Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis

Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Determination of ubiquinone and related metabolites in zebrafish embryos by LC-HRMS.

Introduction Characterization of lipidome is an emerging topic in metabolomics and the contribution of HPLC-HRMS could be an important tool in the elucidation of biochemical pathways. One goal of this work is to provide a rapid, selective and sensitive method to measure ubiquinone and related lipids concentration in zebrafish embryos and to apply it to metabolism studies. Subsequently untargeted analysis of related lipophilic compounds becomes achievable. Ubiquinone (also known as coenzyme Q10) plays an essential role in the mitochondria electron-transport chain. It is a interesting molecule shown to play an important antioxidant role in the cardiovascular system and it is the only endogenously synthesized lipid-soluble antioxidant. Zebrafish (Danio rerio) is an established model for studying toxicology and understanding human diseases. Methods Embryos and adult fishes were raised and maintained under standard laboratory conditions. Biological samples were extracted by LLE with various organic solvents. HPLC-HRMS analyses were accomplished on a Dionex Ultimate 3000 LC system coupled with a LTQ-Orbitrap instrument, with ESI and APCI interfaces. C4, C8 and C18 RP columns were tested for separation. Here we investigate the ionization modes, the ex vivo analytical sensitivity and the fragmentation mechanisms of ubiquinone and related compounds. The developed methodology will be applied to study samples of zebrafish embryos. Preliminary data In the first steps of method development we optimized the chromatographic separation of highly lipophilic ubiquinone related compounds on RP-HPLC columns and studied the sensitivity of different ionization modes (positive vs. negative ion mode, APCI vs. ESI, proton vs ammonium vs. sodium vs. lithium adducts formation). A fragmentation study of the analytes was also done evidencing a double neutral loss of formaldehyde as major pathway. A full validation study was then completed to make possible quantitative determination on biological samples. Accuracy, precision, LLOQ, linearity, extraction recovery were evaluated. Then to confirm the high similarity between zebrafish and higher vertebrates at the cellular and physiological levels we determined the variation of concentration of coenzyme Q10 at various zebrafish growth steps and compared it with similar metabolites as coenzymes with shorter isoprene chain (Q9 to Q6). Finally a study to characterize the untargeted lipidome of embryo samples is ongoing to resolve the complex mixture of compounds giving mono-charged ions in the range 700-1000 m/z. Novel aspects Rapid quantification of ubiquinone and untargeted lipidomic analysis in ex vivo zebrafish samples for biochemical studies