The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.     

Small molecule modulators of aggregation in synthetic melanin polymerizations

There are numerous potential applications for melanin-binding compounds, and new methods are of interest to identify melanin-binding agents. A portion of the polymerization to eumelanin, the black to brown pigment in humans, is thought to be supramolecular aggregation of nanoparticles derived from dihydroxyindoles. Starting with chloroquine, a known eumelanin-binding compound, the ability of small molecules to influence aggregation in synthetic eumelanin polymerizations was investigated. Twenty-eight compounds were tested, including pharmaceuticals, dyes, aromatics, and amines. Compounds that either accelerate or delay the appearance of macroscopic particles in synthetic eumelanin polymerizations were uncovered.     

Baseline mechanical characterization of J774 macrophages

Macrophage cell lines like J774 cells are ideal model systems for establishing the biophysical foundations of autonomous deformation and motility of immune cells. To aid comparative studies on these and other types of motile cells, we report measurements of the cortical tension and cytoplasmic viscosity of J774 macrophages using micropipette aspiration. Passive J774 cells cultured in suspension exhibited a cortical resting tension of ∼0.14 mN/m and a viscosity (at room temperature) of 0.93 kPa·s. Both values are about one order of magnitude higher than the respective values obtained for human neutrophils, lending support to the hypothesis that a tight balance between cortical tension and cytoplasmic viscosity is a physical prerequisite for eukaryotic cell motility. The relatively large stiffness of passive J774 cells contrasts with their capacity for a more than fivefold increase in apparent surface area during active deformation in phagocytosis. Scanning electron micrographs show how microscopic membrane wrinkles are smoothed out and recruited into the apparent surface area during phagocytosis of large targets.     

RNA Editing Genes Associated with Extreme Old Age in Humans and with Lifespan in C. elegans

Background

The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered.

Methodology/Principal Findings

Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function.

Conclusions/Significance

Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan.     

Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part II: Development of a potent and highly selective M1 PAM

This Letter describes a chemical lead optimization campaign directed at VU0119498, a pan G_q mAChR M₁, M₃, M₅ positive allosteric modulator (PAM) with the goal of developing a selective M₁ PAM. An iterative library synthesis approach delivered a potent (M₁ EC₅₀ = 830 nM) and highly selective M₁ PAM (>30 μM vs M₂–M₅).     

Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part I: Development of the first highly selective M5 PAM

This Letter describes a chemical lead optimization campaign directed at VU0238429, the first M₅-preferring positive allosteric modulator (PAM), discovered through analog work around VU0119498, a pan G_q mAChR M₁, M₃, M₅ PAM. An iterative library synthesis approach delivered the first selective M₅ PAM (no activity at M₁–M₄ @ 30 μM), and an important tool compound to study the role of M₅ in the CNS.     

Vesicle trafficking maintains nuclear shape in Saccharomyces cerevisiae during membrane proliferation

The parameters that control nuclear size and shape are poorly understood. In yeast, unregulated membrane proliferation, caused by deletion of the phospholipid biosynthesis inhibitor SPO7, leads to a single nuclear envelope "flare" that protrudes into the cytoplasm. This flare is always associated with the asymmetrically localized nucleolus, which suggests that the site of membrane expansion is spatially confined by an unknown mechanism. Here we show that in spo7Δ cells, mutations in vesicle-trafficking genes lead to multiple flares around the entire nucleus. These mutations also alter the distribution of small nucleolar RNA-associated nucleolar proteins independently of their effect on nuclear shape. Both single- and multi-flared nuclei have increased nuclear envelope surface area, yet they maintain the same nuclear/cell volume ratio as wild-type cells. These data suggest that, upon membrane expansion, the spatial confinement of the single nuclear flare is dependent on vesicle trafficking. Moreover, flares may facilitate maintenance of a constant nuclear/cell volume ratio in the face of altered membrane proliferation.     

Evaluating support for the current classification of eukaryotic diversity

Perspectives on the classification of eukaryotic diversity have changed rapidly in recent years, as the four eukaryotic groups within the five-kingdom classification--plants, animals, fungi, and protists--have been transformed through numerous permutations into the current system of six "supergroups." The intent of the supergroup classification system is to unite microbial and macroscopic eukaryotes based on phylogenetic inference. This supergroup approach is increasing in popularity in the literature and is appearing in introductory biology textbooks. We evaluate the stability and support for the current six-supergroup classification of eukaryotes based on molecular genealogies. We assess three aspects of each supergroup: (1) the stability of its taxonomy, (2) the support for monophyly (single evolutionary origin) in molecular analyses targeting a supergroup, and (3) the support for monophyly when a supergroup is included as an out-group in phylogenetic studies targeting other taxa. Our analysis demonstrates that supergroup taxonomies are unstable and that support for groups varies tremendously, indicating that the current classification scheme of eukaryotes is likely premature. We highlight several trends contributing to the instability and discuss the requirements for establishing robust clades within the eukaryotic tree of life.     

Pair of unusual GCN5 histone acetyltransferases and ADA2 homologues in the protozoan parasite Toxoplasma gondii

GCN5 is a histone acetyltransferase (HAT) essential for development in mammals and critical to stress responses in yeast. The protozoan parasite Toxoplasma gondii is a serious opportunistic pathogen. The study of epigenetics and gene expression in this ancient eukaryote has pharmacological relevance and may facilitate the understanding of these processes in higher eukaryotes. Here we show that the disruption of T. gondii GCN5 yields viable parasites, which were subsequently employed in a proteomics study to identify gene products affected by its loss. Promoter analysis of these TgGCN5-dependent genes, which were mostly parasite specific, reveals a conserved T-rich element. The loss of TgGCN5 does not attenuate virulence in an in vivo mouse model. We also discovered that T. gondii is the only invertebrate reported to date possessing a second GCN5 (TgGCN5-B). TgGCN5-B harbors a strikingly divergent N-terminal domain required for nuclear localization. Despite high homology between the HAT domains, the two TgGCN5s exhibit differing substrate specificities. In contrast to TgGCN5-A, which exclusively targets lysine 18 of H3, TgGCN5-B acetylates multiple lysines in the H3 tail. We also identify two ADA2 homologues that interact differently with the TgGCN5s. TgGCN5-B has the potential to compensate for TgGCN5-A, which probably arose from a gene duplication unique to T. gondii. Our work reveals an unexpected complexity in the GCN5 machinery of this primitive eukaryote.     

The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the Golgi in yeast

Gcs1 is an Arf GTPase-activating protein (Arf-GAP) that mediates Golgi-ER and post-Golgi vesicle transport in yeast. Here we show that the Snc1,2 v-SNAREs, which mediate endocytosis and exocytosis, interact physically and genetically with Gcs1. Moreover, Gcs1 and the Snc v-SNAREs colocalize to subcellular structures that correspond to the trans-Golgi and endosomal compartments. Studies performed in vitro demonstrate that the Snc-Gcs1 interaction results in the efficient binding of recombinant Arf1Delta17N-Q71L to the v-SNARE and the recruitment of purified coatomer. In contrast, the presence of Snc had no effect on Gcs1 Arf-GAP activity in vitro, suggesting that v-SNARE binding does not attenuate Arf1 function. Disruption of both the SNC and GCS1 genes results in synthetic lethality, whereas overexpression of either SNC gene inhibits the growth of a distinct subset of COPI mutants. We show that GFP-Snc1 recycling to the trans-Golgi is impaired in gcs1Delta cells and these COPI mutants. Together, these results suggest that Gcs1 facilitates the incorporation of the Snc v-SNAREs into COPI recycling vesicles and subsequent endosome-Golgi sorting in yeast.