Tumourigenesis Driven by the Human Papillomavirus Type 16 Asian-American E6 Variant in a Three-Dimensional Keratinocyte Model

Infection with a transforming human papillomavirus (HPV) such as type 16 (of species Alphapapillomavirus 9) causes ano-genital and oral tumours via viral persistence in human squamous cell epithelia. Epidemiological studies showed that the naturally occurring HPV16 Asian-American (AA) variant (sublineage D2/D3) is found more often than the European Prototype (EP) (sublineage A1) in high-grade cervical neoplasia and tumours compared to non-cancer controls. Just three amino acid changes within the early gene, E6, of HPV16 AA have been linked to this augmented tumourigenicity. The AAE6 variant''s greater immortalizing and transforming potential over EPE6 has recently been confirmed in retrovirally-transduced keratinocytes expressing the E6 gene only. However, the tumourigenic role of the full-length viral genome of HPV16 has not yet been addressed with regard to these E6 variants. To investigate this process in the context of these two HPV16 E6 genotypes, an organotypic tissue culture model was used to simulate the HPV infectious life cycle. The AAE6 variant demonstrated an enhanced ability over EPE6 to drive the viral life cycle toward tumourigenesis, as evidenced phenotypically—by a more severe grade of epithelial dysplasia with higher proliferation and deregulated differentiation, and molecularly—by high viral oncogene E6 and E7 expression, but lack of productive viral life cycle markers. In contrast, EPE6 had low E6 and E7 but high E1∧E4 expression, indicative of a productive life cycle. We suggest increased viral integration into the host genome for AAE6 as one possible mechanism for these observed differences from EPE6. Additionally, we found downstream effects on immortalization and host innate immune evasion. This study highlights how minor genomic variations in transforming viruses can have a significant affect on their tumourigenic ability.     

Gender Specific Reproductive Strategies of an Arctic Key Species (Boreogadus saida) and Implications of Climate Change

The Arctic climate is changing at an unprecedented rate. What consequences this may have on the Arctic marine ecosystem depends to a large degree on how its species will respond both directly to elevated temperatures and more indirectly through ecological interactions. But despite an alarming recent warming of the Arctic with accompanying sea ice loss, reports evaluating ecological impacts of climate change in the Arctic remain sparse. Here, based upon a large-scale field study, we present basic new knowledge regarding the life history traits for one of the most important species in the entire Arctic, the polar cod (Boreogadus saida). Furthermore, by comparing regions of contrasting climatic influence (domains), we present evidence as to how its growth and reproductive success is impaired in the warmer of the two domains. As the future Arctic is predicted to resemble today''s Atlantic domains, we forecast changes in growth and life history characteristics of polar cod that will lead to alteration of its role as an Arctic keystone species. This will in turn affect community dynamics and energy transfer in the entire Arctic food chain.     

In silico analysis of the adenylation domains of the freestanding enzymes belonging to the eucaryotic nonribosomal peptide synthetase-like family

This work presents a computational analysis of the molecular characteristics shared by the adenylation domains from traditional nonribosomal peptide synthetases (NRPSs) and the group of the freestanding homologous enzymes: alpha-aminoadipate semialdehyde dehydrogenase, alpha-aminoadipate reductase and the protein Ebony. The results of systematic sequence comparisons allow us to conclude that a specificity-conferring code, similar to that described for the NRPSs, can be recognized in such enzymes. The structural and functional roles of the residues involved in the substrate selection and binding are proposed through the analysis of the predicted interactions of the model active sites and their respective substrates. The indications deriving from this study can be useful for the programming of experiments aimed at a better characterization and at the engineering of this emerging group of single NRPS modules that are responsible for amino acid selection, activation and modification in the absence of other NRPS assembly line components.     

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for G_M1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, G_Sα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.     

IGF-binding proteins 3 and 4 are regulators of sprouting angiogenesis

Purpose

We have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis.

Methods

Effects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34⁺ tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum.

Results

Knockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling.

Conclusions

Serum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling.

     

Correction to: Cattle slurry acidification and application method can improve initial phosphorus availability for maize

Plant and Soil - The article Cattle slurry acidification and application method can improve initial phosphorus availability for maize, written by Ingeborg F. Pedersen, Gitte H. Rubæk and Peter...