Integrating metabolic profiling of pancreatic juice with transcriptomic analysis of pancreatic cancer tissue identifies distinct clinical subgroups.

Introduction: Metabolic reprogramming is a hallmark feature of pancreatic ductal adenocarcinoma (PDAC). A pancreatic juice (PJ) metabolic signature has been reported to be prognostic of oncological outcome for PDAC. Integration of PJ profiling with transcriptomic and spatial characterization of the tumor microenvironment would help in identifying PDACs with peculiar vulnerabilities. Methods: We performed a transcriptomic analysis of 26 PDAC samples grouped into 3 metabolic clusters (M_CL) according to their PJ metabolic profile. We analyzed molecular subtypes and transcriptional differences. Validation was performed by multidimensional imaging on tumor slides. Results: Pancreatic juice metabolic profiling was associated with PDAC transcriptomic molecular subtypes (p=0.004). Tumors identified as M_CL1 exhibited a non-squamous molecular phenotype and demonstrated longer survival. Enrichment analysis revealed the upregulation of immune genes and pathways in M_CL1 samples compared to M_CL2, the group with worse prognosis, a difference confirmed by immunofluorescence on tissue slides. Enrichment analysis of 39 immune signatures by xCell confirmed decreased immune signatures in M_CL2 compared to M_CL1 and allowed a stratification of patients associated with longer survival. Discussion: PJ metabolic fingerprints reflect PDAC molecular subtypes and the immune microenvironment, confirming PJ as a promising source of biomarkers for personalized therapy.

Transcriptomic and metabolomic changes might predict frailty in SAMP8 mice.

Frailty is a geriatric, multi-dimensional syndrome that reflects multisystem physiological change and is a transversal measure of reduced resilience to negative events. It is characterized by weakness, frequent falls, cognitive decline, increased hospitalization and dead and represents a risk factor for the development of Alzheimer's disease (AD). The fact that frailty is recognized as a reversible condition encourages the identification of earlier biomarkers to timely predict and prevent its occurrence. SAMP8 (Senescence-Accelerated Mouse Prone-8) mice represent the most appropriate preclinical model to this aim and were used in this study to carry transcriptional and metabolic analyses in the brain and plasma, respectively, upon a characterization at cognitive, motor, structural, and neuropathological level at 2.5, 6, and 9 months of age. At 2.5 months, SAMP8 mice started displaying memory deficits, muscle weakness, and motor impairment. Functional alterations were associated with a neurodevelopmental deficiency associated with reduced neuronal density and glial cell loss. Through transcriptomics, we identified specific genetic signatures well distinguishing SAMP8 mice at 6 months, whereas plasma metabolomics allowed to segregate SAMP8 mice from SAMR1 already at 2.5 months of age by detecting constitutively lower levels of acylcarnitines and lipids in SAMP8 at all ages investigated correlating with functional deficits and neuropathological signs. Our findings suggest that specific genetic alterations at central level, as well as metabolomic changes in plasma, might allow to early assess a frail condition leading to dementia development, which paves the foundation for future investigation in a clinical setting.

Green-house gas fluxes and soil microbial functional genes abundance in saturated and drained peatlands in South-West Iceland.

The drainage of peatlands followed by land use conversion significantly impacts on the fluxes of green-house gases (GHGs, i.e. CO2, CH4, and N2O) to and from the atmosphere, driven by changes in soil properties and microbial communities. In this study, we compared saturated peatlands with drained ones used for sheep grazing or cultivated, which are common in South-West Iceland. These areas exhibit different degrees of soil saturation and nitrogen (N) content, reflecting the anthropic pressure gradient. We aimed at covering knowledge gaps about lack of estimates on N2O fluxes and drainage, by assessing the emissions of GHGs, and the impact of land conversion on these emissions. Moreover, we investigated soil microbial community functional diversity, and its connection with processes contributing to GHGs emission. GHGs emissions differed between saturated and drained peatlands, with increased soil respiration rates (CO2 emissions) and N mineralization (N2O), consistent with the trend of anthropogenic pressure. Drainage drastically reduced methane (CH4) emissions but increased CO2 emissions, resulting in a higher global warming potential (GWP). Cultivation, involving occasional tillage and fertilization, further increased N2O emissions, mediated by higher N availability and conditions favorable to nitrification. Functional genes mirrored the overall trend, showing a shift from prevalent methanogenic archaea (mcrA) in saturated peatlands to nitrifiers (amoA) in drained-cultivated areas. Environmental variables and nutrient content were critical factors affecting community composition in both environments, which overall affected the GHGs emissions and the relative contribution of the three gases.

Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases.

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME). In colorectal liver metastasis (CLM), TAM morphology correlates with prognosis, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger TAMs (L-TAMs). However, the metabolic profile of in vivo human TAM populations remains unknown. Multiparametric flow cytometry was used to freshly isolate S- and L-TAMs from surgically resected CLM patients (n = 14S-, 14L-TAMs). Mass spectrometry-based metabolomics analyses were implemented for the metabolic characterization of TAM populations. Gene expression analysis and protein activity were used to support the biochemical effects of the enzyme-substrate link between riboflavin and (lysine-specific demethylase 1A, LSD1) with TAM morphologies. L-TAMs were characterized by a positive correlation and a strong association between riboflavin and TAM morphologies. Riboflavin in both L-TAMs and in-vitro M2 polarized macrophages modulates LSD1 protein expression and activity. The inflammatory stimuli promoted by TNFα induced the increased expression of riboflavin transporter SLC52A3 and LSD1 in M2 macrophages. The modulation of the riboflavin-LSD1 axis represents a potential target for reprogramming TAM subtypes, paving the way for promising anti-tumor therapeutic strategies.

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth.

BACKGROUND: The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches. METHODS AND RESULTS: Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5ß1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models. CONCLUSIONS: This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.

Anti-VEGF therapy selects for clones resistant to glucose starvation in ovarian cancer xenografts.

BACKGROUND: Genetic and metabolic heterogeneity are well-known features of cancer and tumors can be viewed as an evolving mix of subclonal populations, subjected to selection driven by microenvironmental pressures or drug treatment. In previous studies, anti-VEGF therapy was found to elicit rewiring of tumor metabolism, causing marked alterations in glucose, lactate ad ATP levels in tumors. The aim of this study was to evaluate whether differences in the sensitivity to glucose starvation existed at the clonal level in ovarian cancer cells and to investigate the effects induced by anti-VEGF therapy on this phenotype by multi-omics analysis. METHODS: Clonal populations, obtained from both ovarian cancer cell lines (IGROV-1 and SKOV3) and tumor xenografts upon glucose deprivation, were defined as glucose deprivation resistant (GDR) or glucose deprivation sensitive (GDS) clones based on their in vitro behaviour. GDR and GDS clones were characterized using a multi-omics approach, including genetic, transcriptomic and metabolic analysis, and tested for their tumorigenic potential and reaction to anti-angiogenic therapy. RESULTS: Two clonal populations, GDR and GDS, with strikingly different viability following in vitro glucose starvation, were identified in ovarian cancer cell lines. GDR clones survived and overcame glucose starvation-induced stress by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and both pyruvate and lipids uptake, whereas GDS clones were less able to adapt and died. Treatment of ovarian cancer xenografts with the anti-VEGF drug bevacizumab positively selected for GDR clones that disclosed increased tumorigenic properties in NOD/SCID mice. Remarkably, GDR clones were more sensitive than GDS clones to the mitochondrial respiratory chain complex I inhibitor metformin, thus suggesting a potential therapeutic strategy to target the OXPHOS-metabolic dependency of this subpopulation. CONCLUSION: A glucose-deprivation resistant population of ovarian cancer cells showing druggable OXPHOS-dependent metabolic traits is enriched in experimental tumors treated by anti-VEGF therapy.

Implication of metabolism in the polarization of tumor-associated-macrophages: the mass spectrometry-based point of view.

Tumor-associated macrophages (TAMs) represent one of the main tumor-infiltrating immune cell types and are generally categorized into either of two functionally contrasting subtypes, namely classical activated M1 macrophages and alternatively activated M2 macrophages. TAMs showed different activation states that can be represent by the two extremes of the complex profile of macrophages biology, the M1-like phenotype (pro-inflammatory activity) and the M2-like phenotype (anti-inflammatory activity). Based on the tumor type, and grades, TAMs can acquire different functions and properties; usually, the M1-like phenotype is typical of early tumor stages and is associated to an anti-tumor activity, while M2-like phenotype has a pro-inflammatory activity and is related to a poor patients' prognosis. The classification of macrophages into M1/M2 groups based on well-defined stimuli does not model the infinitely more complex tissue milieu where macrophages (potentially of different origin) would be exposed to multiple signals in different sequential order. This review aims to summarize the recent mass spectrometry-based (MS-based) metabolomics findings about the modifications of metabolism in TAMs polarization in different tumors. The published data shows that MS-based metabolomics is a promising tool to help better understanding TAMs metabolic phenotypes, although it is still poorly applied for TAMs metabolism. The knowledge of key metabolic alterations in TAMs is an essential step for discovering TAMs polarization novel biomarkers and developing novel therapeutic approaches targeting TAM metabolism to repolarize TAMs towards their anti-tumor phenotype.

Brain-Derived Neurotrophic Factor, Kynurenine Pathway, and Lipid-Profiling Alterations as Potential Animal Welfare Indicators in Dairy Cattle.

Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.

Diversity of the Bacterial Community Associated with Hindgut, Malpighian Tubules, and Foam of Nymphs of Two Spittlebug Species (Hemiptera: Aphrophoridae).

Spittlebugs are xylem-sap feeding insects that can exploit a nutrient-poor diet, thanks to mutualistic endosymbionts residing in various organs of their body. Although obligate symbioses in some spittlebug species have been quite well studied, little is known about their facultative endosymbionts, especially those inhabiting the gut. Recently, the role played by spittlebugs as vectors of the phytopathogenetic bacterium Xylella fastidiosa aroused attention to this insect group, boosting investigations aimed at developing effective yet sustainable control strategies. Since spittlebug nymphs are currently the main target of applied control, the composition of gut bacterial community of the juveniles of Philaenus spumarius and Lepyronia coleoptrata was investigated using molecular techniques. Moreover, bacteria associated with their froth, sampled from different host plants, were studied. Results revealed that Sodalis and Rickettsia bacteria are the predominant taxa in the gut of P. spumarius and L. coleoptrata nymphs, respectively, while Rhodococcus was found in both species. Our investigations also highlighted the presence of recurring bacteria in the froth. Furthermore, the foam hosted several bacterial species depending on the host plant, the insect species, or on soil contaminant. Overall, first findings showed that nymphs harbor a large and diverse bacterial community in their gut and froth, providing new accounts to the knowledge on facultative symbionts of spittlebugs.

NSCLC Cells Resistance to PI3K/mTOR Inhibitors Is Mediated by Delta-6 Fatty Acid Desaturase (FADS2).

Hyperactivation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most common events in human cancers. Several efforts have been made toward the identification of selective PI3K pathway inhibitors. However, the success of these molecules has been partially limited due to unexpected toxicities, the selection of potentially responsive patients, and intrinsic resistance to treatments. Metabolic alterations are intimately linked to drug resistance; altered metabolic pathways can help cancer cells adapt to continuous drug exposure and develop resistant phenotypes. Here we report the metabolic alterations underlying the non-small cell lung cancer (NSCLC) cell lines resistant to the usual PI3K-mTOR inhibitor BEZ235. In this study, we identified that an increased unsaturation degree of lipid species is associated with increased plasma membrane fluidity in cells with the resistant phenotype and that fatty acid desaturase FADS2 mediates the acquisition of chemoresistance. Therefore, new studies focused on reversing drug resistance based on membrane lipid modifications should consider the contribution of desaturase activity.

Metabolites Concentration in Plasma and Heart Tissue in Relation to High Sensitive Cardiac Troponin T Level in Septic Shock Pigs.

Elevated circulating cardiac troponin T (cTnT) is frequent in septic shock patients. Signs of myocardial ischemia and myocyte necrosis are not universally present, but the precise mechanism for elevated cTnT is unknown. We investigated plasma and heart tissue metabolites concentration in six septic shock (SS) and three sham swine undergoing a protocol of polymicrobial septic shock and resuscitation, in order to highlight possible pathways and biomarkers involved in troponin release (high sensitive cardiac troponin T, hs-cTnT). The animals were divided into two groups: the high cTnT group (n = 3) were pigs showing a significantly higher concentration of cTnT and lactate after resuscitation; the low cTnT group (n = 6, three sham and three septic shock) characterized by a lower value of cTnT and a lactate level < 2 mmol/L. Spearman correlation was assessed on plasma fold-change of cTnT, cytokines (TNF-α and IL-10), and metabolites. Finally, the fold-change between the end of resuscitation and baseline values (Res./BL) of plasma metabolites was used to perform a partial least square discriminant analysis (PLS-DA) with three latent variables. Before building the model, the number of features was reduced by summing up the metabolites of the same class that resulted similarly correlated to cTnT fold-change. Proline and glycine were significantly higher in the high cTnT group at the end of experiment both in the myocardium and plasma analyses. Moreover, plasma proline fold-change was found to be positively correlated with cTnT and cytokine fold-changes, and trans-4-hydroxyproline (t4-OH-Pro) fold-change was positively correlated with cTnT fold-change. The PLS-DA model was able to separate the two groups and, among the first ranked features based on VIP score, we found sugars, t4-OH-Pro, proline, creatinine, total amount of sphingomyelins, and glycine. Proline, t4-OH-Pro, and glycine are very abundant in collagen, and our results may suggest that collagen degradation could represent a possible mechanism contributing to septic myocardial injury. The common phenotype of septic cardiomyopathy could be associated to dysregulated collagen metabolism and/or degradation, further exacerbated by higher inflammation and oxidative stress.

PGC1α/ß Expression Predicts Therapeutic Response to Oxidative Phosphorylation Inhibition in Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1ß. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1ß (PGC1α/ß) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling and ATP depletion that consequently delayed malignant progression and prolonged the lifespan of high PGC1α/ß-expressing OC-PDX-bearing mice. Conversely, low PGC1α/ß OC-PDXs were not affected by IACS-010759, thus pinpointing a selective antitumor effect of OXPHOS inhibition. The clinical relevance of these findings was substantiated by analysis of ovarian cancer patient datasets, which showed that 25% of all cases displayed high PGC1α/ß expression along with an activated mitochondrial gene program. This study endorses the use of OXPHOS inhibitors to manage ovarian cancer and identifies the high expression of both PGC1α and ß as biomarkers to refine the selection of patients likely to benefit most from this therapy. SIGNIFICANCE: OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/ß expression and offers an effective approach to manage patients on the basis of PGC1α/ß expression.

EASY-FIA: A Readably Usable Standalone Tool for High-Resolution Mass Spectrometry Metabolomics Data Pre-Processing.

Flow injection analysis coupled with high-resolution mass spectrometry (FIA-HRMS) is a fair trade-off between resolution and speed. However, free software available for data pre-processing is few, web-based, and often requires advanced user specialization. These tools rarely embedded blank and noise evaluation strategies, and direct feature annotation. We developed EASY-FIA, a free standalone application that can be employed for FIA-HRMS metabolomic data pre-processing by users with no bioinformatics/programming skills. We validated the tool's performance and applicability in two clinical metabolomics case studies. The main functions of our application are blank subtraction, alignment of the metabolites, and direct feature annotation by means of the Human Metabolome Database (HMDB) using a minimum number of mass spectrometry parameters. In a scenario where FIA-HRMS is increasingly recognized as a reliable strategy for fast metabolomics analysis, EASY-FIA could become a standardized and feasible tool easily usable by all scientists dealing with MS-based metabolomics. EASY-FIA was implemented in MATLAB with the App Designer tool and it is freely available for download.

Application of an Exploratory Knowledge-Discovery Pipeline Based on Machine Learning to Multi-Scale OMICS Data to Characterise Myocardial Injury in a Cohort of Patients with Septic Shock: An Observational Study.

Currently, there is no therapy targeting septic cardiomyopathy (SC), a key contributor to organ dysfunction in sepsis. In this study, we used a machine learning (ML) pipeline to explore transcriptomic, proteomic, and metabolomic data from patients with septic shock, and prospectively collected measurements of high-sensitive cardiac troponin and echocardiography. The purposes of the study were to suggest an exploratory methodology to identify and characterise the multiOMICs profile of (i) myocardial injury in patients with septic shock, and of (ii) cardiac dysfunction in patients with myocardial injury. The study included 27 adult patients admitted for septic shock. Peripheral blood samples for OMICS analysis and measurements of high-sensitive cardiac troponin T (hscTnT) were collected at two time points during the ICU stay. A ML-based study was designed and implemented to untangle the relations among the OMICS domains and the aforesaid biomarkers. The resulting ML pipeline consisted of two main experimental phases: recursive feature selection (FS) assessing the stability of biomarkers, and classification to characterise the multiOMICS profile of the target biomarkers. The application of a ML pipeline to circulate OMICS data in patients with septic shock has the potential to predict the risk of myocardial injury and the risk of cardiac dysfunction.

Hippuric acid: Could became a barometer for frailty and geriatric syndromes?

Aging is a natural biological event that has some downsides such as increased frailty, decline in cognitive and physical functions leading to chronical diseases, and lower quality of life. There is therefore a pressing need of reliable biomarkers to identify populations at risk of developing age-associated syndromes in order to improve their quality of life, promote healthy ageing and a more appropriate clinical management, when needed. Here we discuss the importance of hippuric acid, an endogenous co-metabolite, as a possible hallmark of human aging and age-related diseases, summarizing the scientific literature over the last years. Hippuric acid, the glycine conjugate of benzoic acid, derives from the catabolism by means of intestinal microflora of dietary polyphenols found in plant-based foods (e.g. fruits, vegetables, tea and coffee). In healthy conditions hippuric acid levels in blood and/or urine rise significantly during aging while its excretion drops in conditions related with aging, including cognitive impairments, rheumatic diseases, sarcopenia and hypomobility. This literature highlights the utility of hippuric acid in urine and plasma as a plausible hallmark of frailty, related to low fruit and vegetable intake and changes in gut microflora.

Persistent hyperammonia and altered concentrations of urea cycle metabolites in a 5-day swine experiment of sepsis.

We measured plasma and cerebrospinal fluid (CSF) metabolite concentrations in a 5-day porcine sepsis model of fecal peritonitis. The objectives were: (i) to verify whether the expected pathways that had emerged in previous studies pertain only to the early inflammatory response or persist for the subsequent days; (ii) to identify metabolic derangements that arise later; (iii) to verify whether CSF metabolite concentrations were altered and if these alterations were similar to those in the blood or delayed. We observed an early response to inflammation and cytokine storms with alterations in lipid and glucose metabolism. The arginine/asymmetric dimethylarginine (ADMA) and phenylalanine/tyrosine balances changed 24 h after resuscitation in plasma, and later in CSF. There was a rise in ammonia concentration, with altered concentrations of metabolites in the urea cycle. Whether persistent derangement of these pathways have a role not only on short-term outcomes but also on longer-term comorbidities, such as septic encephalopathy, should be addressed in further studies.

Plasmatic Hippuric Acid as a Hallmark of Frailty in an Italian Cohort: The Mediation Effect of Fruit-Vegetable Intake.

Frailty syndrome is an age-related condition involving a loss of resilience, susceptibility to adverse health outcomes, and poor quality of life. This study was conducted in the framework of InveCe.Ab, an ongoing longitudinal population-based study. Plasma from 130 older individuals (older adults aged 76-78 years) was analyzed and validated (on 303 participants) using mass spectrometry-based metabolomics approaches. Equivalence tests showed that metabolites from the central cellular metabolic pathways were equivalent in frail and fit participants. Hippuric acid was the only cometabolite that distinguished fit from frail older adults. Logistic regression analysis indicated that high hippuric acid levels are significantly associated with a reduction of the risk of frailty after 4 years. Mediation analysis using a Frailty Index, hippuric acid, and fruit-vegetable intake supported the role of fruit-vegetable consumption in the hippuric acid relationship with the Frailty Index. These data point to low plasma hippuric acid as a plausible hallmark of frailty status, associated with lower fruit-vegetable intakes.

Decoding distinctive features of plasma extracellular vesicles in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease's complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell's physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification. METHODS: We analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis. RESULTS: Our procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs. CONCLUSIONS: Our analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

Mitochondrial structural alterations in ovarian cancer patient-derived xenografts resistant to cisplatin.

Mitochondria have attracted attention in cancer research as organelles associated with tumor development and response to therapy. We recently reported acquisition of resistance to cisplatin (DDP) associated with a metabolic rewiring in ovarian cancer patient-derived xenografts (PDXs) models. DDP-resistant PDXs models were obtained mimicking the clinical setting, treating mice bearing sensitive-DDP tumors with multiple cycles of DDP until the development of resistance. To further characterize the metabolic rewiring, the present study focused on tumor mitochondria. We analysed by transmission electron microscopy the mitochondria structure in two models of DDP-resistant and the corresponding DDP-sensitive PDXs and evaluated tumor mDNA content, the expression of genes and proteins involved in mitochondria functionality, and mitochondria fitness-related processes, such as autophagy. We observed a decrease in the number of mitochondria paralleled by an increased volume in DDP-resistant versus DDP-sensitive PDXs. DDP-resistant PDXs presented a higher percentage of damaged mitochondria, in particular of type 2 (concave-shape), and type 3 (cristolysis) damage. We found no difference in the mDNA content, and the expression of genes involved in mitochondrial biogenesis was similar between the sensitive and resistant PDXs. An upregulation of some genes involved in mitochondrial fitness in DDP-R versus DDP-S PDXs was observed. At protein level, no difference in the expression of proteins involved in mitochondrial function and biogenesis, and in autophagy/mitophagy was found. We here reported that the acquisition of DDP resistance is associated with morphological alterations in mitochondria, even if we couldn't find any dysregulation in the studied genes/proteins that could explain the observed differences.