Prolonged exposure to simulated microgravity promotes stemness impairing morphological, metabolic and migratory profile of pancreatic cancer cells: a comprehensive proteomic, lipidomic and transcriptomic analysis

Background: The impact of the absence of gravity on cancer cells is of great interest, especially today that space is more accessible than ever. Despite advances, few and contradictory data are available mainly due to different setup, experimental design and time point analyzed. Methods: Exploiting a Random Positioning Machine, we dissected the effects of long-term exposure to simulated microgravity (SMG) on pancreatic cancer cells performing proteomic, lipidomic and transcriptomic analysis at 1, 7 and 9 days. Results: Our results indicated that SMG affects cellular morphology through a time-dependent activation of Actin-based motility via Rho and Cdc42 pathways leading to actin rearrangement, formation of 3D spheroids and enhancement of epithelial-to-mesenchymal transition. Bioinformatic analysis reveals that SMG may activates ERK5/NF-κB/IL-8 axis that triggers the expansion of cancer stem cells with an increased migratory capability. These cells, to remediate energy stress and apoptosis activation, undergo a metabolic reprogramming orchestrated by HIF-1α and PI3K/Akt pathways that upregulate glycolysis and impair β-oxidation, suggesting a de novo synthesis of triglycerides for the membrane lipid bilayer formation. Conclusions: SMG revolutionizes tumor cell behavior and metabolism leading to the acquisition of an aggressive and metastatic stem cell-like phenotype. These results dissect the time-dependent cellular alterations induced by SMG and pave the base for altered gravity conditions as new anti-cancer technology

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction

Metabolomics Diagnosis of COVID-19 from Exhaled Breath Condensate

Infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to severe respiratory tract damage and acute lung injury. Therefore, it is crucial to study breath-associated biofluids not only to investigate the breath’s biochemical changes caused by SARS-CoV-2 infection, but also to discover potential biomarkers for the development of new diagnostic tools. In the present study, we performed an untargeted metabolomics approach using a bidimensional gas chromatography mass spectrometer (GCxGC-TOFMS) on exhaled breath condensate (EBC) from COVID-19 patients and negative healthy subjects to identify new potential biomarkers for the noninvasive diagnosis and monitoring of the COVID-19 disease. The EBC analysis was further performed in patients with acute or acute-on-chronic cardiopulmonary edema (CPE) to assess the reliability of the identified biomarkers. Our findings demonstrated that an abundance of EBC fatty acids can be used to discriminate COVID-19 patients and that they may have a protective effect, thus suggesting their potential use as a preventive strategy against the infection

New non-invasive approaches for proteomics and metabolomics analyses

Recent technological developments in analytical chemistry spurred the analysis of historical, archaeological and paleontological objects. The Identification of proteins and small molecules from cultural heritage objects is crucial to characterize the materials used by the artists and it can provide invaluable information for designing restoration interventions. All most the developed analytical procedures require at least a micro sampling from the object. However, non-invasive techniques are always preferred for the analysis of precious and unique objects. A part of this PhD research focused on the development and application of new non-invasive methods for the analysis of cultural heritage. A new method for the non-invasive analysis of proteins and small molecules with mass spectrometry from cultural heritage objects was discussed; the results obtained using a non-invasive imaging instrument on ancient Egyptian mural paintings were also presented; the development and application of non-invasive methods that use portable infrared spectroscopy instrumentation were shown. The recent revolution in mass spectrometry technology with the introduction of high throughput instruments and techniques has led to the widespread expansion of advanced analytical methods in health science. But today, the main target of modern mass spectrometry analysis in biomedical research can be summarize as the development of effective and reliable approaches able of discriminating diseased conditions at their earliest stage, in a non or minimally-invasive manner. The aim of the second part of this PhD research was the development and application of non-invasive methods for the analysis of biological materials. A new method for the non-invasive analysis and characterization of adenoma in colon rectal cancer was presented and a combined bi and mono-dimensional gas chromatography mass spectrometry approach for the identification of new biomarkers for prostate cancer in serum was discussed

Non-invasive characterization of colorants by portable diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and chemometrics

During the last years the need for non-invasive and non-destructive analytical methods brought to the development and application of new instrumentation and analytical methods for the in-situ analysis of cultural heritage objects. In this work we present the application of a portable diffuse reflectance infrared Fourier transform (DRIFT) method for the non-invasive characterization of colorants prepared according to ancient recipes and using egg white and Gum Arabic as binders. Approximately 50 colorants were analyzed with the DRIFT spectroscopy: we were able to identify and discriminate the most used yellow (i.e. yellow ochres, Lead-tin Yellow, Orpiment, etc.), red (i.e. red ochres, Hematite) and blue (i.e. Lapis Lazuli, Azurite, indigo) colorants, creating a complete DRIFT spectral library. The Principal Component Analysis\u2013Discriminant Analysis (PCA\u2013DA) was then employed for the colorants classification according to the chemical/mineralogical composition. The DRIFT analysis was also performed on a gouache painting of the artist Sutherland\u201d and the colorants used by the painter were identified directly in-situ and in a non-invasive manner