Glycation Interferes with the Activity of the Bi-Functional UDP-N-Acetylglucosamine 2-Epimerase/N-Acetyl-mannosamine Kinase (GNE)

Mutations in the gene coding for the bi-functional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of the sialic acid biosynthesis, are responsible for autosomal-recessive GNE myopathy (GNEM). GNEM is an adult-onset disease with a yet unknown exact pathophysiology. Since the protein appears to work adequately for a certain period of time even though the mutation is already present, other effects appear to influence the onset and progression of the disease. In this study, we want to investigate whether the late onset of GNEM is based on an age-related effect, e.g., the accumulation of post-translational modifications (PTMs). Furthermore, we also want to investigate what effect on the enzyme activity such an accumulation would have. We will particularly focus on glycation, which is a PTM through non-enzymatic reactions between the carbonyl groups (e.g., of methylglyoxal (MGO) or glyoxal (GO)) with amino groups of proteins or other biomolecules. It is already known that the levels of both MGO and GO increase with age. For our investigations, we express each domain of the GNE separately, treat them with one of the glycation agents, and determine their activity. We demonstrate that the enzymatic activity of the N-acetylmannosamine kinase (GNE-kinase domain) decreases dramatically after glycation with MGO or GO—with a remaining activity of 13% ± 5% (5 mM MGO) and 22% ± 4% (5 mM GO). Whereas the activity of the UDP-N-acetylglucosamine 2-epimerase (GNE-epimerase domain) is only slightly reduced after glycation—with a remaining activity of 60% ± 8% (5 mM MGO) and 63% ± 5% (5 mM GO).Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)Deutsche ForschungsgemeinschaftPeer Reviewe

Scientific Opinion addressing the state of the science on risk assessment of plant protection products for in-soil organisms

Following a request from EFSA, the Panel on Plant Protection Products and their Residues developed an opinion on the science behind the risk assessment of plant protection products for in-soil organisms. The current risk assessment scheme is reviewed, taking into account new regulatory frameworks and scientific developments. Proposals are made for specific protection goals for in-soil organisms being key drivers for relevant ecosystem services in agricultural landscapes such as nutrient cycling, soil structure, pest control and biodiversity. Considering the time-scales and biological processes related to the dispersal of the majority of in-soil organisms compared to terrestrial non-target arthropods living above soil, the Panel proposes that in-soil environmental risk assessments are made at in- and off-field scale considering field boundary levels. A new testing strategy which takes into account the relevant exposure routes for in-soil organisms and the potential direct and indirect effects is proposed. In order to address species recovery and long-term impacts of PPPs, the use of population models is also proposed

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Modulation of the Sialic Acid Biosynthesis and its Medical and Biotechnical Relevance

Titelblatt und Inhalt Einleitung Ergebnisse Diskussion Zusammenfassung / Summary Literatur AnhangIn dieser Arbeit wurden die regulatorischen Auswirkungen einer veränderten Sialinsäurebiosynthese und die damit verbundenen Veränderung der intrazellulären Sialinsäurekonzentration näher charakterisiert. Hierzu wurde untersucht, ob eine Erhöhung der Sialinsäurekonzentration sich auf die Polysialylierung des neuralen Zelladhäsionsmoleküls (NCAM) auswirkt. Es wurden zwei CHO-Zelllinien, die jeweils nur eine der beiden Polysialyltransferasen ST8SiaII, ST8SiaIV exprimieren, mit N-Acetyl-D-Mannosamin (ManNAc), einem Intermediat der Sialinsäurebiosynthese, supplementiert. Die Supplementation mit ManNAc führte zu einem mehr als dreifachen Anstieg der intrazellulären Sialinsäurekonzentration. In beiden Zelllinien kam es daraufhin zu einer Zunahme von polysialyliertem NCAM (PSA-NCAM). Die Sialylierung anderer Glycokonjugate blieb jedoch unverändert. Die Zelllinien wurden außerdem mit einem Expressionsvektor transfiziert, der für eine Sialuriemutation tragende UDP-GlcNAc-2-Epimerase/ManNAc-Kinase (GNE-R263L) kodiert. Die Mutation hat einen Defekt in der "feedback"-Regulierung der GNE zur Folge und führt bei Sialurie-Patienten zu einer stark erhöhten Produktion von Sialinsäure. Die transfizierten Zelllinien zeigten, wie die mit ManNAc supplementierten Zellen, eine erhöhte intrazelluläre Sialinsäurekonzentration und eine Zunahme an PSA- NCAM. Es konnte somit gezeigt werden, dass die Polysialylierung von NCAM von der intrazellulären Sialinsäurekonzentration und der Regulation der Sialinsäurebiosynthese abhängig ist. Die erhöhte Polysialylierung von NCAM könnte damit eine mögliche Ursache für die bei Sialurie-Patienten beschriebene Entwicklungsverzögerung sein. Die Anzahl an rekombinanten Glycoproteinen, die zu Therapiezwecken eingesetzt werden, ist in den letzten Jahren stark angestiegen. Um einen hohen Wirkungsgrad zu erzielen, müssen diese Glycoproteine möglichst vollständig sialyliert sein. Die Produktion rekombinanter Glycoproteine durch Überexpression in eukaryotischen Zellen liefert jedoch eine heterogene und unvollständige Sialylierung. Aus diesem Grund wurde in einem weiteren Teil der Arbeit untersucht, ob durch Expression der GNE-R263L in eukaryontischen Produktionszelllinien die Produktion von hoch sialylierten Glycoproteinen verbessert werden kann. Die Expression der GNE- R263L in CHO-Zellen führte zu einer homogenen Expression der hochsialylierten Form des rekombinanten Glycoproteins Erythropoetin. Eine Expression der GNE- R263L in eukaryontischen Produktionszelllinien stellt somit eine effiziente Möglichkeit zur Verbesserung der Sialylierung von rekombinanten Glycoproteinen dar. In einem weiteren Teil der Arbeit wurde die Dopaminsekretion in PC12-Zellen nach Supplementation mit N-Propanoylmannosamin (ManNProp) untersucht. ManNProp ist ein unnatürlicher Sialinsäurevorläufer, der im Gegensatz zum physiologischen Sialinsäurevorläufer (ManNAc) eine zusätzliche Methylengruppe in der N Acetylseitenkette besitzt. Nach Behandlung mit ManNProp stieg die Dopaminsekretion um 38%. Es konnte gezeigt werden, dass nach ManNProp-Behandlung die Menge an O-GlcNAc (N-Acetylglucosamin) modifizierter Tyrosin-3-Monooxygenase (T3M) ab- und im Gegenzug die Phosphorylierung von T3M zunimmt. Die T3M ist das Schlüsselenzym der Dopaminsynthese. Durch die Phosphorylierung wird die T3M aktiviert und es kommt zu einer vermehrten Synthese und Sekretion von Dopamin. Die aufgezeigte Regulation der Dopaminsekretion über eine O GlcNAc-Modifizierung der T3M könnte einen neuen Ansatzpunkt für die Therapie der Parkinson-Krankheit und anderer neurodegenerativer Erkrankungen mit gestörter Dopaminsekretion bieten.In this thesis, the regulatory role of an altered sialic acid biosynthesis was characterized. Therefore, it was investigated if an enhanced intracellular sialic acid concentration leads to a modified polysialylation of neural cell adhesion molecule (NCAM). CHO cells were supplemented with N-acetyl-D-mannosamine (ManNAc), an intermediate of the sialic acid biosynthesis. Supplementation with ManNAc led to an increased intracellular sialic acid concentration and polysialylation of NCAM (PSA-NCAM). However, the concentration of other membrane bound sialic acid was not increased. Moreover, CHO cells were transfected with an expression vector encoding for the key enzyme of the sialic acid biosynthesis the UDP-GlcNAc-2-epimerase/ManNAc- kinase carrying a sialuria mutation (GNE-R263L). The sialuria mutation inhibits the feedback mechanism of the enzyme and leads to an unregulated synthesis of sialic acid in sialuria patients. The transfected cells showed, like the ManNAc supplemented cells, an increased intracellular concentration of sialic acid and an enhanced polysialylation of NCAM. This clarified that the polysialylation of NCAM depends on the intracellular concentration of sialic acid and therefore on the regulation of the sialic acid biosynthesis. An increased polysialylation of NCAM could hence be a cause for the developmental delay of sialuria patients. The number of recombinant glycoproteins used in therapy has increased over the past years and many of the high-value therapeutic proteins in the market today are glycoproteins. A complete sialylation of therapeutic glycoproteins is essential for optimum efficiency. However, the sialylation of over-expressed glycoproteins in mammalian cell lines like CHO cells used for the production of therapeutic glycoproteins is incomplete. Therefore it was investigated, if the utilization of the GNE sialuria mutation (GNE-R263L) leads to an enhanced sialylation of recombinant glycoproteins. As a model system we chose CHO cells producing recombinant human erythropoietin (rhEPO). Overexpression of the GNE-R263L results in an increased intracellular sialic acid concentration and an increased sialylation of rhEPO. This shows that sialuria-mutated-GNE over- expressing cells are the perfect platform to express highly sialylated therapeutic proteins, such as rhEPO. In an additional experimental approach the dopamine secretion after application of N-propanoylmannosamine (ManNProp) was investigated. ManNProp is a novel, synthetic sialic acid precursor. After treatment with ManNProp the dopamine secretion increased by 38% in PC12-cells. Further experiments showed that tyrosine 3-monooxygenase is modified by O-GlcNAc and that application of ManNProp decreases its content of O-GlcNAc and leads to an increased phosphorylation of tyrosine 3-monooxygenase, which in turn results in activation of tyrosine 3-monooxygenase, leading to an increased synthesis of dopamine by PC12-cells. The described regulation of tyrosine 3-monooxygenase by O-GlcNAc modification is new starting-point for the therapy of the Parkinson's disease or other neurodegenerative disorders with altered dopamine synthesis

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth

International audienceThe cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth

Investigation of the Neuroprotective Impact of Nimodipine on Neuro2a Cells by Means of a Surgery-Like Stress Model

Nimodipine is well characterized for the management of SAH (subarachnoid hemorrhage) and has been shown to promote a better outcome and less DIND (delayed ischemic neurological deficits). In rat experiments, enhanced axonal sprouting and higher survival of motoneurons was demonstrated after cutting or crushing the facial nerve by nimodipine. These results were confirmed in clinical trials following vestibular Schwannoma surgery. The mechanism of the protective competence of nimodipine is unknown. Therefore, in this study, we established an in vitro model to examine the survival of Neuro2a cells after different stress stimuli occurring during surgery with or without nimodipine. Nimodipine significantly decreased ethanol-induced cell death of cells up to approximately 9% in all tested concentrations. Heat-induced cell death was diminished by approximately 2.5% by nimodipine. Cell death induced by mechanical treatment was reduced up to 15% by nimodipine. Our findings indicate that nimodipine rescues Neuro2a cells faintly, but significantly, from ethanol-, heat- and mechanically-induced cell death to different extents in a dosage-dependent manner. This model seems suitable for further investigation of the molecular mechanisms involved in the neuroprotective signal pathways influenced by nimodipine

Disturbance of Key Cellular Subproteomes upon Propofol Treatment Is Associated with Increased Permeability of the Blood-Brain Barrier

Background: Propofol is a short-acting anesthetic, which is often used for induction and maintenance of general anesthesia, sedation for mechanically ventilated adults and procedural sedation. Several side effects of propofol are known and a substantial number of patients suffer from post-operative delirium after propofol application. In this study, we analyzed the effect of propofol on the function and protein expression profile on a proteome-wide scale. Methods: We cultured human brain microvascular endothelial cells in absence and presence of propofol and analyzed the permeability of the blood-brain barrier (BBB) by fluorescein passage and protein abundance on a proteome-wide scale by mass spectrometry. Results: Propofol interfered with the function of the blood-brain barrier. This was not due to decreased adhesion of propofol-treated human brain microvascular endothelial cells. The proteomic analysis revealed that some key pathways in these cells were disturbed, such as oxygen metabolism, DNA damage recognition and response to stress. Conclusions: Propofol has strong effects on protein expression which could explain several side effects of propofol