CMV immune evasion and manipulation of the immune system with aging

Human cytomegalovirus (HCMV) encodes numerous proteins and microRNAs that function to evade the immune response and allow the virus to replicate and disseminate in the face of a competent innate and acquired immune system. The establishment of a latent infection by CMV, which if completely quiescent at the level of viral gene expression would represent an ultimate in immune evasion strategies, is not sufficient for lifelong persistence and dissemination of the virus. CMV needs to reactivate and replicate in a lytic cycle of infection in order to disseminate further, which occurs in the face of a fully primed secondary immune response. Without reactivation, latency itself would be redundant for the virus. It is also becoming clear that latency is not a totally quiescent state, but is characterized by limited viral gene expression. Therefore, the virus also needs immune evasion strategies during latency. An effective immune response to CMV is required or viral replication will cause morbidity and ultimately mortality in the host. There is clearly a complex balance between virus immune evasion and host immune recognition over a lifetime. This poses the important question of whether long-term evasion or manipulation of the immune response driven by CMV is detrimental to health. In this meeting report, three groups used the murine model of CMV (MCMV) to examine if the contribution of the virus to immune senescence is set by the (i) initial viral inoculum, (ii) inflation of T cell responses, (iii) or the balance between functionally distinct effector CD4+ T cells. The work of other groups studying the CMV response in humans is discussed. Their work asks whether the ability to make immune responses to new antigens is compromised by (i) age and HCMV carriage, (ii) long-term exposure to HCMV giving rise to an overall immunosuppressive environment and increased levels of latent virus, or (iii) adapted virus mutants (used as potential vaccines) that have the capacity to elicit conventional and unconventional T cell responses.DvB and SPHVdB are funded by a strategic program grant RIVM. MRW and SEJ are funded by the Medical Research Council Grant (GB) [MR/K021087/1]. The work summarized in the section titled BThe impact of aging on IL-10 secreting HCMV latent antigen specific T cells and latent viral load^ was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. We gratefully acknowledge the participation of all Cambridge NIHR BioResource volunteers, and we thank the Cambridge BioResource staff for their help with volunteer recruitment. The Cambridge BioResource is funded by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (BRC) and the NHS Blood and Transplant (NHSBT). CAB is funded by an NIH grant AI101423. LCS was funded in part by grants from the Helmholtz Association (HGFVI-424) and the German Scientific Council (SFB900 TP B2)

Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activityope

Diagnosis and management of Cornelia de Lange syndrome:first international consensus statement

Cornelia de Lange syndrome (CdLS) is an archetypical genetic syndrome that is characterized by intellectual disability, well-defined facial features, upper limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in any one of seven genes, all of which have a structural or regulatory function in the cohesin complex. Although recent advances in next-generation sequencing have improved molecular diagnostics, marked heterogeneity exists in clinical and molecular diagnostic approaches and care practices worldwide. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria, both for classic CdLS and non-classic CdLS phenotypes, molecular investigations, long-term management and care planning

Interaction of contaminated sediment from a salt marsh with estuarine water: evaluation by leaching and ecotoxicity assays and salts from leachate evaporation

Purpose Wastes from a former Portuguese steel plant were deposited between 1961 and 2001 on the riverbank of a tributary of the Tagus River creating a landfill connected to the river, posing a potential contamination risk to the Tagus estuary ecosystem. This study aims to assess the transfer of chemical elements from contaminated sediments to the estuarine water from cycles of sediment leaching so as to evaluate the ecotoxicity of the leachates, and to analyze the solid phases crystallized from those leachates. Materials and methods Landfill sediment and estuarine water samples were collected during low tide. Sediment samples were analyzed for pH, electric conductivity (EC), C-org, NPK, and iron oxides. Leaching assays (four replicates) were done using estuarine water (200 cm(3)/replicate) and 1.5 kg of sediment per reactor. Each reactor was submitted to four leaching processes (0, 28, 49, and 77 days). The sediment was kept moist between leaching processes. Sediment (total (acid digestion) and available fraction (diluted organic acid extraction- Rhizo)) elemental concentrations were determined by inductively coupled plasma-instrumental neutron activation analysis (ICP/INAA). Leachates, and estuarine and sediment pore waters were analyzed for metals/metalloids by ICP/mass spectrometry (MS) and carbonates/sulfate/chloride by standard methodologies. Ecotoxicity assays were performed in leachates and estuarine and pore waters using Artemia franciscana and Brachionus plicatillis. Aliquots of the leachates were evaporated to complete dryness (23-25 degrees C) and crystals analyzed by X-ray powder diffraction (XRD). Results and discussion Sediment with pH=8 and high EC and Corg was contaminated with As, Cd, Cr, Cu, Pb, and Zn. The element concentrations in the available fraction of the sediment were low compared to the sediment total concentrations (<1 % for Rhizo extraction). The concentrations of potentially hazardous elements in the estuarine water were relatively low, except for Cd. Concentrations of hazardous elements in the leachates were very low. Calcium, K, Mg, Na, and chloride concentrations were high but did not vary significantly among the four leaching experiments. Total concentrations of carbonate were much higher in leachates than in estuarine water. Both estuarine water and leachates showed negligible toxicity. Crystals identified in the solids obtained from the leachates by evaporation were halite, anhydrite, epsomite, dolomite, and polyhalite. Conclusions The sediment showed the capacity to retain the majority of the potentially hazardous chemical elements. Remobilization of chemical elements from sediment by leaching was essentially negligible. The variation of total concentrations of Ca, carbonate, and sulfate in leachates indicates that the sediment contained reactive sulfides. Due to its composition, the sediment seems to be a dynamic system of pollution control, which should not be disturbed