Evelien Wynendaele | Universiteit Gent (original) (raw)

Papers by Evelien Wynendaele

ABSTRACTBackgroundColorectal cancer, one of the most common malignancies worldwide, is associated... more ABSTRACTBackgroundColorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide analyses between healthy individuals and cancer patients suggest a role for the human intestinal microbiota. Nevertheless, which microbial molecules are involved in this communication is largely unknown, with current studies mainly focusing on short chain fatty acids and amino acid metabolites as potential mediators. However, quorum sensing peptides are not yet considered in this microbiome-host interaction: their in vivo presence nor any in vivo host-effect have been reported.ResultsFor the first time, we showed that a quorum sensing peptide metabolite, EntF* produced by intestinal microbiota (E. faecium), is present in the blood circulation of mice. Moreover, it significantly promotes colorectal cancer metastasis in vivo, with metastatic lesions found in both liver and lung tissues, using an orthotopi...

Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They ha... more Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They have already been successfully applied as carriers for several problematic cargoes, like e.g. plasmid DNA and (si)RNA, opening doors for new therapeutics. Although several hundreds of CPPs are already described in the literature, only a few commercial applications of CPPs are currently available. Cellular uptake studies of these peptides suffer from inconsistencies in used techniques and other experimental conditions, leading to uncertainties about their uptake mechanisms and structural properties. To clarify the structural characteristics influencing the cell-penetrating properties of peptides, the chemical-functional space of peptides, already investigated for cellular uptake, was explored. For 186 peptides, a new cell-penetrating (CP)-response was proposed, based upon the scattered quantitative results for cellular influx available in the literature. Principal component analysis (PCA) a...

Bioanalysis

Background: Bacteria coordinate their behavior as a group via communication with their peers, kno... more Background: Bacteria coordinate their behavior as a group via communication with their peers, known as ‘ quorum sensing’. Enterococcus faecalis employs quorum sensing via RNPP-peptides which were not yet reported to be present in mammalian biofluids. Results: Solid phase extraction of murine feces was performed, followed by ultra high performance liquid chromatography (UHPLC–MS/MS) in multiple reaction monitoring (MRM) mode (in total <90 min/sample) for the nine known RNPP peptides. Limits of detection ranged between 0.045 and 52 nM. Adequate identification criteria allowed detection of RNPP quorum sensing peptides in 2/20 wild-type murine feces samples (i.e., cAM373 and cOB1). Conclusion: A fit-for-purpose UHPLC–MS/MS method detected these RNPP peptides in wild-type murine feces samples.

This article describes the performance of a laboratory exercise, the Slug Mucosal Irritation (SMI... more This article describes the performance of a laboratory exercise, the Slug Mucosal Irritation (SMI) assay, carried out by third year undergraduate students, to investigate the local tolerance of an ethanolic plant extract. The plant extract, Spilanthes acmella, contains various bio-active compounds, such as the N-alkylamide spilanthol. After administration of the plant extract to the slugs, they were observed for possible discomfort and tissue damage. When slugs are exposed to a substance with irritant properties, the mucus production of the slugs will increase. Furthermore, slugs will release proteins, including enzymes, when tissue damage occurs. This laboratory experiment is a practically feasible in vivo test using slugs which are invertebrates that are not protected by the legislation on animal testing. Students were supervised by lab instructors who encouraged students to actively contribute in their groups, to think about the experimental design of the laboratory test, and to ...

Lieselotte Veryser1, Lien Taevernier1, Evelien Wynendaele1, Nathalie Roche2 and Bart De Spiegelee... more Lieselotte Veryser1, Lien Taevernier1, Evelien Wynendaele1, Nathalie Roche2 and Bart De Spiegeleer1,* 1Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. 2Department of Plastic and Reconstructive Surgery, University Hospital Ghent, De Pintelaan 185, B-9000 Ghent, Belgium. * Corresponding author: bart.despiegeleer@ugent.be (O. Ref.: 2015-202b) DruQuaR

Since the discovery that peptides can cross the blood-brain barrier (BBB), doors have been opened... more Since the discovery that peptides can cross the blood-brain barrier (BBB), doors have been opened to new therapeutics for CNS diseases and pain management. Recently, we have constructed the Brainpeps database (brainpeps.ugent.be) to give an overview of the available BBB transport data of peptides, which are scattered in the literature [1]. One possible application of the Brainpeps database is the study of structure-property relationships (QSPRs). Before peptides can be used as drugs, their impurity profile needs to be examined as part of the International Conference on Harmonization (ICH) risk assessment of peptide drugs. Compared to small molecules, no in-silico predictive programs are available for toxicity screening of the different peptide impurities towards passing the BBB. To predict the BBB-behaviour of peptides as well as their impurities, we explored the Brainpeps database. During this presentation, the first results of the modelling experiments are presented. Our starting ...

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous huma... more Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a...

REFERENCES The human microbiota, the collection of trillions of microbes in and on the human body... more REFERENCES The human microbiota, the collection of trillions of microbes in and on the human body, has already been indicated to be beneficial for health. Commensal gut bacteria for example supply nutrients, help metabolizing indigestible compounds and defend against colonization by opportunistic pathogens or damage by dietary toxins and carcinogens. However, the alteration of the human microbiota composition can be associated with different diseases as well: e.g. psoriasis, colorectal cancer, gastric adenocarcinoma and autism were found to be linked to several bacterial compounds. One specific group of bacterial compounds are the quorum sensing peptides (QSP), communication molecules between mainly Gram-positive bacteria. In our laboratory, we investigate the role of these QSP in different diseases by using both in vitro and in vivo studies. INTRODUCTION

Human beings live in a co-evolutionary association with microorganisms. The collection of these m... more Human beings live in a co-evolutionary association with microorganisms. The collection of these microorganisms is termed the microbiome. Bacteria are able to communicate with each other by quorum sensing molecules. An important group of these molecules are the oligopeptides, mainly produced by Gram-positive bacteria. These quorum sensing peptides (QSP) are able to interact with human eukaryotic cells [1,2]. The pharmacokinetic properties of these peptides are still to be explored.

Quorum-sensing (QS) enables bacterial cells to establish cell-cell communication and to regulate ... more Quorum-sensing (QS) enables bacterial cells to establish cell-cell communication and to regulate the expression of specific genes in response to local changes in cell density. It provides a means to coordinate the activities of cells in order to function as a multicellular unit. Quorum-sensing chemicals up till now identified as key components in bacterial cell-cell communication can broadly be divided in three groups, which contain peptidic-derived structural characteristics: N-acyl homoserine lactone derivatives (auto-inducer-1), larger quorum-sensing peptides and boron-furanone derivatives (auto-inducer-2). A quorum-sensing peptide database (Quorumpeps®) was constructed encompassing the structures as well as functionality responses. After three-dimensional optimisation of these structures, chem-informatic molecular descriptors were calculated. The resulting data-matrix was subjected to diverse multivariate analyses to cluster these peptides as well as to extract fundamental relat...

Most naturally occurring opioid peptides, such as the endomorphins, are not highly selective for ... more Most naturally occurring opioid peptides, such as the endomorphins, are not highly selective for a single opioid receptor type. However, it was observed that dermorphins show a very high µ-opioid receptor selectivity compared to most other natural peptides. The 3+ charged tetrapeptide Dmt-DALDA (Dmt-D(Arg)-Phe-Lys-NH2), an analogue of the µ-selective dermorphin, is of particular interest since it has been identified as a more potent μ-opioid receptor agonist compared to dermorphin. The present study describes the ADME behaviour of four related Dmt-DALDA analogues including their blood-brain barrier (BBB) transport properties. The influx transfer constant from serum into mouse brain was determined by multiple time regression while the efflux kinetics were investigated with the intracerebroventricular injection technique. Brain parenchyma-capillary cell distribution was evaluated by capillary depletion. In addition, the functional drugability of these µ-opioid peptides was evaluated u...

Medicine in Drug Discovery

BackgroundQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteri... more BackgroundQuorum 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.MethodsGastro-intestinal transport of the peptide is investigated using the in vitro Caco-2 model while transport over the blood-brain barrier is investigated in mice using multiple time regression experiments. Microglial activation is assessed using ELISA, fluorometry, immunoblotting, qPCR and phase-contrast microscopy. In vivo plasma detection and ex vivo metabolization experiments are performed using UHPLC-MS2 and UHPLC-...

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

Skeletal muscle makes up the largest part of human body mass and a good maintenance of this organ... more Skeletal muscle makes up the largest part of human body mass and a good maintenance of this organ is essential for general health. In accordance, muscle wasting, a frequent phenomenon in many diseases, is associated with functional decline and a decrease in quality of life. Unfortunately, due to a lack of knowledge of the underlying pathophysiology, no targeted therapies exist today to encounter muscle wasting. Recent studies suggest a role for the gut microbiome in muscle wasting, without the mediators of this gut-muscle axis being identified. Here we evaluated the possible effects of 75 quorum sensing molecules (QSM), traditionally only seen as intra-bacterial communication molecules, on C2C12 muscle cells, studying viability, differentiation, inflammation, mitochondrial changes and protein degradation as biological outcomes. The responses were evaluated using different approaches: median absolute deviation, quartiles, strictly standardized mean difference and robust strictly standardized mean difference. This study resulted in 30 QSM, with effects observed on C2C12 cells. Known producers of the 27 peptide QSM belong to species of the genus Staphylococcus, Streptococcus, Enterococcus, Bacillus, Lactobacillus and Escherichia, while the 3 non-peptide QSM are produced by a broad range of Gram-positive and Gram-negative bacteria. Altogether, these proof-of-concept findings provide the first evidence that QSM produced by microbiota play a role in the gut-muscle axis, opening new perspectives for diagnostic and therapeutic targets in muscle wasting diseases.

Drug Testing and Analysis

Thermal sensitivity as a practical measure of thermostability is an interesting quality attribute... more Thermal sensitivity as a practical measure of thermostability is an interesting quality attribute that can be used in the quality control (QC) release of biopharmaceuticals. This paper investigates circular dichroism (CD) spectroscopy and nano-differential scanning fluorimetry (nano-DSF) to evaluate the thermal stability of E.coli L-asparaginase (L-ASNase) for QC purposes. In CD, molar ellipticity as a function of temperature (from 20 to 80°C) was measured at 222 nm. Different L-ASNase samples dissolved in different diluents were investigated by determining the melting temperature (Tm ) from the first derivative curve as well as the slope of the fitted sigmoidal function of the temperature gradient CD data. The obtained Tm values could be correlated with the L-ASNase sample origin as well as with the pH of the diluent. The Tm values obtained from the CD data were moreover consistent with the Tm values determined by nano-DSF, confirming their reliability. Next to the Tm value, also the slope of the fitted sigmoidal CD-function was able to differentiate different L-ASNase samples, including unstressed from stressed protein. By using both the Tm and the curve slope, the thermal stability of L-ASNase was investigated, demonstrating and recommending the use of this heat-stress characteristic as a QC quality attribute of proteins, which can be applied to detect substandard and falsified proteins.

Talanta

Biosensors are rising technologies in the pharmaceutical field for medicine discovery, developmen... more Biosensors are rising technologies in the pharmaceutical field for medicine discovery, development and Quality Control (QC) stages. Surface acoustic wave (SAW) biosensor employs acoustic waves generated by oscillating a piezoelectric crystal quartz plate to meas. mass and viscosity, and allows to detect and quantify binding events between the analyte and an immobilized interacting ligand. We present here a SAW biosensor based approach for the functional quantification of Escherichia colil-asparaginase (E. colil-ASNase), using polyclonal antibody (pAb) as the interaction partner immobilized on the chip. Different immobilization strategies of pAb were initially evaluated, resulting in the BS3 activated amide coupling via protein G strategy as the final immobilization method. The method was validated by evaluating the selectivity, linearity, as well as accuracy (a recovery of 102.4%) and precision (RSD of 8.5%). The application of the validated method on different samples encompassing different lots of E. colil-ASNase, deamidated E. colil-ASNase and dry-heated E. colil-ASNase (80 °C, 10 min) indicated the suitability of the developed SAW method to quantify E. colil-ASNase. We suggest this SAW method can be adopted as a pharmaceutical QC method.

ABSTRACTBackgroundColorectal cancer, one of the most common malignancies worldwide, is associated... more ABSTRACTBackgroundColorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide analyses between healthy individuals and cancer patients suggest a role for the human intestinal microbiota. Nevertheless, which microbial molecules are involved in this communication is largely unknown, with current studies mainly focusing on short chain fatty acids and amino acid metabolites as potential mediators. However, quorum sensing peptides are not yet considered in this microbiome-host interaction: their in vivo presence nor any in vivo host-effect have been reported.ResultsFor the first time, we showed that a quorum sensing peptide metabolite, EntF* produced by intestinal microbiota (E. faecium), is present in the blood circulation of mice. Moreover, it significantly promotes colorectal cancer metastasis in vivo, with metastatic lesions found in both liver and lung tissues, using an orthotopi...

Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They ha... more Cell-penetrating peptides (CPPs) are a promising tool to overcome cell membrane barriers. They have already been successfully applied as carriers for several problematic cargoes, like e.g. plasmid DNA and (si)RNA, opening doors for new therapeutics. Although several hundreds of CPPs are already described in the literature, only a few commercial applications of CPPs are currently available. Cellular uptake studies of these peptides suffer from inconsistencies in used techniques and other experimental conditions, leading to uncertainties about their uptake mechanisms and structural properties. To clarify the structural characteristics influencing the cell-penetrating properties of peptides, the chemical-functional space of peptides, already investigated for cellular uptake, was explored. For 186 peptides, a new cell-penetrating (CP)-response was proposed, based upon the scattered quantitative results for cellular influx available in the literature. Principal component analysis (PCA) a...

Bioanalysis

Background: Bacteria coordinate their behavior as a group via communication with their peers, kno... more Background: Bacteria coordinate their behavior as a group via communication with their peers, known as ‘ quorum sensing’. Enterococcus faecalis employs quorum sensing via RNPP-peptides which were not yet reported to be present in mammalian biofluids. Results: Solid phase extraction of murine feces was performed, followed by ultra high performance liquid chromatography (UHPLC–MS/MS) in multiple reaction monitoring (MRM) mode (in total <90 min/sample) for the nine known RNPP peptides. Limits of detection ranged between 0.045 and 52 nM. Adequate identification criteria allowed detection of RNPP quorum sensing peptides in 2/20 wild-type murine feces samples (i.e., cAM373 and cOB1). Conclusion: A fit-for-purpose UHPLC–MS/MS method detected these RNPP peptides in wild-type murine feces samples.

This article describes the performance of a laboratory exercise, the Slug Mucosal Irritation (SMI... more This article describes the performance of a laboratory exercise, the Slug Mucosal Irritation (SMI) assay, carried out by third year undergraduate students, to investigate the local tolerance of an ethanolic plant extract. The plant extract, Spilanthes acmella, contains various bio-active compounds, such as the N-alkylamide spilanthol. After administration of the plant extract to the slugs, they were observed for possible discomfort and tissue damage. When slugs are exposed to a substance with irritant properties, the mucus production of the slugs will increase. Furthermore, slugs will release proteins, including enzymes, when tissue damage occurs. This laboratory experiment is a practically feasible in vivo test using slugs which are invertebrates that are not protected by the legislation on animal testing. Students were supervised by lab instructors who encouraged students to actively contribute in their groups, to think about the experimental design of the laboratory test, and to ...

Lieselotte Veryser1, Lien Taevernier1, Evelien Wynendaele1, Nathalie Roche2 and Bart De Spiegelee... more Lieselotte Veryser1, Lien Taevernier1, Evelien Wynendaele1, Nathalie Roche2 and Bart De Spiegeleer1,* 1Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. 2Department of Plastic and Reconstructive Surgery, University Hospital Ghent, De Pintelaan 185, B-9000 Ghent, Belgium. * Corresponding author: bart.despiegeleer@ugent.be (O. Ref.: 2015-202b) DruQuaR

Since the discovery that peptides can cross the blood-brain barrier (BBB), doors have been opened... more Since the discovery that peptides can cross the blood-brain barrier (BBB), doors have been opened to new therapeutics for CNS diseases and pain management. Recently, we have constructed the Brainpeps database (brainpeps.ugent.be) to give an overview of the available BBB transport data of peptides, which are scattered in the literature [1]. One possible application of the Brainpeps database is the study of structure-property relationships (QSPRs). Before peptides can be used as drugs, their impurity profile needs to be examined as part of the International Conference on Harmonization (ICH) risk assessment of peptide drugs. Compared to small molecules, no in-silico predictive programs are available for toxicity screening of the different peptide impurities towards passing the BBB. To predict the BBB-behaviour of peptides as well as their impurities, we explored the Brainpeps database. During this presentation, the first results of the modelling experiments are presented. Our starting ...

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous huma... more Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a...

REFERENCES The human microbiota, the collection of trillions of microbes in and on the human body... more REFERENCES The human microbiota, the collection of trillions of microbes in and on the human body, has already been indicated to be beneficial for health. Commensal gut bacteria for example supply nutrients, help metabolizing indigestible compounds and defend against colonization by opportunistic pathogens or damage by dietary toxins and carcinogens. However, the alteration of the human microbiota composition can be associated with different diseases as well: e.g. psoriasis, colorectal cancer, gastric adenocarcinoma and autism were found to be linked to several bacterial compounds. One specific group of bacterial compounds are the quorum sensing peptides (QSP), communication molecules between mainly Gram-positive bacteria. In our laboratory, we investigate the role of these QSP in different diseases by using both in vitro and in vivo studies. INTRODUCTION

Human beings live in a co-evolutionary association with microorganisms. The collection of these m... more Human beings live in a co-evolutionary association with microorganisms. The collection of these microorganisms is termed the microbiome. Bacteria are able to communicate with each other by quorum sensing molecules. An important group of these molecules are the oligopeptides, mainly produced by Gram-positive bacteria. These quorum sensing peptides (QSP) are able to interact with human eukaryotic cells [1,2]. The pharmacokinetic properties of these peptides are still to be explored.

Quorum-sensing (QS) enables bacterial cells to establish cell-cell communication and to regulate ... more Quorum-sensing (QS) enables bacterial cells to establish cell-cell communication and to regulate the expression of specific genes in response to local changes in cell density. It provides a means to coordinate the activities of cells in order to function as a multicellular unit. Quorum-sensing chemicals up till now identified as key components in bacterial cell-cell communication can broadly be divided in three groups, which contain peptidic-derived structural characteristics: N-acyl homoserine lactone derivatives (auto-inducer-1), larger quorum-sensing peptides and boron-furanone derivatives (auto-inducer-2). A quorum-sensing peptide database (Quorumpeps®) was constructed encompassing the structures as well as functionality responses. After three-dimensional optimisation of these structures, chem-informatic molecular descriptors were calculated. The resulting data-matrix was subjected to diverse multivariate analyses to cluster these peptides as well as to extract fundamental relat...

Most naturally occurring opioid peptides, such as the endomorphins, are not highly selective for ... more Most naturally occurring opioid peptides, such as the endomorphins, are not highly selective for a single opioid receptor type. However, it was observed that dermorphins show a very high µ-opioid receptor selectivity compared to most other natural peptides. The 3+ charged tetrapeptide Dmt-DALDA (Dmt-D(Arg)-Phe-Lys-NH2), an analogue of the µ-selective dermorphin, is of particular interest since it has been identified as a more potent μ-opioid receptor agonist compared to dermorphin. The present study describes the ADME behaviour of four related Dmt-DALDA analogues including their blood-brain barrier (BBB) transport properties. The influx transfer constant from serum into mouse brain was determined by multiple time regression while the efflux kinetics were investigated with the intracerebroventricular injection technique. Brain parenchyma-capillary cell distribution was evaluated by capillary depletion. In addition, the functional drugability of these µ-opioid peptides was evaluated u...

Medicine in Drug Discovery

BackgroundQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteri... more BackgroundQuorum 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.MethodsGastro-intestinal transport of the peptide is investigated using the in vitro Caco-2 model while transport over the blood-brain barrier is investigated in mice using multiple time regression experiments. Microglial activation is assessed using ELISA, fluorometry, immunoblotting, qPCR and phase-contrast microscopy. In vivo plasma detection and ex vivo metabolization experiments are performed using UHPLC-MS2 and UHPLC-...

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

Skeletal muscle makes up the largest part of human body mass and a good maintenance of this organ... more Skeletal muscle makes up the largest part of human body mass and a good maintenance of this organ is essential for general health. In accordance, muscle wasting, a frequent phenomenon in many diseases, is associated with functional decline and a decrease in quality of life. Unfortunately, due to a lack of knowledge of the underlying pathophysiology, no targeted therapies exist today to encounter muscle wasting. Recent studies suggest a role for the gut microbiome in muscle wasting, without the mediators of this gut-muscle axis being identified. Here we evaluated the possible effects of 75 quorum sensing molecules (QSM), traditionally only seen as intra-bacterial communication molecules, on C2C12 muscle cells, studying viability, differentiation, inflammation, mitochondrial changes and protein degradation as biological outcomes. The responses were evaluated using different approaches: median absolute deviation, quartiles, strictly standardized mean difference and robust strictly standardized mean difference. This study resulted in 30 QSM, with effects observed on C2C12 cells. Known producers of the 27 peptide QSM belong to species of the genus Staphylococcus, Streptococcus, Enterococcus, Bacillus, Lactobacillus and Escherichia, while the 3 non-peptide QSM are produced by a broad range of Gram-positive and Gram-negative bacteria. Altogether, these proof-of-concept findings provide the first evidence that QSM produced by microbiota play a role in the gut-muscle axis, opening new perspectives for diagnostic and therapeutic targets in muscle wasting diseases.

Drug Testing and Analysis

Thermal sensitivity as a practical measure of thermostability is an interesting quality attribute... more Thermal sensitivity as a practical measure of thermostability is an interesting quality attribute that can be used in the quality control (QC) release of biopharmaceuticals. This paper investigates circular dichroism (CD) spectroscopy and nano-differential scanning fluorimetry (nano-DSF) to evaluate the thermal stability of E.coli L-asparaginase (L-ASNase) for QC purposes. In CD, molar ellipticity as a function of temperature (from 20 to 80°C) was measured at 222 nm. Different L-ASNase samples dissolved in different diluents were investigated by determining the melting temperature (Tm ) from the first derivative curve as well as the slope of the fitted sigmoidal function of the temperature gradient CD data. The obtained Tm values could be correlated with the L-ASNase sample origin as well as with the pH of the diluent. The Tm values obtained from the CD data were moreover consistent with the Tm values determined by nano-DSF, confirming their reliability. Next to the Tm value, also the slope of the fitted sigmoidal CD-function was able to differentiate different L-ASNase samples, including unstressed from stressed protein. By using both the Tm and the curve slope, the thermal stability of L-ASNase was investigated, demonstrating and recommending the use of this heat-stress characteristic as a QC quality attribute of proteins, which can be applied to detect substandard and falsified proteins.

Talanta

Biosensors are rising technologies in the pharmaceutical field for medicine discovery, developmen... more Biosensors are rising technologies in the pharmaceutical field for medicine discovery, development and Quality Control (QC) stages. Surface acoustic wave (SAW) biosensor employs acoustic waves generated by oscillating a piezoelectric crystal quartz plate to meas. mass and viscosity, and allows to detect and quantify binding events between the analyte and an immobilized interacting ligand. We present here a SAW biosensor based approach for the functional quantification of Escherichia colil-asparaginase (E. colil-ASNase), using polyclonal antibody (pAb) as the interaction partner immobilized on the chip. Different immobilization strategies of pAb were initially evaluated, resulting in the BS3 activated amide coupling via protein G strategy as the final immobilization method. The method was validated by evaluating the selectivity, linearity, as well as accuracy (a recovery of 102.4%) and precision (RSD of 8.5%). The application of the validated method on different samples encompassing different lots of E. colil-ASNase, deamidated E. colil-ASNase and dry-heated E. colil-ASNase (80 °C, 10 min) indicated the suitability of the developed SAW method to quantify E. colil-ASNase. We suggest this SAW method can be adopted as a pharmaceutical QC method.