Structural Plasticity of the Semliki Forest Virus Glycome upon Interspecies Transmission

Cross-species viral transmission subjects parent and progeny alphaviruses to differential post-translational processing of viral envelope glycoproteins. Alphavirus biogenesis has been extensively studied, and the Semliki Forest virus E1 and E2 glycoproteins have been shown to exhibit differing degrees of processing of N-linked glycans. However the composition of these glycans, including that arising from different host cells, has not been determined. Here we determined the chemical composition of the glycans from the prototypic alphavirus, Semliki Forest virus, propagated in both arthropod and rodent cell lines, by using ion-mobility mass spectrometry and collision-induced dissociation analysis. We observe that both the membrane-proximal E1 fusion glycoprotein and the protruding E2 attachment glycoprotein display heterogeneous glycosylation that contains N-linked glycans exhibiting both limited and extensive processing. However, E1 contained predominantly highly processed glycans dependent on the host cell, with rodent and mosquito-derived E1 exhibiting complex-type and paucimannose-type glycosylation, respectively. In contrast, the protruding E2 attachment glycoprotein primarily contained conserved under-processed oligomannose-type structures when produced in both rodent and mosquito cell lines. It is likely that glycan processing of E2 is structurally restricted by steric-hindrance imposed by local viral protein structure. This contrasts E1, which presents glycans characteristic of the host cell and is accessible to enzymes. We integrated our findings with previous cryo-electron microscopy and crystallographic analyses to produce a detailed model of the glycosylated mature virion surface. Taken together, these data reveal the degree to which virally encoded protein structure and cellular processing enzymes shape the virion glycome during interspecies transmission of Semliki Forest virus

Ammonium excess leads to Ca restrictions, morphological changes, and nutritional imbalances in tomato plants, which can be monitored by the N/Ca ratio.

Both nitrogen and calcium fertilization management are vital for crops, where an imbalance of these elements can cause both physiological and yield problems. It has been proposed that nitrogen absorption, particularly ammonium, is in part dependent on calcium supply. Moreover, the balance between these two nutrients could be a key indicator of plant growth in some species. Tomato, one of the most cultivated crops worldwide, can also be widely affected by nutritional imbalance. Using large amounts of N fertilizers could lead to an imbalance with other nutrients and, thus, detrimental effects in terms of plant development and yield. Here we show that ammonium excess has a negative impact on plant development and results in calcium deficiency. Moreover, a deficit in calcium nutrition not only affects calcium concentration but also leads to a restriction in N uptake and reduced N concentration in the plant. These effects were evident at the seedling stage and also during flowering/fruit set. Using PCA analysis, we integrated both phenotypic and nutritional imbalances in seedlings and grown plants. Interestingly, the Ca/N ratio appears to be a key indicator to monitor appropriate N and calcium nutrition and more importantly the balance between both. Maintaining this balance could be an essential element for tomato crop production

Plasmatic Soluble Receptor for Advanced Glycation End Products as a New Oxidative Stress Biomarker in Patients with Prosthetic-Joint-Associated Infections?

Prosthetic joint infection (PJI) is the most common cause of failure of total joint arthroplasty, but a gold standard for PJI diagnosis is still lacking. Advanced glycation end products (AGEs) are proinflammatory molecules inducing intracellular oxidative stress (OS) after binding to their cell membrane receptors (RAGE). The aim of this study was to evaluate plasmatic soluble receptor for advanced glycation end products (sRAGE), as a new OS and infection marker correlating sRAGE to the level of OS and antioxidant defenses, in PJI, in order to explore the possible application of this new biomarker in the early diagnosis of PJI. Plasmatic sRAGE levels (by ELISA assay), plasma antioxidant total defenses (by lag time method), plasma reactive oxygen species (ROS), and thiobarbituric acid reactive substance (TBARS) levels (by colorimetric assay) were evaluated in 11 PJI patients and in 30 matched controls. ROS and TBARS were significantly higher (p < 0 001) while plasma total antioxidant capacity and sRAGE were significantly lower (p < 0 01) in patients with PJI compared to controls. Our results confirm the OS in PJI and show a strong negative correlation between the level of sRAGE and oxidative status, suggesting the plasmatic sRAGE as a potential marker for improving PJI early diagnosis

The Glycan Shield of HIV Is Predominantly Oligomannose Independently of Production System or Viral Clade

The N-linked oligomannose glycans of HIV gp120 are a target for both microbicide and vaccine design. The extent of cross-clade conservation of HIV oligomannose glycans is therefore a critical consideration for the development of HIV prophylaxes. We measured the oligomannose content of virion-associated gp120 from primary virus from PBMCs for a range of viral isolates and showed cross-clade elevation (62–79%) of these glycans relative to recombinant, monomeric gp120 (∼30%). We also confirmed that pseudoviral production systems can give rise to notably elevated gp120 oligomannose levels (∼98%), compared to gp120 derived from a single-plasmid viral system using the HIVLAI backbone (56%). This study highlights differences in glycosylation between virion-associated and recombinant gp120

Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera

Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studiesinfo:eu-repo/semantics/publishedVersio

Uukuniemi Phlebovirus Assembly and Secretion Leave a Functional Imprint on the Virion Glycome

Uukuniemi virus (UUKV) is a model system for investigating the genus Phlebovirus of the Bunyaviridae. We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly-N-acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leave a functional imprint on the phleboviral glycome

Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/ or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and Cterminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn136 in V1 (T138N mutation) inconjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons inV1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection

Test beam performance of a CBC3-based mini-module for the Phase-2 CMS Outer Tracker before and after neutron irradiation

The Large Hadron Collider (LHC) at CERN will undergo major upgrades to increase the instantaneous luminosity up to 5–7.5×10$^{34}$ cm$^{-2}$s$^{-1}$. This High Luminosity upgrade of the LHC (HL-LHC) will deliver a total of 3000–4000 fb-1 of proton-proton collisions at a center-of-mass energy of 13–14 TeV. To cope with these challenging environmental conditions, the strip tracker of the CMS experiment will be upgraded using modules with two closely-spaced silicon sensors to provide information to include tracking in the Level-1 trigger selection. This paper describes the performance, in a test beam experiment, of the first prototype module based on the final version of the CMS Binary Chip front-end ASIC before and after the module was irradiated with neutrons. Results demonstrate that the prototype module satisfies the requirements, providing efficient tracking information, after being irradiated with a total fluence comparable to the one expected through the lifetime of the experiment