Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

BACKGROUND: The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer. RESULTS: With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively. CONCLUSIONS: Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells

Etablierung adenoviraler Genexpression zum antifibrotischen Einsatz bei der Leberfibrose

Liver fibrosis results from an imbalance between synthesis and degradation of extracellular matrix components due to a variety of deseases such as virus hepatitis, alcoholic abuse or metabolic dysfunction. The cytokine TGF-ß plays an important role in so far it interferes during hepatic injury with the transcriptional regulation of genes which are responsible for the production and degradation of matrix proteins. A simplified model to investigate liver fibrosis in vitro is the spontaneous activation of cultured hepatic stellate cells (HSC) on a plastic surface to myofibroblasts (MFB) which synthesize the components of the connective tissue. This process of transdifferentiation plays the key role in developing liver fibrosis. In order to prevent proceeding liver fibrogenesis it may be useful to interact with the gene expression of HSC and MFB. The use of adenoviral-mediated gene transfer proved that heterologic genes could be introduced into these cells. A protein cassette is inserted into an E1 – region-deleted adenoviral type 5 genome which now expresses recombinant, replication-deficient adenoviruses. Enhanced green fluorescent protein (EGFP) was used as a reporter construct under the transcriptional control of the human cytomegalovirus promotor (CMV). Both cell types could completely be infected in cell culture by this adenoviral construct. The morphological integrity of adenovirus was shown by electronic microscopy. In order to use this gene transfer system in vitro against liver fibrosis the reporter gene GFP was replaced by a protein with antifibrotic potential, a soluble TGF-ß type II receptor which is fusioned with the Fc-region of human immunogobuline IgG. The CMV-promotor was retained. The expression of this receptor shall interrupt the signal cascade of TGF-ß, thus inhibiting more fibrotic effects by TGF-ß. The existence of this chimeric fusion protein was assessed by Northern Blot, Western Blot and immunoprecipitation, the biological activity was confirmed by proliferation assay

An IIoT-Device for Acquisition and Analysis of High-Frequency Data Processed by Artificial Intelligence

This publication presents the development of an Industrial-Internet-of-Things device. The device is capable of completing several tasks, such as the acquisition of high-frequency measurement data and evaluating data via machine learning methods in an artificial intelligence application. The installed measurement technology generates data which is comparable to data generated by costly laboratory equipment, meaning that it can be used as a low-budget and open-source alternative. A workflow method has been designed that promotes experimental work and simplifies the effort required to implement artificial intelligence solutions. At the end of this paper, the results of the experiment, which aimed to collect measurement data, extract suitable features, and train artificial intelligence models, are presented. Techniques from vibration analysis were used for feature extraction, and concepts for the extrapolation and enhancement of data sets were investigated. The test results have proven that the development is comparable with high-end laboratory equipment. The created application has demonstrated sufficient accuracy in predictions, and the designed process can be used for arbitrary, artificial intelligence-based rapid prototyping

Struggle in the bubble - a prospective study on the effect of remote learning and distance education on confidence in practical surgical skills acquired during COVID-19

Abstract Background The coronavirus disease (COVID-19) has significantly changed healthcare systems and medical education. Universities were required to develop innovative curricula based on remote and distance education to continue medical education. This prospective questionnaire-based study aimed to investigate the impact of COVID-19-associated remote learning on the surgical training of medical students. Methods A 16-item questionnaire-based survey was distributed to medical students at the University Hospital of Münster before and after a surgical skills laboratory (SSL). Two cohorts were included: summer semester 2021 (COV-19), with rigorous social-distancing restrictions requiered SSL to be remotely, and winter semester 2021 (postCOV-19), in which the SSL was provided as a face-to-face, hands-on course. Results Both, cohorts showed a significant improvement in self-assessment of pre- and post-course confidence. While no significant difference in the average gain in self-confidence for sterile working was observed between the two cohorts, improvement in self-confidence was significantly higher in the COV-19 cohort regarding skin suturing and knot tying (p < 0.0001). However the average improvement regarding history and physical was significantly higher in the postCOV-19 cohort (p < 0.0001). In subgroup analysis, gender-associated differences varied in the two cohorts and were not related to specific subtasks, while age-stratified analysis revealed superior results for younger students. Conclusion The results of our study underline the usability, feasibility, and adequacy of remote learning for the surgical training of medical students. The on-site distance education version, presented in the study, allows the continuing of hands-on experience in a safe environment in compliance with governmental social-distancing restrictions