DNA Methylation Profiling of Embryonic Stem Cell Differentiation into the Three Germ Layers

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes

Functional genomics screen with pooled shRNA library and gene expression profiling with extracts of Azadirachta indica identify potential pathways for therapeutic targets in head and neck squamous cell carcinoma

Tumor suppression by the extracts of Azadirachta indica (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that neem extracts and their associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell lines treated with crude neem leaf extracts, known for their anti-tumorigenic activity. We analyzed differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing. We found 225 genes affected the cancer cell growth in the shRNA-infected cells treated with neem extract. Pathway enrichment analyses of whole-genome gene expression data from cells temporally treated with neem extract revealed important roles played by the TGF-β pathway and HSF-1-related gene network. Our results indicate that neem extract affects various important molecular signaling pathways in head and neck cancer cells, some of which may be therapeutic targets for this devastating tumor

Multiomics Investigation Revealing the Characteristics of HIV-1-Infected Cells In Vivo

For eradication of HIV-1 infection, it is important to elucidate the detailed features and heterogeneity of HIV-1-infected cells in vivo. To reveal multiple characteristics of HIV-1-producing cells in vivo, we use a hematopoietic-stem-cell-transplanted humanized mouse model infected with GFP-encoding replication-competent HIV-1. We perform multiomics experiments using recently developed technology to identify the features of HIV-1-infected cells. Genome-wide HIV-1 integration-site analysis reveals that productive HIV-1 infection tends to occur in cells with viral integration into transcriptionally active genomic regions. Bulk transcriptome analysis reveals that a high level of viral mRNA is transcribed in HIV-1-infected cells. Moreover, single-cell transcriptome analysis shows the heterogeneity of HIV-1-infected cells, including CXCL13high cells and a subpopulation with low expression of interferon-stimulated genes, which can contribute to efficient viral spread in vivo. Our findings describe multiple characteristics of HIV-1-producing cells in vivo, which could provide clues for the development of an HIV-1 cure

Comprehensive Investigation on the Interplay between Feline APOBEC3Z3 Proteins and Feline Immunodeficiency Virus Vif Proteins

As the hosts of lentiviruses, almost 40 species of felids (family Felidae) are distributed around the world, and more than 20 feline species test positive for feline immunodeficiency virus (FIV), a lineage of lentiviruses. These observations suggest that FIVs globally infected a variety of feline species through multiple cross-species transmission events during a million-year history. Cellular restriction factors potentially inhibit lentiviral replication and limit cross-species lentiviral transmission, and cellular APOBEC3 deaminases are known as a potent restriction factor. In contrast, lentiviruses have evolutionary-acquired viral infectivity factor (Vif) to neutralize the APOBEC3-mediated antiviral effect. Because the APOBEC3-Vif interaction is strictly specific for viruses and their hosts, a comprehensive investigation focusing on Vif-APOBEC3 interplay can provide clues that will elucidate the roles of this virus-host interplay on cross-species transmission of lentiviruses. Here, we performed a comprehensive investigation with 144 patterns of a round robin test using 18 feline APOBEC3Z3 genes, an antiviral APOBEC3 gene in felid, and 8 FIV Vifs and derived a matrix showing the interplay between feline APOBEC3Z3 and FIV Vif. We particularly focused on the interplay between the APOBEC3Z3 of three felids (domestic cat, ocelot, and Asian golden cat) and an FIV Vif (strain Petaluma), and revealed that residues 65 and 66 of the APOBEC3Z3 protein of multiple felids are responsible for the counteraction triggered by FIV Petaluma Vif. Altogether, our findings can be a clue to elucidate not only the scenarios of the cross-species transmissions of FIVs in felids but also the evolutionary interaction between mammals and lentiviruses

Ongoing EEG artifact correction using blind source separation

Objective: Analysis of the electroencephalogram (EEG) for epileptic spike and seizure detection or brain-computer interfaces can be severely hampered by the presence of artifacts. The aim of this study is to describe and evaluate a fast automatic algorithm for ongoing correction of artifacts in continuous EEG recordings, which can be applied offline and online. Methods: The automatic algorithm for ongoing correction of artifacts is based on fast blind source separation. It uses a sliding window technique with overlapping epochs and features in the spatial, temporal and frequency domain to detect and correct ocular, cardiac, muscle and powerline artifacts. Results: The approach was validated in an independent evaluation study on publicly available continuous EEG data with 2035 marked artifacts. Validation confirmed that 88% of the artifacts could be removed successfully (ocular: 81%, cardiac: 84%, muscle: 98%, powerline: 100%). It outperformed state-of-the-art algorithms both in terms of artifact reduction rates and computation time. Conclusions: Fast ongoing artifact correction successfully removed a good proportion of artifacts, while preserving most of the EEG signals. Significance: The presented algorithm may be useful for ongoing correction of artifacts, e.g., in online systems for epileptic spike and seizure detection or brain-computer interfaces.Comment: 16 pages, 4 figures, 3 table

The antitumor activity of xanthohumol

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells

Change in brain plasmalogen composition by exposure to prenatal undernutrition leads to behavioral impairment of rats.

Epidemiological studies suggest that poor nutrition during pregnancy influences offspring predisposition to experience developmental and psychiatric disorders. Animal studies have shown that maternal undernutrition leads to behavioral impairment, which is linked to alterations in monoaminergic systems and inflammation in the brain. In this study, we focused on the ethanolamine plasmalogen of the brain as a possible contributor to behavioral disturbances observed in offspring exposed to maternal undernutrition. Maternal food or protein restriction between gestational day (GD) 5.5 and GD 10.5 resulted in hyperactivity of rat male adult offspring. Genes related to the phospholipid biosynthesis were found to be activated in the prefrontal cortex (PFC), but not in the nucleus accumbens or striatum, in the offspring exposed to prenatal undernutrition. Corresponding to these gene activations, increased ethanolamine plasmalogen (18:0p-22:6) was observed in the PFC using mass spectrometry imaging. A high number of crossings and the long time spent in the center area was observed in the offspring exposed to prenatal undernutrition and was mimicked in adult rats via the intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome. Additionally, plasmalogen (18:0p-22:6) increased only in the PFC, and not in the nucleus accumbens or striatum. These results suggest that brain plasmalogen is one of the key molecules to control behavior and its injection using liposome is a potential therapeutic approach for cognitive impairment.Significance Statement:Maternal undernutrition correlates to developmental and psychiatric disorders. Here, we found that maternal undernutrition in early pregnancy led to hyperactivity in rat male offspring and induced gene activation of phospholipid-synthesizing enzyme and elevation of ethanolamine plasmalogen (18:0p-22:6) level in the prefrontal cortex (PFC). Intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome maintained crossing activity and was circumscribed to the center area for a long time period, in prenatally undernourished offspring with aberrant behavior. Furthermore, the amount of ethanolamine plasmalogen (18:0p-22:6) increased in the PFC of the rat after injection. Our result suggests that brain plasmalogen is one of the key molecules to control behavior and that its injection using liposome is a potential therapeutic approach for cognitive impairment