Discovery of (S)-3′-hydroxyblebbistatin and (S)-3′-aminoblebbistatin : polar myosin II inhibitors with superior research tool properties

In search of myosin II inhibitors with superior research tool properties, a chemical optimization campaign of the blebbistatin scaffold was conducted in this paper. (S)-Blebbistatin is the best known small-molecule inhibitor of myosin II ATPase activity. Unfortunately, as a research tool this compound has several deficiencies: it is photolabile and (photo) toxic, has low water solubility, and its (fluorescent) precipitates interfere in (fluorescence) readouts. In view of obtaining tool compounds with improved properties, both enantiomers of a series of D-ring modified polar analogs were prepared. We identified (S)-3'-hydroxyblebbistatin (S)-2 and (S)-3'-aminoblebbistatin (S)-3 as two myosin II inhibitors with a 30-fold higher water solubility than (S)-blebbistatin. These molecules furthermore do not cause interference in (fluorescence) readouts. (S)-2 and (S)-3 thus are superior alternatives to (S)-blebbistatin as research tools to study myosin II

Sputum Induction in Children Is Feasible and Useful in a Bustling General Hospital Practice

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The Jeroen Bosch Hospital funded this study.Peer reviewedPublisher PD

Evaluation of local tolerance of a plant extract by the slug mucosal irritation (SMI) assay

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 facilitate scientific discussions, but the majority of the ideas had to come from the students themselves. Hence, this biomedical in vivo experiment offered a great opportunity for students to learn to work in group, to critically interpret and report their results, to gain knowledge about the subject, and to communicate and discuss with other students as well as with the lab instructors. Furthermore, this experiment teaches students current toxicological methodologies encompassing principles and their application of biochemistry, analytical chemistry, toxicology, animal experimentation and data handling. This way of interdisciplinary teaching is especially important for last year undergraduate students, as this is a good preparation for the Masters dissertation. At the end of the laboratory exercise, students received a questionnaire and most of the students indicated that they gained valuable knowledge and skills. This laboratory exercise can be incorporated into chemical, biological, pharmaceutical, toxicological and medical disciplines

Evaluation of local tolerance of a plant extract by the slug mucosal irritation (SMI) assay

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 facilitate scientific discussions, but the majority of the ideas had to come from the students themselves. Hence, this biomedical in vivo experiment offered a great opportunity for students to learn to work in group, to critically interpret and report their results, to gain knowledge about the subject, and to communicate and discuss with other students as well as with the lab instructors. Furthermore, this experiment teaches students current toxicological methodologies encompassing principles and their application of biochemistry, analytical chemistry, toxicology, animal experimentation and data handling. This way of interdisciplinary teaching is especially important for last year undergraduate students, as this is a good preparation for the Masters dissertation. At the end of the laboratory exercise, students received a questionnaire and most of the students indicated that they gained valuable knowledge and skills. This laboratory exercise can be incorporated into chemical, biological, pharmaceutical, toxicological and medical disciplines

Microglial activation arises after aggregation of phosphorylated-tau in a neuron-specific P301S tauopathy mouse model

Alzheimer's disease, progressive supranuclear palsy and frontotemporal dementia are characterized by neuronal expression of aberrant tau protein, tau hyperphosphorylation (pTAU), tau aggregation and neurofibrillary tangle formation sequentially culminating into neuronal cell death, a process termed tauopathy. Our aim was to address at which tauopathy stage neuroinflammation starts and to study the related microglial phenotype. We used Thy1-hTau.P301S (PS) mice expressing human tau with a P301S mutation specifically in neurons. Significant levels of cortical pTAU were present from 2 months onwards. Dystrophic morphological complexity of cortical microglia arose after pTAU accumulation concomitant with increased microglial lysosomal volumes and a significant loss of homeostatic marker Tmem119. Interestingly, we detected increases in neuronal pTAU and postsynaptic structures in the lysosomes of PS microglia. Moreover, the overall cortical postsynaptic density was decreased in 6-month-old PS mice. Together, our results indicate that microglia adopt a pTAU-associated phenotype, and are morphologically and functionally distinct from wild-type microglia after neuronal pTAU accumulation has initiated

Retrospective analysis of the Draize test for serious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN GHS/EU CLP for the development and evaluation of in vitro test methods

For more than two decades, scientists have been trying to replace the regulatory in vivo Draize eye test by in vitro methods, but so far only partial replacement has been achieved. In order to better understand the reasons for this, historical in vivo rabbit data were analysed in detail and resampled with the purpose of (1) revealing which of the in vivo endpoints are most important in driving United Nations Globally Harmonized System/European Union Regulation on Classification, Labelling and Packaging (UN GHS/EU CLP) classification for serious eye damage/eye irritation and (2) evaluating the method’s within-test variability for proposing acceptable and justifiable target values of sensitivity and specificity for alternative methods and their combinations in testing strategies. Among the Cat 1 chemicals evaluated, 36–65 % (depending on the database) were classified based only on persistence of effects, with the remaining being classified mostly based on severe corneal effects. Iritis was found to rarely drive the classification (<4 % of both Cat 1 and Cat 2 chemicals). The two most important endpoints driving Cat 2 classification are conjunctiva redness (75–81 %) and corneal opacity (54–75 %). The resampling analyses demonstrated an overall probability of at least 11 % that chemicals classified as Cat 1 by the Draize eye test could be equally identified as Cat 2 and of about 12 % for Cat 2 chemicals to be equally identified as No Cat. On the other hand, the over-classification error for No Cat and Cat 2 was negligible (<1 %), which strongly suggests a high over-predictive power of the Draize eye test. Moreover, our analyses of the classification drivers suggest a critical revision of the UN GHS/EU CLP decision criteria for the classification of chemicals based on Draize eye test data, in particular Cat 1 based only on persistence of conjunctiva effects or corneal opacity scores of 4. In order to successfully replace the regulatory in vivo Draize eye test, it will be important to recognise these uncertainties and to have in vitro tools to address the most important in vivo endpoints identified in this paper.JRC.I.5-Systems Toxicolog

The transcriptome of lung tumor-infiltrating dendritic cells reveals a tumor-supporting phenotype and a microRNA signature with negative impact on clinical outcome

Targeting immunomodulatory pathways has ushered a new era in lung cancer therapy. Further progress requires deeper insights into the biology of immune cells in the lung cancer micro-environment. Dendritic cells (DCs) represent a heterogeneous and highly plastic immune cell system with a central role in controlling immune responses. The intratumoral infiltration and activation status of DCs are emerging as clinically relevant parameters in lung cancer. In this study, we used an orthotopic preclinical model of lung cancer to dissect how the lung tumor micro-environment affects tissue-resident DCs and extract novel biologically and clinically relevant information. Lung tumor-infiltrating leukocytes expressing generic DC markers were found to predominantly consist of CD11b(+) cells that, compare with peritumoral lung DC counterparts, strongly overexpress the T-cell inhibitory molecule PD-L1 and acquire classical surface markers of tumor-associated macrophages (TAMs). Transcriptome analysis of these CD11b(+) tumor-infiltrating DCs (TIDCs) indicates impaired antitumoral immunogenicity, confirms the skewing toward TAM-related features, and indicates exposure to a hypoxic environment. In parallel, TIDCs display a specific microRNA (miRNA) signature dominated by the prototypical lung cancer oncomir miR-31. In vitro, hypoxia drives intrinsic miR-31 expression in CD11b(+) DCs. Conditioned medium of miR-31 overexpressing CD11b(+) DCs induces pro-invasive lung cancer cell shape changes and is enriched with pro-metastatic soluble factors. Finally, analysis of TCGA datasets reveals that the TIDC-associated miRNA signature has a negative prognostic impact in non-small cell lung cancer. Together, these data suggest a novel mechanism through which the lung cancer micro-environment exploits the plasticity of the DC system to support tumoral progression