Analysis of iodinated quorum sensing peptides by LC-UV/ESI ion trap mass spectrometry

Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C-18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono- and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low (2%) to high (57%)

Residual active pharmaceutical ingredient on the primary packaging of medicines : a valuable GMP-marker

Medicines are meant to help people and treat their conditions and to promote general well-being of all members of the society. Unfortunately, this is being compromised by the distribution and sale of poor-quality medicines around the world, being a consequence of non-GMP manufacturing. In this study, the contamination of the outer primary packaging with active pharmaceutical ingredient (API, i.e. artemether) is investigated as a possible and objective, quantifiable marker for GMP-compliance. First, an analytical UPLC-MS method was developed and verified for artemether, with emphasis on the quantification in the lower concentration range. Second, a swabbing procedure for the outer surface of plastic bottles (powders for suspension) was developed, including a swabbing recovery of the API from the bottle surfaces. Finally, twenty antimalarial samples were investigated. All of them showed some degree of outer contamination; however, large differences in the amount of API contamination between the different samples was observed, ranging between 4 and 144 ng/cm(2). A positive correlation was found between the amount of artemether on the packaging and the number of information elements missing on the packaging or leaflet, which was used as one of the tools to evaluate the GMP status of the manufacturer

Quality of isotopically labelled internal standards for peptide quantification

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Influence of blood collection methods and long-term plasma storage on quorum-sensing peptide stability

Finding adequate biomarkers for rapid and accurate disease detection, prognosis, and therapy is increasingly important. Quorum-sensing peptides are herein a new emerging group, produced by bacteria, fungi, protozoa, and viruses, with blood being the most straightforward sample type to detect/quantitate them. However, detailed information about suitable blood sample collection methods and storage conditions for measuring these quorum-sensing peptides hampers further clinical research and development. Here, we first tested the time-dependent stability of a set of chemically diverse quorum-sensing peptides, spiked in blood at different temperatures (4, 21, and 37 °C) in four different ethylenediamine tetraacetic acid (EDTA)-containing plasma tubes (with different protein-stabilizing additives) over a period of up to 7.5 h. Next, we determined the storage stability of these quorum-sensing peptides in plasma at different temperatures (4, −35, and −80 °C). UPLC/MS–MS was used to selectively detect and quantify the spiked quorum-sensing peptides. The results of this study indicate that a cost-effective tube, designed for traditional proteomics and stored at 4 °C, is the preferred collection condition when quorum-sensing peptides need to be detected/quantified in human plasma. When the tubes are handled at room temperature (21 °C), a more specialized tube is required. Long-term storage of plasma samples, even under low-temperature conditions (−80 °C), indicates rapid degradation of certain quorum-sensing peptides

MEK1/2 inhibition in murine heart and aorta after oral administration of refametinib supplemented drinking water

Upregulation of the RAS-RAF-MEK-ERK-MAPK pathway is involved in the development of several human tumors, aortic aneurysms, atherosclerosis, and cardiomyopathy. Refametinib, a highly selective MEK-inhibitor, has already shown antineoplastic activity in phase II trials. Furthermore, it showed potency to attenuate aortic root growth in murine models. Current formulations of this drug however necessitate oral gavage as a delivery method for long-term studies, which is labor-intensive and induces stress and occasional injury, potentially confounding results. Therefore, we developed a novel oral administration method for refametinib. A 2-hydroxypropyl-beta-cyclodextrin (HPBCD) based drinking water preparation of refametinib was formulated, for which a selective, analytical UHPLC-UV method was developed to assess the in-use stability. Next, 16 week old male wild-type C57Bl/6J mice received either a daily dose of 50 or 75 mg/kg/day refametinib or were given regular drinking water during 7 days. In both dosage groups the refametinib plasma levels were measured (n = 10 or 7, respectively). Furthermore, pERK/total ERK protein levels were calculated in the myocardial and aortic tissue of mice receiving a daily dose of 50 mg/kg/day refametinib and untreated mice (n = 4/group). After 7 days no significant degradation of refametinib was observed when dissolved in drinking water provided that drinking bottles were protected from UV/visible light. Furthermore, a dose-dependent increase in refametinib plasma levels was found whereby active plasma levels (> 1.2 mu g/mL) were obtained even in the lowest dose-group of 50 mg/kg/day. A significant reduction of pERK/total ERK protein levels compared to untreated mice was observed in aortic and myocardial tissue of mice receiving a daily dose of 50 mg/kg/day refametinib. Importantly, a relatively high mortality rate was noted in the highest dose group (n = 5). This approach provides a valid alternative oral administration method for refametinib with a reduced risk of complications due to animal manipulation and without loss of functionality, which can be implemented in future research regarding the malignant upregulation of the RAS-RAF-MEK-ERK-MAPK pathway. However, care must be taken not to exceed the toxic dose

Disbiome database : linking the microbiome to disease

Background: Recent research has provided fascinating indications and evidence that the host health is linked to its microbial inhabitants. Due to the development of high-throughput sequencing technologies, more and more data covering microbial composition changes in different disease types are emerging. However, this information is dispersed over a wide variety of medical and biomedical disciplines. Description: Disbiome is a database which collects and presents published microbiota-disease information in a standardized way. The diseases are classified using the MedDRA classification system and the micro-organisms are linked to their NCBI and SILVA taxonomy. Finally, each study included in the Disbiome database is assessed for its reporting quality using a standardized questionnaire. Conclusions: Disbiome is the first database giving a clear, concise and up-to-date overview of microbial composition differences in diseases, together with the relevant information of the studies published. The strength of this database lies within the combination of the presence of references to other databases, which enables both specific and diverse search strategies within the Disbiome database, and the human annotation which ensures a simple and structured presentation of the available data

PapRIV, a BV-2 microglial cell activating quorum sensing peptide

Quorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction