Identification and drug-induced reversion of molecular signatures of Alzheimer's disease onset and progression in AppNL-G-F, AppNL-F, and 3xTg-AD mouse models.

BACKGROUND: In spite of many years of research, our understanding of the molecular bases of Alzheimer's disease (AD) is still incomplete, and the medical treatments available mainly target the disease symptoms and are hardly effective. Indeed, the modulation of a single target (e.g., ß-secretase) has proven to be insufficient to significantly alter the physiopathology of the disease, and we should therefore move from gene-centric to systemic therapeutic strategies, where AD-related changes are modulated globally. METHODS: Here we present the complete characterization of three murine models of AD at different stages of the disease (i.e., onset, progression and advanced). We combined the cognitive assessment of these mice with histological analyses and full transcriptional and protein quantification profiling of the hippocampus. Additionally, we derived specific Aß-related molecular AD signatures and looked for drugs able to globally revert them. RESULTS: We found that AD models show accelerated aging and that factors specifically associated with Aß pathology are involved. We discovered a few proteins whose abundance increases with AD progression, while the corresponding transcript levels remain stable, and showed that at least two of them (i.e., lfit3 and Syt11) co-localize with Aß plaques in the brain. Finally, we found two NSAIDs (dexketoprofen and etodolac) and two anti-hypertensives (penbutolol and bendroflumethiazide) that overturn the cognitive impairment in AD mice while reducing Aß plaques in the hippocampus and partially restoring the physiological levels of AD signature genes to wild-type levels. CONCLUSIONS: The characterization of three AD mouse models at different disease stages provides an unprecedented view of AD pathology and how this differs from physiological aging. Moreover, our computational strategy to chemically revert AD signatures has shown that NSAID and anti-hypertensive drugs may still have an opportunity as anti-AD agents, challenging previous reports.

Down-regulation of the expression of alcohol dehydrogenase 4 and CYP2E1 by the combination of α-endosulfan and dioxin in HepaRG human cells.

Pesticides and other persistent organic pollutants are considered as risk factors for liver diseases. We treated the human hepatic cell line HepaRG with both 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the organochlorine pesticide, α-endosulfan, to evaluate their combined impact on the expression of hepatic genes involved in alcohol metabolism. We show that the combination of the two pollutants (25nM TCDD and 10µM α-endosulfan) led to marked decreases in the amounts of both the mRNA (up to 90%) and protein (up to 60%) of ADH4 and CYP2E1. Similar results were obtained following 24h or 8days of treatment with lower concentrations of these pollutants. Experiments with siRNA and AHR agonists and antagonist demonstrated that the genomic AHR/ARNT pathway is necessary for the dioxin effect. The PXR, CAR and estrogen receptor alpha transcription factors were not modulators of the effects of α-endosulfan, as assessed by siRNA transfection. In another human hepatic cell line, HepG2, TCDD decreased the expression of ADH4 and CYP2E1 mRNAs whereas α-endosulfan had no effect on these genes. Our results demonstrate that exposure to a mixture of pollutants may deregulate hepatic metabolism.

A proteomic study of mesenchymal stem cells from equine umbilical cord.

To the best of our knowledge, this is the first study describing the proteome of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs) in a global and functional manner. The aim of this work was to analyze the proteome of previously characterized UCIM-MSCs to determine protein abundance and classify the identified proteins according to Gene Ontology (GO) terms. Protein classification analysis according to biological process, molecular function and cellular component was performed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System, which revealed enrichment for 42 biological processes, 23 molecular functions and 18 cellular components. Protein abundance was estimated according to the emPAI method (Exponential Modified Protein Abundance Index). The two most abundant proteins in the proteome of UCIM-MSCs were the cytoskeletal proteins actin and vimentin, which have important roles in cell stability and motility. Additionally, we identified 14 cell surface antigens. Three of them, CD44, CD90 and CD105, had been previously validated by flow cytometry. In the present study, we also identified important information about the biological properties of UCIM-MSCs such as differentiation potential, low immunogenicity (low MHC-II expression) and chromosomal stability, which reinforces their use for cell therapy. Together with the proteomic findings, this information allowed us to infer the functional relevance of several activities related to primary metabolic processes, protein synthesis, production of vesicle coats, vesicle-mediated transport and antioxidant activity. In addition, the identification of different cell surface markers may help establish an immunophenotypic panel suitable for the characterization of MSCs from equine fetal membranes.

Shotgun proteomic analysis of the secretome of bovine endometrial mesenchymal progenitor/stem cells challenged or not with bacterial lipopolysaccharide.

The use of the conditioned medium (CM) for diseases treatment is based on its enrichment with biomolecules with therapeutic properties and themselves have a beneficial effect. Secretome of bovine endometrial mesenchymal progenitor/stem cells (eMSCs) using a proteomics approach is until now unknown. This work aimed to evaluate the secretome of bovine eMSCs-CM challenged or not with lipopolysaccharide (LPS). For this, eMSCs characterized were challenged (TG) or not (CG). The CM was collected 12h after stimulation and submitted to mass spectrometry analysis. The classification of identified proteins was done by PANTHER according to biological processes, molecular function, cellular component and protein class. 397 protein groups were identified in TG and 302 in CG. We observed positive enrichment for antibacterial response proteins, macrophage activation function, receptor-mediated endocytosis, hydrolase activity, inhibitory enzyme in TG, and for activity structural molecule and intermediate filament cytoskeleton in the CG. Our experimental model shows that eMSCs respond to LPS in the concentration used and can be used to study immune-inflammatory response, besides of the secretion of proteins mainly related to tissue remodeling, immune response and angiogenesis which is an interesting feature for use in cell therapy.

ShockOmics: multiscale approach to the identification of molecular biomarkers in acute heart failure induced by shock.

BACKGROUND: The ShockOmics study (ClinicalTrials.gov identifier NCT02141607) is a multicenter prospective observational trial aimed at identifying new biomarkers of acute heart failure in circulatory shock, by means of a multiscale analysis of blood samples and hemodynamic data from subjects with circulatory shock. METHODS AND DESIGN: Ninety septic shock and cardiogenic shock patients will be recruited in three intensive care units (ICU) (Hôpital Erasme, Université Libre de Bruxelles, Belgium; Hospital Universitari Mutua Terrassa, Spain; Hôpitaux Universitaires de Genève, Switzerland). Hemodynamic signals will be recorded every day for up to seven days from shock diagnosis (time T0). Clinical data and blood samples will be collected for analysis at: i) T1 < 16 h from T0; ii) T2 = 48 h after T0; iii) T3 = day 7 or before discharge or before discontinuation of therapy in case of fatal outcome; iv) T4 = day 100. The inclusion criteria are: shock, Sequential Organ Failure Assessment (SOFA) score > 5 and lactate levels ≥ 2 mmol/L. The exclusion criteria are: expected death within 24 h since ICU admission; > 4 units of red blood cells or >1 fresh frozen plasma transfused; active hematological malignancy; metastatic cancer; chronic immunodepression; pre-existing end stage renal disease requiring renal replacement therapy; recent cardiac surgery; Child-Pugh C cirrhosis; terminal illness. Enrollment will be preceded by the signature of the Informed Consent by the patient or his/her relatives and by the physician in charge. Three non-shock control groups will be included in the study: a) healthy blood donors (n = 5); b) septic patients (n = 10); c) acute myocardial infarction or patients with prolonged acute arrhythmia (n = 10). The hemodynamic data will be downloaded from the ICU monitors by means of dedicated software. The blood samples will be utilized for transcriptomics, proteomics and metabolomics ("-omics") analyses. DISCUSSION: ShockOmics will provide new insights into the pathophysiological mechanisms underlying shock as well as new biomarkers for the timely diagnosis of cardiac dysfunction in shock and quantitative indices for assisting the therapeutic management of shock patients.

Gilthead sea bream liver proteome altered at low temperatures by oxidative stress.

Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold-stress response was reproduced in gilthead sea bream acclimated to 20 degrees C (Warm group) when the water temperature was lowered to 8 degrees C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2-DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine-homocysteine-methyl transferase, glutathione-S-transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin-methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen-like protein indicated an increase in proteolysis. Increases in elongation factor-1alpha, the GAPDH oxidative form, tubulin and Raf-kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.

Cyclization-activated prodrugs. Synthesis, reactivity and toxicity of dipeptide esters of paracetamol.

Dipeptide esters of paracetamol were prepared in high yields. These compounds are quantitatively hydrolyzed to paracetamol and corresponding 2,5-diketopiperazines at pH 7.4 and 37 degrees C. The reactivity is increased in sarcosine and proline peptides and decreased by bulky side chains at both the N- and C-terminal residues of the dipeptide carrier. Moreover, dipeptide esters of paracetamol did not affect the levels of hepatic glutathione. Thus, dipeptides seem promising candidates as carriers for cyclization-activated prodrugs.

Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII.

Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.

Synthetic approaches to multivalent lipopeptide dendrimers containing cyclic disulfide epitopes of foot-and-mouth disease virus.

The synthesis of a multiantigenic peptide dendrimer incorporating four copies of a cyclic disulfide epitope has been undertaken. Since standard chemoselective ligation procedures involving thioether formation are inadvisable in the presence of a preformed disulfide, conjugation through a peptide bond between the lipidated branched lysine scaffold and a suitably protected version of the cyclic disulfide has been used instead. Several synthetic approaches to the partially protected cyclic disulfide peptide have been explored. The most effective involves building a minimally protected version of the peptide by Boc solid phase synthesis, using fluorenyl-based anchorings and cysteine protecting groups. Peptide-resin cleavage and cysteine deprotection/oxidation are performed simultaneously by base-promoted elimination. The cyclic disulfide epitope is readily obtained in sufficient amounts by this procedure and subsequently incorporated to the lipidated lysine core by peptide bond formation in solution. A final acid deprotection step in anhydrous HF yields a peptide construction containing a maximum of three copies of the cyclic disulfide epitope, the lower substitution being attributable to steric constraints. This immunogen has been successfully used in an experimental vaccination trial against foot-and-mouth disease virus.