Expression of the murine cytomegalovirus glycoprotein H by recombinant vaccinia virus

The sequence of the gene encoding glycoprotein H (gH) of murine cytomegalovirus (MCMV) strain Smith was determined and compared with the sequence of the gH of MCMV strain K181. Transcriptional analysis showed that gH is encoded by a large mRNA of 5.0 kb, which is synthesized late in infection. A recombinant vaccinia virus expressing the MCMV gH open reading frame was constructed (Vac-gH). Anti-MCMV serum precipitated a protein of 87K from Vac-gH-infected cells. Reactivity with a monoclonal antibody showed the identity of the MCMV gH with a 87K envelope glycoprotein described previously by Loh and Qualtiere. Immunization of mice with the Vac-gH recombinant gave rise to an anti-gH serum, which neutralized MCMV without complement in vitro

Genome-wide multi-parametric analysis of H2AX or γH2AX distributions during ionizing radiation-induced DNA damage response

Background: After induction of DNA double strand breaks (DSBs), the DNA damage response (DDR) is activated. One of the earliest events in DDR is the phosphorylation of serine 139 on the histone variant H2AX (gH2AX) catalyzed by phosphatidylinositol 3-kinases-related kinases. Despite being extensively studied, H2AX distribution[1] across the genome and gH2AX spreading around DSBs sites[2] in the context of different chromatin compaction states or transcription are yet to be fully elucidated. Materials and methods: gH2AX was induced in human hepatocellular carcinoma cells (HepG2) by exposure to 10 Gy X-rays (250 kV, 16 mA). Samples were incubated 0.5, 3 or 24 hours post irradiation to investigate early, intermediate and late stages of DDR, respectively. Chromatin immunoprecipitation was performed to select H2AX, H3 and gH2AX-enriched chromatin fractions. Chromatin-associated DNA was then sequenced by Illumina ChIP-Seq platform. HepG2 gene expression and histone modification (H3K36me3, H3K9me3) ChIP-Seq profiles were retrieved from Gene Expression Omnibus (accession numbers GSE30240 and GSE26386, respectively). Results: First, we combined G/C usage, gene content, gene expression or histone modification profiles (H3K36me3, H3K9me3) to define genomic compartments characterized by different chromatin compaction states or transcriptional activity. Next, we investigated H3, H2AX and gH2AX distributions in such defined compartments before and after exposure to ionizing radiation (IR) to study DNA repair kinetics during DDR. Our sequencing results indicate that H2AX distribution followed H3 occupancy and, thus, the nucleosome pattern. The highest H2AX and H3 enrichment was observed in transcriptionally active compartments (euchromatin) while the lowest was found in low G/C and gene-poor compartments (heterochromatin). Under physiological conditions, the body of highly and moderately transcribed genes was devoid of gH2AX, despite presenting high H2AX levels. gH2AX accumulation was observed in 5’ or 3’ flanking regions, instead. The same genes showed a prompt gH2AX accumulation during the early stage of DDR which then decreased over time as DDR proceeded. Finally, during the late stage of DDR the residual gH2AX signal was entirely retained in heterochromatic compartments. At this stage, euchromatic compartments were completely devoid of gH2AX despite presenting high levels of non-phosphorylated H2AX. Conclusions: We show that gH2AX distribution ultimately depends on H2AX occupancy, the latter following H3 occupancy and, thus, nucleosome pattern. Both H2AX and H3 levels were higher in actively transcribed compartments. However, gH2AX levels were remarkably low over the body of actively transcribed genes suggesting that transcription levels antagonize gH2AX spreading. Moreover, repair processes did not take place uniformly across the genome; rather, DNA repair was affected by genomic location and transcriptional activity. We propose that higher H2AX density in euchromaticcompartments results in high relative gH2AXconcentration soon after the activation of DDR, thus favoring the recruitment of the DNA repair machinery to those compartments. When the damage is repaired and gH2AX is removed, its residual fraction is retained in the heterochromatic compartments which are then targeted and repaired at later times

The Conditions of Primary Infection Define the Load of Latent Viral Genome in Organs and the Risk of Recurrent Cytomegalovirus Disease

Recurrence of cytomegalovirus (CMV) from latency is a frequent cause of disease in immunocompromised patients. To date, there is no explanation for the diversity in the clinical manifestations. Primary infection can occur perinatally or later in life, and inevitably results in latent infection. Seropositivity for antibodies against CMV is indicative of latent infection, but is insufficient as a predictor for the risk of recurrence. As a model for this important medical problem, we compared the risks of murine CMV recurrence from latency established after neonatal primary infection and after infection at adult age. The risk of CMV recurrence was high only after neonatal infection. The copy number of latent viral genome in tissues was identified as the key parameter that determines the overall and organ-specific risks of recurrence. Latent CMV burden and risk of recurrence were related to the extent of virus multiplication during primary infection. The presence of latent CMV in multiple organs provides the molecular basis for stochastic events of recurrence in single organs or in any combination thereof. These findings are discussed as a concept of multifocal CMV latency and recurrence. It provides a rationale for the diversity in the clinical outcome of CMV disease

Analysis of the Relationship between Frugal Innovation and Sustainable Development

The relation between Frugal Innovation and Sustainability is a widely discussed topic in current academic literature. To assess the correlation between these two concepts, a sample of the most frequently mentioned Frugal Innovations was selected. The criteria to evaluate the sustainability potential for each of these cases was built upon the UN Sustainable Development Goals, a global reference indicator. Frugal Innovations among the banking, transport, energy, ICT, water, appliances and healthcare sectors were evaluated. The results suggested a positive contribution and impact of Frugal Innovations towards economic, social and environmental development

The impact of sequence database choice on metaproteomic results in gut microbiota studies

Background: Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics, the study of the whole protein complement of a microbial community, can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Results: Here, we present a systematic investigation of variables concerning database construction and annotation and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. In particular, the contribution of experimental metagenomic databases was revealed to be mandatory when dealing with mouse samples. Moreover, the use of a "merged" database, containing all metagenomic sequences from the population under study, was found to be generally preferable over the use of sample-matched databases. We also observed that taxonomic and functional results are strongly database-dependent, in particular when analyzing the mouse gut microbiota. As a striking example, the Firmicutes/Bacteroidetes ratio varied up to tenfold depending on the database used. Finally, assembling reads into longer contigs provided significant advantages in terms of functional annotation yields. Conclusions: This study contributes to identify host- and database-specific biases which need to be taken into account in a metaproteomic experiment, providing meaningful insights on how to design gut microbiota studies and to perform metaproteomic data analysis. In particular, the use of multiple databases and annotation tools has to be encouraged, even though this requires appropriate bioinformatic resources

Small Satellite Constellations, Infrastructure Shift and Space Market Regulation

With the commissioning of the first constellations of hundreds or even thousands of small satellites, we are witnessing today an infrastructure shift. While it has not completely distanced the exploration and use of outer space from the dramatic geopolitical and military implications they once entailed for States, it is undoubtedly transforming this realm into a new economic frontier of competition, with its predominant players, this time being private profit-driven actors sensitive to market forces. As the exploitation of outer space becomes more economically viable, new commercial services should emerge through the deployment of SmallSat constellations and the provision of services by means thereof, creating a risk of increased dependency of the services consumed on earth on these newinfrastructures. Therefore, new legal challenges pertaining to competition, foreign investment and the global economy as a whole do arise

Small Satellite Constellations, Infrastructure Shift and Space Market Regulation

With the commissioning of the first constellations of hundreds or even thousands of small satellites, we are witnessing today an infrastructure shift. While it has not completely distanced the exploration and use of outer space from the dramatic geopolitical and military implications they once entailed for States, it is undoubtedly transforming this realm into a new economic frontier of competition, with its predominant players, this time being private profit-driven actors sensitive to market forces. As the exploitation of outer space becomes more economically viable, new commercial services should emerge through the deployment of SmallSat constellations and the provision of services by means thereof, creating a risk of increased dependency of the services consumed on earth on these newinfrastructures. Therefore, new legal challenges pertaining to competition, foreign investment and the global economy as a whole do arise

Reinventing treaty compliant ‘‘safety zones’’ in the context of space sustainability

This paper questions the concept of safety zones, tracing it to maritime and military law, in order to unpack its potential legal uses, applications and implications in the current context of the New Space economy. To achieve this, it starts by investigating the historical foundations of safety zones rooted in the Cold-War-era legal literature and then move on to a larger discussion of how such zones can be balanced with the non-appropriation principle. Then, this paper examines a number of legal analogies, as they appear in maritime law, aviation law as well as relevant examples in outer-space law. Lastly, it attempts to sketch the outline of a “solution ”that identifies the criteria, to be used in order to model a potential safety zone that strikes a balance between the industry’s growing concerns and requirements on one hand, and the legal prohibitions that preclude sovereign claims on outer space on the other hand. Particular attention is given to the special case of the United States’ extraterritorial jurisdiction in outer space

Reinventing treaty compliant ‘‘safety zones’’ in the context of space sustainability

This paper questions the concept of safety zones, tracing it to maritime and military law, in order to unpack its potential legal uses, applications and implications in the current context of the New Space economy. To achieve this, it starts by investigating the historical foundations of safety zones rooted in the Cold-War-era legal literature and then move on to a larger discussion of how such zones can be balanced with the non-appropriation principle. Then, this paper examines a number of legal analogies, as they appear in maritime law, aviation law as well as relevant examples in outer-space law. Lastly, it attempts to sketch the outline of a “solution ”that identifies the criteria, to be used in order to model a potential safety zone that strikes a balance between the industry’s growing concerns and requirements on one hand, and the legal prohibitions that preclude sovereign claims on outer space on the other hand. Particular attention is given to the special case of the United States’ extraterritorial jurisdiction in outer space