Variations in pre-analytical FFPE sample processing and bioinformatics: challenges for next generation molecular diagnostic testing in clinical pathology

Advances in cellular pathology techniques will improve diagnostic medicine. However, such improvements have to overcome many challenges including variations in pre-analytical sample processing, bioinformatics data analysis and clinical interpretation of data. In order to resolve such challenges, bioinformatics needs to become more tightly coupled to the experimental methodology development

Insights into Exosome Transport through the Blood–Brain Barrier and the Potential Therapeutical Applications in Brain Diseases

Drug delivery to the central nervous system (CNS) is limited due to the presence of the blood–brain barrier (BBB), a selective physiological barrier located at the brain microvessels that regulates the flow of cells, molecules and ions between the blood and the brain. Exosomes are nanosized extracellular vesicles expressed by all cell types and that function as cargos, allowing for communication between the cells. The exosomes were shown to cross or regulate the BBB in healthy and disease conditions. However, the mechanistic pathways by which exosomes cross the BBB have not been fully elucidated yet. In this review, we explore the transport mechanisms of exosomes through the BBB. A large body of evidence suggests that exosome transport through the BBB occurs primarily through transcytosis. The transcytosis mechanisms are influenced by several regulators. Inflammation and metastasis also enhance exosome trafficking across the BBB. We also shed light on the therapeutical applications of exosomes for treating brain diseases. Further investigations are essential to provide clearer insights related to trafficking of exosomes across the BBB and disease treatment

Protagonisten und Antagonisten : Eine Studie über Schillers Tragödientheorie und ihre Verwendung in drei seiner Dramen ; Die Räuber, Maria Stuart und Die Braut von Messina

Es handelt sich bei dieser Dissertation um die Tragödientheorie von Friedrich Schiller. Die Arbeit ist in einer Einleitung, drei Kapiteln und einem Schlusswort untertitelt. In der Einleitung behandle ich die verschiedenen Theorien und Philosophien von Aristoteles über Schakespear, die französischen Klassiker, Kant bis hin zur Tragödientheorie von Schiller. Im ersten Kapitel analysiere ich das Drama "Die Räuber" insbesondere die Charaktere Karl und Franz Moor, die gestörte Vaterwelt und die religiösen Anspielungen im Drama. Im zweiten Kapitel behandle ich das Drama "Maria Stuart". Neben einer Analyse der beiden Hauptcharaktere (Elisabeth und Maria) behandle ich die Begegnung der beiden Königinnen und versuche am Ende zu zeigen, warum und wie Maria als eine erhabene Figur und eine shcöne Seele gilt. Im dritten Kapitel behandle ich das Drama "Die Braut von Messina". Hier erforsche ich die Rolle des Chores im Drama und versuche zu zeigen, dass Don Cesar keine erhabene Figur ist. Im Schlusswort legte ich alle Bemerkungen und Schlussfolgerungen, die ich gesammelt habe, dar

Identifying Patterns of Breast Cancer Genetic Signatures using Unsupervised Machine Learning

Deploying machine learning to improve medical diagnosis is a promising area. The purpose of this study is to identify and analyze unique genetic signatures for breast cancer grades using publicly available gene expression microarray data. The classification of cancer types is based on unsupervised feature learning. Unsupervised clustering use matrix algebra based on similarity measures which made it suitable for analyzing gene expression. The main advantage of the proposed approach is the ability to use gene expression data from different grades of breast cancer to generate features that automatically identify and enhance the cancer diagnosis. In this paper, we tested different similarity measures in order to find the best way that identifies the sets of genes with a common function using expression microarray data

miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function

The Effect of Melatonin Treatment and Exposure to Continuous Darkness on the Reactivity of Smooth Muscles to Drugs in Rats

Melatonin is the major secretory product of the pineal gland, playing an integral role in numerous metabolic, physiological and behavioral processes. This study represents an attempt to understand the effect of environmental changes on the responses of a selected isolated tissue to pharmacologically active substances in certain species which may lead to important experimental and clinical implications in future. Twenty male albino wistar rats were divided into three experimental groups: Group 1: Control animals, comprising 6 rats were exposed to 12:12 hour light: dark cycle for 4 weeks. Group 2: Composed of 8 rats, was kept in a dark environment for 4 weeks. Group 3: Consisted of 6 rats aged 6-7 weeks, subjected to continuous darkness for 2 weeks, at the end of the first week the rats treated daily with s.c melatonin injection at 1.00 p.m. for 7 consecutive days. The result of this study showed that the vas deferens preparation from the third group reacted with significant increase in reactivity to nor-adrenaline and 5-HT than that of control and continuous dark animals. The limitation of time and shortage of materials may be blamed for the kind and quantity of results obtained and the conclusions made. Therefore, further studies with regard to the exposure period have to be undertaken in future to extensively elaborate on this point

Systems Immunology Analysis Reveals an Immunomodulatory Effect of Snail-p53 Binding on Neutrophil- and T Cell-Mediated Immunity in KRAS Mutant Non-Small Cell Lung Cancer

Immunomodulation and chronic inflammation are important mechanisms utilized by cancer cells to evade the immune defense and promote tumor progression. Therefore, various efforts were focused on the development of approaches to reprogram the immune response to increase the immune detection of cancer cells and enhance patient response to various types of therapy. A number of regulatory proteins were investigated and proposed as potential targets for immunomodulatory therapeutic approaches including p53 and Snail. In this study, we investigated the immunomodulatory effect of disrupting Snail-p53 binding induced by the oncogenic KRAS to suppress p53 signaling. We analyzed the transcriptomic profile mediated by Snail-p53 binding inhibitor GN25 in non-small cell lung cancer cells (A549) using Next generation whole RNA-sequencing. Notably, we observed a significant enrichment in transcripts involved in immune response pathways especially those contributing to neutrophil (IL8) and T-cell mediated immunity (BCL6, and CD81). Moreover, transcripts associated with NF-κB signaling were also enriched which may play an important role in the immunomodulatory effect of Snail-p53 binding. Further analysis revealed that the immune expression signature of GN25 overlaps with the signature of other therapeutic compounds known to exhibit immunomodulatory effects validating the immunomodulatory potential of targeting Snail-p53 binding. The effects of GN25 on the immune response pathways suggest that targeting Snail-p53 binding might be a potentially effective therapeutic strategy

Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections

To investigate intracellular heterogeneity, cell capture of particular cell populations followed by transcriptome analysis has been highly effective in freshly isolated tissues. However, this approach has been quite challenging in immunostained formalin-fixed paraffin-embedded (FFPE) sections. This study aimed at combining the standard pathology techniques, immunostaining and laser capture microdissection, with whole RNA-sequencing and bioinformatics analysis to characterize FFPE breast cancer cell populations with heterogeneous expression of progesterone receptor (PR). Immunocytochemical analysis revealed that 60% of MCF-7 cells admixture highly express PR. Immunocytochemistry-based targeted RNA-seq (ICC-RNAseq) and in silico functional analysis revealed that the PR-high cell population is associated with upregulation in transcripts implicated in immunomodulatory and inflammatory pathways (e.g. NF-κB and interferon signaling). In contrast, the PR-low cell population is associated with upregulation of genes involved in metabolism and mitochondrial processes as well as EGFR and MAPK signaling. These findings were cross-validated and confirmed in FACS-sorted PR high and PR-low MCF-7 cells and in MDA-MB-231 cells ectopically overexpressing PR. Significantly, ICC-RNAseq could be extended to analyze samples captured at specific spatio-temporal states to investigate gene expression profiles using diverse biomarkers. This would also facilitate our understanding of cell population-specific molecular events driving cancer and potentially other diseases