Gene Expression Analysis in Four Dogs With Canine Pemphigus Clinical Subtypes Reveals B Cell Signatures and Immune Activation Pathways Similar to Human Disease

Pemphigus is a group of autoimmune-mediated mucocutaneous blistering diseases characterized by acantholysis. Pemphigus has also been recognized in dogs and shares similar clinical characteristics and variants with human pemphigus. While relationships between human and canine pemphigus have been reported, gene expression patterns across species have not been described in the literature. We sought to perform gene expression analysis of lesional skin tissue from four dogs with various forms of pemphigus to examine gene expression during spontaneous disease in dogs. We found increased T and B cell signatures in canine pemphigus lesions compared to controls, as well as significant upregulation of CCL3, CCL4, CXCL10, and CXCL8 (IL8), among other genes. Similar chemokine/cytokine expression patterns and immune infiltrates have been reported in humans, suggesting that these genes play a role in spontaneous disease. Direct comparison of our dataset to previously published human pemphigus datasets revealed five conserved differentially expressed genes: CD19, WIF1, CXCL10, CD86, and S100A12. Our data expands our understanding of pemphigus and facilitates identification of biomarkers for prediction of disease prognosis and treatment response, which may be useful for future veterinary and human clinical trials.

The Role of Thyroid Hormone 3,3 ‘, 5 - Triiodothyronine (T3) in the Expression of Osteocalcin and Lipocalin 2 (LCN2) in Osteoblasts

Introduction: The thyroid hormone 3,3 ‘, 5 - triiodothyronine (T3) has an important role in bone physiology and metabolism, stimulating osteodifferentiation and bone homeostasis. Although there is this evidence, little is known about the synergistic events of T3 together with the endocrine role of other players that interfere with osteoblast metabolism, such as Lipocalin 2 (LCN2) and its hypothalamic-located MCR4 receptor. It is also known that LCN2 interferes at a physiological level with the parameters of food intake. Objective: To evaluate the role of the thyroid hormone 3,3 ‘, 5 - Triiodothyronine (T3) in the control of the expression of both Osteocalcin (OCN) and Lipocalin 2 (LCN2) in osteoblasts. Materials and Methods:. In order to understand the molecular and physiological mechanisms about that probable synergism, the experiments were as follows: Mouse pre-osteoblasts were challenged with T3 hormone treatments in three levels (1nM; 1.5nM; 2.0nM) and different times (0h; 24h; 72h). The samples were collected to perform gene expression of the main osteodifferentiation markers (RUNX2, OTX, BSP), matrix remodeling (MMPs, TIMPs, BMPII) and zymography assay for analysis of metalloproteinase activities (MMPs). Results and Discussion: Preliminarily, it was founded a synergism between the hormone T3 and LCN2 expression in osteoblasts, occurring the modulation of the marker genes of differentiation, extracellular matrix and hormonal synthesis. The T3 hormone acts in the modulation of RUNX2, OTX, BMP2, favoring osteodifferentiation and the remodeling of the matrix, probably activating the TIMP1-MMP9 and TIMP2-MMP2 complexes, mainly in hyperthyroid conditions. In addition, we note a direct influence of T3 on the both expression of both Osteocalcin (OCN) and Lipocalin 2 (LCN2). Final Considerations: Our preliminary data indicates that the hormone T3 acts on the metabolism of osteoblasts through the modulation of LCN2 and BMPII. These molecular findings need to be confronted with further analyzes in vivo for more conclusive physiological conclusions.

The Effectiveness of Dietary Byproduct Antioxidants on Induced CYP Genes Expression and Histological Alteration in Piglets Liver and Kidney Fed with Aflatoxin B1 and Ochratoxin A

The purpose of this study was to investigate the potential of a byproduct mixture derived from grapeseed and sea buckthorn oil industry to mitigate the harmful damage produced by ochratoxin A and aflatoxin B1 at hepatic and renal level in piglets after weaning. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three experimental groups (E1, E2, E3) and one control group (C), and fed with experimental diets for 30 days. The basal diet was served as a control and contained normal compound feed for starter piglets without mycotoxins. The experimental groups were fed as follows: E1—basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal); E2—the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1); and E3—basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform gene expression and histological analysis. The gene expression analysis showed that when weaned piglets were fed with contaminated diet, the expression of most analyzed genes was downregulated. Among the CYP450 family, CYP1A2 was the gene with the highest downregulation. According to these results, in liver, we found that mycotoxins induced histomorphological alterations in liver and kidney and had an effect on the expression level of CYP1A2, CYP2A19, CYP2E1, and CYP3A29, but we did not detect important changes in the expression level of CY4A24, MRP2 and GSTA1 genes.

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems

Cell-free transcription-translation systems are a versatile tool to study gene expression, enzymatic reactions and biochemical regulation mechanisms. Because cell-free transcription-translation systems are often derived from cell lysates, many different substances, among them amino acids, are present. However, experiments concerning the incorporation of non-canonical amino acids into proteins require a system with negligible amounts of canonical analogs. Here we propose a two-step method for the removal of residual free lysine in an all Escherichia coli-based cell-free expression system. The first step consists of the expression of a high-lysine dummy protein. The second step consists of direct removal via binding between lysine and DNA. The presented method is an efficient, fast and simple way to remove residual lysine without altering the system ability to perform gene expression.

Anatomical and genetic bases underlying convergent evolution of fleshy and dry dehiscent fruits in Cestrum and Brugmansia (Solanaceae)

Background. The mechanisms controlling evolutionary shifts between dry and fleshy fruits in angiosperms are poorly understood. In Solanaceae, Cestrum and Brugmansia represent cases of convergent evolution of fleshy and dry fruits, respectively. Here we study the anatomical and genetic bases of the independent origin of fleshy fruits in Cestrum and the reversion to dry dehiscent fruits in Brugmansia. We also characterize the expression of candidate fruit development genes, including ALCATRAZ/SPATULA, FRUITFULL, HECATE1/2/3, REPLUMLESS and SHATTERPROOF.Methods. We identify anatomical changes to establish developmental stages in the ovary-to-fruit transition in Cestrum nocturnum and Brugmansia suaveolens. We generate reference transcriptomes for both species, isolate homologs for all genes in the fruit genetic regulatory network (GRN) and perform gene expression analyses for ALC/SPT, FUL, HEC1/2/3, RPL and SHP throughout fruit development. Finally, we compare our results to expression patterns found in typical capsules of Nicotiana tabacum and berries of Solanum lycopersicum available in public repositories.Results. We have identified homologous, homoplasious and unique anatomical features in C.nocturnum and B. suaveolens fruits, resulting in their final appearance. Expression patterns suggest that FUL, SHP and SPT might control homologous characteristics, while ALC and RPL likely contribute to homoplasious anatomical features.Conclusions. The convergent anatomical features in Cestrum and Brugmansia fruits are likely the result of changes in ALC and RPL expression patterns. The fruit GRN changes considerably in these genera when compared to typical capsules and berries of Solanaceae, particularly in B. suaveolens, where expression of FUL2 and RPL1 is lacking.

NetworkAnalyst 3.0: a visual analytics platform for comprehensive gene expression profiling and meta-analysis

The growing application of gene expression profiling demands powerful yet user-friendly bioinformatics tools to support systems-level data understanding. NetworkAnalyst was first released in 2014 to address the key need for interpreting gene expression data within the context of protein-protein interaction (PPI) networks. It was soon updated for gene expression meta-analysis with improved workflow and performance. Over the years, NetworkAnalyst has been continuously updated based on community feedback and technology progresses. Users can now perform gene expression profiling for 17 different species. In addition to generic PPI networks, users can now create cell-type or tissue specific PPI networks, gene regulatory networks, gene co-expression networks as well as networks for toxicogenomics and pharmacogenomics studies. The resulting networks can be customized and explored in 2D, 3D as well as Virtual Reality (VR) space. For meta-analysis, users can now visually compare multiple gene lists through interactive heatmaps, enrichment networks, Venn diagrams or chord diagrams. In addition, users have the option to create their own data analysis projects, which can be saved and resumed at a later time. These new features are released together as NetworkAnalyst 3.0, freely available at https://www.networkanalyst.ca.

Michigan vs. Pittsburgh Style GA Optimisation of Fuzzy Rule Bases for Gene Expression Analysis

Microarray studies and gene expression analysis have received a lot of attention and provide many promising avenues towards the understanding of fundamental questions in biology and medicine. In this paper, the authors perform gene expression analysis and apply two hybrid GA-fuzzy approaches to classify gene expression data. Both are based on fuzzy if-then rule bases but they differ in the way these rule bases are optimised. The authors employ both a Michigan style approach, where single rules are handled as individuals in the population of the genetic algorithm, and a Pittsburgh type algorithm, which treats whole rule sets as individuals. Experimental results show that both approaches achieve good classification accuracy but that the Michigan style algorithm clearly outperforms the Pittsburgh classifier.

Effects of acute doxorubicin exposure on caspase-mediated apoptosis in cardiomyocytes.

e12036 Background: Doxorubicin binds to DNA-associated enzymes, intercalates the base pairs of the DNA and induces apoptosis in cancerous and healthy tissues especially in cardiomyocytes. Caspase mediated apoptosis in cardiomyocytes remains largely unknown. We investigated the role of doxorubicin via caspase system on apoptosis of cardiomyocytes. Methods: H9C2ratcardiomyocytes were incubated with doxorubicin a concentration of 10-6 Mfor 4 or 24 hours to perform gene expression and activity of caspases, respectively. Total RNA isolated and expression analysis were performed by real time PCR assay. Caspase activity was determined by colorimetric caspase assay kit. At least two fold increases in gene expression were accepted statistically significant. Results: In comparison to controls, bothof caspase expression and activity increased in doxorubicin-treated samples. Increase in expression of Caspase 9 and 2 as initiator caspases and caspases 7 and 3 as effector caspases were detected statistically higher than control values. In contrast, expression of caspase 1, 4, 6, 8 and 12 were detected lower than controls. Activity of both caspase 3 and 9 increased significantly. Conclusions: Acute doxorubicin administration caused a significant caspase activation on apoptosis of cardiomyocytes.