The N276 Glycosylation Site Is Required for HIV-1 Neutralization by the CD4 Binding Site Specific HJ16 Monoclonal Antibody

Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb), HJ16 recognizes a new epitope in the CD4 binding site (CD4bs) region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs) of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb.     

The impact of fossil stratigraphic ranges on tip-calibration,and the accuracy and precision of divergence time estimates

The molecular clock provides the only viable means of establishing realistic evolutionary timescales but it remains unclear how best to calibrate divergence time analyses. Calibrations can be applied to the tips and/or to the nodes of a phylogeny. Tip-calibration is an attractive approach since it allows fossil species to be included alongside extant relatives in molecular clock analyses. However, most fossil species are known from multiple stratigraphical horizons and it remains unclear how such age ranges should be interpreted to codify tip-calibrations. We use simulations and empirical data to explore the impact on precision and accuracy of different approaches to informing tip-calibrations. In particular, we focus on the effect of using tip-calibrations defined using the oldest vs youngest stratigraphic occurrences, the full stratigraphical range, as well as confidence intervals on these data points. The results of our simulations show that using different calibration approaches leads to different divergence-time estimates and demonstrate that concentrating tip-calibrations near the root of the dated phylogeny improves both precision and accuracy of estimated divergence times. Finally, our results indicate that the highest levels of accuracy and precision are achieved when fossil tips are calibrated based on the fossil occurrence from which the morphological data were derived. These trends were corroborated by analysis of an empirical dataset for Ursidae. Overall, we conclude that tip-dating analyses should, in particular, employ tip calibrations close to the root of the tree and they should be calibrated based on the age of the fossil used to inform the morphological data used in Total Evidence Dating.     

Investigating the heat shock protein response involved in coral bleaching across scleractinian species in the central Red Sea

Coral Reefs - Coral bleaching represents the most serious threat to contemporary coral reefs. In response, focus is being laid on understanding the cellular processes involved in the response of...     

Neuroprotective activities of bacopa,lycopene, astaxanthin, and vitamin B12 combination on oxidative stress-dependent neuronal death

Oxidative stress is considered the common effector of the cascade of degenerative events in many neurological conditions. Thus, in this paper we tested different nutraceuticals in H₂O₂ in vitro model to understand if could represent an adjuvant treatment for neurological diseases. In this study, nutraceuticals bacopa, lycopene, astaxanthin, and vitamin B12 were used alone or in combination in human neuronal differentiated SH-SY5Y cells upon hydrogen peroxide-induced injury and neuroprotective, neuronal death pathways were analyzed. The nutraceuticals analyzed were able to protect H₂O₂ cytotoxic effects, through increasing cell viability and proteins involved in neuroprotection pathways and restoring proteins involved in cell death pathways. On this basis, it is possible to propose the use of these compounds as dietary supplement for the prevention or as adjuvant to the only symptomatic treatments so far available for neurodegenerative diseases.     

Structural model of the full-length Ser/Thr protein kinase StkP from S. pneumoniae and its recognition of peptidoglycan fragments

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.     

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.     

Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53‐dependent pathway

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53‐wild type U2OS cells (and not of p53‐null Saos and p53‐mutant MG63 cells) by slowing‐down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin‐induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub‐G1 population, Bcl‐2 downregulation, caspase‐3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination‐induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine‐alpha. Moreover, the doxorubicin‐induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53‐dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy. J. Cell. Physiol. 228: 198–206, 2013. © 2012 Wiley Periodicals, Inc.     

Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.     

Fast Two-Dimensional Bubble Analysis of Biopolymer Filamentous Networks Pore Size from Confocal Microscopy Thin Data Stacks

The average pore size ξ₀ of filamentous networks assembled from biological macromolecules is one of the most important physical parameters affecting their biological functions. Modern optical methods, such as confocal microscopy, can noninvasively image such networks, but extracting a quantitative estimate of ξ₀ is a nontrivial task. We present here a fast and simple method based on a two-dimensional bubble approach, which works by analyzing one by one the (thresholded) images of a series of three-dimensional thin data stacks. No skeletonization or reconstruction of the full geometry of the entire network is required. The method was validated by using many isotropic in silico generated networks of different structures, morphologies, and concentrations. For each type of network, the method provides accurate estimates (a few percent) of the average and the standard deviation of the three-dimensional distribution of the pore sizes, defined as the diameters of the largest spheres that can be fit into the pore zones of the entire gel volume. When applied to the analysis of real confocal microscopy images taken on fibrin gels, the method provides an estimate of ξ₀ consistent with results from elastic light scattering data.