Quercetin has been shown to act as an anticarcinogen in experimental colorectal cancer (CRC). The aim of the present study was to characterize transcriptome and proteome changes occurring in the ...distal colon mucosa of rats supplemented with 10 g quercetin/kg diet for 11 wk. Transcriptome data analyzed with Gene Set Enrichment Analysis showed that quercetin significantly downregulated the potentially oncogenic mitogen-activated protein kinase (Mapk) pathway. In addition, quercetin enhanced expression of tumor suppressor genes, including Pten, Tp53, and Msh2, and of cell cycle inhibitors, including Mutyh. Furthermore, dietary quercetin enhanced genes involved in phase I and II metabolism, including Fmo5, Ephx1, Ephx2, and Gpx2. Quercetin increased PPARα target genes, and concomitantly enhanced expression of genes involved in mitochondrial fatty acid (FA) degradation. Proteomics performed in the same samples revealed 33 affected proteins, of which four glycolysis enzymes and three heat shock proteins were decreased. A proteome-transcriptome comparison showed a low correlation, but both pointed out toward altered energy metabolism. In conclusion, transcriptomics combined with proteomics showed that dietary quercetin evoked changes contrary to those found in colorectal carcinogenesis. These tumor-protective mechanisms were associated with a shift in energy production pathways, pointing at decreased cytoplasmic glycolysis and toward increased mitochondrial FA degradation.
HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from ...drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 microL plasma and 1x10(6) PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.
The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study ...was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.
The relative affinity of the monovalent metal ions Li+, Na+, Cu+ and Ag+ towards a series of aliphatic alkyl acetates and some selected 1-alkenes (P) was examined using the kinetic method. A detailed ...analysis of the dissociation characteristics of a series of mixed metal-bound dimer ions of the type P1-M+-P2 and the evaluated proton affinities (PAs) of the monomers shows that the affinity of the cation towards long-chain alkyl acetates and alkenes (having a chain length ? C4) is markedly enhanced. In line with recent studies of nitriles, alcohols and methyl alkanoates, this is attributed to a bidentate interaction of the metal ion with the functional group or double bond and the aliphatic chain. In particular, the longer chain alkyl acetates, methyl alkanoates and alcohols show a remarkably similar behaviour with respect to silver ion hydrocarbon bonding. The Ag+ adducts of the alkyl acetates dissociate by loss of CH3COOH. This reaction becomes more pronounced at longer chain lengths, which points to metal ion bidentate formation in Ag+···1-alkene product ions having a long hydrocarbon chain. In the same vein, the heterodimers 1-hexene···Ag+···1-heptene and 1-heptene·Ag+···1-octene dissociate primarily into Ag+···1-heptene and Ag+···1-octene ions, respectively. Hydrocarbon bidentate formation in Ag+···1-octene also reveals itself by the reluctance of this ion to react with water in an ion trap, as opposed to Ag+···1-hexene which readily undergoes hydration.
The goal of this manuscript is to explore the role of clinical proteomics for detecting mutations in chronic obstructive pulmonary disease (COPD) and lung cancer by mass spectrometry‐based ...technology. COPD and lung cancer caused by smoke inhalation are most likely linked by challenging the immune system via partly shared pathways. Genome‐wide association studies have identified several single nucleotide polymorphisms which predispose an increased susceptibility to COPD and lung cancer. In lung cancer, this leads to coding mutations in the affected tissues, development of neoantigens, and different functionality and abundance of proteins in specific pathways. If a similar reasoning can also be applied in COPD will be discussed. The technology of mass spectrometry has developed into an advanced technology for proteome research detecting mutated peptides or proteins and finding relevant molecular mechanisms that will enable predicting the response to immunotherapy in COPD and lung cancer patients.
Abstract Background Deprescribing of medication for cardiovascular risk factors and diabetes has been incorporated in clinical guidelines but proves to be difficult to implement in primary care. ...Training of healthcare providers is needed to enhance deprescribing in eligible patients. This study will examine the effects of a blended training program aimed at initiating and conducting constructive deprescribing consultations with patients. Methods A cluster-randomized trial will be conducted in which local pharmacy-general practice teams in the Netherlands will be randomized to conducting clinical medication reviews with patients as usual (control) or after receiving the CO-DEPRESCRIBE training program (intervention). People of 75 years and older using specific cardiometabolic medication (diabetes drugs, antihypertensives, statins) and eligible for a medication review will be included. The CO-DEPRESCRIBE intervention is based on previous work and applies models for patient-centered communication and shared decision making. It consists of 5 training modules with supportive tools. The primary outcome is the percentage of patients with at least 1 cardiometabolic medication deintensified. Secondary outcomes include patient involvement in decision making, healthcare provider communication skills, health/medication-related outcomes, attitudes towards deprescribing, medication regimen complexity and health-related quality of life. Additional safety and cost parameters will be collected. It is estimated that 167 patients per study arm are needed in the final intention-to-treat analysis using a mixed effects model. Taking loss to follow-up into account, 40 teams are asked to recruit 10 patients each. A baseline and 6-months follow-up assessment, a process evaluation, and a cost-effectiveness analysis will be conducted. Discussion The hypothesis is that the training program will lead to more proactive and patient-centered deprescribing of cardiometabolic medication. By a comprehensive evaluation, an increase in knowledge needed for sustainable implementation of deprescribing in primary care is expected. Trial registration The study is registered at ClinicalTrials.gov (identifier: NCT05507177).
In cancer and autoimmune diseases, immunoglobulins with a specific molecular signature that could potentially be used as diagnostic or prognostic markers are released into body fluids. An immunomics ...approach based on this phenomenon relies on the ability to identify the specific amino acid sequences of the complementarity-determining regions (CDR) of these immunoglobulins, which in turn depends on the level of accuracy, resolution, and sensitivity that can be achieved by advanced mass spectrometry. Reproducible isolation and sequencing of antibody fragments (e.g., Fab) by high-resolution mass spectrometry (MS) from seven healthy donors revealed 43 217 MS signals: 225 could be associated with CDR1 peptides, 513 with CDR2 peptides, and 19 with CDR3 peptides. Seventeen percent of the 43 217 MS signals did not overlap between the seven donors. The Fab isolation method used is reproducible and fast, with a high yield. It provides only one Fab sample fraction for subsequent characterization by high-resolution MS. In 17% and 4% of these seven healthy donors, qualitative (presence/absence) and quantitative (intensity) differences in Fab fragments could be demonstrated, respectively. From these results, we conclude that the identification of a CDR signature as biomarker for autoimmune diseases and cancer without prior knowledge of the antigen is feasible.
Osteoblasts are the bone forming cells, capable of secreting an extracellular matrix with mineralization potential. The exact mechanism by which osteoblasts differentiate and form a mineralized ...extracellular matrix is presently not fully understood. To increase our knowledge about this process, we conducted proteomics analysis in human immortalized preosteoblasts (SV-HFO) able to differentiate and mineralize. We identified 381 proteins expressed during the time course of osteoblast differentiation. Gene ontology analysis revealed an overrepresentation of protein categories established as important players for osteoblast differentiation, bone formation, and mineralization such as pyrophosphatases. Proteins involved in antigen presentation, energy metabolism and cytoskeleton rearrangement constitute other overrepresented processes, whose function, albeit interesting, is not fully understood in the context of osteoblast differentiation and bone formation. Correlation analysis, based on quantitative data, revealed a biphasic osteoblast differentiation, encompassing a premineralization and a mineralization period. Identified differentially expressed proteins between mineralized and nonmineralized cells include cytoskeleton (e.g., CCT2, PLEC1, and FLNA) and extracellular matrix constituents (FN1, ANXA2, and LGALS1) among others. FT-ICR-MS data obtained for FN1, ANXA2, and LMNA shows a specific regulation of these proteins during the different phases of osteoblast differentiation. Taken together, this study increases our understanding of the proteomics changes that accompany osteoblast differentiation and may permit the discovery of novel modulators of bone formation.
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