CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins—high physical stability, specificity and low production costs—with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development

Higuchi's fractal dimension, but not frontal or posterior alpha asymmetry, predicts PID-5 anxiousness more than depressivity

Depression is a major cause of health disability. EEG measures may provide one or more economical biomarkers for the diagnosis of depression. Here we compared frontal alpha asymmetry (FAA), posterior alpha asymmetry (PAA), and Higuchi's fractal dimension (HFD) for their capacity to predict PID-5 depressivity and for the specifcity of these predictions relative to PID-5 anxiousness. University students provided 8 or 10minutes of resting EEG and PID-5 depressivity and PID-5 anxiousness questionnaire scores. FAA and PAA had no signifcant correlations with the measures at any electrode pair. There were distinct frontal and posterior factors underlying HFD that correlated signifcantly with anxiousness and with each other. Posterior HFD also correlated signifcantly with depressivity, though this was weaker than the correlation with anxiousness. The portion of depressivity variance accounted for by posterior HFD was not unique but shared with anxiousness. Inclusion of anxiety disorder patients into the sample rendered the frontal factor somewhat more predictive than the posterior one but generally strengthened the prior conclusions. Contrary to our predictions, none of our measures specifcally predicted depressivity. Previous reports of links with depression may involve confounds with concurrent anxiety. Indeed, HFD may be a better measure of anxiety than depression" and its previous linkage to depression may be due to a confound between the two, given the high incidence of depression in cases of severe anxiety

Analysis of the mineral composition of Vitellaria paradoxa seed as potential feed source for livestock

No Abstract. Animal Production Research Avancees Vol. 3 (3) 2007: pp. 216-21

Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size

Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced