detection and scavenging of hydroxyl radical via d phenylalanine hydroxylation in human fluids

Abstract Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .OH attack, using a detection system based on Fenton reaction-mediated D -phenylalanine ( D -Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D -Phe on .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N′-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl- L -cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30 g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 ± 0.12 times, p D -Phe hydroxylation is a suitable method for routine and non-invasive evaluation of .OH detection and its scavenging in human biological fluids

Proteomic analysis of plasma derived from platelet buffy coats during storage at room temperature. An application of ProteoMiner™ technology

The present study was aimed at revealing new insights into the analysis of storage-related processes occurring in the supernatants of platelet concentrates (PCs) derived from pooled buffy coats suspended in whole plasma. To reduce the dynamic range of plasma protein concentrations and access low-abundance proteins, we made use of a solid-phase combinatorial peptide ligand library, known under the trade name of ProteoMiner™. Afterwards, two-dimensional electrophoresis (2-DE) was coupled with mass spectrometry (MS) to reveal changes in proteomic profiles. Several storage-induced protein alterations were identified including changes to major plasma proteins. In particular, a precursor of the secretory form of clusterin was shown to accumulate during storage of PC supernatants, together with platelet-derived tropomyosin, suggesting a progressive loss of platelet integrity. Platelet-released proteins following activation have also been detected (alpha-1-B-glycoprotein, kininogen-1, and serpin proteinase inhibitor 8). Moreover, specific protein fragments (vitronectin, plakoglobin, hornerin, and apolipoprotein A-IV) were found to be modulated upon storage, possibly indicating a time-dependent buffy-coat PC deterioration. Globally, our findings provided the disclosure of unique proteins in PC supernatants with respect to previous studies conducted in similar experimental conditions, suggesting ProteoMiner enrichment technology to be a possible complementary tool in the identification of diagnostically relevant proteins as age/quality biomarkers of therapeutic products. © 2011 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications

We aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confirmed benign prostatic hyperplasia (BPH). The stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the effect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutoff of acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments

Proteomics and Transcriptomics Investigation on longissimus Muscles in Large White and Casertana Pig Breeds

L'articolo è disponibile sul sito dell'editore http://pubs.acs.org/Consumer complaints against the blandness of modern lean meat and the frequent reference to the more strongly flavored meat that was available years ago have prompted reconsideration of high fatdepositing typical pig breeds. Casertana and Large White pig breeds are characterized by a different tendency toward fat accumulation as they exhibit opposite genetic and physiological traits with respect to the energy metabolism. These physiological differences were investigated in longissimus lumborum muscles through proteomics (2-DE, MS/MS) and microarray approaches. Data were analyzed for pathway and network analyses, as well as GO term enrichment of biological functions. As a result, Casertana showed a greater amount of proteins involved in glycolitic metabolism and mainly rely on fast-mobilizable energy sources. Large White overexpressed cell cycle and skeletal muscle growth related genes. Metabolic behavior and other implications are discussed

Circulating microRNAs and Kallikreins before and after Radical Prostatectomy: Are They Really Prostate Cancer Markers?

The aim of our study was to monitor serum levels of two miRNAs (miR-21 and miR-141) and three KLKs (hK3/PSA, hK11, and hK13) before and 1, 5, and 30 days after radical prostatectomy, in order to characterize their fluctuations after surgery. 38 patients with prostate cancer were included. miR-21 and miR-141 were quantified through real-time PCR, while ELISA assays were used to quantify hK3 (PSA), hK11, and hK13. Both miR-21 and miR-141 showed a significant increase at the 5th postoperative day, after which a gradual return to the preoperative levels was recorded. These findings suggest that miR-21 and miR-141 could be involved in postsurgical inflammatory processes and that radical prostatectomy does not seem to alter their circulating levels. Postoperative serum kallikreins showed a significant decrease, highlighting the potential usefulness of kallikreins apart from PSA as potential prostate cancer markers

Socioeconomic inequalities in health status and survival: a cohort study in Rome

Objectives: To analyse the association between individual and contextual socioeconomic position (SEP) with health status and to investigate the role of SEP and baseline health status on survival. Design: Cross-sectional and cohort study. Setting: Rome, Italy. Participants, primary and secondary outcomes: We selected the 25–99 year- olds included in the Rome 2011 census cohort. As a measure of health status on the census reference date (09 October 2011), we used the presence of chronic or rare conditions from the Disease-Related Co-payment Exemption Registry, a database implemented to provide free care to people with chronic or rare diseases. We used logistic regression to analyse the association between both individual (educational attainment) and contextual SEP (neighbourhood real estate price quintiles) with baseline health status. We analysed the role of SEP and the presence of chronic or rare conditions on 5- year survival (until 31 December 2016) using accelerated failure time models with Weibull distribution, reporting time ratios (TRs; 95% CI). Results: In middle-aged, subjects with low SEP (either individual or contextual) had a prevalence of chronic conditions comparable with the prevalence in high SEP individuals 10 years older. Adjusted logistic models confirmed the direct association between SEP and baseline health status in both women and men. The lowest educated were up to 67% more likely to have a chronic condition than the highest educated, while the difference was up to 86% for lowest versus highest contextual SEP. Low SEP and the presence of chronic conditions were associated with shorter survival times in both sexes, lowest versus highest educated TR was TR=0.79 for women (95% CI: 0.77 to 0.81) and TR=0.71 for men (95% CI: 0.70 to 0.73). The contextual SEP shrunk survival times by about 10%. Conclusion: Inequalities were present in both baseline health and survival. The association between SEP and survival was independent of baseline health status

Renal artery embolization before radical nephrectomy for complex renal tumour: which are the true advantages?

Renal artery embolization is performed before radical nephrectomy (RN) for renal mass in order to induce preoperative infarction and to facilitate surgical intervention through decrease of intraoperative bleeding. Moreover, in metastatic renal cancer it seems to stimulate tumour-specific antibodies, even if no established benefits in clinical response or survival have been reported. The role of preoperative renal artery embolization (PRAE) in management of renal masses has been often debated and its real benefits are still unclear. Nevertheless, in huge and complex renal masses, which are often characterized by a high and anarchic blood supply and rapid local invasion, radical nephrectomy can be challenging even for skilled surgeons. The aim of this prospective randomized study was to evaluate the effectiveness and safety of PRAE in complex masses by comparing perioperative outcomes of RN with and without PRAE