Side-chain Oxidation Research Articles

Molecular Dynamics Calculations for the Temperature Response of Poly(alkylated tri(ethylene oxide)isocyanate) Aqueous Solution

Aqueous solutions of conventional temperature-responsive amphiphilic polymers undergo a coil–globule conformational transition around the lower critical solution temperature (LCST) that causes the polymer surfaces to become hydrophobic and the polymers to aggregate together. Isocyanate polymers with alkylated oligo(ethylene oxide) side chains are expected to have rigid main chains and, thus, do not undergo the coil–globule structural transition, but they have recently been reported to exhibit temperature-responsive properties. In this study, molecular dynamics was used to calculate the agglomeration tendencies of two chains of poly(alkylated tri(ethylene oxide)isocyanate) (PRTEOIC, where R = methyl (Me) or ethyl (Et)) in aqueous solution to elucidate the LCST phenomenon in the absence of coil–globule conformational transition. Our MD simulations showed that aggregation also occurs in rod polymers. Furthermore, we found that both (PMeTEOIC)2 and (PEtTEOIC)2 showed parallel agglomeration of the two molecular chains with increasing temperature, but only (PMeTEOIC)2 showed a metastable T-shaped agglomeration in the middle temperature range. The crossing-point temperature (TCRP) at which the density of the first hydrophobic hydration shell around the sidechain alkyl group equals the bulk water density is a useful indicator for predicting the LCST of rod polymers with dense side chains terminated by alkyl groups.

Fine-tuning morphology via oligo(ethylene oxide) side chain engineering for squaraine-based organic solar cells

Two squaraine dyes (SQs), USQ-O bearing oligo(ethylene oxide) (OE) chain, and USQ-C bearing alkyl chain, were synthesized. The influence of OE side chain on photovoltaic performances was systematically investigated. USQ-C and USQ-O show similar absorption spectra in dilute solutions, energy levels, charge mobility and the molecular electrostatic potential (ESP) on the conjugated skeleton. However, USQ-O exhibits hypsochromic absorption band compared to that of USQ-C in the film, and the distribution of positive and negative alternating ESP on the OE side chain. Significantly different electrostatic interactions and multiple hydrogen bonds among OE side chains could greatly affect the behaviors of intermolecular interactions, and, in turn, influence the photophysical properties of pristine/blend films and the miscibility with PC71BM acceptor. USQ-O-based devices exhibit higher short circuit current than that of USQ-C-based (13.94 vs 12.36 mA cm−2), which could be principally put down to the effective improvement of the blending morphology. These results indicate that simply substituted alkyl chain by OE chain for SQ is an effective strategy for optimizing morphology.

Lobosteroids A-F: Six New Highly Oxidized Steroids from the Chinese Soft Coral Lobophytum sp.

To explore the steroidal constituents of the soft coral Lobophytum sp. at the coast of Xuwen County, Guangdong Province, China, a chemical investigation of the above-mentioned soft coral was carried out. After repeated column chromatography over silica gel, Sephadex LH-20, and reversed-phase HPLC, six new steroids, namely lobosteroids A-F (1-6), along with four known compounds 7-10, were obtained. Their structures were determined by extensive spectroscopic analysis and comparison with the spectral data reported in the literature. Among them, the absolute configuration of 1 was determined by X-ray diffraction analysis using Cu Kα radiation. These steroids were characterized by either the presence of an α,β-α',β'-unsaturated carbonyl, or an α,β-unsaturated carbonyl moiety in ring A, or the existence of a 5α,8α-epidioxy system in ring B, as well as diverse oxidation of side chains. The antibacterial bioassays showed that all isolated steroids exhibited significant inhibitory activities against the fish pathogenic bacteria Streptococcus parauberis FP KSP28, Phoyobacterium damselae FP2244, and Streptococcus parauberis SPOF3K, with IC90 values ranging from 0.1 to 11.0 µM. Meanwhile, compounds 2 and 6-10 displayed potent inhibitory effects against the vancomycin-resistant Enterococcus faecium bacterium G7 with IC90 values ranging from 4.4 to 18.3 µM. Therefore, ten highly oxidized steroids with strong antibacterial activities were isolated from the Chinese soft coral Lobophytum sp., which could be developed as new chemotypes of antibacterial drug leads.

Comparative Study on UV Degradation of Black Chinese Lacquers with Different Additives.

This study investigates the UV degradation of black Chinese lacquer by incorporating carbon black and ferrous hydroxide as additives. The purpose of this research is to understand the effects of these additives on the degradation behavior of the lacquer film. Different concentrations of carbon black powder (1%, 3%, and 5%) and Fe(OH)2 (10%, 20%, and 30%) were added to the lacquer following traditional techniques. The main methods employed for analysis were gloss loss measurement, color change assessment, SEM imaging, FTIR spectroscopy, and XPS analysis. The results demonstrate a significant decrease in gloss levels and an increase in lightness values with increasing ultraviolet exposure time. SEM images reveal the formation of cracks in the lacquer film. FTIR analysis indicates oxidation of the urushiol side chain and an increase in oxidation products. The infrared difference spectrum highlights the differences between the additives, with Fe(OH)2 showing a lower impact on the spectra compared to carbon black. XPS analysis confirms the oxidation of the C-H functional group and the presence of C-O-C and C-OH groups. In conclusion, this study sheds light on the influence of carbon black and ferrous hydroxide additives on the UV degradation of black Chinese lacquer and suggests the protective effect of Fe(OH)2 against UV aging. These findings contribute to a better understanding of the degradation mechanisms and provide insights for improving the UV resistance of Chinese lacquer coatings. Further research can explore alternative additives and optimization strategies to mitigate UV-induced degradation.

Thermal degradation and oxidation of glucosinolates in model systems and Brassica vegetable broth is mediated by redox-active compounds

Glucosinolates (GLSs) are secondary plant metabolites with health-promoting effects found in Brassica vegetables. Recently, next to non-enzymatic degradation yielding nitriles, 4-(methylthio)butyl GLS (4MTB-GLS) was shown to undergo side chain oxidation during thermal treatment, forming 4-(methylsulfinyl)butyl GLS (4MSOB-GLS). Here, we investigated natural plant components and artificial analogs on their capability of altering the thermal reactivity of 4MTB-GLS in vegetable broths and model systems using buffers. Addition of ascorbic acid and dehydroascorbic acid caused varying effects: in broth samples, it increased nitrile formation, while in buffer, 4MSOB-GLS was formed. In further experiments, the antioxidant compounds quercetin and Trolox triggered the side chain oxidation of 4MTB-GLS, while H2S terminated its degradation. A synergistic effect of ascorbic acid and Fe2+ was observed, degrading 98% of 4MTB-GLS to the nitrile after 60 min of boiling. Deepening the understanding of factors that influence the non-enzymatic degradation of GLSs will help to preserve their health-promoting effects.

Плазмохимический синтез нитропроизводных аценафтена в поле токов высокой частоты при пониженном давлении

Plasma-chemical processing devices are widely used in practice and are an integral part in microelectronics, medicine, light industry and nanotechnology. In addition, they are used as methods for modifying the modern materials surfaces and in liquids processing. The paper considers preparation of the aromatic compound nitro derivatives under the action of capacitive high-frequency electrodeless discharge at the gas--liquid interface with the reduced pressure. During the plasma-chemical treatment of the reaction mixture containing the 1,2-dihydroacenaphthylene (acenaphthene), its complete conversion was observed, which was confirmed by detection of the acenaphthene mono- and dinitro-derivatives, as well as of the side chain oxidation products, in the reaction products. The proposed method of implementing nitration reaction of the aromatic substances, which belongs to the new instrumental methods of organic synthesis along with photochemistry and electrochemical synthesis, allows direct binding of the molecular nitrogen and oxygen into the valuable nitrogen-containing organic compounds. Study results demonstrate effective introduction of the plasma chemistry methods in chemical technology not only for the purposes of oxidative destruction of the organic substances, but also as the physicochemical tools for the fine organic synthesis allowing selective functionalization of the polynuclear aromatic compounds

Inactivation of vitamin D2 metabolites by human CYP24A1

Vitamin D is found in two forms in humans, D3 produced in the skin and D2 solely from the diet. Both 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) are oxidised and inactivated by CYP24A1, a tightly regulated mitochondrial enzyme that controls serum levels of these secosteroids. The pathways of oxidation of 25(OH)D2 and 1,25(OH)2D2, particularly 25(OH)D2, by human CYP24A1 are not well characterized. The aim of this study was to further elucidate these pathways, and to compare the kinetics of metabolism of 25(OH)D2 and 1,25(OH)2D2 with their vitamin D3 counterparts. We used expressed and partially purified human CYP24A1 with substrates dissolved in the membrane of phospholipid vesicles, to mimic the inner mitochondrial membrane. We found that the major pathways for side chain oxidation of 25(OH)D2 and 1,25(OH)2D2 were identical and that predominant intermediates of 25(OH)D2 metabolism could be converted to the corresponding intermediates in the pathway of 1,25(OH)2D2 oxidation by 1α-hydroxylation by CYP27B1. The initial steps in the CYP24A1-mediated oxidation involved hydroxylation at the C24R position, and another unknown position where the alcohol was oxidised to an aldehyde. The 24R-hydroxylation was followed by hydroxylation at C26 or C28, or cleavage between C24 and C25 to produce the 24-oxo-25,26,27-trinor derivative. All of these products were further oxidised, with 24-oxo-25,26,27-trinor-1(OH)D2 giving a product tentatively identified as 24-oxo-25,26,27-trinor-1,28(OH)2D2. The catalytic efficiency (kcat/Km) of CYP24A1 for initial 25(OH)D2 hydroxylation was similar to that for 25(OH)D3, indicating that they have similar rates of inactivation at low substrate concentrations, supporting that vitamins D2 and D3 are equally effective in maintaining serum 25(OH)D concentrations. In contrast, the kcat/Km value for 1,25(OH)2D3 was almost double that for 1,25(OH)2D2 indicating a lower rate of inactivation of 1,25(OH)2D2 at a low substrate concentration, suggesting that it has increased metabolic stability in vivo.

Highly Conductive Polyoxanorbornene-Based Polymer Electrolyte for Lithium-Metal Batteries.

This present study illustrates the synthesis and preparation of polyoxanorbornene-based bottlebrush polymers with poly(ethylene oxide) (PEO) side chains by ring-opening metathesis polymerization for solid polymer electrolytes (SPE). In addition to the conductive PEO side chains, the polyoxanorbornene backbones may act as another ion conductor to further promote Li-ion movement within the SPE matrix. These results suggest that these bottlebrush polymer electrolytes provide impressively high ionic conductivity of 7.12 × 10-4 S cm-1 at room temperature and excellent electrochemical performance, including high-rate capabilities and cycling stability when paired with a Li metal anode and a LiFePO4 cathode. The new design paradigm, which has dual ionic conductive pathways, provides an unexplored avenue for inventing new SPEs and emphasizes the importance of molecular engineering to develop highly stable and conductive polymer electrolytes for lithium-metal batteries (LMB).

Novel concrete superplasticizers with sterically hindered amines as stabilizing pendant side chains

In this work, a sterically hindered amine, 2,2,6,6-tetramethyl-4-piperidyl methacrylate (TMPMA), is introduced as a bulky pendant group to achieve steric hindrance, replacing the traditional poly(ethylene oxide) (PEO) side chains of traditional polycarboxylate ether (PCE) superplasticizers (SPs). Note that PCE is the common name for concrete superplasticizers having both carboxylic acid and PEO side-chains in which the bulky PEO pendant group exert large steric hindrance leading to good dispersion and stabilization of cement particles. In this work, the PEO side chains are replaced by a sterically hindered amine. Therefore, copolymers of methacrylic acid (MAA) and TMPMA with different molar ratios were prepared by Reversible-Addition Fragmentation Chain Transfer polymerization (RAFT). The prepared copolymers were characterized by proton nuclear magnetic resonance (1H NMR) and size exclusion chromatography (SEC) to elucidate the chemical structure and molecular weights. Rheological studies and fluidity measurements revealed that poly(MAA-co-TMPMA) SPs, which we introduce here as polycarboxylate amine (PCA) SPs, enhanced cement flowability, workability, and slump retention. Free radical polymerization (FRP) was also employed, as an industrially more relevant, more straightforward and more cost-effective synthesis technique, for the preparation of PCA SPs to reflect the possibility of scaling up these SPs via conventional polymerization methods. The resulting FRP PCA analog exhibited the same plasticizing efficacy as those prepared by RAFT polymerization, demonstrating that these polymers could have high potential in the concrete industry.

Spectral Features Differentiate Aging-Induced Changes in Parchment-A Combined Approach of UV/VIS, µ-ATR/FTIR and µ-Raman Spectroscopy with Multivariate Data Analysis.

From the moment of production, artworks are constantly exposed to changing environmental factors potentially inducing degradation. Therefore, detailed knowledge of natural degradation phenomena is essential for proper damage assessment and preservation. With special focus on written cultural heritage, we present a study on the degradation of sheep parchment employing accelerated aging with light (295-3000 nm) for one month, 30/50/80% relative humidity (RH) and 50 ppm sulfur dioxide with 30/50/80%RH for one week. UV/VIS spectroscopy detected changes in the sample surface appearance, showing browning after light-aging and increased brightness after SO2-aging. Band deconvolution of ATR/FTIR and Raman spectra and factor analysis of mixed data (FAMD) revealed characteristic changes of the main parchment components. Spectral features for degradation-induced structural changes of collagen and lipids turned out to be different for the employed aging parameters. All aging conditions induced denaturation (of different degrees) indicated by changes in the secondary structure of collagen. Light treatment resulted in the most pronounced changes for collagen fibrils in addition to backbone cleavage and side chain oxidations. Additional increased disorder for lipids was observed. Despite shorter exposure times, SO2-aging led to a weakening of protein structures induced by transitions of stabilizing disulfide bonds and side chain oxidations.

Multi-Detection Size Exclusion Chromatography as an Advanced Tool for Monitoring Enzyme-Antibody Conjugation Reaction and Quality Control of a Final Product.

The multi-detection size exclusion chromatography (SEC) has been recognized as an advanced analytical technique for the characterization of macromolecules and process control, as well as the manufacturing and formulation of biotechnology products. It reveals reproducible molecular characterization data, such as molecular weight and its distribution, and the size, shape, and composition of the sample peaks. The aim of this work was to investigate the potential and suitability of the multi-detection SEC as a tool for surveillance over the molecular processes during the conjugation reaction between the antibody (IgG) and horseradish peroxidase (HRP), and demonstrate the plausibility of its application in the quality control of the final product, the IgG-HRP conjugate. Guinea pig anti-Vero IgG-HRP conjugate was prepared using a modified periodate oxidation method, based on periodate oxidation of the carbohydrate side chains of HRP, followed by the formation of Schiff bases between the activated HRP and amino groups of the IgG. The quantitative molecular characterization data of the starting samples, intermediates, and final product were obtained by multi-detection SEC. Titration of the prepared conjugate was performed by the ELISA and its optimal working dilution was determined. This methodology proved to be a promising and powerful technology for the IgG-HRP conjugate process control and development, as well as for the quality control of the final product, as verified by the analysis of several commercially available reagents.

Iron Availability Influences Protein Carbonylation in Arabidopsis thaliana Plants.

Protein carbonylation is an irreversible form of post-translational modification triggered by reactive oxygen species in animal and plant cells. It occurs either through the metal-catalyzed oxidation of Lys, Arg, Pro, and Thr side chains or the addition of α, β-unsaturated aldehydes and ketones to the side chains of Cys, Lys, and His. Recent genetic studies concerning plants pointed to an implication of protein carbonylation in gene regulation through phytohormones. However, for protein carbonylation to stand out as a signal transduction mechanism, such as phosphorylation and ubiquitination, it must be controlled in time and space by a still unknown trigger. In this study, we tested the hypothesis that the profile and extent of protein carbonylation are influenced by iron homeostasis in vivo. For this, we compared the profile and the contents of the carbonylated proteins in the Arabidopsis thaliana wild-type and mutant-deficient in three ferritin genes under normal and stress conditions. Additionally, we examined the proteins specifically carbonylated in wild-type seedlings exposed to iron-deficient conditions. Our results indicated that proteins were differentially carbonylated between the wild type and the triple ferritin mutant Fer1-3-4 in the leaves, stems, and flowers under normal growth conditions. The profile of the carbonylated proteins was also different between the wild type and the ferritin triple mutant exposed to heat stress, thus pointing to the influence of iron on the carbonylation of proteins. Consistent with this, the exposure of the seedlings to iron deficiency and iron excess greatly influenced the carbonylation of certain proteins involved in intracellular signal transduction, translation, and iron deficiency response. Overall, the study underlined the importance of iron homeostasis in the occurrence of protein carbonylation in vivo.

Synthesis of Diastereomeric 8‐Fluoro‐ABC‐Steroid Building Blocks

AbstractAn eight‐step linear sequence for the preparation of two diastereomers of an 8‐fluoro‐ABC‐steroid building block was developed. Key step was an intramolecular Diels–Alder reaction of an intermediate o‐quinodimethane formed from a benzocyclobutene substituted with a 5‐fluorohex‐5‐en‐4‐one chain. This side chain was prepared from 6‐chlorohex‐1‐ene by bromofluorination, elimination of HBr, Finkelstein reaction and alkylation of a literature‐known benzocyclobutene derivative with the thus‐formed 6‐iodo‐2‐fluorohex‐1‐ene. Allylic oxidation of side chain's fluorovinyl moiety to an α‐fluoro‐α,β‐unsaturated ketone completed the preparation of the precursor for the [4+2]‐cycloaddition.

Adsorption and Photo-Degradation of Organophosphates on Sulfate-Terminated Anatase TiO2 Nanoparticles

The adsorption and photocatalytic degradation of trimethyl phosphate (TMP) and triethyl phosphate (TEP), two environmentally relevant model pollutants, have been studied on commercial anatase TiO2 and sulfate-terminated anatase TiO2 nanoparticles by means of operando diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and 2D correlation spectroscopy (2D COS). It is concluded that both TMP and TEP adsorb dissociatively on anatase TiO2, while on the sulfate-terminated anatase TiO2, TMP and TEP adsorb associatively. Upon UV illumination, TMP and TEP are completely oxidized on sulfate-terminated anatase TiO2, as evidenced by the evolution of the IR bands characteristic for water and carbon dioxide. In contrast, on anatase TiO2, UV illumination leads to the formation of stable surface-coordinated carboxylate products, which impedes complete oxidation. 2D COS analysis suggests that parallel reaction pathways occur during oxidation under UV illumination, viz. methoxide/ethoxide (ads) → carboxylates (ads) and methoxide/ethoxide (ads) → aldehydes (ads) → carboxylates (ads). A parallel reaction occurs on sulfated TiO2 that yields CO2 and H2O by direct radical reactions with the methoxide groups with little, or no, formation of surface-coordinated intermediates. Sulfated TiO2 favor the formation of aldehyde intermediates, with reaction rates 10 times and 30 times faster for TMP and TEM, respectively, compared with commercial anatase TiO2. About 37% (33%) and 32% (24%) of TMP (TEP) were degraded on sulfated-terminated TiO2 and pure TiO2, respectively, after the first 9 min of UV illumination. We show that the sulfate-functionalization of TiO2 has two main functions. First, it prevents the formation of strongly bonded bridging carboxylates, thereby alleviating deactivation. Second, it promotes full oxidation of the organic side-chains into carbon dioxide and water. Improved electron-hole separation by the electrophilic S(VI) in combination with the blocking of bridging reaction intermediates is proposed to contribute to the improved activity. The presented results give insights into how acidic surface modifications change adsorbate surface chemistries, which can be used to increase the sustained activity of low-temperature photocatalysts.

Polyethylenimine-Conjugated Hydroxyethyl Cellulose for Doxorubicin/Bcl-2 siRNA Co-Delivery Systems

Hydroxyethyl cellulose (HEC), widely known for its biocompatibility and water solubility, is a polysaccharide with potential for pharmaceutical applications. Here, we synthesized polyethylenimine2k (PEI2k)-conjugated hydroxyethyl cellulose (HECP2k) for doxorubicin/Bcl-2 siRNA co-delivery systems. HECP2ks were synthesized by reductive amination of PEI2k with periodate-oxidized HEC. The synthesis of the polymers was characterized using 1H NMR, 13C NMR, primary amine quantification, FT-IR, and GPC. Via agarose gel electrophoresis and Zeta-sizer measurement, it was found that HECP2ks condensed pDNA to positively charged and nano-sized complexes (100-300 nm, ~30 mV). The cytotoxicity of HECP2ks was low and HECP2k 10X exhibited higher transfection efficiency than PEI25k even in serum condition, showing its high serum stability from ethylene oxide side chains. Flow cytometry analysis and confocal laser microscopy observation verified the superior cellular uptake and efficient endosome escape of HECP2k 10X. HECP2k 10X also could load Dox and Bcl-2 siRNA, forming nano-particles (HECP2k 10X@Dox/siRNA). By median effect analysis and annexin V staining analysis, it was found that HECP2k 10X@Dox/siRNA complexes could cause synergistically enhanced anti-cancer effects to cancer cells via induction of apoptosis. Consequently, it was concluded that HECP2k possesses great potential as a promising Dox/Bcl-2 siRNA co-delivery carrier.

Tetrafluorinated versus hydrogenated azobenzene polymers in water: Access to visible-ligh stimulus at the expense of responsiveness

Photo-responsive polymers are at the core of numerous applications as actuators in biology but often exhibit the drawback of activation in the UV range. This work describes a methacrylate-based terpolymer presenting tetra-ortho-fluoroazobenzene groups showing activation through visible light illumination. The presence of poly(ethylene oxide) side chains and maleimide functions ensures that it can be easily used for biological applications in aqueous media. The terpolymer was shown to form unimolecular micelles in water. The tetra-ortho-fluoroazobenzene moiety was reversibly switched from trans to cis upon visible light irradiation without significant modification of the unimolecular morphology. The deprotection of the maleimide function resulted in a reactive polymer which was used to obtain hydrogels by reaction with poly(ethylene oxide) tetra-thiols or bioconjugates by reaction with a model peptide or a poly(ethylene oxide) mono-thiol exhibiting a biotin group. Hydrogels incorporating β-cyclodextrin molecules were also obtained. Contrary to polymers bearing hydrogenated azobenzenes, the terpolymer bearing tetra-ortho-fluoroazobenzene moieties did not form a complex with β-cyclodextrin and none of the presented hydrogels exhibited macroscopic changes upon irradiation although the trans/cis isomerization occurred. This was explained by the burial of tetra-ortho-fluoroazobenzene groups towards the polymer backbone, therefore completely hindering its access.

Complementation between microbial necromass and plant debris governs the long-term build-up of the soil organic carbon pool in conservation agriculture

Plant residue is ultimately the source of soil organic matter but occurs as a continuum from relatively fresh plant tissue to highly evolved components. Nevertheless, the extent to which crop residue accumulates as plant tissue versus microbial necromass in conservation agricultural systems is still uncertain. A 12-year conservation tillage experiment with maize additions of zero, half and full harvest was conducted in Northeast China. Soil samples in the 0–10 cm depth were collected at 1–4 year intervals. The microbial residue biomarker amino sugar and plant component biomarker lignin phenol were quantified to trace the dynamics of microbial necromass and plant debris. Compared with the initial value, the content of amino sugar increased exponentially to 21–45% and approached steady-state equilibrium after 12 years of maize straw mulching, indicating the existence of “microbial carrying capacity” under repeated plant residue addition. With prolonged duration of conservation tillage and increasing rate of maize straw return, the ratio of amino sugar to lignin phenol decreased from 6.2 to 1.7, accompanied by a decreased degree of lignin side-chain oxidation, indicating that organic input enhanced the selective retention of fresh plant lignin to a larger extent than microbial necromass. The build-up of the SOC pool under conservation tillage was critically controlled by the functional complementation of microbial- and plant-derived components. Maize straw input was able to enhance the contribution of microbial necromass to SOC stabilization by increasing fungal necromass accumulation, while the retention of lignin-like plant debris enlarged the SOC pool capacity and potentially maintained the decomposability of SOC in the long term.

The bacterial cytochrome P450 (CYP) CYP125 enzymes can competitively oxidise sitosterol in the presence of cholesterol.

Cholesterol catabolism is an important survival mechanism for the pathogenic Mycobacterium tuberculosis. Various other mycobacteria degrade not only cholesterol but plant sterols such as sitosterol and campesterol. In this work we demonstrate that the cytochrome P450 (CYP) CYP125 enzyme family is capable of sitosterol and campesterol side-chain oxidation and activation in these bacteria. We also show that the CYP142 and CYP124 cholesterol hydroxylating enzyme families are significantly less active for sitosterol hydroxylation compared to CYP125 enzymes.

Insight into Different Stages of Steroid Degradation in Thermophilic Saccharopolyspora hirsuta VKM Ac-666T Strain.

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT-qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts.

The cysteine residue in beta-lactoglobulin reacts with oxidized tyrosine residues in beta-casein to give casein-lactoglobulin dimers

Proteins are modified during milk processing and storage, with sidechain oxidation and crosslinking being major consequences. Despite the prevalence and importance of proteins in milk, and particularly caseins (∼80% of total content), the nature of the cross-links formed by oxidation, and their mechanisms of formation, are poorly characterized. In this study, we investigated the formation and stability of cross-links generated by the nucleophilic addition of Cys residues to quinones generated on oxidation of Tyr residues. The mechanisms and stability of these adducts was explored using ubiquitin as a model protein, and β-casein. Ubiquitin and β-casein were oxidized using a rose Bengal/visible light/O2 system, or by the enzyme tyrosinase. The oxidized proteins were incubated with glutathione or β-lactoglobulin (non-oxidized, but unfolded by treatment at 70 °C), before analysis by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that Cys-quinone adducts are readily-formed, and are stable for >48 h. Thus, oxidized β-casein reacts efficiently with the thermally unfolded β-lactoglobulin, likely via Michael addition of the exposed Cys to a Tyr-derived quinone. These data provide a novel, and possibly general, mechanism of protein cross-link formation, and provides information of the stability of these species that have potential as markers of protein quality.