ß-Caryophyllene Inhibits Monoacylglycerol Lipase Activity and Increases 2-Arachidonoyl Glycerol Levels In Vivo: A New Mechanism of Endocannabinoid-Mediated Analgesia?

The mechanisms of ß-caryophyllene (BCP)-induced analgesia are not well studied. Here, we tested the efficacy of BCP in an acute postsurgical pain model and evaluated its effect on the endocannabinoid system. Rats were treated with vehicle and 10, 25, 50, and 75 mg/kg BCP. Paw withdrawal responses to mechanical stimuli were evaluated using an electronic von Frey anesthesiometer. Endocannabinoids, including 2-arachidonoylglycerol (2-AG), were also evaluated in plasma and tissues using high-performance liquid chromatography-tandem mass spectrometry. Monoacylglycerol lipase (MAGL) activity was evaluated in vitro as well as ex vivo. We observed a dose-dependent and time-dependent alleviation of hyperalgesia in incised paws up to 85% of the baseline value at 30 minutes after administration of BCP. We also observed dose-dependent increases in the 2-AG levels of about threefold after administration of BCP as compared with vehicle controls. Incubations of spinal cord tissue homogenates from BCP-treated rats with isotope-labeled 2-arachidonoylglycerol-d8 revealed a reduced formation of the isotope-labeled MAGL product 2-AG-d8 as compared with vehicle controls, indicating MAGL enzyme inhibition. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 15.8 µM for MAGL inhibition using BCP. These data showed that BCP inhibits MAGL activity in vitro and in vivo, causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose that 2-AG-mediated cannabinoid receptor activation contributes to BCP's mechanism of analgesia. SIGNIFICANCE STATEMENT: ß-Caryophyllene (BCP) consumption is relatively safe and is approved by the Food and Drug Administration as a flavoring agent, which can be used in cosmetic and food additives. BCP is a potent anti-inflammatory agent that showed substantial antihyperalgesic properties in this study of acute pain suggesting that BCP might be an alternative to opioids. This study shows an additive mechanism (monoacylglycerol lipase inhibition) by which BCP might indirectly alter CB1 and CB2 receptor activity and exhibit its pharmacological properties.

Clostridium botulinum C3bot mediated effects on cytokine-induced psoriasis-like phenotype in full-thickness skin model.

Clostridium botulinum C3 exoenzyme (C3bot) exclusively inhibits RhoA, B and C by ADP-ribosylation and is therefore used as a cell-permeable tool for investigating the cellular role of these Rho-GTPases. Rho-GTPases represent a molecular switch integrating different receptor signalling to downstream cascades including transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton and cell proliferation. C3bot-induced inhibition of RhoA leads to reorganization of the actin cytoskeleton, morphological changes, and inhibition of cell proliferation as well as modulation of inflammatory response. In this study, we characterized the C3bot-mediated effects on a full-thickness skin model exhibiting a psoriasis-like phenotype through the addition of cytokines. Indeed, after the addition of cytokines, a decrease in epidermal thickness, parakeratosis, and induction of IL-6 was detected. In the next step, it was studied whether C3bot caused a reduction in the cytokine-induced psoriasis-like phenotypes. Basal addition of C3bot after cytokine induction of the full-thickness skin models caused less epidermal thinning and reduced IL-6 abundance. Simultaneous basal incubation with cytokines and C3bot, IL-6 abundance was inhibited, but epidermal thickness was only moderately affected. When C3bot was added apically to the skin model, IL-6 abundance was reduced, but no further effects on the psoriasis-like phenotype of the epidermis were observed. In summary, C3bot inhibits the cytokine-induced expression of IL-6 and thus may have an impact on the pro-inflammatory immune response in the psoriasis-like phenotype.

Evaluation of the National Competence Based Catalogue of Learning Objectives (NKLM 2.0) for undergraduate medical education at the Medical School Hannover.

Background: The National Competence-Based Catalogue of Learning Objectives for Undergraduate Medical Education (NKLM) serves as the foundation for curricular development in undergraduate medical education in Germany. A new version of the NKLM was launched in 2021, and medical faculties are now evaluating the learning objectives (LOs). This paper describes the evaluation process used at Hannover Medical School. Methods: The evaluation process was structured in three steps. LOs were rated as "keep", "modify" or "delete". First, the 1133 LOs were compared with the mapping of the Hannover curriculum from 2017. Then, a small team from the Curricular Development Department conducted a pre-evaluation of the 1133 LOs. Finally, a group of clinical experts and students discussed and agreed on the ratings. Results: For 868 LOs, one or more counterparts were found in the mapping, but 265 new LOs were not found and thus, classified as new. In the first rating, 779 LOs were kept, 300 were modified (172 due to wording), 45 were deleted, and there was no rating for 9 LOs. The expert group changed 47 of the pre-evaluation decisions. The final rating was to keep 738 LOs, modify 356, and delete 39 LOs. Conclusion: This method effectively evaluated the LOs from NKLM 2.0 while balancing expert knowledge and an overview of the curriculum.

Corrigendum: Potentiation of brain-derived neurotrophic factor-induced protection of spiral ganglion neurons by C3 exoenzyme/Rho inhibitor.

[This corrects the article DOI: 10.3389/fncel.2021.602897.].

Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide.

In primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot156-181 peptide resulted in the upregulation of glutamate uptake by up to 22% compared to untreated cells. A similar enhancement of glutamate transport was also achieved by the classical phorbol-ester-mediated activation of protein kinase C subtypes. Yet comparable, effects elicited by C3 preparations seemed not to rely on PKCα, γ, ε, or ζ activation. Blocking of tyrosine phosphorylation by tyrosine kinase inhibitors prevented the observed effect mediated by C3bot and C3bot 26mer. By using biochemical and molecular biological assays we could rule out that the observed C3bot and C3bot 26mer-mediated effects solely resulted from enhanced transporter expression or translocation to the neuronal surface but was rather mediated by transporter phosphorylation at tyrosine residues that was found to be significantly enhanced following incubation with either full length protein or the 26mer C3 peptide.

Potentiation of Brain-Derived Neurotrophic Factor-Induced Protection of Spiral Ganglion Neurons by C3 Exoenzyme/Rho Inhibitor.

Preservation of the excitability of spiral ganglion neurons (SGN) may contribute to an improved speech perception after cochlear implantation. Thus, the application of exogenous neurotrophic factors such as the neurotrophin brain-derived neurotrophic factor (BDNF) to increase SGN survival in vitro and in vivo is a promising pharmacological approach in cochlear implant (CI) research. Due to the difficult pharmacokinetic profile of proteins such as BDNF, there is a quest for small molecules to mediate the survival of SGN or to increase the efficacy of BDNF. The C3 exoenzyme from Clostridium botulinum could be a potential new candidate for the protection and regeneration of SGN. Inhibition of the RhoA GTPase pathway which can be mediated by C3 is described as a promising strategy to enhance axonal regeneration and to exert pro-survival signals in neurons. Nanomolar concentrations of C3, its enzymatically inactive form C3E174Q, and a 26mer C-terminal peptide fragment covering amino acid 156-181 (C3156-181) potentiated the neuroprotective effect on SGN mediated by BDNF in vitro. The neuroprotective effect of C3/BDNF was reduced to the neuroprotective effect of BDNF alone after the treatment with wortmannin, an inhibitor of the phosphatidylinositol-3-kinase (PI3K).The exoenzyme C3 (wild-type and enzyme-deficient) and the C3 peptide fragment C3154-181 present novel biologically active compounds for the protection of the SGN. The exact underlying intracellular mechanisms that mediate the neuroprotective effect are not clarified yet, but the combination of BDNF (TrkB stimulation) and C3 exoenzyme (RhoA inhibition) can be used to protect SGN in vitro.

Classroom teaching with simulated patients during COVID-19: the communication skills course in the second year of the model medical curriculum HannibaL.

Objective: In the spring of 2020 in response to the COVID-19 pandemic, the question arose at Hannover Medical School as to how simulated patients (SP) could still be utilized in the communication course that is part of the module "Diagnostic methods" taught in the second year of the model medical curriculum known as HannibaL. Methods: This short report summarizes the process of implementing the utilization of SP in analog classroom teaching and describes the relevant results on the concluding Objective Structured Clinical Examination (OSCE) in comparison to the previous year. Results: Overall, the analog SP deployments were practicable under local conditions and in compliance with precautionary measures to curb the risk of infection, whereby the OSCE scores did not deviate significantly from those in the prior year. Conclusion: During the COVID-19 pandemic and perhaps other epidemics as well, it will continue to be important in the future to make locally adapted, purpose-oriented, and preventively effective decisions regarding university didactics in undergraduate studies.

The Higher Sensitivity of GABAergic Compared to Glutamatergic Neurons to Growth-Promoting C3bot Treatment Is Mediated by Vimentin.

The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of ß1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects.

HannibaL - the model curriculum at Hannover Medical School: targets, implementation and experiences.

Aim: The model curriculum known as HannibaL is an integrated, professionally-based adaptive curriculum that began at the Hannover Medical School (MHH) during the 2005/06 academic year. HannibaL turns medical students into competent physicians through its patient-based interdisciplinary instruction. This paper provides an overview of the curriculum's creation, educational content and philosophy and reflects on the experience that has been gathered. Also described are organizational and quality assurance measures which were also employed to implement the model curriculum. Method: The central ideas and processes are reported in a primarily narrative manner in an attempt to present the information coherently. The aspects discussed are setting up the model curriculum, central features of teaching and exams with their underlying educational premises; organization and evaluation are also covered in the context of the research literature on curriculum and faculty development. Developing the teaching and learning culture of the model curriculum is also explored. Results: The basic objectives were realized, including the design of learning spirals and intensifying the inclusion of patients and practical elements at the beginning of study. However, plans to allow students more freedom to pursue their own learning and research interests have not yet been satisfactorily implemented. Key areas to support teaching have been expanded (teacher training for instructors, student advising, course evaluations). Conclusion: The model curriculum and its aims are widely recognized and supported not only by medical students and instructors, but also external committees and experts. As a consequence, HannibaL will be developed further in upcoming years to implement the objectives which have not yet been met and to master new challenges faced by medical education.

[Examination facility at Hannover Medical School]. / Prüfungen an der Medizinischen Hochschule Hannover ­ Wie kommt man vom Ist zum Soll?

Dissection of the examination system of Hannover Medical School has identified potential for improvement of the complete procudere. Five scopes has been identified: 1. advancement of electronic examinations, 2. improvement of quality control, 3. central management of all exams, 4. more transperancy, 5. establishment of an incentive structure. The strategies for improvement were presented.

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons.

Clostridium botulinum C3 transferase (C3bot) ADP-ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild-type with Vim-/- mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP-ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin-binding RGD motif (C3bot-G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross-talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild-type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim-/- astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.

Evidence for an Adaptation of a Phage-Derived Holin/Endolysin System to Toxin Transport in Clostridioides difficile.

The pathogenicity locus (PaLoc) of Clostridioides difficile usually comprises five genes (tcdR, tcdB, tcdE, tcdA, tcdC). While the proteins TcdA and TcdB represent the main toxins of this pathogen, TcdR and TcdC are involved in the regulation of their production. TcdE is a holin family protein, members of which are usually involved in the transport of cell wall-degrading enzymes (endolysins) for phage-induced lysis. In the past, TcdE has been shown to contribute to the release of TcdA and TcdB, but it is unclear whether it mediates a specific transport or rather a lysis of cells. TcdE of C. difficile strains analyzed so far can be produced in three isoforms that are initiated from distinct N-terminal ATG codons. When produced in Escherichia coli, we found that the longest TcdE isoform had a moderate effect on cell growth, whereas the shortest isoform strongly induced lysis. The effect of the longest isoform was inhibitory for cell lysis, implying a regulatory function of the N-terminal 24 residues. We analyzed the PaLoc sequence of 44 C. difficile isolates and found that four of these apparently encode only the short TcdE isoforms, and the most closely related holins from C. difficile phages only possess one of these initiation codons, indicating that an N-terminal extension of TcdE evolved in C. difficile. All PaLoc sequences comprised also a conserved gene encoding a short fragment of an endolysin remnant of a phage holin/endolysin pair. We could produce this peptide, which we named TcdL, and demonstrated by bacterial two-hybrid analysis a self-interaction and an interaction with TcdB that might serve to mediate TcdE-dependent transport.

The impact of the program structure at Hannover Medical School on academic success in medical studies.

Aim: The classical course structure for medicine in Germany is separated by three sections of the medical state examination. This structure is generally regarded as sensible and unchangeable. Because the special program structure at Hannover Medical School (MHH) has one integrated, rather than two separate study blocks, it is possible to examine the influence of structural modifications on the study success of different admission groups. Methods: The data was obtained from students admitted to the MHH between 2006 and 2008 in different admission quotas. Study success was defined as the successful completion of the entire program, but completion of the first section of the state examination was also analysed. Results: More students from the best "Abitur" (school leaving examinations) quota successfully completed their studies than those accepted via the selection process of the universities. The latter were more successful than students from the waiting list quota. However the successful graduates of this last group completed their studies more often within the prescribed period of study, although they needed more time for completing all parts of the first section of the state examination. Conclusion: The data shows that an integrated course structure can offer, in particular, students from the waiting list quota, the opportunity to compensate for delays in the first years of study. However, they do not provide any evidence which applicants are best suited to meet the social and professional requirements of trained doctors. Due to the complex structure of such longitudinal studies, our results allow more than one plausible interpretation.

MS-based quantification of RhoA/B and RhoC ADP-ribosylation.

Mono ADP-ribosylation is a common characteristic of bacterial toxins resulting to aberrant activation or inactivation of target proteins. The C3 exoenzyme of Clostridium botulinum (C3bot) ADP-ribosylates the small GTPases RhoA, RhoB and RhoC, leading to inactivation of these important regulators and impaired down-stream signaling. Quantification of ADP-ribosylation using gel migration assays, antibodies, and radioactivity-based methods are limited. Therefore a novel LC-MS-based method to specifically determine and quantify ADP-ribosylation of Rho GTPases was established. A heavy labeled protein standard that contained ADP-ribosylation specific peptides in similar amounts in ADP ribosylated and non ADP ribosylated form was used for relative quantification in vivo. In a proof of principle experiment HT22 cells were treated with C3bot and the kinetics of RhoA/B and RhoC ADP-ribosylation were determined in vivo.

Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018.

Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin.

Quantitative Phosphoproteome Analysis of Clostridioides difficile Toxin B Treated Human Epithelial Cells.

The large clostridial glucosylating toxin B (TcdB) is a major virulence factor of the nosocomial pathogen Clostridioides difficile. TcdB inhibits small GTPases by glucosylation leading to impaired downstream signaling. TcdB also possesses a glucosyltransferase independent effect described as pyknosis. To elucidate the impact of TcdB and its glucosylation-inactive mutant TcdBNXN on the kinome of human cells, SILAC labeled HEp-2 cells were treated with 2 nM TcdB for 8 h. Phosphopeptides were enriched using SCX chromatography, IMAC and TiO2 followed shotgun mass spectrometry analysis. Overall 4,197 phosphopeptides were identified; more than 1,200 phosphosites responded to treatment with TcdB or TcdBNXN. The data suggested that predominantly stress-activated MAPK-dependent signaling pathways were triggered by toxin B treatment.

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ß1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.

Glucosyltransferase-dependent and -independent effects of TcdB on the proteome of HEp-2 cells.

Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase-dependent and -independent effect on treated HEp-2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase-deficient TcdBNXN and their proteomes were analyzed by LC-MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdBNXN treatment and 49 by both. TcdB mainly led to changes in proteins that are related to "GTPase mediated signaling" and the "cytoskeleton" while "chromatin" and "cell cycle" related proteins were altered by both, TcdB and TcdBNXN . The obtained dataset of HEp-2 cell proteome helps us to better understand glucosyltransferase-dependent and -independent mechanisms of TcdB and TcdBNXN , particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 (https://proteomecentral.proteomexchange.org/dataset/PXD006658).