Phenylalanine meta-Hydroxylase: A Single Residue Mediates Mechanistic Control of Aromatic Amino Acid Hydroxylation

The rare nonproteinogenic amino acid, meta-l -tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta-hydroxylation is unknown. Herein, we report the analysis of 3-hydroxylase (Phe3H) from Streptomyces coeruleorubidus. Insights from kinetic analyses of the wild-type enzyme and key mutants as well as of the biocatalytic conversion of synthetic isotopically labelled substrates and fluorinated substrate analogues advance understanding of the process by which meta-hydroxylation is mediated, revealing T202 to play an important role. In the case of the WT enzyme, a deuterium label at the 3-position is lost, whereas in in the T202A mutant 75 % retention is observed, with loss of stereospecificity. These data suggest that one of two possible mechanisms is at play; direct, enzyme-catalysed deprotonation following electrophilic aromatic substitution or stereospecific loss of one proton after a 1,2-hydride shift. Furthermore, our kinetic parameters for Phe3H show efficient regiospecific generation of meta-l -tyrosine from phenylalanine and demonstrate the enzyme's ability to regiospecifically hydroxylate unnatural fluorinated substrates.     

Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe³⁺ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.     

Thymosin β4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis

Thymosin β4 (Tβ4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tβ4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tβ4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tβ4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron²⁺ and iron³⁺ binding regions along the peptide and show that the presence of Tβ4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tβ4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tβ4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tβ4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tβ4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.     

Difference in miRNA Expression in Functioning and Silent Corticotroph Pituitary Adenomas Indicates the Role of miRNA in the Regulation of Corticosteroid Receptors

Corticotroph pituitary adenomas commonly cause Cushing’s disease (CD), but some of them are clinically silent. The reason why they do not cause endocrinological symptoms remains unclear. We used data from small RNA sequencing in adenomas causing CD (n = 28) and silent ones (n = 20) to explore the role of miRNA in hormone secretion and clinical status of the tumors. By comparing miRNA profiles, we identified 19 miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. The analysis of their putative target genes indicates a role of miRNAs in regulation of the corticosteroid receptors expression. Adenomas causing CD have higher expression of hsa-miR-124-3p and hsa-miR-135-5p and lower expression of their target genes NR3C1 and NR3C2. The role of hsa-miR-124-3p in the regulation of NR3C1 was further validated in vitro using AtT-20/D16v-F2 cells. The cells transfected with miR-124-3p mimics showed lower levels of glucocorticoid receptor expression than control cells while the interaction between miR-124-3p and NR3C1 3′ UTR was confirmed using luciferase reporter assay. The results indicate a relatively small difference in miRNA expression between clinically functioning and silent corticotroph pituitary adenomas. High expression of hsa-miR-124-3p in adenomas causing CD plays a role in the regulation of glucocorticoid receptor level and probably in reducing the effect of negative feedback mediated by corticosteroids.     

Optimization of the Synthesis of 2,4,6,8,10,12‐Hexaallyl‐ 2,4,6,8,10,12‐Hexaazaisowurtzitane

2,4,6,8,10,12‐Hexaallyl‐2,4,6,8,10,12‐hexaazaisowurtzitane (HALLIW) was obtained in a condensation reaction of glyoxal with allylamine in the presence of a protonic acid as a catalyst. Optimization of the synthesis was accomplished by means of a mathematical experiment planning theory with the steepest descent method. The effect of the following parameters was examined: process temperature, amount of the catalyst, amount of the solvent, glyoxal addition time, reaction time, and the ratio of the substrates. As a result of the study an improved yield was achieved from 20 to 66.5%. The highest yield for the reaction was found for the conditions (mole ratios relative to glyoxal): catalyst (formic acid) 0.1 : 1; solvents: acetonitrile 9.35 : 1, water 0.18 : 1; allylamine 2.2 : 1; temperature 15 °C; glyoxal addition time 15 min, reaction time 60 min. The studies were conducted on a small laboratory scale where the yield and purity of the product obtained were examined. A method was developed for purification of HALLIW. NMR, IR spectroscopy, DSC, and TG analyses were employed in the study and their results are reported.     

An Antiapoptotic Neuroprotective Role for Neuroglobin

Cell death associated with mitochondrial dysfunction is common in acute neurological disorders and in neurodegenerative diseases. Neuronal apoptosis is regulated by multiple proteins, including neuroglobin, a small heme protein of ancient origin. Neuroglobin is found in high concentration in some neurons, and its high expression has been shown to promote survival of neurons in vitro and to protect brain from damage by both stroke and Alzheimer’s disease in vivo. Early studies suggested this protective role might arise from the protein’s capacity to bind oxygen or react with nitric oxide. Recent data, however, suggests that neither of these functions is likely to be of physiological significance. Other studies have shown that neuroglobin reacts very rapidly with cytochrome c released from mitochondria during cell death, thus interfering with the intrinsic pathway of apoptosis. Systems level computational modelling suggests that the physiological role of neuroglobin is to reset the trigger level for the post-mitochondrial execution of apoptosis. An understanding of the mechanism of action of neuroglobin might thus provide a rational basis for the design of new drug targets for inhibiting excessive neuronal cell death.     

Development of Microporosity in Mesoporous Carbons

Monolithic carbons with uniform and spherical mesopores can be easily obtained by filling the pores of colloidal silica monoliths with carbon precursors followed by carbonization and silica dissolution. In this study three different phenolic resin blends: resorcinol and crotonaldehyde (MC-RC), phenol and paraformaldehyde (MC-PP), and resorcinol and furfural (MC-RF) were used as carbon precursors. Subsequent heating and carbonization of the resulting silica–phenolic resin nanocomposites followed by silica dissolution afforded monolithic carbons with spherical mesopores matching the size of the silica colloids used. Development of microporosity in these carbons was achieved by post-synthesis KOH activation. This study shows that the combination of colloidal templating with post-synthesis activation affords monolithic micro–mesoporous carbons with large specific surface area and well-developed accessible porosity for adsorption, catalysis, environmental and energy-related applications.     

Microstructural Control of Colloidal‐Based Ceramics by Directional Solidification Under Weak Magnetic Fields

The use of weak magnetic fields to control the microstructural evolution of colloidal‐based systems in conjunction with directional solidification is demonstrated as a convenient processing route to fabricate anisotropic ceramic scaffolds with complex microarchitectures. A variety of graded and aligned microstructures were formed by applying external static magnetic fields oriented radially, axially, and transversely with respect to the solidification direction of freezing slurries containing micro/nanoparticles of ZrO₂ and Fe₃O₄. The graded structures, formed by the radial and axial fields, resemble core–shell architectures composed of dense outer perimeters surrounding porous inner cores. The aligned structures, formed by transverse fields, exhibit two modes of microstructural alignment: lamellar walls aligned by the growing ice crystals and mineral bridges aligned by the magnetic fields. The alignment of mineral bridges that connect adjacent lamellae, provide these scaffolds enhanced strength and stiffness when compressed parallel to their orientation (parallel to the direction of the magnetic field).     

Spectroscopic and structural characterization of low molecular weight fractions of polyaniline

Detailed characterization of low molecular weight byproducts of oxidative polymerization of aniline is given. Presence of these compounds in the final product of polymerization strongly influences its macroscopic properties, such as density, solubility, etc., and they should be removed for the sake of reproducibility of properties of the polymer. We focused our research on separation and identification of the low molecular weight compounds, which are created during the polymerization process. We also compared the results obtained for room temperature and 0 °C polymerization products. Polymerization at −30 °C was also carried out but only very small amounts of the low molecular weight products were produced.