Innovative e-Learning Training Modules to Improve Animal Welfare during Transport and Slaughter of Pigs: A Pretest-Posttest Study to Pre-Evaluate the General Didactical Concept.

In addition to the information on the possession of a certificate of competence, there are no concrete obligations for repetitive training for personnel handling live animals at transport and slaughter. Deficiencies in the animal-welfare-friendly handling of pigs are known. The developed pilot modules "Handling of pigs" and "Electrical stunning" were tested in a pretest-posttest study in German and Romanian using questions of knowledge before and after the implementation of the modules. In this study, 45 and 46 datasets of participants could be analyzed. The mean percentages of correctly answered questions in the posttest increased by 5.6% in the module "Handling of pigs" and by 10.6% in the module "Electrical stunning". A significant interaction was found for the language match and trend categories in the module "Handling of pigs". No Romanian native speaker had a positive trend in this module. For both modules separately, participant education level significantly interacted with the language match and the presence or absence of a certificate of competence. Comparing the percentages of the correct given answers, significant interactions in the subgroups were more common in the module "Electrical stunning". One question in "Electrical stunning" was correctly answered significantly more often in the posttest. Because of the positive mean trends of knowledge within this pre-evaluation, we assume the didactical concept was suitable for our target groups. Holders of a certificate of competence also gave more correct answers in the post-test. This underlines the importance of repetitive training. Differences in the trends of knowledge gain seem to be topic and experience related.

Creating Veterinary Public Health Online Cases by Students for Students.

Online-based processing of case reports is often used and well accepted in veterinary medical education. However, lecturers usually develop cases from their own point of view, without input from students. In order to give students the chance to create online cases for students, an elective course Creative Workshop Case Creation, was held three times between 2017 and 2019 at the Faculty of Veterinary Medicine, Freie Universität Berlin. During this course, students created cases based on animal welfare and epizootics issues through a problem-based blended learning approach. In this approach, students worked on an assigned veterinary public health problem and actively solved it in small groups in class and then used the issue as the basis to create cases for their fellow students. The cases were implemented in interdisciplinary lectures, which are mandatory for every student in semesters six to eight. After taking these classes, fellow students evaluated one of these cases, specifically, on animal welfare and another one on epizootics. Evaluations showed these cases were received well. Moreover, we received excellent feedback from students participating in the elective course, and working with a proactive and motivated group of six students throughout the course was a very productive experience. The course made it possible to create cases that are more accurately tailored to the needs of students. The students' good ideas and preparatory work also saved time in the preparation of cases for lecturers.

Teaching Outbreak Investigations with an Interactive Blended Learning Approach.

Public health is a central but often neglected component of veterinary education. German veterinary public health (VPH) education includes substantial theory-focused lectures, but practical case studies are often missing. To change this, we combined the advantages of case-based teaching and blended learning to teach these topics in a more practical and interactive way. Blended learning describes the combination of online and classroom-based teaching. With it, we created an interdisciplinary module for outbreak investigations and zoonoses, based on the epidemiology, food safety, and microbiology disciplines. We implemented this module within the veterinary curriculum of the seventh semester (in the clinical phase of the studies). In this study, we investigated the acceptance of this interdisciplinary approach and established a framework for the creation of interactive outbreak investigation cases that can serve as a basis for further cases. Over a period of 3 years, we created three interactive online cases and one interactive in-class case and observed the student-reported evaluation of the blended learning concept and self-assessed learning outcomes. Results show that 80% (75-89) of students evaluated the chosen combination of case-based and blended learning for interdisciplinary teaching positively and therefore accepted it well. Additionally, 76% (70-98) of students evaluated their self-assessed learning outcomes positively. Our results suggest that teaching VPH through interdisciplinary cases in a blended learning approach can increase the quality of teaching VPH topics. Moreover, it provides a framework to incorporate realistic interdisciplinary VPH cases into the curriculum.

Students' acceptance of case-based blended learning in mandatory interdisciplinary lectures for clinical medicine and veterinary public health.

BACKGROUND: In German veterinary education interdisciplinary lectures (ILs) are an important and mandatory part of the curriculum as their merging character builds a useful preparation for the future profession as a veterinarian. These lectures should enable students to work on practically-relevant and interdisciplinary cases, which should ideally be defined jointly by lecturers from different disciplines. METHODS: In order to give students the opportunity to work on these cases and at the same time have contact with their lecturers and fellow students, the Faculty of Veterinary Medicine, Freie Universität Berlin, has converted its former in-class ILs (face-to-face delivery format) into a blended learning format. The mandatory lectures comprise 196 curricular hours and are delivered over the course of three semesters within the veterinary curriculum. The new concept was developed over a period of three academic years and extensively evaluated (old-new-comparison) with regard to its acceptance and compliance with national requirements for interdisciplinary teaching. RESULTS: A total of 306 students were asked to evaluate different aspects of the newly implemented format. Overall, more than 79% of the students attending the newly implemented blended learning format responded positively, and the evaluation showed a significant improvement of learning motivation and acceptance when compared to the traditional teaching format. CONCLUSION: The results indicated that blended learning is a suitable option for teaching mandatory ILs in clinical medicine and veterinary public health.

Teaching small animal reproduction via virtual patients.

Virtual patients have become an interesting alternative in medical education. Due to increasing demands regarding theoretical and clinical teaching and to improve an interdisciplinary approach, a new blended learning concept including virtual patients was developed and implemented in the veterinary curriculum of the Freie Universität Berlin. In the presented project, three virtual patients from the field of canine reproduction were developed. They focus on pregnancy diagnosis with suspected luteal insufficiency, pyometra and benign prostatic hyperplasia, respectively. The results of an evaluation by veterinary students of the 7th semester showed a high acceptance of virtual patients in a blended learning reproduction module in the interdisciplinary lectures. Students especially preferred videos, such as video lectures, hands-on videos and animations as well as a glossary for background information, to successfully and autonomously work on a virtual case. The content covered by the new modules that were developed in the context of this project is part of a spiral curriculum; they will be revised and enhanced during the clinical year.

Activation and molecular recognition of the GPCR rhodopsin--insights from time-resolved fluorescence depolarisation and single molecule experiments.

The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin.

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.

MeCP2 Rett mutations affect large scale chromatin organization.

Rett syndrome is a neurological, X chromosomal-linked disorder associated with mutations in the MECP2 gene. MeCP2 protein has been proposed to play a role in transcriptional regulation as well as in chromatin architecture. Since MeCP2 mutant cells exhibit surprisingly mild changes in gene expression, we have now explored the possibility that Rett mutations may affect the ability of MeCP2 to bind and organize chromatin. We found that all but one of the 21 missense MeCP2 mutants analyzed accumulated at heterochromatin and about half of them were significantly affected. Furthermore, two-thirds of all mutants showed a significantly decreased ability to cluster heterochromatin. Three mutants containing different proline substitutions (P101H, P101R and P152R) were severely affected only in heterochromatin clustering and located far away from the DNA interface in the MeCP2 methyl-binding domain structure. MeCP2 mutants affected in heterochromatin accumulation further exhibited the shortest residence time on heterochromatin, followed by intermediate binding kinetics for clustering impaired mutants. We propose that different interactions of MeCP2 with methyl cytosines, DNA and likely other heterochromatin proteins are required for MeCP2 function and their dysfunction lead to Rett syndrome.

3D-Image analysis platform monitoring relocation of pluripotency genes during reprogramming.

Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization. We performed a comparative analysis of the relationship between spatial positioning of pluripotency genes with their genomic activity and determined the degree of similarity between fibroblasts, induced pluripotent stem cells and embryonic stem cells. Our analysis revealed a preferred positioning of actively transcribed Sox2, Oct4 and Nanog away from the nuclear periphery, but not from pericentric heterochromatin. Moreover, in the silent state, we found no common nuclear localization for any of the genes. Our results suggest that the surrounding gene density hinders relocation from an internal nuclear position. Altogether, our data do not support the hypothesis that the nuclear periphery acts as a general transcriptional silencer, rather suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts. Thus, our computational approach enables comparative analysis of topological relationships in spite of stark morphological variability typical of biological data sets.

Fast live simultaneous multiwavelength four-dimensional optical microscopy.

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

Functional interaction structures of the photochromic retinal protein rhodopsin.

We studied functional interaction structures of the vertebrate membrane photoreceptor rhodopsin containing retinal as a chromophore. Using time-resolved fluorescence depolarization we analyzed real-time dynamics and conformational changes of the cytoplasmic helix 8 (H8) preceding the long C-terminal tail of rhodopsin. H8 runs parallel to the membrane surface and extends from transmembrane helix 7 whose highly conserved NPxxY(x)F motif connects that region of rhodopsin with the retinal binding pocket. Our measurements indicate that photo-induced retinal isomerization from 11-cis to all-trans provokes conformational changes of H8, including slower motion and reduced flexibility, that are specific for the active metarhodopsin-II photo-intermediate. These conformational changes are absent in the retinal-devoid state opsin and in the phosphorylated metarhodopsin-II state upon receptor deactivation. Furthermore we show that membrane rim effects can influence interfacial reactions at the cytoplasmic rhodopsin surface such as proton transfer reactions between surface and aqueous bulk phase or binding of the signaling protein transducin visualized with single-molecule widefield microscopy. These findings are important for an understanding of the effects of membrane structure on the photo-transduction mechanism.

Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.

AIDA: an adaptive image deconvolution algorithm with application to multi-frame and three-dimensional data.

We describe an adaptive image deconvolution algorithm (AIDA) for myopic deconvolution of multi-frame and three-dimensional data acquired through astronomical and microscopic imaging. AIDA is a reimplementation and extension of the MISTRAL method developed by Mugnier and co-workers and shown to yield object reconstructions with excellent edge preservation and photometric precision [J. Opt. Soc. Am. A21, 1841 (2004)]. Written in Numerical Python with calls to a robust constrained conjugate gradient method, AIDA has significantly improved run times over the original MISTRAL implementation. Included in AIDA is a scheme to automatically balance maximum-likelihood estimation and object regularization, which significantly decreases the amount of time and effort needed to generate satisfactory reconstructions. We validated AIDA using synthetic data spanning a broad range of signal-to-noise ratios and image types and demonstrated the algorithm to be effective for experimental data from adaptive optics-equipped telescope systems and wide-field microscopy.

Wavelet-based characterization of vertebral trabecular bone structure from magnetic resonance images at 3 T compared with micro-computed tomographic measurements.

Trabecular bone structure and bone density contribute to the strength of bone and are important in the study of osteoporosis. Wavelets are a powerful tool in characterizing and quantifying texture in an image. The purpose of this study was to validate wavelets as a tool in computing trabecular bone thickness directly from gray-level images. To this end, eight cylindrical cores of vertebral trabecular bone were imaged using 3-T magnetic resonance imaging (MRI) and micro-computed tomography (microCT). Thickness measurements of the trabecular bone from the wavelet-based analysis were compared with standard 2D structural parameters analogous to bone histomorphometry (MR images) and direct 3D distance transformation methods (microCT images). Additionally, bone volume fraction was determined using each method. The average difference in trabecular thickness between the wavelet and standard methods was less than the size of 1 pixel size for both MRI and microCT analysis. A correlation (R) of .94 for microCT measurements and that of .52 for MRI were found for the bone volume fraction. Based on these results, we conclude that wavelet-based methods deliver results comparable with those from established MR histomorphometric measurements. Because the wavelet transform is more robust with respect to image noise and operates directly on gray-level images, it could be a powerful tool for computing structural bone parameters from MR images acquired using high resolution and thus limited signal scenarios.