Yorick Janssens

Quorum Sensing Peptides Selectively Penetrate the Blood-Brain Barrier

Bacteria communicate with each other by the use of signaling molecules, a process called ‘quorum sensing’. One group of quorum sensing molecules includes the oligopeptides, which are mainly produced by Gram-positive bacteria. Recently, these quorum sensing peptides were found to biologically influence mammalian cells, promoting i.a. metastasis of cancer cells. Moreover, it was found that bacteria can influence different central nervous system related disorders as well, e.g. anxiety, depression and autism. Research currently focuses on the role of bacterial metabolites in this bacteria-brain interaction, with the role of the quorum sensing peptides not yet known. Here, three chemically diverse quorum sensing peptides were investigated for their brain influx (multiple time regression technique) and efflux properties in an in vivo mouse model (ICR-CD-1) to determine blood-brain transfer properties: PhrCACET1 demonstrated comparatively a very high initial influx into the mouse brain (Kin = 20.87 μl/(g×min)), while brain penetrabilities of BIP-2 and PhrANTH2 were found to be low (Kin = 2.68 μl/(g×min)) and very low (Kin = 0.18 μl/(g×min)), respectively. All three quorum sensing peptides were metabolically stable in plasma (in vitro) during the experimental time frame and no significant brain efflux was observed. Initial tissue distribution data showed remarkably high liver accumulation of BIP-2 as well. Our results thus support the potential role of some quorum sensing peptides in different neurological disorders, thereby enlarging our knowledge about the microbiome-brain axis.

Peptides as quorum sensing molecules : measurement techniques and obtained levels in vitro and in vivo

The expression of certain bacterial genes is regulated in a cell-density dependent way, a phenomenon called quorum sensing. Both Gram-negative and Gram-positive bacteria use this type of communication, though the signal molecules (auto-inducers) used by them differ between both groups: Gram-negative bacteria use predominantly N-acyl homoserine lacton (AHL) molecules (autoinducer-1, AI-1) while Gram-positive bacteria use mainly peptides (autoinducer peptides, AIP or quorum sensing peptides). These quorum sensing molecules are not only involved in the inter-microbial communication, but can also possibly cross-talk directly or indirectly with their host. This review summarizes the currently applied analytical approaches for quorum sensing identification and quantification with additionally summarizing the experimentally found in vivo concentrations of these molecules in humans.

Chromatography of Quorum Sensing Peptides: An Important Functional Class of the Bacterial Peptidome

Quorum sensing is the process by which bacteria communicate with each other to regulate the expression of certain genes in a cell density-dependent manner. One group of signalling molecules used in this process are the quorum sensing peptides, which are primarily produced by Gram-positive bacteria. Aside from their known role in bacterial physiology, it was recently found that these peptides are also able to influence human cells in a direct or indirect manner. To fully elucidate their biological and clinical significance in the microbiome, the ability to detect and quantify these active peptides in cell media and in vivo is of utmost importance. Several different methods, primarily for bacterial cell culture media, have already been developed. Standard procedure employs traditional techniques such as solid-phase extraction during sample preparation, a critical step in the analysis. A suitable sample preparation to extract the entire peptidome, prior to untargeted mass spectrometry detection, has not yet been proposed. Following sample preparation, liquid chromatography is coupled with different detection methods with mass spectrometry as the most commonly used. The aim of this review is to summarise and critically discuss current analytical methods for quorum sensing peptide identification and quantification.

Blood-brain barrier transport kinetics of NOTA-modified proteins: the somatropin case

BACKGROUND Chemical modifications such as PEG, polyamine and radio labeling on proteins can alter their pharmacokinetic behaviour and their blood-brain barrier (BBB) transport characteristics. NOTA, i.e. 1,4,7-triazacyclononane-1,4,7-triacetic acid, is a bifunctional chelating agent that has attracted the interest of the scientific community for its high complexation constant with metals like gallium. Until now, the comparative BBB transport characteristics of NOTA-modified proteins versus unmodified proteins are not yet described. METHODS Somatropin (i.e. recombinant human growth hormone), NOTA-conjugated somatropin and gallium-labelled NOTA-conjugated somatropin were investigated for their brain penetration characteristics (multiple time regression and capillary depletion) in an in vivo mice model to determine the blood-brain transfer properties. RESULTS The three compounds showed comparable initial brain influx, with Kin = 0.38 ± 0.14 μL/(g×min), 0.36 ± 0.16 μL/(g×min) and 0.28 ± 0.18 μL/(g×min), respectively. Capillary depletion indicated that more than 80% of the influxed compounds reached the brain parenchyma. All three compounds were in vivo stable in serum and brain during the time frame of the experiments. CONCLUSIONS Our results show that modification of NOTA as well as gallium chelation onto proteins, in casu somatropin, does not lead to a significantly changed pharmacokinetic profile at the blood-brain barrier.

Development of a capillary zone electrophoresis method to quantify E. coli l -asparaginase and its acidic variants

A capillary zone electrophoresis (CZE) method with UV detection was developed for the quantification of the E.colil-asparaginase (l-ASNase) and its acidic variants. During the initial method development, a variety of experimental conditions were screened. Subsequently, a Design of Experiments (DoE) was used to optimize the pH and concentration of the selected background electrolyte (BGE) containing both TRIS and boric acid. Optimization was performed taking into account both the separation efficiency of l-ASNase and its acidic variants as well as overall method robustness. A repeatable separation between E.colil-ASNase and its acidic variants was achieved on a bare fused silica capillary in combination with a BGE consisting of both 400 mM TRIS and boric acid. The method was validated for linearity, accuracy, precision, LOD, LOQ and robustness. The recovery for l-ASNase was 97.9-104.4% with a precision RSD of 1.5-3.2%, while the recovery of impurities was 92.1-109.8% with a RSD of 1.7-4.6%. The quantification limit was 1.9% (m/m). Moreover, the CZE-UV method was applied to determine the degradation rate in the presence of ammonium bicarbonate, confirming the suitability of the method. The degraded, partially charged l-ASNase was evaluated for its in-vitro enzymatic activity showing an insignificant different enzyme activity compared to the unmodified sample.

Regulatory status of N-alkylamide containing health products

N-alkylamides (NAAs) are secondary metabolites occurring in more than 25 plant families. Plants containing NAAs are traditionally used in food for flavouring, tingling, pungent and saliva-enhancing properties but also to treat various diseases. NAA containing products are abundantly available on the market as food, cosmetics, medical devices and medicinal products. However, no unambiguous legal product classification is applied for these products. In this study, the different health product classes from a European viewpoint are discussed in relation to the pharmacokinetic and pharmacodynamic properties of the NAAs, their applied dosage and claimed usage.

Screening of quorum sensing peptides for biological effects in neuronal cells

HighlightsQuorum sensing peptides were screened to investigate effects on neuronal cells.Twenty‐two peptides showed effects on different neuronal cell types.Effects are seen on viability, morphology, differentiation and pro‐inflammatory markers.Majority of these active peptides originate from bacilli species.Our findings may explain the role of microbiome in neuropsychiatric disorders. &NA; Quorum sensing peptides (QSP) are an important class of bacterial peptides which can have an effect on human host cells. These peptides are used by bacteria to communicate with each other. Some QSP are able to cross the blood‐brain barrier and reach the brain parenchyma. However, nothing is known about the effects of these peptides in the brain. Therefore, 85 quorum sensing peptides were screened on six different neuronal cell lines using MTT toxicity, neurite differentiation, cytokine production and morphology as biological outcomes. This primary screening resulted in 22 peptides with effects observed on neuronal cell lines, indicating a possible role in the gut‐brain axis. Four peptides (Q138, Q143, Q180 and Q212) showed induction of neurite outgrowth while two peptides (Q162 and Q208) inhibited NGF‐induced neurite outgrowth in PC12 cells. Eight peptides (Q25, Q135, Q137, Q146, Q151, Q165, Q208 and Q298) induced neurite outgrowth in human SH‐SY5Y neuroblastoma cells. Two peptides (Q13 and Q52) were toxic for SH‐SY5Y cells and one (Q123) for BV‐2 microglia cells based on the MTT assay. Six peptides had an effect on BV‐2 microglia, Q180, Q184 and Q191 were able to induce IL‐6 expression and Q164, Q192 and Q208 induced NO production. Finally, Q75 and Q147 treated C8D1A astrocytes demonstrated a higher fraction of round cells. Overall, these in vitro screening study results indicate for the first time possible effects of QSP on neuronal cells.

Detection and quantification of Enterococcus faecalis RNPP-type quorum sensing peptides in bacterial culture media by UHPLC-MS.

HIGHLIGHTSCovering all RNPP E. faecalis quorum sensing peptides.A straight‐forward sample preparation method.Sensitive and selective UHPLC‐MS/MS method for quantification.Short turnaround time and limited sample quantity. ABSTRACT Bacteria communicate with each other using quorum sensing; i.e. the production and sensing of signalling molecules. Enterococcus faecalis, a Gram‐positive bacterium, employs peptides as quorum sensing molecules. These peptides have previously been isolated from culture media by elaborate, time and medium‐consuming sample preparation approaches and specific bacteria‐based bio‐sensors. Here, a method for the detection and quantification of all nine currently reported E. faecalis quorum sensing peptides belonging to the RNPP family in bacterial cell culture medium was developed. The approach developed consists of solid‐phase extraction (SPE) sample preparation followed by a UHPLC‐triple quadrupole mass spectroscopic method, operated in Multiple Reaction Monitoring (MRM) mode. All nine peptides were quantified with a total analysis time below 90 min per sample and limited cell culture medium volumes of only 1 ml per sample. A method verification, performed in uniplicate, was carried out to obtain an idea of the method performance. The recovery varied between 19.9 and 119.0%, and the limit of detection is in the low nM range. Analytical stability, carry‐over and dilution integrity were investigated and were acceptable. This method will be a useful tool in the investigation of the roles of the RNPP‐type quorum sensing peptides in microbial processes.

Disbiome database: linking the microbiome to disease

BackgroundRecent 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.DescriptionDisbiome 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.ConclusionsDisbiome 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.

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 C18 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%).