trioxide singly doped with lanthanides (Eu3+) and transition metals (Cr3+) ions are research subjects. Optical spectroscopy was the major research tool used to record excitation and emission spectra in a wide spectral range for studied systems. The emitted radiation of glasses doped with Cr3+ ions is dominated by broadband luminescence centered at 770 nm and 1050 nm (4T2 → 4A2). Interestingly, the increase of concentration of one of the oxides contributed to the detectable changes of the R-line (2E → 4A2) of Cr3+ ions. Moreover, EPR spectroscopy confirmed the paramagnetic properties of the obtained glasses. The influence of molar ratio GeO2:B2O3 on spectroscopic properties for Eu3+ ions is discussed. The intensity of luminescence bands due to transitions of trivalent europium ions as well as the ratio R/O decrease with the increase of B2O3. On the other hand, the increase in concentration B2O3 influences the increasing tendency of luminescence lifetimes for the 5D0 state of Eu3+ ions. The results will contribute to a better understanding of the role of the glass host and thus the prospects for new optical materials.

Longevity, clonal relationship, and transcriptional program of celiac disease-specific plasma cells.

Disease-specific plasma cells (PCs) reactive with transglutaminase 2 (TG2) or deamidated gluten peptides (DGPs) are abundant in celiac disease (CeD) gut lesions. Their contribution toward CeD pathogenesis is unclear. We assessed expression of markers associated with PC longevity in 15 untreated and 26 treated CeD patients in addition to 13 non-CeD controls and performed RNA sequencing with clonal inference and transcriptomic analysis of 3,251 single PCs. We observed antigen-dependent V-gene selection and stereotypic antibodies. Generation of recombinant DGP-specific antibodies revealed a key role of a heavy chain residue that displays polymorphism, suggesting that immunoglobulin gene polymorphisms may influence CeD-specific antibody responses. We identified transcriptional differences between CeD-specific and non-disease-specific PCs and between short-lived and long-lived PCs. The short-lived CD19+CD45+ phenotype dominated in untreated and short-term-treated CeD, in particular among disease-specific PCs but also in the general PC population. Thus, the disease lesion of untreated CeD is characterized by massive accumulation of short-lived PCs that are not only directed against disease-specific antigens.

Novel Multicomponent Titanate-Germanate Glasses: Synthesis, Structure, Properties, Transition Metal, and Rare Earth Doping.

Novel multicomponent titanate-germanate glasses singly doped with transition metal (Cr3+) and rare earth ions (Eu3+) were synthesized and the glass transition temperatures and thermal stability parameters were determined using DSC measurements. X-ray diffraction analysis confirmed fully amorphous nature of the received samples. Their structural and optical properties were compared with germanate glasses without TiO2. Correlation between local structure and optical properties in titanate-germanate glasses is well evidenced by FT-IR, Raman, EPR, and luminescence spectroscopy. In particular, luminescence spectra and their decays are examined for glass samples, where GeO2 was partially substituted by TiO2.

Antioxidant Properties of Various Alcoholic Beverages: Application of a Semiempirical Equation.

Presented here is a comprehensive study on the antioxidant properties of various alcoholic beverages: beers, wines, tinctures, and strong spirits, including whisky, brandy, cognac, vodkas, and liquors. The Trolox equivalent antioxidant capacity (TEAC) of each of these various alcoholic beverages is determined using an electron paramagnetic resonance (EPR) method, which is based on a semiempirical correlation. Moreover, the EC50 parameter was determined on the basis of the dependences of the TEAC values obtained by this mathematical equation. Moreover, the total phenolic content (TP) and color index, which have a direct influence on the antioxidant properties of the alcoholic beverages, were identified. The results showed that all the investigated alcoholic beverages (ABs), with the exception for vodkas, exhibit antioxidant properties. By performing a statistical analysis, it was found that the TEAC value significantly depends on the TP, production method, and raw material from which the alcohol was made. However, it is assumed that the TEAC value of alcoholic beverages is independent of the alcohol content.

Comprehensive comparison of antioxidant properties of tinctures.

Homemade tinctures, traditional Polish alcoholic beverages called "nalewkas" (similar to alcohol herbal tinctures), which antioxidant capacity have never been studied before, were characterized by electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR) and ultraviolet visible (UV-vis) spectroscopy. The antioxidant properties of nalewkas made according to homemade recipes were compared to commercially produced nalewkas. The impact of aging on antioxidant properties of nalewkas was investigated. The results showed that all of examined nalewkas exhibited strong antioxidant properties (antioxidant capacity TEACDPPH 466 µmol TE/100 mL - 11890 µmol TE/100 mL). It was found that the value of the antioxidant capacity corresponds to the total phenolic and aromatic proton content. The impact of the production method and the type of fruit used on the TEACDPPH value was also noted. The unripe walnuts with green husks has the highest value of the antioxidant capacity TEACDPPH (11890 µM/100 mL) not only for alcoholic beverages, but also among food products.

Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells.

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.

B-cell composition in the blood and cerebrospinal fluid of multiple sclerosis patients treated with dimethyl fumarate.

BACKGROUND: B cells may contribute to the immunopathogenesis of multiple sclerosis (MS). Dimethyl fumarate (DMF) has recently been shown to reduce the frequency of memory B cells in blood, but it is not known whether the drug influences the cellular composition in the cerebrospinal fluid (CSF). METHODS: A cross-sectional study examining the cellular composition in blood and cerebrospinal fluid (CSF) from 10 patients treated with DMF and 18 patients receiving other disease modifying drugs or no treatment. RESULTS: Patients treated with DMF had reduced proportions of memory B cells in blood compared to other MS patients (p = 0.0007), and the reduction correlated with treatment duration (rs = -0.75, p = 0.021). In the CSF, the absolute number of mononuclear cells were significantly lower in DMF-treated patients compared to the other patients (p = 0.023), and there was a disproportionate decrease of plasmablasts (p = 0.031). CONCLUSION: The results of this exploratory study support a B-cell mediated mechanism of action for DMF in both blood and CSF.

A new equation for converting the parameter EC50 into the total antioxidant capacity TEAC and vice versa.

A new equation for converting EC50 into TEAC values and TEAC into EC50 is presented in this paper. The model was fitted to 180 data points of electron paramagnetic resonance (EPR) spectroscopy measurements of various food products. The model was tested basing on 75 EPR spectroscopy measurements. Moreover, the equation was tested on literature data. The determination coefficient between EC50cal and EC50exp was found to be R2 = 0.9762, while the determination coefficient between TEACcal and TEACexp was found to be R2 = 0.9686. Based on obtained results it can be concluded that the proposed model for converting EC50 into TEAC values and TEAC into EC50 values works properly. This will enable a comparison of the antioxidant properties of products identified in literature through the use of different parameters (TEAC, EC50).

Thermal Stability and Flame Retardancy of Polypropylene Composites Containing Siloxane-Silsesquioxane Resins.

A novel group of silsesquioxane derivatives, which are siloxane-silsesquioxane resins (S4SQ), was for the first time examined as possible flame retardants in polypropylene (PP) materials. Thermal stability of the PP/S4SQ composites compared to the S4SQ resins and neat PP was estimated using thermogravimetric (TG) analysis under nitrogen and in air atmosphere. The effects of the non-functionalized and n-alkyl-functionalized siloxane-silsesquioxane resins on thermostability and flame retardancy of PP materials were also evaluated by thermogravimetry-Fourier transform infrared spectrometry (TG-FTIR) and by cone calorimeter tests. The results revealed that the functionalized S4SQ resins may form a continuous ceramic layer on the material surface during its combustion, which improves both thermal stability and flame retardancy of the PP materials. This beneficial effect was observed especially when small amounts of the S4SQ fillers were applied. The performed analyses allowed us to propose a possible mechanism for the degradation of the siloxane-silsesquioxane resins, as well as to explain their possible role during the combustion of the PP/S4SQ composites.

A comparison of antioxidative capacities of fruit juices, drinks and nectars, as determined by EPR and UV-vis spectroscopies.

The differences in the Trolox Equivalent Antioxidant Capacity (TEAC) values at the same incubation time obtained by two different techniques: electron paramagnetic resonance (EPR) spectroscopy and ultraviolet visible (UV-vis) spectroscopy, which use the same antioxidant-free radical reaction mechanism, were determined for fruit juices, nectars and drinks. For this study, the stable free radical 1,1-Diphenyl-2-picryl-hydrazyl (DPPH(•)) was used. The antioxidant capacity was presented in Trolox Equivalents, e.g., µM trolox per 100 ml of sample. All of the studied fruit juices, drinks and nectars showed antioxidative properties. Dependencies between TEAC values and the percent fruit content and sample color were observed for the studied beverages. It was found that EPR spectroscopy is the more adequate method for determining TEAC values for these kinds of samples.

Investigation of Antioxidant Activity of Pomegranate Juices by Means of Electron Paramagnetic Resonance and UV-Vis Spectroscopy.

Pomegranate fruit (Punica granatum L.) is a source of numerous phenolic compounds, and it contains flavonoids such as anthocyanins, anthocyanidins, cyanidins, catechins and other complexes of flavonoids, ellagitannins, and hydrolyzed tannins. Pomegranate juice shows antioxidant, antiproliferative, and anti-atherosclerotic properties. The antioxidant capacity (TEAC) of the pomegranate juices was measured using electron paramagnetic resonance (EPR) spectroscopy and 1,1-diphenyl-2-picrylhydrazyl (DPPH•) as a source of free radicals, and the total phenolic (TP) content was measured using UV-Vis spectroscopy. All the examined pomegranate juices exhibited relatively high antioxidant properties. The TEAC values determined by means of EPR spectroscopy using Trolox (TE) as a free radical scavenger were in the range of 463.12 to 1911.91 µmol TE/100 mL juice. The TP content measured by the Folin-Ciocalteu method, using gallic acid (GA) as a free radical scavenger, widely varied in the investigated pomegranate juice samples and ranged from 1673.62 to 5263.87 mg GA/1 L juice. The strongest antioxidant properties were observed with the fresh pomegranate juices obtained from the fruits originating from Israel, Lebanon, and Azerbaijan. Correlation analysis of numerical data obtained by means of EPR spectroscopy (TEAC) and UV-Vis spectroscopy (TP) gave correlation coefficient (r)=0.90 and determination coefficient (r2)=0.81 (P<0.05).

Application of Electron Paramagnetic Resonance Spectroscopy for Investigating Antioxidant Activity of Selected Herbs.

Antioxidant properties of selected herbs were investigated using electron paramagnetic resonance (EPR) spectroscopy. This was possible by measuring changes in the intensity of the EPR spectrum that resulted from the interaction of the stable radical 1,1-diphenyl-2-picrylhydrazyl with antioxidants found in herbal samples. Moreover, the total phenolic content (TPC) was measured using UV-Vis spectroscopy. The values of trolox equivalent antioxidant capacity were in the range of 10.95 to 505.95 µmol Trolox/1 g of dry weight of sample. TPC values were in the range of 3.38 to 63.13 mg of gallic acid/1 g of dry weight. The results showed that all of the investigated herbs exhibit antioxidant properties. A positive and significant correlation between antioxidant activity and total phenolic content was observed. The studied herbs could be a good source of natural antioxidants.

Antioxidant Activity of Selected Thyme (Thymus L.) Species and Study of the Equivalence of Different Measuring Methodologies.

This study presents the results of comparative evaluation of the antioxidant activity of the phenolic fraction exhaustively extracted with aqueous methanol from 18 different thyme (Thymus L.) specimens and species. This evaluation is made with use of the same free radical source (DPPH• radical), three different free radical scavenging models (gallic acid, ascorbic acid, and Trolox), and three different measuring techniques (the dot blot test, UV-Vis spectrophotometry, and electron paramagnetic resonance spectroscopy, EPR). A comparison of the equivalence of these three different measuring techniques (performed with use of hierarchical clustering with Euclidean distance as a similarity measure and Ward's linkage) is particularly important in view of the fact that different laboratories use different antioxidant activity measuring techniques, which makes any interlaboratory comparison hardly possible. The results obtained confirm a semiquantitative equivalence among the three compared methodologies, and a proposal is made of a simple and cost-effective dot blot test that uses the DPPH• radical and provides differentiation of antioxidant activity of herbal matter comparable with the results of the UV-Vis spectrophotometry and EPR.

Antioxidant Capacity: Experimental Determination by EPR Spectroscopy and Mathematical Modeling.

A new method of determining antioxidant capacity based on a mathematical model is presented in this paper. The model was fitted to 1000 data points of electron paramagnetic resonance (EPR) spectroscopy measurements of various food product samples such as tea, wine, juice, and herbs with Trolox equivalent antioxidant capacity (TEAC) values from 20 to 2000 µmol TE/100 mL. The proposed mathematical equation allows for a determination of TEAC of food products based on a single EPR spectroscopy measurement. The model was tested on the basis of 80 EPR spectroscopy measurements of herbs, tea, coffee, and juice samples. The proposed model works for both strong and weak antioxidants (TEAC values from 21 to 2347 µmol TE/100 mL). The determination coefficient between TEAC values obtained experimentally and TEAC values calculated with proposed mathematical equation was found to be R(2) = 0.98. Therefore, the proposed new method of TEAC determination based on a mathematical model is a good alternative to the standard EPR method due to its being fast, accurate, inexpensive, and simple to perform.

Structural factors affecting cytotoxic activity of (E)-1-(Benzo[d ][1,3]oxathiol-6-yl)-3-phenylprop-2-en-1-one derivatives.

Derivatives of (E)-1-(5-alkoxybenzo[d][1,3]oxathiol-6-yl)-3-phenylprop-2-en-1-one demonstrated exceptionally high in vitro cytotoxic activity, with IC50 values of the most active derivatives in the nanomolar range. To identify structural fragments necessary for the activity, several analogs deprived of selected fragments were prepared, and their cytotoxic activity was tested. It was found that the activity depends on combined effects of (i) the heterocyclic ring, (ii) the alkoxy group at position 5 of the benzoxathiole ring, and (iii) the substituents in the phenyl ring B. Replacement of the sulfur atom by oxygen does not influence the activity. None of the listed structural fragments alone assured high cytotoxic activity.

A study of the antioxidant properties of beers using electron paramagnetic resonance.

The antioxidant properties of various kinds of beers were investigated using electron paramagnetic resonance (EPR) spectroscopy. This was possible by measuring the changes in the intensity of the EPR spectrum that resulted from the interaction of the stable radical DPPH (1,1-diphenyl-2-picrylhydrazyl) with the antioxidants found in a beer sample. The antioxidant capacity was then presented in Trolox Equivalents, e.g. µM trolox in a beer sample of 100ml. The influence of the type, colour, the content of the extract and alcohol on the antioxidant activities of commercial beer samples was investigated using two-way hierarchical clustering and analysis of variance. The results showed that all of the beers investigated exhibit antioxidant properties. By performing an analysis of variance, it was found that the value of the antioxidant capacity significantly (0.05 level of significance) depends on the content of the extract and the colour of the beer. It seems that additives also influence the antioxidant properties to some extent, but neither the alcohol content nor the kind of fermentation affects the antioxidant properties of beer.