Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4⁺ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Proteomic Identification of a Candidate Sequence of Wheat Cytokinin-Binding Protein 1

Our knowledge on the principle mechanisms of cytokinin action has been significantly deepened over the last years, but several weakly explored areas still remain on the map of cytokinin cellular physiology. Cytokinin-binding proteins could also be included in this pending field of cytokinin research. Probably, the best explored representative of this group is the wheat cytokinin-binding protein 1 (CBP-1). The role of this germ-allocated protein as a presumable regulator of free aromatic cytokinin levels during grain germination has been discussed intensively. To dig deeper into this interesting protein, this study was aimed at the identification of the CBP-1 amino acid sequence. A combination of in silico BLAST search, classical biochemical CBP-1 purification based on isoelectric point precipitation and ion-exchange chromatography, and proteomic analysis of the isolated protein by ultra-high resolution tandem mass spectrometry allowed us to uncover and validate two CBP-1 subunit candidate sequences with molecular masses of 56.2 and 55.0 kDa, respectively. Interestingly, we found the latter sequence alternated in two amino acids in the putative cytokinin-binding motive in comparison to the composition of this domain reported in the original studies. A BLAST search for the amino acid sequence of the binding region among plant proteomes revealed several highly related protein sequences, all originating from the Poaceae family. This piece of information could give support to the elucidation of the role of CBP-1 in physiological processes mediated by aromatic cytokinins.     

Behavioural Tests Reveal Severe Visual Deficits in the Strictly Subterranean African Mole‐Rats (Bathyergidae) but Efficient Vision in the Fossorial Rodent Coruro (Spalacopus cyanus,Octodontidae)

Vision has long been considered purposeless in the dark underground ecotope. However, recent anatomical studies revealed an unexpected diversity of ocular and retinal features and various degrees of development of the visual system in mammals with predominantly subterranean activity, and have suggested retention of basic visual capabilities even in some strictly subterranean mammals such as the African mole‐rats. Behavioural tests assessing image‐forming vision have not yet been conducted in subterranean mammals. Here, we investigated the visual capacities in three species of the African mole‐rats, namely the giant mole‐rat Fukomys mechowii, the Mashona mole‐rat Fukomys darlingi and the silvery mole‐rat Heliophobius argenteocinereus, in the fossorial coruro Spalacopus cyanus and the inbred C57L/J mouse. The behavioural assays performed in this study revealed severe visual deficits in all three species of mole‐rats. The absence of the visual placing reflex suggested impairment of either image‐forming vision or visuomotor integration. The random choice between the shallow and the deep side of a visual cliff clearly demonstrated inability of mole‐rats to perceive depth. The nesting assay did not yield conclusive evidence regarding the capacity for visually guided spatial orientation in the only tested species, the giant mole‐rat. In contrast, both the coruro and the mouse exhibited a clear placing reaction and preferred the shallow side of the visual cliff, implying functional image‐forming vision. Thus, the behavioural data gathered in this study show that vision is seriously compromised in the strictly subterranean, congenitally microphthalmic African mole‐rats but efficient (i.e. comparable to that of surface‐dwelling rodents) in a species with regular surface activity, the coruro.     

The Oomycete <Emphasis Type="Italic">Pythium oligandrum</Emphasis> Can Suppress and Kill the Causative Agents of Dermatophytoses

Pythium oligandrum (Oomycota) is known for its strong mycoparasitism against more than 50 fungal and oomycete species. However, the ability of this oomycete to suppress and kill the causal agents of dermatophytoses is yet to be studied. We provide a complex study of the interactions between P. oligandrum and dermatophytes representing all species dominating in the developed countries. We assessed its biocidal potential by performing growth tests, on both solid and liquid cultivation media and by conducting a pilot clinical study. In addition, we studied the molecular background of mycoparasitism using expression profiles of genes responsible for the attack on the side of P. oligandrum and the stress response on the side of Microsporum canis. We showed that dermatophytes are efficiently suppressed or killed by P. oligandrum in the artificial conditions of cultivations media between 48 and 72 h after first contact. Significant intra- and interspecies variability was noted. Of the 69 patients included in the acute regimen study, symptoms were completely eliminated in 79% of the patients suffering from foot odour, hyperhidrosis disappeared in 67% of cases, clinical signs of dermatomycoses could no longer be observed in 83% of patients, and 15% of persons were relieved of symptoms of onychomycosis. Our investigations provide clear evidence that the oomycete is able to recognize and kill dermatophytes using recognition mechanisms that resemble those described in oomycetes attacking fungi infecting plants, albeit with some notable differences.     

Activity of free‐living subterranean blind mole rats Spalax galili (Rodentia: Spalacidae) in an area of supposed sympatric speciation

Subterranean rodents forage underground, which is energetically costly. Therefore, they can be expected to economize burrowing activity in response to food supply and soil characteristics. We analyzed the activity of radio‐tracked blind mole rats, Spalax galili, on a locality sharply subdivided into harder but relatively food‐rich, basaltic soil and softer, relatively food‐poor rendzina. It was recently proposed that the mole rats in this locality are undergoing sympatric ecological speciation. We predicted that mole rats from basaltic soil would be less active than those from rendzina as a result of the reduced need for burrowing to reach food. By contrast to our predictions, mole rats from basaltic soil were more frequently located outside the nest and observed pushing soil above ground. We suggest that this is a result of territorial behaviour due to high population density. All mole rats exhibited a unimodal daily activity pattern likely related to temperature. Large males had large but gradually decreasing home‐ranges, likely indicating the end of the mating season. We conclude that the ecological differences between the habitats cause behavioural differences in the mole rats, which indicates different selection pressures. The genetic divergence previously found between the populations might have arisen via density‐dependent selection.