Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library

Phage display technology is a powerful selection approach to identify strong and specific binders to a large variety of targets. In this study, we compared the efficacy of a phage library displaying human heavy chain complementarity determining region 3 (HCDR3) repertoires with a set of conventional random peptide libraries for the identification of CXCR4 antagonists using a peptide corresponding to the second extracellular loop of the receptor CXCR4 as target. A total of 11 selection campaigns on this target did not result in any specific ligand from the random peptide libraries. In contrast, a single selection campaign with an HCDR3 library derived from the IgM repertoire of a nonimmunized donor resulted in nine specific peptides with lengths ranging from 10 to 19 residues. Four of these HCDR3 sequences interacted with native receptor and the most frequently isolated peptide displayed an affinity of 5.6 μm and acted as a CXCR4 antagonist (IC(50) = 23 μm). To comprehend the basis of the highly efficient HCDR3 library selection, its biochemical properties were investigated. The HCDR3 length varied from 3 to 21 residues and displayed a biased amino acid content with a predominant proportion of Tyr, Gly, Ser and Asp. Repetitive and conserved motifs were observed in the majority of the HCDR3 sequences. The strength and efficacy of the HCDR3 libraries reside in the combination of multiple size peptides and a naturally biased sequence variation. Therefore, HCDR3 libraries represent a powerful and versatile alternative to fully randomized peptide libraries, in particular for difficult targets.     

Impact of the HIV-1 env genetic context outside HR1-HR2 on resistance to the fusion inhibitor enfuvirtide and viral infectivity in clinical isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.     

Equivalence of multibreed animal models and hierarchical Bayes analysis for maternally influenced traits

Background

It has been argued that multibreed animal models should include a heterogeneous covariance structure. However, the estimation of the (co)variance components is not an easy task, because these parameters can not be factored out from the inverse of the additive genetic covariance matrix. An alternative model, based on the decomposition of the genetic covariance matrix by source of variability, provides a much simpler formulation. In this study, we formalize the equivalence between this alternative model and the one derived from the quantitative genetic theory. Further, we extend the model to include maternal effects and, in order to estimate the (co)variance components, we describe a hierarchical Bayes implementation. Finally, we implement the model to weaning weight data from an Angus × Hereford crossbred experiment.

Methods

Our argument is based on redefining the vectors of breeding values by breed origin such that they do not include individuals with null contributions. Next, we define matrices that retrieve the null-row and the null-column pattern and, by means of appropriate algebraic operations, we demonstrate the equivalence. The extension to include maternal effects and the estimation of the (co)variance components through the hierarchical Bayes analysis are then straightforward. A FORTRAN 90 Gibbs sampler was specifically programmed and executed to estimate the (co)variance components of the Angus × Hereford population.

Results

In general, genetic (co)variance components showed marginal posterior densities with a high degree of symmetry, except for the segregation components. Angus and Hereford breeds contributed with 50.26% and 41.73% of the total direct additive variance, and with 23.59% and 59.65% of the total maternal additive variance. In turn, the contribution of the segregation variance was not significant in either case, which suggests that the allelic frequencies in the two parental breeds were similar.

Conclusion

The multibreed maternal animal model introduced in this study simplifies the problem of estimating (co)variance components in the framework of a hierarchical Bayes analysis. Using this approach, we obtained for the first time estimates of the full set of genetic (co)variance components. It would be interesting to assess the performance of the procedure with field data, especially when interbreed information is limited.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Interactions of exercise training and lipoic acid on skeletal muscle glucose transport in obese Zucker rats.

Exercise training (ET) or the antioxidant R(+)-alpha-lipoic acid (R-ALA) individually increases insulin action in the insulin-resistant obese Zucker rat. The purpose of the present study was to determine the interactions of ET and R-ALA on insulin action and oxidative stress in skeletal muscle of the obese Zucker rat. Animals either remained sedentary, received R-ALA (30 mg x kg body wt(-1) x day(-1)), performed ET (treadmill running), or underwent both R-ALA treatment and ET for 6 wk. During an oral glucose tolerance test, ET alone or in combination with R-ALA resulted in a significant lowering of the glucose (26-32%) and insulin (29-30%) responses compared with sedentary controls. R-ALA alone decreased (19%) the glucose-insulin index (indicative of increased insulin sensitivity), and this parameter was reduced (48-52%) to the greatest extent in the ET and combined treatment groups. ET or R-ALA individually increased insulin-mediated glucose transport activity in isolated epitrochlearis (44-48%) and soleus (37-57%) muscles. The greatest increases in insulin action in these muscles (80 and 99%, respectively) were observed in the combined treatment group. Whereas the improvement in insulin-mediated glucose transport in soleus due to R-ALA was associated with decreased protein carbonyl levels (an index of oxidative stress), improvement because of ET was associated with decreased protein carbonyls as well as enhanced GLUT-4 protein. However, there was no interactive effect of ET and R-ALA on GLUT-4 protein or protein carbonyl levels. These results indicate that ET and R-ALA interact in an additive fashion to improve insulin action in insulin-resistant skeletal muscle. Because the further improvement in muscle glucose transport in the combined group was not associated with additional upregulation of GLUT-4 protein or a further reduction in oxidative stress, the mechanism for this interaction must be due to additional, as yet unidentified, factors.