To understand functional consequences of genetic and transcriptional aberrations in prostate cancer, the proteomic changes during disease formation and progression need to be revealed. Here we report ...high-throughput mass spectrometry on clinical tissue samples of benign prostatic hyperplasia (BPH), untreated primary prostate cancer (PC) and castration resistant prostate cancer (CRPC). Each sample group shows a distinct protein profile. By integrative analysis we show that, especially in CRPC, gene copy number, DNA methylation, and RNA expression levels do not reliably predict proteomic changes. Instead, we uncover previously unrecognized molecular and pathway events, for example, several miRNA target correlations present at protein but not at mRNA level. Notably, we identify two metabolic shifts in the citric acid cycle (TCA cycle) during prostate cancer development and progression. Our proteogenomic analysis uncovers robustness against genomic and transcriptomic aberrations during prostate cancer progression, and significantly extends understanding of prostate cancer disease mechanisms.
Ceramides and phosphatidylcholines (PCs) are bioactive lipids and lipid bilayer membrane components. Distinct ceramides/PCs (ratios) predict cardiovascular outcome in patients with coronary artery ...disease. Extracellular vesicles (EVs) are proposed biomarkers for cardiovascular disease and contain ceramides/PCs. Ceramides/PCs have not been studied in patients undergoing carotid endarterectomy (CEA) nor in EVs. We therefore investigated whether levels of ceramides/PCs in plasma and EVs are associated with postoperative risk of major adverse cardiovascular events (MACE) following CEA. In 873 patients undergoing CEA of the Athero-Express biobank, we quantitatively measured seven ceramides/PCs in preoperative blood samples: Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1), PC(14:0/22:6), PC(16:0/16:0) and PC(16:0/22:5) in plasma and two plasma EV-subfractions (LDL and TEX). We analyzed the association of ceramides, PCs and their predefined ratios with the three-year postoperative risk of MACE (including stroke, myocardial infarction and cardiovascular death). A total of 138 patients (16%) developed MACE during the three-year follow-up. In the LDL-EV subfraction, higher levels of Cer(d18:1/24:1) and Cer(d18:1/16:0)/PC(16:0/22:5) ratio were significantly associated with an increased risk of MACE (adjusted HR per SD 95% CI 1.24 1.01-1.53 and 1.26 1.04-1.52, respectively). In the TEX-EV subfraction, three ratios Cer(d18:1/16:0)/Cer(d18:1/24:0), Cer(d18:1/18:0)/Cer(d18:1/24:0) and Cer(d18:1/24:1)/Cer(d18:1/24:0) were positively associated with MACE (adjusted HR per SD 1.34 1.06-1.70, 1.24 1.01-1.51 and 1.31 1.08-1.58, respectively). In conclusion, distinct ceramides and PCs in plasma EVs determined in preoperative blood were independently associated with an increased 3-year risk of MACE after CEA. These lipids are therefore potential markers to identify high-risk CEA patients qualifying for secondary preventive add-on therapy.
Ceramides are sphingolipids that play roles as structural lipids and as second messengers in biological processes. Circulating ceramides are influenced by diet/food and predict major cardiovascular ...(CV) events, such as atrial fibrillation (AF). In 1227 patients with symptomatic chronic heart failure (HF), an association between diet and ceramides was found for coffee consumption of ≥3 cups and Cer(d18:1/24:0). Increased Cer(d18:1/24:0) was associated with lower incident AF (24.3% vs 15.4% tertile 1 vs 3, P = 0.016) and lower CV mortality (28.4% vs 12.0% tertile 1 vs 3, P < 0.0001). For coffee consumption, only an association with incident AF was found (24.5% never, 5.2% ≥3 cups). These inverse associations with AF were confirmed in survival analyses corrected for biomarkers (Cer(d18:1/24:0) HR: 0.79, P = 0.018; coffee consumption HR: 0.22, P = 0.001). In conclusion, higher coffee intake was associated with a lower risk of incident AF and with higher concentrations of Cer(d18:1/24:0). Cer(d18:1/24:0) was inversely associated to risk of AF.
Abstract
Ceramides contribute to the development of type 2 diabetes but it is uncertain whether they predict gestational diabetes (GDM). In this multicentre case–control study including 1040 women ...with GDM and 958 non-diabetic controls, early pregnancy (mean 10.7 gestational weeks) concentrations of four ceramides—Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)—were determined by a validated mass-spectrometric method from biobanked serum samples. Traditional lipids including total cholesterol, LDL, HDL and triglycerides were measured. Logistic and linear regression and the LASSO logistic regression were used to analyse lipids and clinical risk factors in the prediction of GDM. The concentrations of four targeted ceramides and total cholesterol, LDL and triglycerides were higher and HDL was lower among women with subsequent GDM than among controls. After adjustments, Cer(d18:1/24:0), triglycerides and LDL were independent predictors of GDM, women in their highest quartile had 1.44-fold (95% CI 1.07–1.95), 2.17-fold (95% CI 1.57–3.00) and 1.63-fold (95% CI 1.19–2.24) odds for GDM when compared to their lowest quartiles, respectively. In the LASSO regression modelling ceramides did not appear to markedly improve the predictive performance for GDM alongside with clinical risk factors and triglycerides. However, their adverse alterations highlight the extent of metabolic disturbances involved in GDM.
Prevalence of many eye and ocular surface diseases increases with age. While the clinical characteristics and pathophysiologic mechanisms of these conditions are often either known or extensively ...studied, the effects of normal aging on tear film and ocular surface have not been as widely researched.
In order to examine the effects of aging on tear fluid proteomics, tear fluid samples were collected preoperatively from 115 subjects undergoing strabismus or refractive surgery using glass microcapillary tubes. In addition to their refractive error or strabismus, the subjects did not have any other current, known eye diseases. The non-pooled samples were analysed using NanoLC-TripleTOF implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry, resulting in quantified data of 849 proteins.
According to correlation results, 17 tear proteins correlated significantly with increased age and many of these proteins were connected to inflammation, immune response and cell death. According to enrichment analysis, growth and survival of cells decreased while immune response and inflammation increased with aging. We also discovered several well-known, activated and inhibited upstream regulators, e.g. NF-κB, which has been previously connected to aging in numerous previous studies.
Overall, the results show the common age-dependent alterations in tear fluid protein profile, which demonstrate similar age-associated alterations of biological functions previously shown in other tissue and sample types.
Background Identification of patients with stable coronary heart disease who are at significant residual risk could be helpful for targeted prevention. Our aim was to determine the prognostic value ...of the recently introduced ceramide- and phospholipid-based risk score, the Cardiovascular Event Risk Test (CERT2), in patients with stable coronary heart disease on optimal medical therapy and to identify biological processes that contribute to the CERT2 score. Methods and Results Plasma samples (n=11 222) obtained from the STABILITY (Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy) trial were analyzed using a tandem liquid chromatography-mass spectrometry method. STABILITY was a trial in patients with stable coronary heart disease randomized to the lipoprotein-associated phospholipase A2 inhibitor darapladib or placebo on optimized medical therapy at baseline, with a median follow-up of 3.7 years. Hazard ratios per SD for the CERT2 risk score were 1.32 (95% CI, 1.25-1.39) for major adverse cardiovascular event, 1.47 (95% CI, 1.35-1.59) for cardiovascular death, 1.32 (95% CI, 1.16-1.49) for stroke, 1.23 (95% CI, 1.14-1.33) for myocardial infarction, and 1.56 (95% CI, 1.39-1.76) for hospitalization due to heart failure, when adjusted for traditional cardiovascular risk factors. CERT2 showed correlation (
<0.001,
>0.2) with inflammatory markers high-sensitivity C-reactive protein, interleukin 6, the heart failure marker N-terminal pro-B-type natriuretic peptide, and low-density lipoprotein cholesterol. After also adjusting for levels of other prognostic biomarkers, the CERT2 score was still independently related to the risk of cardiovascular death but not to nonfatal events. Conclusions The CERT2 risk score can detect residual risk in patients with stable coronary heart disease and is associated with biomarkers indicating inflammation, myocardial necrosis, myocardial dysfunction, renal dysfunction, and dyslipidemia. REGISTRATION URL: https://www.clinicaltrials.gov. Unique identifier: NCT00799903.
Advances in mass spectrometry have accelerated biomarker discovery in many areas of medicine. The purpose of this study was to compare two mass spectrometry (MS) methods, isobaric tags for relative ...and absolute quantitation (iTRAQ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), for analytical efficiency in biomarker discovery when there are multiple methodological constraints such as limited sample size and several time points for each patient to be analyzed.
A total of 140 tear samples were collected from 28 glaucoma patients at 5 time points in a glaucoma drug switch study. Samples were analyzed with iTRAQ and SWATH methods using NanoLC-MSTOF mass spectrometry.
We discovered that even though iTRAQ is faster than SWATH with respect to analysis time per sample, it loses in sensitivity, reliability and robustness. While SWATH analysis yielded complete data of 456 proteins in all samples, with iTRAQ we were able to quantify 477 proteins in total but on average only 125 proteins were quantified in a sample. 283 proteins were common in the datasets produced by the two methods. Repeatability of the methods was assessed by calculating percent relative standard deviation (% RSD) between replicate MS analyses: SWATH was more repeatable (56% of proteins < 20% RSD), compared to iTRAQ (43% of proteins < 20% RSD). Despite the overall benefits of SWATH, both methods showed less than 1 log fold change difference in the expression of 74% common proteins. In addition, comparison to MS/MS peptide results using 8 isotopically labeled peptide standards, SWATH and iTRAQ showed similar results in terms of accuracy. Moreover, both methods detected similar trends in a longitudinal analysis of protein expression of two known tear biomarkers.
Overall, we conclude that SWATH should be preferred for biomarker discovery studies when analyzing limited volumes of clinical samples collected at multiple time points.
The study was approved by the Ethics Committee at Tampere University Hospital and was registered in EU clinical trials register (EudraCT Number: 2010-021039-14).
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide an unlimited cell source for retinal cell replacement therapies. Clinical trials using hESC-RPE to treat diseases ...such as age-related macular degeneration (AMD) are currently underway. Human ESC-RPE cells have been thoroughly characterized at the gene level but their protein expression profile has not been studied at larger scale. In this study, proteomic analysis was used to compare hESC-RPE cells differentiated from two independent hESC lines, to primary human RPE (hRPE) using Isobaric tags for relative quantitation (iTRAQ). 1041 common proteins were present in both hESC-RPE cells and native hRPE with majority of the proteins similarly regulated. The hESC-RPE proteome reflected that of normal hRPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No signs of increased stress, apoptosis, immune response, proliferation, or retinal degeneration related changes were noted in hESC-RPE, while important RPE specific proteins involved in key RPE functions such as visual cycle and phagocytosis, could be detected in the hESC-RPE. Overall, the results indicated that the proteome of the hESC-RPE cells closely resembled that of their native counterparts.
Voltage-gated sodium (Na
) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Na
channels in several non-excitable cells, ...such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Na
channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Na
channels in RPE both ex vivo and in vitro.
Our work demonstrates that several subtypes of Na
channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Na
1.4, Na
1.6, and Na
1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Na
subtypes. Importantly, we show that the Na
channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Na
1.4 and Na
1.8 accumulate on phagosomes and that pharmacological inhibition of Na
channels as well as silencing the expression of Na
1.4 with shRNA impairs the phagocytosis process.
Taken together, our study shows that Na
channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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