Front Matter

This file contains cover for Volume II, Issue II, Editorial Board, Acknowledgements

RNA Sequencing Reveals the Alteration of the Expression of Novel Genes in Ethanol-Treated Embryoid Bodies.

Fetal alcohol spectrum disorder is a collective term representing fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not well characterized. In this present study, our aim is to profile important genes that regulate cellular development during fetal development. Human embryonic carcinoma cells (NCCIT) are cultured to form embryoid bodies and then treated in the presence and absence of ethanol (50 mM). We employed RNA sequencing to profile differentially expressed genes in the ethanol-treated embryoid bodies from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH data sets. A total of 632, 205 and 517 differentially expressed genes were identified from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. Functional annotation using bioinformatics tools reveal significant enrichment of differential cellular development and developmental disorders. Furthermore, a group of 42, 15 and 35 transcription factor-encoding genes are screened from all of the differentially expressed genes obtained from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. We validated relative gene expression levels of several transcription factors from these lists by quantitative real-time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanism underlying the pathology of alcohol-mediated anomalies and ease further research

Effect of Aβ₄₂ on the expressions of inflammatory mediators in microglial cells.

<p>Quantitative real-time reverse transcriptase-PCR analysis of inflammatory gene expression in BV-2 and primary microglial cells stimulated with Aβ₄₂. The expression of inflammatory genes were significantly up-regulated in primary microglial cells treated with Aβ₄₂ compared with untreated cells (<i>*P<0</i>.<i>05</i>, <i>**P<0</i>.<i>001</i> and ns stands for no significant difference compared with control) at the indicated times. Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are shown as the means ± SD of triplicate wells.</p

Top network and canonical pathway analyses at each time point (2 h and 4 h) in LPS-stimulated BV-2 microglial cells.

<p>(A) Ingenuity Bioinformatics pathway analysis of gene network with connections to nf-κb, stat1-stat2, irf1 and irf9 and differentially expressed genes in LPS-stimulated BV-2 microglia cells. (B) The most highly represented canonical pathways for differentially expressed genes in BV-2 microglial cells.</p

Dual RNA Sequencing Reveals the Expression of Unique Transcriptomic Signatures in Lipopolysaccharide-Induced BV-2 Microglial Cells

<div><p>Microglial cells become rapidly activated through interactions with pathogens, and the persistent activation of these cells is associated with various neurodegenerative diseases. Previous studies have investigated the transcriptomic signatures in microglia or macrophages using microarray technologies. However, this method has numerous restrictions, such as spatial biases, uneven probe properties, low sensitivity, and dependency on the probes spotted. To overcome this limitation and identify novel transcribed genes in response to LPS, we used RNA Sequencing (RNA-Seq) to determine the novel transcriptomic signatures in BV-2 microglial cells. Sequencing assessment and quality evaluation showed that approximately 263 and 319 genes (≥ 1.5 log₂-fold), such as cytokines and chemokines, were strongly induced after 2 and 4 h, respectively, and the induction of several genes with unknown immunological functions was also observed. Importantly, we observed that previously unidentified transcription factors (TFs) (irf1, irf7, and irf9), histone demethylases (kdm4a) and DNA methyltransferases (dnmt3l) were significantly and selectively expressed in BV-2 microglial cells. The gene expression levels, transcription start sites (TSS), isoforms, and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with LPS. In addition, gene ontology, molecular networks and pathway analyses identified the top significantly regulated functional classification, canonical pathways and network functions at each activation status. Moreover, we further analyzed differentially expressed genes to identify transcription factor (TF) motifs (−950 to +50 bp of the 5’ upstream promoters) and epigenetic mechanisms. Furthermore, we confirmed that the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in LPS treated primary microglial cells. This transcriptomic analysis is the first to show a comparison of the family-wide differential expression of most known immune genes and also reveal transcription evidence of multiple gene families in BV-2 microglial cells. Collectively, these findings reveal unique transcriptomic signatures in BV-2 microglial cells required for homeostasis and effective immune responses.</p></div

RNA-Seq analysis reveals that LPS-stimulated pro-inflammatory gene expression in BV-2 microglial cells.

<p>(A) Heat map representing RNA-Seq gene expression of up-regulated (≥ 1.5 log₂-fold) inflammatory genes at 2 and 4 h after LPS stimulation in BV-2 microglia cells compared with controls. (B) Venn diagram displaying the number of inducible or repressible (≥ 1.5 log₂-fold) genes after LPS stimulation in BV-2 microglia cells. (C, D) UCSC Browser images representing normalized RNA-Seq read densities at 2 and 4 h after LPS stimulation in BV-2 microglia cells compared with controls. (E, F) Gene Ontology analysis of functional annotations associated with up-regulated genes at 2 and 4 h after LPS stimulation in BV-2 microglia.</p

Transcriptomic analysis of selected TF families in BV-2 microglial cells.

<p>(A) Heat map represents differential expression of TF families of nfκb, irf, stat, klf, and other genes at 2 and 4 h after LPS stimulation in BV-2 microglial cells. (B) UCSC Browser images representing normalized RNA-Seq read densities for TF expression at 2 and 4 h after LPS stimulation in BV-2 microglia cells compared with controls. (C) Quantitative real-time reverse transcriptase-PCR analysis of TF and inflammatory gene expression in RAW 264.7 mouse macrophage cells stimulated with LPS (10 ng/ml) at the indicated times. (D) Patterns of transcription factor motif enrichments within the promoters of the genes in LPS-stimulated BV-2 microglia cells. (E) Transcriptional and post-transcriptional regulatory effects on overall transcript output in LPS-stimulated BV-2 microglial cells.</p

Confirmation of differential gene expression via qRT-PCR analysis.

<p>A) Validation of the relative mRNA expression of the TF-encoding genes that are randomly selected from the listed 35 genes found in EB vs. EB+EtOH dataset. B) Validation of the relative mRNA expression of the TF-encoding genes in different concentrations of ethanol. The expression value was normalized to the GAPDH expression level. Values are represented as average mRNA expression ± SEM bars, n = 3 replicates. Asterisks indicate statistically significant changes based on adjusted <i>p</i>-values < 0.05.</p

List of primers used in q-RT-PCR studies.

<p>List of primers used in q-RT-PCR studies.</p