Recent advances in applying mass spectrometry and systems biology to determine brain dynamics

Neurological disorders encompass various pathologies which disrupt normal brain physiology and function. Poor understanding of their underlying molecular mechanisms and their societal burden argues for the necessity of novel prevention strategies, early diagnostic techniques and alternative treatment options to reduce the scale of their expected increase. Areas covered: This review scrutinizes mass spectrometry based approaches used to investigate brain dynamics in various conditions, including neurodegenerative and neuropsychiatric disorders. Different proteomics workflows for isolation/enrichment of specific cell populations or brain regions, sample processing; mass spectrometry technologies, for differential proteome quantitation, analysis of post-translational modifications and imaging approaches in the brain are critically deliberated. Future directions, including analysis of cellular sub-compartments, targeted MS platforms (selected/parallel reaction monitoring) and use of mass cytometry are also discussed. Expert commentary: Here, we summarize and evaluate current mass spectrometry based approaches for determining brain dynamics in health and diseases states, with a focus on neurological disorders. Furthermore, we provide insight on current trends and new MS technologies with potential to improve this analysis.Peer reviewe

Isotope‐labeled amyloid‐β does not transmit to the brain in a prion‐like manner after peripheral administration

Findings of early cerebral amyloid-β deposition in mice after peripheral injection of amyloid-β-containing brain extracts, and in humans following cadaveric human growth hormone treatment raised concerns that amyloid-β aggregates and possibly Alzheimer’s disease may be transmissible between individuals. Yet, proof that Aβ actually reaches the brain from the peripheral injection site is lacking. Here, we use a proteomic approach combining stable isotope labeling of mammals and targeted mass spectrometry. Specifically, we generate 13C-isotope-labeled brain extracts from mice expressing human amyloid-β and track 13C-lysine-labeled amyloid-β after intraperitoneal administration into young amyloid precursor protein-transgenic mice. We detect injected amyloid-β in the liver and lymphoid tissues for up to 100 days. In contrast, injected 13C-lysine-labeled amyloid-β is not detectable in the brain whereas the mice incorporate 13C-lysine from the donor brain extracts into endogenous amyloid-β. Using a highly sensitive and specific proteomic approach, we demonstrate that amyloid-β does not reach the brain from the periphery. Our study argues against potential transmissibility of Alzheimer’s disease while opening new avenues to uncover mechanisms of pathophysiological protein deposition.Peer reviewe

Lactate-Induced Glucose Output Is Unchanged by Metformin at a Therapeutic Concentration - A Mass Spectrometry Imaging Study of the Perfused Rat Liver

Metformin is the first line drug for type 2 diabetes but its molecular mechanisms remain unclear. Here, we have studied the acute effect of a therapeutically relevant intrahepatic concentration of metformin on glucose production from lactate. We selected the perfused rat liver as experimental system since it enables the complete control of drug dosage. We used MALDI (matrix-assisted laser desorption/ionization) mass spectrometry imaging to estimate the concentration of metformin in the livers and we measured the concentration of glucose in the effluent medium under basal conditions and following lactate addition. MALDI mass spectra of thin-sections of freeze-clamped rat liver perfused with metformin showed a peak at 130.16 m/z which was unambiguously assigned to metformin. The mass spectrometric detection limit was at a tissue concentration of about 250 nM, and uptake of metformin from the perfusion medium to the liver occurred with a K-m of 0.44 mM. Metformin was evenly distributed in the liver irrespective of its concentration in the perfusion medium and the duration of a perfusion. At a parenchymal concentration of 30 mu M, metformin did not induce any significant suppression of the basal or lactate-induced glucose release from the liver. These results show that matrix-assisted laser desorption/ionization mass spectrometry imaging can be applied to estimate the tissue concentration and distribution of metformin in a therapeutically relevant micromolar concentration range. Our findings challenge the view that metformin causes an inhibition of glucose release from the liver by an acute inhibition of mitochondrial glycerol 3-phosphate dehydrogenase (EC 1.1.5.3).Peer reviewe

Characterizing the Key Metabolic Pathways of the Neonatal Mouse Heart Using a Quantitative Combinatorial Omics Approach

The heart of a newborn mouse has an exceptional capacity to regenerate from myocardial injury that is lost within the first week of its life. In order to elucidate the molecular mechanisms taking place in the mouse heart during this critical period we applied an untargeted combinatory multiomics approach using large-scale mass spectrometry-based quantitative proteomics, metabolomics and mRNA sequencing on hearts from 1-day-old and 7-day-old mice. As a result, we quantified 1.937 proteins (366 differentially expressed), 612 metabolites (263 differentially regulated) and revealed 2.586 differentially expressed gene loci (2.175 annotated genes). The analyses pinpointed the fructose-induced glycolysis-pathway to be markedly active in 1-day-old neonatal mice. Integrated analysis of the data convincingly demonstrated cardiac metabolic reprogramming from glycolysis to oxidative phosphorylation in 7-days old mice, with increases of key enzymes and metabolites in fatty acid transport (acylcarnitines) and beta-oxidation. An upsurge in the formation of reactive oxygen species and an increase in oxidative stress markers, e.g., lipid peroxidation, altered sphingolipid and plasmalogen metabolism were also evident in 7-days mice. In vitro maintenance of physiological fetal hypoxic conditions retained the proliferative capacity of cardiomyocytes isolated from newborn mice hearts. In summary, we provide here a holistic, multiomics view toward early postnatal changes associated with loss of a tissue regenerative capacity in the neonatal mouse heart. These results may provide insight into mechanisms of human cardiac diseases associated with tissue regenerative incapacity at the molecular level, and offer a prospect to discovery of novel therapeutic targets.Peer reviewe

Tumor Infiltrating Neutrophils Are Enriched in Basal-Type Urothelial Bladder Cancer

15noBackground: Urothelial bladder cancers (UBCs) are distinct in two main molecular subtypes, namely basal and luminal type. Subtypes are also diverse in term of immune contexture, providing a rationale for patient selection to immunotherapy. Methods: By digital microscopy analysis of a muscle-invasive BC (MIBC) cohort, we explored the density and clinical significance of CD66b(+) tumor-associated-neutrophils (TAN) and CD3(+) T cells. Bioinformatics analysis of UBC datasets and gene expression analysis of UBC cell lines were additionally performed. Results: Basal type BC contained a significantly higher density of CD66b(+) TAN compared to the luminal type. This finding was validated on TCGA, GSE32894 and GSE124305 datasets by computing a neutrophil signature. Of note, basal-type MIBC display a significantly higher level of chemokines (CKs) attracting neutrophils. Moreover, pro-inflammatory stimuli significantly up-regulate CXCL1, CXCL2 and CXCL8 in 5637 and RT4 UBC cell lines and induce neutrophil chemotaxis. In term of survival, a high density of T cells and TAN was significantly associated to a better outcome, with TAN density showing a more limited statistical power and following a non-linear predicting model. Conclusions: TAN are recruited in basal type MIBC by pro-inflammatory CKs. This finding establishes a groundwork for a better understanding of the UBC immunity and its relevance.openopenMandelli, Giulio Eugenio; Missale, Francesco; Bresciani, Debora; Gatta, Luisa Benerini; Scapini, Patrizia; Caveggion, Elena; Roca, Elisa; Bugatti, Mattia; Monti, Matilde; Cristinelli, Luca; Belotti, Sandra; Simeone, Claudio; Calza, Stefano; Melocchi, Laura; Vermi, WilliamMandelli, Giulio Eugenio; Missale, Francesco; Bresciani, Debora; Gatta, Luisa Benerini; Scapini, Patrizia; Caveggion, Elena; Roca, Elisa; Bugatti, Mattia; Monti, Matilde; Cristinelli, Luca; Belotti, Sandra; Simeone, Claudio; Calza, Stefano; Melocchi, Laura; Vermi, Willia

Proteomic and functional analyses in disease models reveal CLN5 protein involvement in mitochondrial dysfunction

CLN5 disease is a rare form of late-infantile neuronal ceroid lipofuscinosis (NCL) caused by mutations in theCLN5genethat encodes a protein whose primary function and physiological roles remains unresolved. Emerging lines of evidence point to mitochondrial dysfunction in the onset and progression of several forms of NCL, offering new insights into putative biomarkers and shared biological processes. In this work, we employed cellular and murine models of the disease, in an effort to clarify disease pathways associated with CLN5 depletion. A mitochondria-focused quantitative proteomics approach followed by functional validations using cell biology and immunofluorescence assays revealed an impairment of mitochondrial functions in different CLN5 KO cell models and inCln5−/−cerebral cortex, which well correlated with disease progression. A visible impairment of autophagy machinery coupled with alterations of key parameters of mitophagy activation process functionally linked CLN5 protein to the process of neuronal injury. The functional link between impaired cellular respiration and activation of mitophagy pathways in the human CLN5 disease condition was corroborated by translating organelle-specific proteome findings to CLN5patients’fibroblasts. Our study highlights the involvement of CLN5 in activation of mitophagy and mitochondrial homeostasis offering new insights into alternative strategies towards the CLN5 disease treatment.Peer reviewe

Does the rapid initiation of antiretroviral therapy at HIV diagnosis impact virological response in a real-life setting? A single-centre experience in Northern Italy

: Rapid initiation of antiretroviral therapy (ART) has been proven efficacious and safe, but more investigations are needed to define feasibility of rapid ART approach in real-life settings.We conducted a retrospective, observational study on newly HIVdiagnosed patients referred to our Infectious Diseases Department from September 1st, 2015, to July 31st, 2019. According to the timing of ART initiation, we distinguished 3 groups of patients (rapid, intermediate and late group) and represented the trend of virological response during a 400-days-period. The hazard ratios of each predictor on viral suppression were estimated through the Cox proportional hazard model.The median time from HIV diagnosis to the first medical referral was 15 days and the median time from the first care access to therapy start was 24 days. Among patients, 37.6% started ART within 7 days, 20.6% between 8 and 30 days, and 41.8% after 30 days. Longer time to ART start and higher baseline viral load were associated with a lower probability of viral suppression. After one year, all groups showed a high viral suppression rate (99%). In a high-income setting the rapid ART approach seems useful to accelerate viral suppression which is great over time regardless of ART initiation timing

Multidimensional Prognostic Index (MPI) score has the major impact on outcome prediction in elderly surgical patients with colorectal cancer: The FRAGIS study

Background This study aims (I) to evaluate whether the Multidimensional Prognostic Index (MPI) score is associated with postoperative outcomes and (II) to develop a prognostic model for individual complication-risk prediction following colorectal cancer (CRC) surgery.Method This is a prospective multicentric cohort study. Consecutive >= 75-year-old candidates for elective CRC surgery were enrolled from October 2017 to August 2019. Patients underwent standardized preoperative geriatric assessment including the MPI. Patients with MPI score > 0.33 were classified as frail. Logistic regression models were employed to evaluate variables associated with major postoperative complications and mortality, using 10-fold cross-validated LASSO (least absolute shrinkage and selection operator) for model selection.Results In all, 104 patients were included, 34 (33%) had MPI score > 0.33. Major postoperative complications occurred in 52% of frail versus 16% of fit (MPI score <= 0.33) patients (p < .01). Both 30-day (9% vs. 0%; p = .033) and 90-day mortality (18% vs. 1%; p < .01) were higher among frail patients. In multivariate analysis, MPI score was associated with adverse outcomes. A final postoperative complication predictive model was created, including MPI score, gait-speed test, ASA (American Society of Anesthesiology) score, surgical approach, and stoma creation.Conclusion MPI score is strongly associated with postoperative major complications in CRC elderly patients and it is a primary component of an individual prediction model