Lipid imaging for visualizing cilastatin amelioration of cisplatin-induced nephrotoxicity

Nephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species. Lipid imaging was performed with MALDI mass spectrometry imaging (MALDI-MSI) in kidney sections from treated rats. Cilastatin was proved to significantly diminish the lipid distribution alterations caused by cisplatin, lipid levels being almost completely recovered to those of control samples. The extent of recovery of cisplatin-altered lipids by cilastatin turned out to be relevant for discriminating direct or secondary lipid alterations driven by cisplatin. Lipid peroxidation induced by cisplatin was also shown to be reduced when cilastatin was administered. Importantly, significant groups separation was achieved during multivariate analysis of cortex and outer-medullary lipids, indicating that damaged kidney can be discerned from the nephroprotected and healthy groups and classified according to lipid distribution. Therefore, we propose MALDI-MSI as a powerful potential tool offering multimolecule detection possibilities to visualize and evaluate nephrotoxicity and nephroprotection based on lipid analysis

Bridging the gap between molecular and elemental mass spectrometry: Higher energy collisional dissociation (HCD) revealing elemental information

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS

Polyphenols from Tamarix nilotica: LC−ESI-MSn Profiling and In Vivo Antifibrotic Activity

Tamarix nilotica (Ehrenb.) Bunge (Tamaricaceae), an indigenous plant to the Middle East region, is well-known as a medicinal plant for treating many human ailments. The current study aimed at exploring the polyphenol profile of the alcohol soluble fraction of aqueous T. nilotica extract, assessing its in vivo antifibrotic activity and the possible underlying mechanism, to unravel the impact of quantitative difference of sulphated polyphenols content on the antifibrotic activity of T. nilotca grown in two different habitats. Polyphenol profiling of T. nilotica extracts was performed using HPLC-HRESI-QTOF-MS-MS. The major polyphenol components included sulphated flavonoids, phenolic acids and free aglycones. The antifibrotic activity was evaluated through carbon tetrachloride-induced liver fibrosis in rats. Biochemical evaluations revealed that both fractions ameliorated the increased levels of hepatic aminotransferases, lipid peroxidation, hydroxyproline, α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB). Moreover, both fractions reduced catalase activity (CAT) and enhanced hepatic glutathione (GSH) content. Histopathological imaging undoubtedly confirmed such results. In conclusion, the T. nilotica polyphenol-rich fraction exhibited potential antifibrotic activity in rats. Significant alterations in GSH levels were recorded based on the sulphated polyphenol metabolite content.Peer Reviewe

Femtosecond laser-induced dissociation (fs-LID) as an activation method in mass spectrometry

We report the photolysis of biomolecules in a Fourier-transform ion cyclotron resonance mass spectrometer by intense near-infrared femtosecond laser pulses. Photo-fragmentation was accompanied by photo-ionization and created a large number and variety of charged fragments, which could be identified with high confidence due to the outstanding resolving power and mass accuracy of the mass spectrometer. Fragmentation patterns were sufficient for peptide sequence analysis. Fragments formed by non-ergodic cleavage retained labile post-translational modifications

Inductively Coupled Plasma Mass Spectrometry-Based Method for the Specific Quantification of Sulfenic Acid in Peptides and Proteins

A robust ICPMS-based method is introduced to obtain relative and absolute quantification of sulfenic acid (SA) in peptides and proteins. A new metal-containing reagent (Ln-DOTA-Dimedone) devised to react specifically with SA has been developed. The lanthanide-containing metal-coded affinity tag (Ln-MeCAT) was used to quantify thiol residues. We presented two approaches which allow the parallel and consecutive determination of SA and thiols in peptide and protein samples. The high sensitivity, structure-independent signal, and multiplexing capabilities of ICPMS together with the specificity of Ln-DOTA-Dimedone and Ln-MeCAT toward sulfenic acid and thiol residues, respectively, allow the characterization of various biological states and offer closer insight onto thiol-sulphenic acid equilibria which are involved in intracellular redox-mediated events altering structure and function of proteins in important diseases

Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

<div><p>The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM³⁸²↓³⁸³SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473.</p></div

Evaluation of Plant Phenolic Metabolites as a Source of Alzheimer's Drug Leads

Epidemiological studies have proven an association between consumption of polyphenols and prevention of Alzheimer’s disease, the most common form of dementia characterized by extracellular deposition of amyloid beta plaques. The aim of this study is pharmacological screening of the aqueous alcohol extract of Markhamia platycalyx leaves, Schotia brachypetala leaves and stalks, and piceatannol compared to aqueous alcohol extract of Camellia sinensis leaves as potential Alzheimer’s disease drugs. LC-HRESI(-ve)-MSn was performed to identify phenolics’ profile of Schotia brachypetala stalks aqueous alcohol extract and revealed ten phenolic compounds as first report: daidzein, naringin, procyanidin isomers, procyanidin dimer gallate, quercetin 3-O-rhamnoside, quercetin 3-O-glucuronide, quercetin hexose gallic acid, quercetin hexose protocatechuic acid, and ellagic acid. Alzheimer’s disease was induced by a single intraperitoneal injection of LPS. Adult male Swiss albino mice were divided into groups of 8–10 mice each receiving treatment for six days. In vivo behavioral tests (Y maze and object recognition) and in vitro estimation of amyloid beta 42 by ELISA showed significant differences between results of treated and nontreated animals

Origanum majorana L. protects against neuroinflammation-mediated cognitive impairment: a phyto-pharmacological study

Abstract Background Neuroinflammation and oxidative stress are critical players in the pathogenesis of numerous neurodegenerative diseases, such as Alzheimer’s disease (AD) which is responsible for most cases of dementia in the elderly. With the lack of curative treatments, natural phenolics are potential candidates to delay the onset and progression of such age-related disorders due to their potent antioxidant and anti-inflammatory effects. This study aims at assessing the phytochemical characteristics of Origanum majorana L. (OM) hydroalcohol extract and its neuroprotective activities in a murine neuroinflammatory model. Methods OM phytochemical analysis was done by HPLC/PDA/ESI-MSn. Oxidative stress was induced in vitro by hydrogen peroxide and cell viability was measured using WST-1 assay. Swiss albino mice were injected intraperitoneally with OM extract at a dose of 100 mg/kg for 12 days and with 250 μg/kg LPS daily starting from day 6 to induce neuroinflammation. Cognitive functions were assessed by novel object recognition and Y-maze behavioral tests. Hematoxylin and eosin staining was used to assess the degree of neurodegeneration in the brain. Reactive astrogliosis and inflammation were assessed by immunohistochemistry using GFAP and COX-2 antibodies, respectively. Results OM is rich in phenolics, with rosmarinic acid and its derivatives being major constituents. OM extract and rosmarinic acid significantly protected microglial cells against oxidative stress-induced cell death (p < 0.001). OM protected against the LPS-induced alteration of recognition and spatial memory in mice (p < 0.001) and (p < 0.05), respectively. Mice that received OM extract prior to the induction of neuroinflammation showed comparable histology to control brains, with no overt neurodegeneration. Furthermore, OM pre-treatment decreased the immunohistochemistry profiler score of GFAP from positive to low positive and COX-2 from low positive to negative in the brain tissue, compared to the LPS group. Conclusion These findings highlight the potential preventive effects of OM phenolics against neuroinflammation and pave the way toward drug discovery and development for neurodegenerative disorders

Validation of the candidate substrate A1AT of HTRA1.

<p>Purified A1AT from human plasma was incubated with HTRA1 at 37°C for 17 hours. (A) 2-D gel of A1AT in the absence of HTRA1 incubation time 0 h and SDS-PAGE of A1AT and HTRA1. The A1AT preparation contains only very small amount of cleaved A1AT. (B) Incubation over a period of 17 hours in the absence (left site) and presence (right site) of HTRA1. After incubation with HTRA1 a significant amount of A1AT was cleaved. The incubation of plasma A1AT with HTRA1 showed similar fragmentation patterns as the incubation of placental A1AT with HTRA1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109483#pone-0109483-g004" target="_blank">Fig. 4A</a>).</p