NO● Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis

We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO^● suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NO^● interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NO^● suppresses ETE-PE oxidation. Our study reveals that O₂ and NO^● use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O₂ and NO^● to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NO^● donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NO^●, in further support of the ability of NO^● to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NO^●.     

Evaluating dissolved organic carbon-water partitioning using polyparameter linear free energy relationships: Implications for the fate of disinfection by-products

The partitioning of micropollutants to dissolved organic carbon (DOC) can influence their toxicity, degradation, and transport in aquatic systems. In this study carbon-normalized DOC-water partition coefficients (K_DOC-w) were measured for a range of non-polar and polar compounds with Suwannee River fulvic acid (FA) using headspace and solid-phase microextraction (SPME) methods. The studied chemicals were selected to represent a range of properties including van der Waal forces, cavity formation and hydrogen bonding interactions. The K_DOC-w values were used to calibrate a polyparameter linear free energy relationship (pp-LFER). The difference between experimental and pp-LFER calculated K_DOC-w values was generally less than 0.3 log units, indicating that the calibrated pp-LFER could provide a good indication of micropollutant interaction with FA, though statistical analysis suggested that more data would improve the predictive capacity of the model. A pp-LFER was also calibrated for Aldrich humic acid (HA) using K_DOC-w values collected from the literature. Both experimental and pp-LFER calculated K_DOC-w values for Aldrich HA were around one order of magnitude greater than Suwannee River FA. This difference can be explained by the higher cavity formation energy in Suwannee River FA. Experimental and pp-LFER calculated K_DOC-w values were compared for halogenated alkanes and alkenes, including trihalomethane disinfection by-products, with good agreement between the two approaches. Experimental and calculated values show that DOC-water partitioning is generally low; indicating that sorption to DOC is not an important fate process for these chemicals in the environment.     

Synthesis of nitrogen-containing carbon nanofibers by catalytic decomposition of ethylene/ammonia mixture

The formation of carbon nanofibers (CNFs) doped with nitrogen was investigated during decomposition of C₂H₄/NH₃ mixtures at 450-675 °C over metal catalysts: 90Ni-Al₂O₃, 82Ni-8Cu-Al₂O₃, 65Ni-25Cu-Al₂O₃, 45Ni-45Cu-Al₂O₃, 90Fe-Al₂O₃, 85Fe-5Co-Al₂O₃, 62Fe-8Co-Al₂O₃, 62Fe-8Ni-Al₂O₃. It was found that the yield of CNFs, their structural and textural properties, as well as nitrogen content in CNFs are strongly dependent on the synthesis conditions such as: catalyst used, feed composition, temperature and duration. The 65Ni-25Cu-Al₂O₃ was proved to be the most efficient catalyst for the production of nitrogen-containing carbon nanofibers (N-CNFs) with nitrogen content up to 7 wt.%. Ammonia concentration in the feed equal 75 vol.%, temperature 550 °C and duration 1 h were found to be the optimum reaction parameters to reach the maximum nitrogen content in N-CNFs. TEM studies revealed that the nanofibers have a helical morphology and a “herringbone” structure composed of graphite sheets. According to the XPS data, the nitrogen incorporation in the N-CNF structure leads to the formation of two types of nitrogen coordination: pyridinic and quaternary, and their abundance depends on the reaction conditions.     

Comparative Genomics of Seasonal Senescence in Forest Trees

In the course of evolution, both flowering plants and some gymnosperms have developed such an adaptation to winter and unfavorable living conditions as deciduousness. Of particular interest is Siberian larch (Larix sibirica Ledeb.), which is the only species in the pine family (Pinaceae) with a seasonal deciduousness. New generation sequencing technologies make it possible to study this phenomenon at the genomic level and to reveal the genetic mechanisms of leaf and needle aging in angiosperms and gymnosperms. Using a comparative analysis of the genomes of evergreen and deciduous trees, it was found that the genes that control EXORDIUM LIKE 2 (EXL2) and DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) proteins are most represented in Siberian larch, while an excess of genes that control proteins acting as immune receptors were found in evergreens. Orthologs from the family of genes that control leucine-rich repeat receptor-like kinases (LRR-RLK) contributed mostly to the distinction between evergreens and deciduous plants.     

Comparative Analysis of Enzymatic Transglycosylation Using E. coli Nucleoside Phosphorylases: A Synthetic Concept for the Preparation of Purine Modified 2′-Deoxyribonucleosides from Ribonucleosides

A comparative analysis of the transglycosylation conditions catalyzed by E. coli nucleoside phosphorylases, leading to the formation of 2′-deoxynucleosides, was performed. We demonstrated that maximal yields of 2′-deoxynucleosides, especially modified, can be achieved under small excess of glycosyl-donor (7-methyl-2′-deoxyguanosine, thymidine) and a 4-fold lack of phosphate. A phosphate concentration less than equimolar one allows using only a slight excess of the carbohydrate residue donor nucleoside to increase the reaction’s output. A three-step methodology was elaborated for the preparative synthesis of purine-modified 2′-deoxyribonucleosides, starting from the corresponding ribonucleosides.     

Identification of Phytaspase Interactors via the Proximity-Dependent Biotin-Based Identification Approach

Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.     

Increased Susceptibility of the CD57− NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential,Activation Degree,and Death Induction Response

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57⁻NKG2C⁺ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57⁻ NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3⁺NKG2C⁺ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3⁺ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3⁺NKG2C⁺ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.     

Comparative Characterisation of Aqueous Starch Dispersions by Light Microscopy,Rheometry and Iodine Binding Behaviour

Aqueous potato, wheat and pea starch dispersions (2 g dry Starch/100 g dispersion) were prepared at different time-temperature conditions and characterised by light microscopy of stained cryo-sections, amperometric iodine titration and rheometry. Depending on the gelatinisation conditions and the type of starch, a broad range of microstructures was obtained ranging from limited swelling to complete disintegration of starch granules. The swelling was accompanied by leaching of amylose from the granules and accumulation of amylose in the centre of the granules. The iodine binding capacity of the starch systems was related to the extent of swelling and solubilisation of starch and is therefore an appropriate indicator to follow the gelatinisation process. The flow behaviour was also sensitive to differences in the microstructure. The combination of light microscopy, iodometry and rheology successfully allowed the extensive characterisation of the supramolecular structure of low concentration starch systems.     

Scattering of X-ray Ultrashort Pulses by Complex Polyatomic Structures

The scattering of X-ray ultrashort pulses (USPs) is an important aspect of the diffraction analysis of matter using modern USP sources. The theoretical basis, which considers the specifics of the interaction of ultrashort pulses with complex polyatomic structures, is currently not well developed. In general, research is focused on the specifics of the interaction of ultrashort pulses with simple systems—these are atoms and simple molecules. In this work, a theory of scattering of X-ray ultrashort pulses by complex polyatomic structures is developed, considering the specifics of the interaction of ultrashort pulses with such a substance. The obtained expressions have a rather simple analytical form, which allows them to be used in diffraction analysis. As an example, it is shown that the obtained expressions can be used to study the structures of deoxyribonucleic (DNA) and ribonucleic (RNA) acids.     

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.