In this study, 2 chalcone analogues were synthesized and assessed through in silico and experimental methods for their potential to inhibit the lipoxygenase enzyme, which plays a role in the inflammation pathway. The structure elucidation of each chalcone was conducted through a combination of Nuclear Magnetic Resonance (NMR) and Density Functional Theory (DFT). Α “LOX-chalcone” complex, predicted by docking studies, was further examined using Molecular Dynamics (MD) simulations to evaluate the stability of the complex. After fully characterizing the “LOX-chalcone” complexes in silico, the atomic details of each chalcone’s interaction with LOX-1 and 5-LOX were revealed through Saturation Transfer Difference (STD) NMR (Nuclear Magnetic Resonance). Finally, their selectivity profile was investigated for human 15-LOX-1 and general Lipoxidase activity. The in silico methods suggest that chalcones could be promising lead compounds for drug design targeting the LOX enzyme.

Green leaf volatiles (GLVs) are important in giving grape a fresh and green aroma. But the changes in GLVs during the phenological development of grapevines are not well known. This study analysed the GLVs and transcription levels of associated biosynthetic genes in six grape species from the Loess Plateau region at five stages of maturation. Thirteen GLVs were detected, showing unique patterns for each grape type at various growth phases. The primary components in six grapes were (E)-2-Hexenal, (E)-2-Hexen-1-ol, and Hexanal. With the exception of Cabernet Franc in 2019, the overall GLV contents of the six types generally increased during growth and development, peaking or stabilizing at harvest. And Sauvignon Blanc, Cabernet Gernischt, and Cabernet Sauvignon exhibited higher total contents among the varieties. Correlation analysis revealed a strong positive correlation between the levels of Hexanal, 1-Hexanol, (E)-2-Hexen-1-ol, (Z)-3-Hexenyl acetate, Nonanal, and (E, E)-2,6-Nonadienal and the expression of VvHPL and VvAAT genes in the LOX-HPL pathway. Specifically, VvHPL emerges as a potential candidate gene responsible for species-specific differences in GLV compounds. Comprehending the changing patterns in the biosynthesis and accumulation of GLVs offers viticulturists and enologists the opportunity to devise targeted strategies for improving the aromatic profile of grapes and wines.

ADAM10 is a multi-functional proteinase that can cleave approximately 100 different substrates. Previously, we demonstrated that ADAM10 is expressed by ameloblasts responsible for enamel formation. Here we asked if ADAM10 plays a role in enamel development. Deletion of Adam10 in mice is embryonic lethal and deletion of Adam10 from epithelia is perinatal lethal. We therefore deleted Adam10 from ameloblasts. Here, we confirm ameloblast-specific expression of the Tg(Amelx-iCre)872pap construct. We crossed these mice with Adam10 floxed mice to generate Amelx-iCre; Adam10fl/fl mice (Adam10 cKO). We show that Adam10 cKO mice have discolored teeth with softer than normal enamel. Notably, the Adam10 cKO enamel density and volume were significantly reduced in both incisors and molars. Moreover, the incisor enamel rod pattern became progressively more disorganized moving from the DEJ to the outer enamel surface and this disorganized rod structure created large gaps between and among the rods. ADAM10 cleaves proteins essential for cell signaling and for enamel formation such as RELT and COL17A1. ADAM10 also cleaves cell-cell contacts such as E- and N-cadherins that may support ameloblast movement necessary for normal rod patterns. Here, we show for the first time that ADAM10 expressed by ameloblasts is essential for proper enamel formation.

Commonly employed methods for reversibly disrupting gene expression, such as those based on RNAi or CRISPRi, are rarely capable of achieving >80-90% downregulation, making them unsuitable for targeting genes that require more complete disruption to elicit a phenotype. Genetic deletion, on the other hand, while enabling complete disruption of target genes, often produces undesirable irreversible consequences such as cytotoxicity or cell death. Here we describe the design, development and detailed characterization of a dual-function "TRE-Lox" system for effecting either (a) doxycycline (Dox)-mediated downregulation or (b) genetic deletion of cathepsin D (CatD), based on targeted insertion of a tetracycline-response element (TRE) and two LoxP sites into the 5' end of the endogenous gene (CTSD). Using an optimized reverse-tetracycline transrepressor (rtTR) variant fused with the Krüppel-associated box (KRAB) domain, we show that CatD expression can be disrupted by as much as 98% in mouse embryonic fibroblasts (MEFs). This system is highly sensitive to Dox (IC50 = 1.46 ng/mL) and results in rapid (t1/2 = 0.57 d) and titratable downregulation of CatD. Notably, even near-total disruption of CatD expression was completely reversed by withdrawal of Dox. As expected, transient expression of Cre recombinase results in complete deletion of the CTSD gene. The dual functionality of this novel system will facilitate future studies of the involvement of CatD in various diseases, particularly those attributable to partial loss of CatD function. In addition, the TRE-Lox approach should be applicable to the regulation of other target genes requiring more complete disruption than can be achieved by traditional methods.

Leaf mechanical wounding triggers a rapid, within minutes, release of a blend of volatile organic compounds (VOCs). Wounding-induced VOC blend is mainly composed of oxygenated ubiquitous stress volatiles such as methanol and volatile products of lipoxygenase (LOX) pathway (mainly C5 and C6 alcohols and aldehydes and their derivatives), but also includes multiple minor VOCs that collectively act as infochemicals inducing defences in non-damaged plant leaves, neighbouring plants and attracting herbivore enemies. Till present, interspecific variability of the rate of induction and magnitude of wounding-induced emissions, and the extent to which plant structural traits and physiological activity alter these emissions are poorly known. Particularly scarce is the information of the induced emissions in tropical agricultural plant species despite their economic importance and large area of cultivation at regional to global scales. We chose five tropical crops with varying photosynthetic activity and leaf structural characteristics: Abelmoschus esculentus, Amaranthus cruentus, Amaranthus hybridus, Solanum aethiopicum and Telfairia occidentalis to characterize the kinetics and magnitude of wounding-induced emissions, hypothesizing that the induced emission response is greater and faster in physiologically more active species with greater photosynthetic activity than in less active species. Rapid highly repeatable leaf wounds (12-mm cuts) were generated by a within-leaf-chamber cutting knife. Wounding-induced VOC emissions were measured continuously with a proton-transfer reaction time-of-flight mass spectrometer and gas-chromatography mass spectrometry was used to separate isomers. Twenty-three ion VOCs and twelve terpenoid molecule structures were identified, whereas ubiquitous stress volatiles methanol (on average 40% of total emissions), hexenal (24%), and acetaldehyde (11%) were the main compounds across the species. Emissions of low-weight oxygenated compounds (LOC, 70% of total), and LOX products (29%) were positively correlated across species, but minor VOC components, monoterpenoids and benzenoids were negatively correlated with LOC and LOX, indicating a reverse relationship between signal specificity and strength. There was a large interspecific variability in the rate of induction and emission magnitude, but the hypothesis of a stronger emission response in physiologically more active species was only partly supported. In addition, the overall emission levels were somewhat lower with different emission blend compared to the data reported for wild species, as well as different shares for the VOCs in the blend. The study demonstrates that wounding-dependent emissions from tropical agricultural crops can significantly contribute to atmospheric volatiles, and these emissions cannot be predicted based on current evidence of wild plant model systems.

The central nervous system (CNS) is an essential hub for neuronal communication. As a major component of the CNS, glial cells are vital in the maintenance and regulation of neuronal network dynamics. Research on microglia, the resident innate immune cells of the CNS, has advanced considerably in recent years, and our understanding of their diverse functions continues to grow. Microglia play critical roles in the formation and regulation of neuronal synapses, myelination, responses to injury, neurogenesis, inflammation and many other physiological processes. In parallel with advances in microglial biology, cutting-edge techniques for the characterization of microglial properties have emerged with increasing depth and precision. Labeling tools and reporter models are important for the study of microglial morphology, ultrastructure and dynamics, but also for microglial isolation, which is required to glean key phenotypic information through single-cell transcriptomics and other emerging approaches. Strategies for selective microglial depletion and modulation can provide novel insights into microglia-targeted treatment strategies in models of neuropsychiatric and neurodegenerative conditions, cancer and autoimmunity. Finally, fate mapping has emerged as an important tool to answer fundamental questions about microglial biology, including their origin, migration and proliferation throughout the lifetime of an organism. This review aims to provide a comprehensive discussion of these emerging techniques, with applications to the study of microglia in development, homeostasis and CNS pathologies.

Renal cell carcinoma (RCC) represents around 3% of all cancers, within which clear cell RCC (ccRCC) are the most common type (70–75%). The RCC disease regularly progresses asymptomatically and upon presentation is recurrently metastatic, so an early method of detection is necessary. The identification of one or more spe-cific biomarkers measurable in biofluids (i.e urine) by combined approaches could surely be appropriate for this kind of cancer, especially due to easy obtainability by non invasive method. OLR1 is a metabolic gene that encodes for the Lectin-like oxidized low-density lipoprotein re-ceptor-1 (LOX-1), implicated in inflammation, atherosclerosis, ROS and metabolic disor-der-associated carcinogenesis. Specifically, LOX-1 is clearly involved in tumor insurgence and progression of different human cancers. This work reports for the first time the presence of LOX-1 protein in ccRCC urine and its peculiar distribution in tumoral tissues. In parallel, urine samples headspace has been analyzed for the presence of the volatile compounds (VOCs) by SPME-GC/MS and gas sensor array. In particular, it was found by GC/MS analysis that 2-Cyclohexen-1-one,3-methyl-6-(1-methylethyl)- correlates with LOX-1 concentration in urine. Thus, the combined approach of VOCs analysis and protein quantification could led to promis-ing results in terms of diagnostic and prognostic potential for ccRCC tumor.