Urine Proteomics Revealed a Significant Correlation Between Urine-Fibronectin Abundance and Estimated-GFR Decline in Patients with Bardet-Biedl Syndrome.

BACKGROUND: /Aims: Renal disease is a common cause of morbidity in patients with Bardet-Biedl syndrome (BBS), however the severity of kidney dysfunction is highly variable. To date, there is little information on the pathogenesis, the risk and predictor factors for poor renal outcome in this setting. The present study aims to analyze the spectrum of urinary proteins in BBS patients, in order to potentially identify 1) disease-specific proteomic profiles that may differentiate the patients from normal subjects; 2) urinary markers of renal dysfunction. METHODS: Fourteen individuals (7 males and 7 females) with a clinical diagnosis of BBS have been selected in this study. A pool of 10 aged-matched males and 10 aged-matched females have been used as controls for proteomic analysis. The glomerular filtration rate (eGFR) has been estimated using the CKD-EPI formula. Variability of eGFR has been retrospectively assessed calculating average annual eGFR decline (ΔeGFR) in a mean follow-up period of 4 years (3-7). RESULTS: 42 proteins were significantly over- or under-represented in BBS patients compared with controls; the majority of these proteins are involved in fibrosis, cell adhesion and extracellular matrix organization. Statistic studies revealed a significant correlation between urine fibronectin (u-FN) (r2=0.28; p<0.05), CD44 antigen (r2 =0.35; p<0.03) and lysosomal alfa glucosidase ( r20.27; p<0.05) abundance with the eGFR. In addition, u-FN (r2 =0.2389; p<0.05) was significantly correlated with ΔeGFR. CONCLUSION: The present study demonstrates that urine proteome of BBS patients differs from that of normal subjects; in addition, kidney dysfunction correlated with urine abundance of known markers of renal fibrosis.

On the Liquid Chemistry of the Reactive Nitrogen Species Peroxynitrite and Nitrogen Dioxide Generated by Physical Plasmas.

Cold physical plasmas modulate cellular redox signaling processes, leading to the evolution of a number of clinical applications in recent years. They are a source of small reactive species, including reactive nitrogen species (RNS). Wound healing is a major application and, as its physiology involves RNS signaling, a correlation between clinical effectiveness and the activity of plasma-derived RNS seems evident. To investigate the type and reactivity of plasma-derived RNS in aqueous systems, a model with tyrosine as a tracer was utilized. By high-resolution mass spectrometry, 26 different tyrosine derivatives including the physiologic nitrotyrosine were identified. The product pattern was distinctive in terms of plasma parameters, especially gas phase composition. By scavenger experiments and isotopic labelling, gaseous nitric dioxide radicals and liquid phase peroxynitrite ions were determined as dominant RNS. The presence of water molecules in the active plasma favored the generation of peroxynitrite. A pilot study, identifying RNS driven post-translational modifications of proteins in healing human wounds after the treatment with cold plasma (kINPen), demonstrated the presence of in vitro determined chemical pathways. The plasma-driven nitration and nitrosylation of tyrosine allows the conclusion that covalent modification of biomolecules by RNS contributes to the clinically observed impact of cold plasmas.

On a heavy path - determining cold plasma-derived short-lived species chemistry using isotopic labelling.

Cold atmospheric plasmas (CAPs) are promising medical tools and are currently applied in dermatology and epithelial cancers. While understanding of the biomedical effects is already substantial, knowledge on the contribution of individual ROS and RNS and the mode of activation of biochemical pathways is insufficient. Especially the formation and transport of short-lived reactive species in liquids remain elusive, a situation shared with other approaches involving redox processes such as photodynamic therapy. Here, the contribution of plasma-generated reactive oxygen species (ROS) in plasma liquid chemistry was determined by labeling these via admixing heavy oxygen 18O2 to the feed gas or by using heavy water H2 18O as a solvent for the bait molecule. The inclusion of heavy or light oxygen atoms by the labeled ROS into the different cysteine products was determined by mass spectrometry. While products like cysteine sulfonic acid incorporated nearly exclusively gas phase-derived oxygen species (atomic oxygen and/or singlet oxygen), a significant contribution of liquid phase-derived species (OH radicals) was observed for cysteine-S-sulfonate. The role, origin, and reaction mechanisms of short-lived species, namely hydroxyl radicals, singlet oxygen, and atomic oxygen, are discussed. Interactions of these species both with the target cysteine molecule as well as the interphase and the liquid bulk are taken into consideration to shed light onto several reaction pathways resulting in observed isotopic oxygen incorporation. These studies give valuable insight into underlying plasma-liquid interaction processes and are a first step to understand these interaction processes between the gas and liquid phase on a molecular level.

Nitrosylation vs. oxidation - How to modulate cold physical plasmas for biological applications.

Thiol moieties are major targets for cold plasma-derived nitrogen and oxygen species, making CAPs convenient tools to modulate redox-signaling pathways in cells and tissues. The underlying biochemical pathways are currently under investigation but especially the role of CAP derived RNS is barely understood. Their potential role in protein thiol nitrosylation would be relevant in inflammatory processes such as wound healing and improving their specific production by CAP would allow for enhanced treatment options beyond the current application. The impact of a modified kINPen 09 argon plasma jet with nitrogen shielding on cysteine as a thiol-carrying model substance was investigated by FTIR spectroscopy and high-resolution mass spectrometry. The deposition of short-lived radical species was measured by electron paramagnetic resonance spectroscopy, long-lived species were quantified by ion chromatography (NO2-, NO3-) and xylenol orange assay (H2O2). Product profiles were compared to samples treated with the so-called COST jet, being introduced by a European COST initiative as a reference device, using both reference conditions as well as conditions adjusted to kINPen gas mixtures. While thiol oxidation was dominant under all tested conditions, an Ar + N2/O2 gas compositions combined with a nitrogen curtain fostered nitric oxide deposition and the desired generation of S-nitrosocysteine. Interestingly, the COST-jet revealed significant differences in its chemical properties in comparison to the kINPen by showing a more stable production of RNS with different gas admixtures, indicating a different •NO production pathway. Taken together, results indicate various chemical properties of kINPen and COST-jet as well as highlight the potential of plasma tuning not only by gas admixtures alone but by adjusting the surrounding atmosphere as well.

Integration of Proteomics and Metabolomics in Exploring Genetic and Rare Metabolic Diseases.

BACKGROUND: Inherited metabolic disorders or inborn errors of metabolism are caused by deficiency of enzymatic activities in the catabolism of amino acids, carbohydrates, or lipids. These disorders include aminoacidopathies, urea cycle defects, organic acidemias, defects of oxidation of fatty acids, and lysosomal storage diseases. Inborn errors of metabolism constitute a significant proportion of genetic diseases, particularly in children; however, they are individually rare. Clinical phenotypes are very variable, some of them remain asymptomatic, others manifest metabolic decompensation in neonatal age, and others encompass mental retardation at later age. The clinical manifestation of these disorders can involve different organs and/or systems. Some disorders are easily managed if promptly diagnosed and treated, whereas in other cases neither diet, vitamin therapy, nor transplantation appears to prevent multi-organ impairment. SUMMARY: Here, we discuss the principal challenges of metabolomics and proteomics in inherited metabolic disorders. We review the recent developments in mass spectrometry-based proteomic and metabolomic strategies. Mass spectrometry has become the most widely used platform in proteomics and metabolomics because of its ability to analyze a wide range of molecules, its optimal dynamic range, and great sensitivity. The fast measurement of a broad spectrum of metabolites in various body fluids, also collected in small samples like dried blood spots, have been facilitated by the use of mass spectrometry-based techniques. These approaches have enabled the timely diagnosis of inherited metabolic disorders, thereby facilitating early therapeutic intervention. Due to its analytical features, proteomics is suited for the basic investigation of inborn errors of metabolism. Modern approaches enable detailed functional characterization of the pathogenic biochemical processes, as achieved by quantification of proteins and identification of their regulatory chemical modifications. KEY MESSAGE: Mass spectrometry-based "omics" approaches most frequently used to study the molecular mechanisms underlying inherited metabolic disorders pathophysiology are described.

Comparison of the efficacy and safety of two formulations of micronized progesterone (Ellios and Utrogestan) used as luteal phase support after in vitro fertilization.

OBJECTIVE: To compare the efficacy and the tolerability of two different vaginal formulations of micronized progesterone, Ellios and Utrogestan, used for luteal phase support after an in vitro fertilization (IVF) cycle. DESIGN: Cohort study. SETTING: Fertility center in a university hospital. PATIENT(S(: One hundred twenty-three women who underwent IVF/intracytoplasmic sperm injection (ICSI) stimulated cycles from October 1998 to March 2000, who had at least six follicles of > or =14 mm on the day of hCG administration. INTERVENTION(S): Patients received Ellios pessaries (2 times 200-mg pessary/day) or Utrogestan capsules (2 x 100-mg capsules, two times a day). Progesterone was administered from the day of oocyte pickup (day 0) until menses or up to 10 weeks in pregnant patients. MAIN OUTCOME MEASURE(S): Progesterone levels, pregnancy rate, and tolerability tested by patient questionnaire. RESULT(S): The progesterone levels on days 0, 9, 16 were not statistically different between the two formulations. The pregnancy rate were similar in groups 1 and 2 (25.5% vs. 18.6%), whereas tolerance was significantly better in group 1 versus group 2 (vaginal discharge: 43% vs. 82%). CONCLUSION(S): The efficacy of the two formulations of progesterone is comparable, although the patient tolerance for Ellios is better.