Investigation of Microbial Interactions and Ecosystem Dynamics in a Low O2 Cyanobacterial Mat.

Cyanobacteria are believed to be responsible for the oxygenation of the Earth’s atmosphere and oceans, which enabled the evolution of metabolisms that depend on O2. Little is known about cyanobacteria adapted to low-O2, sulfidic conditions, which dominated the oceans when oxygenic photosynthesis first evolved. To better understand how such cyanobacteria function and contribute to biogeochemistry, metagenomics and metatranscriptomics were used to characterize modern cyanobacterial mats that thrive under low-O2, sulfidic conditions in the Middle Island Sinkhole (MIS) of Lake Huron. Metagenomics revealed a consortium of microorganisms that regulate biogeochemical cycling at the sediment/water interface. The mats were dominated by Phormidium, a cyanobacterium that was inferred to perform anoxygenic photosynthesis in the presence of sulfide based on (i) primary production rate experiments, (ii) expression of sulfide quinone reductase, and (iii) a high ratio of transcripts for photosystem I to photosystem II. Combined with excess organic matter, chemical reductants and rapid utilization of O2 by respiration, this anoxygenic photosynthesis makes the MIS mats a net sink for O2. Such anoxygenic cyanobacterial mats were likely widespread under the low-O2 conditions of the Proterozoic, and may help to explain why atmospheric O2 levels remained low for much of Earth’s history. Genome sequences were reconstructed for the dominant mat organisms, and transcript abundance was used to identify organisms expressing metabolic pathways that regulate geochemical cycling at MIS. Desulfobacterales were responsible for mediating production of sulfide, which likely contributes to hypoxia at MIS and regulates oxygenic versus anoxygenic photosynthesis by Phormidium. Members of the Proteobacteria were found to perform aerobic oxidation of various sulfur species, H2 and CO. Viral predation was detected by two way exchange of DNA between Phormidium and PhV1, an abundant virus at MIS. Phormidium used viral DNA within a CRISPR system to defend itself, while PhV1 was found to possess a host derived nblA gene, which breaks down photosynthetic pigments. Overall, this work suggests that ancient cyanobacterial mats were not necessarily a source for O2, and that sulfide concentration, metabolic products from other organisms, viral predation, and light availability could all influence cyanobacterial production of O2 in low-O2 environments.PHDEarth and Environmental SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107234/1/alexav_1.pd

Can Oxygen Set Thermal Limits in an Insect and Drive Gigantism?

Contains fulltext : 111575.pdf (publisher's version ) (Open Access

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing

<div><p>Ebola virus and Marburg virus are members of the <i>Filovirdae</i> family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.</p></div

VP35 proteins from Marburg virus and all five <i>Ebolavirus</i> species antagonize SeV induced promoter activity.

<p>The abilities of the VP35 proteins from Marburgvirus and the five species of Ebolavirus to antagonize the IFN response induced by virus infection were compared in the context of a luciferase reporter under the control of the IFN-β promoter. (A) 293T cells were transfected with increasing amounts (4 ng, 20 ng, or 100 ng) of pCAGGS-based plasmids expressing N-terminally FLAG-tagged filoviral proteins, an empty pCAGGS plasmid to transfect equal amounts between samples, and a plasmid expressing Renilla luciferase as a transfection control. The following day, cells were either mock-infected or infected with SeV to induce IFN-β promoter activity. The third day, cells were harvested and luciferase expression was measured. Error bars represent standard error of the mean of triplicates. MARV, Marburg Virus; EBOV, Ebola Virus (Mayinga); SUDV, Sudan Virus; BDBV, Bundibugyo Virus; RESTV, Reston Virus; TAFV, Taï Forest Virus. (B) Western blot analysis against the FLAG tag shows relative expression of filoviral proteins when 100 ng of each FLAG-tagged protein-expressing plasmid was transfected.</p

An analysis of host RNAs highlights transcripts bound by VP35.

<p>(A) Total number of sequencing reads from triplicate samples of wildtype and mutant VP35 proteins infected in the SeV infected groups. The pie charts depict the percentage of reads that map to the SeV DI genome and the percentage of reads that do not map to the SeV DI. (B) Multidimensional scaling (MDS) plot of triplicate samples from wild-type VP35 (black squares), wild-type VP35 infected with SeV (black circles), mutant VP35 (black triangle) and mutant VP35 infected with SeV (black diamond). Axes in the MDS plot (Leading logFC dim1 and Leading logFC dim2) are arbitrary, and the values on the axes are distance units. (C) Heat map showing the binding of the wild-type and mutant VP35 protein to human mRNA transcripts for all samples included in the analysis. Each row represents an experimental replicate, and each column represents a single transcript. Colors indicate relative abundance for each gene, where orange is low abundance and white is high abundance. (D) Sorting of the 62 most statistically significantly enriched mRNAs associated with VP35 (from the mock-infected wild-type VP35 samples). Y-axis denotes the p-value of each sample and X-axis denotes fold-change of transcript abundance between wild-type and mutant VP35.</p

Mutations in VP35 important for dsRNA binding abrogate its ability to bind the immunostimulatory SeV DI.

<p>(A) Schematic of the EBOV VP35 protein containing a coiled-coil domain important for oligomerization in the N-terminal half of the protein and dsRNA-binding domain in the C-terminal half. Residues K309 and R312 were mutated to alanine to generate the EBOV VP35 RNA-binding mutant. (B) Protein staining of immunoprecipitated FLAG-tagged wild-type and mutant VP35 from which the RNA transfected in (C) and sequenced in (D) were recovered. (C) Immunostimulatory activity of RNA following immunoprecipitation of the pCAGGS empty vector (EV), wild-type EBOV VP35, and mutant EBOV VP35 in cells infected with SeV or mock-infected. (D) Next-generation sequencing and read mapping to the SeV genome. RNA associated with the pCAGGS empty vector, wild-type EBOV VP35, and mutant EBOV VP35 was purified and subjected to Illumina sequencing and the resulting reads were mapped to the SeV genome. The graph depicts nucleotide coverage (Y-axis) at each position of the SeV genome (X-axis).</p

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

A pesar de que la arquitectura actual parece tener una tendencia general hacia lo mediático, aproximándose cada vez más a convertirse en un producto de consumo del mercado del arte, aún existen unos pocos arquitectos que conservan unos valores y una forma de pensar y hacer arquitectura considerados ya por muchos obsoletos. Uno de estos arquitectos es el madrileño Víctor López Cotelo y con este trabajo se pretende estudiar su obra para tratar de mostrar no sólo que no se trata de valores obsoletos, sino que suponen el mejor camino para llegar a la verdadera arquitectura y por ello estarán siempre vigentes, sin importar los cambios que haya en las modas. Para estudiar la obra de este arquitecto se realiza un recorrido por tres de sus edificios, situados en la ribera del río Sarela, en Santiago de Compostela. Tres obras (Ponte Sarela, la Vaquería y Pontepedriña) que parten de ruinas de piedra y variadas topografías para llegar a tres magníficos edificios en los que el tiempo es el protagonista, el pasado y el presente se dan la mano y la arquitectura se pone al servicio de la vida, con el objetivo de mejorar la de quienes la habitan

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes