Histone H2B ubiquitin ligase RNF20 is required for MLL-rearranged leukemia

Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion-mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program

Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming

Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies

Characterization of the Ebola virus nucleoprotein–RNA complex

When Ebola virus nucleoprotein (NP) is expressed in mammalian cells, it assembles into helical structures. Here, the recombinant NP helix purified from cells expressing NP was characterized biochemically and morphologically. We found that the recombinant NP helix is associated with non-viral RNA, which is not protected from RNase digestion and that the morphology of the helix changes depending on the environmental salt concentration. The N-terminal 450 aa residues of NP are sufficient for these properties. However, digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that this RNA is an essential structural component of the helix, binding to individual NP molecules via the N-terminal 450 aa. These findings enhance our knowledge of Ebola virus assembly and understanding of the Ebola virus life cycle

Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galβ1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing β3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines. Of these, SIAT-NExt cells exhibit markedly increased binding of H3 HAs and susceptibility to infection by recent H3N2 virus strains, but without impacting final virus titers. Glycome analysis of these cell lines and allantoic and amniotic egg membranes provide insights into the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for cell- and egg-based vaccines

Multiple reassortment events in the evolutionary history of H1N1 influenza A virus since 1918

The H1N1 subtype of influenza A virus has caused substantial morbidity and mortality in humans, first documented in the global pandemic of 1918 and continuing to the present day. Despite this disease burden, the evolutionary history of the A/H1N1 virus is not well understood, particularly whether there is a virological basis for several notable epidemics of unusual severity in the 1940s and 1950s. Using a data set of 71 representative complete genome sequences sampled between 1918 and 2006, we show that segmental reassortment has played an important role in the genomic evolution of A/H1N1 since 1918. Specifically, we demonstrate that an A/H1N1 isolate from the 1947 epidemic acquired novel PB2 and HA genes through intra-subtype reassortment, which may explain the abrupt antigenic evolution of this virus. Similarly, the 1951 influenza epidemic may also have been associated with reassortant A/H1N1 viruses. Intra-subtype reassortment therefore appears to be a more important process in the evolution and epidemiology of H1N1 influenza A virus than previously realized

Transposable-Element Associated Small RNAs in Bombyx mori Genome

Small RNAs are a group of regulatory RNA molecules that control gene expression at transcriptional or post-transcriptional levels among eukaryotes. The silkworm, Bombyx mori L., genome harbors abundant repetitive sequences derived from families of retrotransposons and transposons, which together constitute almost half of the genome space and provide ample resource for biogenesis of the three major small RNA families. We systematically discovered transposable-element (TE)-associated small RNAs in B. mori genome based on a deep RNA-sequencing strategy and the effort yielded 182, 788 and 4,990 TE-associated small RNAs in the miRNA, siRNA and piRNA species, respectively. Our analysis suggested that the three small RNA species preferentially associate with different TEs to create sequence and functional diversity, and we also show evidence that a Bombyx non-LTR retrotransposon, bm1645, alone contributes to the generation of TE-associated small RNAs in a very significant way. The fact that bm1645-associated small RNAs partially overlap with each other implies a possibility that this element may be modulated by different mechanisms to generate different products with diverse functions. Taken together, these discoveries expand the small RNA pool in B. mori genome and lead to new knowledge on the diversity and functional significance of TE-associated small RNAs

Substitutions near the hemagglutinin receptor-binding site determine the antigenic evolution of influenza A H3N2 viruses in U.S. swine

Swine influenza A virus is an endemic and economically important pathogen in pigs, with the potential to infect other host species. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major component in swine influenza A vaccines. However, as a result of antigenic drift, vaccine strains must be regularly updated to reflect currently circulating strains. Characterizing the cross-reactivity between strains in pigs and seasonal influenza virus strains in humans is also important in assessing the relative risk of interspecies transmission of viruses from one host population to the other. Hemagglutination inhibition (HI) assay data for swine and human H3N2 viruses were used with antigenic cartography to quantify the antigenic differences among H3N2 viruses isolated from pigs in the United States from 1998 to 2013 and the relative cross-reactivity between these viruses and current human seasonal influenza A virus strains. Two primary antigenic clusters were found circulating in the pig population, but with enough diversity within and between the clusters to suggest updates in vaccine strains are needed. We identified single amino acid substitutions that are likely responsible for antigenic differences between the two primary antigenic clusters and between each antigenic cluster and outliers. The antigenic distance between current seasonal influenza virus H3 strains in humans and those endemic in swine suggests that population immunity may not prevent the introduction of human viruses into pigs, and possibly vice versa, reinforcing the need to monitor and prepare for potential incursions

Reversible and Irreversible Interactions of Poly(3-hexylthiophene) with Oxygen Studied by Spin-Sensitive Methods

Understanding of degradation mechanisms in polymer:fullerene bulk-heterojunctions on the microscopic level aimed at improving their intrinsic stability is crucial for the breakthrough of organic photovoltaics. These materials are vulnerable to exposure to light and/or oxygen, hence they involve electronic excitations. To unambiguously probe the excited states of various multiplicities and their reactions with oxygen, we applied combined magneto-optical methods based on multifrequency (9 and 275 GHz) electron paramagnetic resonance (EPR), photoluminescence (PL), and PL-detected magnetic resonance (PLDMR) to the conjugated polymer poly(3-hexylthiophene) (P3HT) and polymer:fullerene bulk heterojunctions (P3HT:PCBM; PCBM = [6,6]-phenyl-C61-butyric acid methyl ester). We identified two distinct photochemical reaction routes, one being fully reversible and related to the formation of polymer:oxygen charge transfer complexes, the other one, irreversible, being related to the formation of singlet oxygen under participation of bound triplet excitons on the polymer chain. With respect to the blends, we discuss the protective effect of the methanofullerenes on the conjugated polymer bypassing the triplet exciton generation

Presence and Persistence of Ebola or Marburg Virus in Patients and Survivors: A Rapid Systematic Review

Background: The 2013-15 Ebola outbreak was unprecedented due to sustainedtransmission within urban environments and thousands of survivors. In 2014 the World Health Organization stated that there was insufficient evidence to give definitive guidance about which body fluids are infectious and when they pose a risk to humans. We report a rapid systematic review of published evidence on the presence of filoviruses in body fluids of infected people and survivors. Methods: Scientific articles were screened for information about filovirus in human body fluids. The aim was to find primary data that suggested high likelihood of actively infectious filovirus in human body fluids (viral RNA). Eligible infections were from Marburg virus (MARV or RAVV) and Zaire, Sudan, Taï Forest and Bundibugyo species of Ebola. [1] Cause of infection had to be laboratory confirmed (in practice either tissue culture or RT-PCR tests), or evidenced by compatible clinical history with subsequent positivity for filovirus antibodies or inflammatory factors. Data were extracted and summarized narratively. Results: 6831 unique articles were found, and after screening, 33 studies were eligible. For most body fluid types there were insufficient patients to draw strong conclusions, and prevalence of positivity was highly variable. Body fluids taken >16 days after onset were usually negative. In the six studies that used both assay methods RT-PCR tests for filovirus RNA gave positive results about 4 times more often than tissue culture. Conclusions: Filovirus was reported in most types of body fluid, but not in every sample from every otherwise confirmed patient. Apart from semen, most non-blood, RT-PCR positive samples are likely to be culture negative and so possibly of low infectious risk. Nevertheless, it is not apparent how relatively infectious many body fluids are during or after illness, even when culture-positive, not least because most test results come from more severe cases. Contact with blood and blood-stained body fluids remains the major risk for disease transmission because of the known high viral loads in blood