ALBERT

Description: Structural evidence for the binding of small organic ligands by the phi class glutathione transferase AtGSTF2 from Arabidopsis is presented. Complexes of AtGSTF2 with indole‐3‐aldehyde, camalexin and the rhamnosylated flavonoid quercetrin are reported in which ligands are found at ‘L1’ sites at the periphery of the dimer, or at an ‘L2’ site at the dimer interface. Glutathione transferases (GSTs) are involved in many processes in plant biochemistry, with their best characterised role being the detoxification of xenobiotics through their conjugation with glutathione. GSTs have also been implicated in noncatalytic roles, including the binding and transport of small heterocyclic ligands such as indole hormones, phytoalexins and flavonoids. Although evidence for ligand binding and transport has been obtained using gene deletions and ligand binding studies on purified GSTs, there has been no structural evidence for the binding of relevant ligands in noncatalytic sites. Here we provide evidence of noncatalytic ligand‐binding sites in the phi class GST from the model plant Arabidopsis thaliana, AtGSTF2, revealed by X‐ray crystallography. Complexes of the AtGSTF2 dimer were obtained with indole‐3‐aldehyde, camalexin, the flavonoid quercetrin and its non‐rhamnosylated analogue quercetin, at resolutions of 2.00, 2.77, 2.25 and 2.38 Å respectively. Two symmetry‐equivalent‐binding sites (L1) were identified at the periphery of the dimer, and one more (L2) at the dimer interface. In the complexes, indole‐3‐aldehyde and quercetrin were found at both L1 and L2 sites, but camalexin was found only at the L1 sites and quercetin only at the L2 site. Ligand binding at each site appeared to be largely determined through hydrophobic interactions. The crystallographic studies support previous conclusions made on ligand binding in noncatalytic sites by AtGSTF2 based on isothermal calorimetry experiments (Dixon et al. (2011) Biochem J 438, 63–70) and suggest a mode of ligand binding in GSTs commensurate with a possible role in ligand transport.

Description: We validated the down‐regulation of miR‐146a‐5p in hepatocellular carcinoma using data from TCGA, GEO, and in‐house qRT‐PCR, which included 762 HCC and 454 noncancerous liver tissues. A total of 104 genes were related to both HCC and miR‐146a‐5p. RAC1 was the most connected hub gene and four enriched pathways were denoted (VEGF signaling pathway, adherens junction, toll‐like receptor signaling pathway, and neurotrophin signaling pathway). Our previous research has demonstrated that miR‐146a‐5p is down‐regulated in hepatocellular carcinoma (HCC) and might play a tumor‐suppressive role. In this study, we sought to validate the decreased expression with a larger cohort and to explore potential molecular mechanisms. GEO and TCGA databases were used to gather miR‐146a‐5p expression data in HCC, which included 762 HCC and 454 noncancerous liver tissues. A meta‐analysis of the GEO‐based microarrays, TCGA‐based RNA‐seq data, and additional qRT‐PCR data validated the down‐regulation of miR‐146a‐5p in HCC and no publication bias was observed. Integrated genes were generated by overlapping miR‐146a‐5p‐related genes from predicted and formerly reported HCC‐related genes using natural language processing. The overlaps were comprehensively analyzed to discover the potential gene signatures, regulatory pathways, and networks of miR‐146a‐5p in HCC. A total of 251 miR‐146a‐5p potential target genes were predicted by bioinformatics platforms and 104 genes were considered as both HCC‐ and miR‐146a‐5p‐related overlaps. RAC1 was the most connected hub gene for miR‐146a‐5p and four pathways with high enrichment (VEGF signaling pathway, adherens junction, toll‐like receptor signaling pathway, and neurotrophin signaling pathway) were denoted for the overlapped genes. The down‐regulation of miR‐146a‐5p in HCC has been validated with the most complete data possible. The potential gene signatures, regulatory pathways, and networks identified for miR‐146a‐5p in HCC could prove useful for molecular‐targeted diagnostics and therapeutics.

Description: Bodak et al. report that murine Dicer is required for exit from pluripotency state of mouse embryonic stem cells (mESCs). Moreover, in Dicer_knockout mESCs, the pluripotency network is reinforced and there is a strong accumulation of LINE‐1 mRNA and protein, leading to an increased retrotransposition rate. Indeed, DICER is essential for stemness and genome integrity. A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer‐deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE‐1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self‐renewal and genome integrity by controlling L1s regulation.

Description: We investigated the effect of hydrogen sulphide on cerebral ischemia/reperfusion injury. Sodium hydrosulfide (NaHS), an H2S donor, attenuated cerebral ischemia/reperfusion injury induced by transient middle cerebral artery occlusion (MCAO) followed by reperfusion in rats. NaHS treatment also reduced cellular injury induced by oxygen–glucose deprivation/re‐oxygenation in PC12 cells. The mechanism by which NaHS attenuates cerebral ischemia/reperfusion injury might involve suppression of overactivated autophagy, which accelerated cellular injury after MCAO in rats. Ischemic stroke is a leading cause of death and disability worldwide, and autophagy may be involved in the pathological process of cerebral ischemia/reperfusion injury. Hydrogen sulfide (H2S) is an endogenous gasotransmitter with protective effects against multiple diseases. Here, we tested the effect of H2S on cerebral ischemia/reperfusion injury in rats. Sodium hydrosulfide (NaHS), an H2S donor, improved neurological function and reduced the size of the infarcts induced by transient middle cerebral artery occlusion (MCAO) followed by reperfusion in rats. NaHS treatment reduced the lactate dehydrogenase (LDH) activity in the serum (a marker of cellular membrane integrity) and the expression of cleaved caspase‐3 (a marker for apoptosis) in the brains of MCAO rats. We also found that autophagy was overactivated in the brains of MCAO rats, as indicated by an increased ratio of LC3 II to I, decreased expression of p62, and transmission electron microscope detection. NaHS treatment significantly inhibited the autophagic activity in the brains of MCAO rats. Furthermore, PC12 cells were subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) to mimic MCAO in vitro. We found that NaHS treatment reduced cellular injury and suppressed overactivated autophagy induced by OGD/R in PC12 cells. An autophagy stimulator (rapamycin) eliminated the protective effect of NaHS against LDH release and caspase‐3 activity induced by OGD/R in PC12 cells. An autophagy inhibitor (3‐methyladenine, 3‐MA) also reduced the cellular injury induced by OGD/R in PC12 cells. In conclusion, the results indicate that overactivated autophagy accelerates cellular injury after MCAO in rats and that exogenous H2S attenuates cerebral ischemia/reperfusion injury via suppressing overactivated autophagy in rats.

Description: In microglia, α7 nicotinic acetylcholine receptors play a critical role in inflammation. We show that direct coupling to Gαi enables α7 nicotinic receptor signaling through the regulation of PLC, intracellular calcium, and TNF‐α release. Acetylcholine activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) in microglia attenuates neuroinflammation and regulates TNF‐α release. We used lipopolysaccharide to model inflammation in the microglial cell line EOC20 and examined signaling by the α7 nAChR. Co‐immunoprecipitation experiments confirm that α7 nAChRs bind heterotrimeric G proteins in EOC20 cells. Interaction with Gαi mediates α7 nAChR signaling via enhanced intracellular calcium release and a decrease in cAMP, p38 phosphorylation, and TNF‐α release. These α7 nAChR effects were blocked by the inhibition of Gαi signaling via pertussis toxin, PLC activity with U73122, and α7 nAChR channel activity with the selective antagonist α‐bungarotoxin. Moreover, α7 nAChR signaling in EOC20 cells was significantly diminished by the expression of a dominant‐negative α7 nAChR, α7345‐8A, shown to be impaired in G protein binding. These findings indicate an essential role for G protein coupling in α7 nAChR function in microglia leading to the regulation of inflammation in the nervous system.

Description: A polydesmid millipede Chamberlinius hualienensis accumulates (R)‐mandelonitrile as a precursor of the defensive substance cyanide. The mechanism of (R)‐mandelonitrile biosynthesis in cyanogenic millipedes, however, remains unclear. We revealed that (E/Z)‐phenylacetaldoxime and phenylacetonitrile are the biosynthetic precursors of (R)‐mandelonitrile. Furthermore, we identified that a cytochrome P450, CYP3201B1, catalyses the stereoselective hydroxylation of phenylacetonitrile into (R)‐mandelonitrile. Specialized arthropods and more than 2500 plant species biosynthesize hydroxynitriles and release hydrogen cyanide as a defensive mechanism. The millipede Chamberlinius hualienensis accumulates (R)‐mandelonitrile as a cyanide precursor. Although biosynthesis of hydroxynitriles in cyanogenic plants and in an insect are extensively studied, (R)‐mandelonitrile biosynthesis in cyanogenic millipedes has remained unclear. In this study, we identified the biosynthetic precursors of (R)‐mandelonitrile and an enzyme involved in (R)‐mandelonitrile biosynthesis. Using deuterium‐labelled compounds, we revealed that (E/Z)‐phenylacetaldoxime and phenylacetonitrile are the biosynthetic precursors of (R)‐mandelonitrile in the millipede as well as other cyanogenic organisms. To identify the enzymes involved in (R)‐mandelonitrile biosynthesis, 50 cDNAs encoding cytochrome P450s were cloned and coexpressed with yeast cytochrome P450 reductase in yeast, as cytochrome P450s are involved in the biosynthesis of hydroxynitriles in other cyanogenic organisms. Among the 50 cytochrome P450s from the millipede, CYP3201B1 produced (R)‐mandelonitrile from phenylacetonitrile but not from (E/Z)‐phenylacetaldoxime, whereas plant and insect cytochrome P450s catalysed the dehydration of aldoximes and hydroxylation of nitriles. CYP3201B1 is not phylogenetically related to cytochrome P450s from other cyanogenic organisms, indicating that hydroxynitrile biosynthetic cytochrome P450s have independently evolved in distant species. Our study will shed light on the evolution of cyanogenesis among plants, insects and millipedes. Database Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers LC125356–LC125405.

Description: The G2 checkpoint is an intricate signaling network that regulates the progression of G2 to mitosis. We propose here a node‐based model of G2 checkpoint regulation that provides an intelligible structure of the complex interactions governing the decision to delay or continue mitotic progression and may also aid in predicting the effects of agents that target these G2 checkpoint nodes. Tight regulation of the eukaryotic cell cycle is paramount to ensure genomic integrity throughout life. Cell cycle checkpoints are present in each phase of the cell cycle and prevent cell cycle progression when genomic integrity is compromised. The G2 checkpoint is an intricate signaling network that regulates the progression of G2 to mitosis (M). We propose here a node‐based model of G2 checkpoint regulation, in which the action of the central CDK1–cyclin B1 node is determined by the concerted but opposing activities of the Wee1 and cell division control protein 25C (CDC25C) nodes. Phosphorylation of both Wee1 and CDC25C at specific sites determines their subcellular localization, driving them either toward activity within the nucleus or to the cytoplasm and subsequent ubiquitin‐mediated proteasomal degradation. In turn, this subcellular balance of the Wee1 and CDC25C nodes is directed by the action of the PLK1 and CHK1 nodes via what we have termed the ‘nuclear and cytoplasmic decision states’ of Wee1 and CDC25C. The proposed node‐based model provides an intelligible structure of the complex interactions that govern the decision to delay or continue G2/M progression. The model may also aid in predicting the effects of agents that target these G2 checkpoint nodes.

Description: GenBank ® ( www.ncbi.nlm.nih.gov/genbank/ ) is a comprehensive database that contains publicly available nucleotide sequences for 370 000 formally described species. These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or the NCBI Submission Portal. GenBank staff assign accession numbers upon data receipt. Daily data exchange with the European Nucleotide Archive (ENA) and the DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through the NCBI Nucleotide database, which links to related information such as taxonomy, genomes, protein sequences and structures, and biomedical journal literature in PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. Recent updates include changes to policies regarding sequence identifiers, an improved 16S submission wizard, targeted loci studies, the ability to submit methylation and BioNano mapping files, and a database of anti-microbial resistance genes.

Description: The abnormal transcriptional regulation of non-coding RNAs (ncRNAs) and protein-coding genes (PCGs) is contributed to various biological processes and linked with human diseases, but the underlying mechanisms remain elusive. In this study, we developed ChIPBase v2.0 ( http://rna.sysu.edu.cn/chipbase/ ) to explore the transcriptional regulatory networks of ncRNAs and PCGs. ChIPBase v2.0 has been expanded with ~10 200 curated ChIP-seq datasets, which represent about 20 times expansion when comparing to the previous released version. We identified thousands of binding motif matrices and their binding sites from ChIP-seq data of DNA-binding proteins and predicted millions of transcriptional regulatory relationships between transcription factors (TFs) and genes. We constructed ‘Regulator’ module to predict hundreds of TFs and histone modifications that were involved in or affected transcription of ncRNAs and PCGs. Moreover, we built a web-based tool, Co-Expression, to explore the co-expression patterns between DNA-binding proteins and various types of genes by integrating the gene expression profiles of ~10 000 tumor samples and ~9100 normal tissues and cell lines. ChIPBase also provides a ChIP-Function tool and a genome browser to predict functions of diverse genes and visualize various ChIP-seq data. This study will greatly expand our understanding of the transcriptional regulations of ncRNAs and PCGs.

Description: We present an update of the Eukaryotic Promoter Database EPD ( http://epd.vital-it.ch ), more specifically on the EPDnew division, which contains comprehensive organisms-specific transcription start site (TSS) collections automatically derived from next generation sequencing (NGS) data. Thanks to the abundant release of new high-throughput transcript mapping data (CAGE, TSS-seq, GRO-cap) the database could be extended to plant and fungal species. We further report on the expansion of the mass genome annotation (MGA) repository containing promoter-relevant chromatin profiling data and on improvements for the EPD entry viewers. Finally, we present a new data access tool, ChIP-Extract, which enables computational biologists to extract diverse types of promoter-associated data in numerical table formats that are readily imported into statistical analysis platforms such as R.