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We combined cell‐free ribosome display and cell‐based yeast display selection to build specific protein binders to the extracellular domain of the human interleukin 9 receptor alpha (IL‐9Rα). The target, IL‐9Rα, is the receptor involved in the signalling pathway of IL‐9, a pro‐inflammatory cytokine medically important for its involvement in respiratory diseases. The successive use of modified protocols of ribosome and yeast displays allowed us to combine their strengths—the virtually infinite selection power of ribosome display and the production of (mostly) properly folded and soluble proteins in yeast display. The described experimental protocol is optimized to produce binders highly specific to the target, including selectivity to common proteins such as BSA, and proteins potentially competing for the binder such as receptors of other cytokines. The binders were trained from DNA libraries of two protein scaffolds called 57aBi and 57bBi developed in our laboratory. We show that the described unconventional combination of ribosome and yeast displays is effective in developing selective small protein binders to the medically relevant molecular target. [ABSTRACT FROM AUTHOR]

The article’s topic is animal in Christian religion and concept of creation. It describes how Christian theology has reflected the status and significance of animals in the historical development, which positions it has created or preferred in this matter and how they have changed from biblical times until our post-modern present age. Methodically this cross-section through history means that each period is conveyed in its basic features and presented through selected authors, works or texts of the given time. Along with the historical line, the text also follows the principal question of the nature of the relation between human and animal from a theological viewpoint. The object of reflection is thus also the theoretical basis for a morally justifiable relation of post-modern people to other living creatures. The text mainly deals with the following key aspects: a) anthropocentrism and its difficulties in relation to the position of animals in the western, traditionally Christian culture, b) possible ethical perspectives and consequences of the theological concept of creation for the status of animal today. [ABSTRACT FROM AUTHOR]

The author examines trust as a term and its meaning in context of sociology, psychology, and mainly philosophy and theology. The text is written from the perspective of Christian philosophical and theological anthropology, or rather philosophy and theology of religion. Trust is thematized in the three typifying contexts (society, life of an individual, and question about God) and an important idea of certain work or thinker within each of them is analysed. Trust is understood as something indigenous and irreducible, an irreplaceable, indispensable, and uniting element of society (Luhmann); personally formative (Erikson); inward and fundamentally corresponding with the empirical structure of human person (Landsberg); deep, borderline, transcending (Schillebeeckx); opening access to reality, lifegiving, and referring to God (Küng). The author systematically reflects the term and its anthropological and theological relevance with respect to historical variability of life and thinking. [ABSTRACT FROM AUTHOR]

We hereby describe the process of design and selection of nonantibody protein binders mimicking cytokine signaling. We chose to mimic signaling of IFN‐λ1, type 3 interferon (also known as IL‐29) for its novelty and the importance of its biological functions. All four known interferons λ signal through binding to the extracellular domains of IL‐28 receptor 1 (IL‐28R1) and IL‐10 receptor 2 (IL‐10R2). Our binders were therefore trained to bind both receptors simultaneously. The bifunctional binder molecules were developed by yeast display, a method of directed evolution. The signaling capacity of the bivalent binders was tested by measuring phosphorylation of the JAK/STAT signaling pathway and production of mRNA of six selected genes naturally induced by IFN‐ λ1 in human cell lines. The newly developed bivalent binders offer opportunities to study cytokine‐related biological functions and modulation of the cell behavior by receptor activation on the cell surfaces alternative to the use of natural IFN‐λ. [ABSTRACT FROM AUTHOR]

Ratzinger's early work Introduction to Christianity presents an attempt at rational understanding of what theology considers to be crucial for Christianity and what should be thought over in the context of the (then) new challenges. The open character of the book can be understood in light of the fact that anthropological discourse was already well established in post-Council theology. For Ratzinger, this seems to be connected with a necessity to outline clear contours for Christianity. The book is one of the author's significant works with a wide impact, and which also represents Ratzinger's way of thinking in its relation to philosophical thought. The title of this contribution is also its point: Ratzinger's book offers a certain interpretation of Christianity and provides at the same time a sort of defence of Christianity against a never-ending chain of interpretations. It is just that which should be subjected to interpretation. [ABSTRACT FROM AUTHOR]

The article deals with the problem of an anthropological understanding of metaphysics in theology and addresses its relevance against a confrontation with an ontological understanding of theological metaphysics. The article format does not allow for a comprehensive treatise but raises the issue as a principal question that can comprehend the problem's basic context and aspire to an answer as to whose conclusiveness should be -- in discussion with philosophical -religious and philosophical -theological theorems -- rationally autonomous. The text is based on the works of authors whose thinking a) enables an identification of a "connecting place" for metaphysics in theology, b) raises the possibility of an "anthropological connection" to theological metaphysics, c) helps recognise what is representative of the current state of reflection on this matter in the field of theological thinking, d) and itself elaborates the anthropological viewpoint for theological metaphysical thinking. The thread of content proceeds from a philosophical -religious typology of talking about God via a presentation of the anthropological viewpoint of metaphysics towards the standing of metaphysics in contemporary theology and the search for points of contact in T. Pröpper's theological anthropology. The conclusion refers to the possibility of process thought for an anthropologically relevant embedding in theological metaphysics. [ABSTRACT FROM AUTHOR]

Interleukin 24 (IL‐24) is a cytokine with the potential to be an effective treatment for autoimmune diseases and cancer. However, its instability and difficulties in its production have hampered detailed biological and biophysical studies. We approached the challenges of IL‐24 production by using the PROSS algorithm to design more stable variants of IL‐24. We used homology models built from the sequences and known structures of IL‐20 and IL‐19 and predicted and produced several extensively mutated IL‐24 variants that were highly stable and produced in large yields; one of them was crystallized (IL‐24B, PDB ID6GG1; 3D Interactive at http://proteopedia.org/w/Journal: FEBS_Journal:1). The mutated variants, however, lost most of their binding capacity to the extracellular parts of cognate receptors. While the affinity to the receptor 2 (IL‐20R2) was preserved, the variants lost affinity to IL‐20R1 and IL‐22R1 (shared receptors 1). Back engineering of the variants revealed that reintroduction of a single IL‐24 wild‐type residue (T198) to the patch interacting with receptors 1 restored 80% of the binding affinity and signaling capacity, accompanied by an acceptable drop in the protein stability by 9 °C. Multiple sequence alignment explains the stabilizing effect of the mutated residues in the IL‐24 variants by their presence in the related and more stable cytokines IL‐20 and IL‐19. Our homology‐based approach can enhance existing methods for protein engineering and represents a viable alternative to study and produce difficult proteins for which only in silico structural information is available, estimated as >40% of all important drug targets. [ABSTRACT FROM AUTHOR]

Interferon gamma (IFN-γ) is one of the key players in the immune system of vertebrates. The evolution and properties of IFN-γ and its receptors in fish species are of special interest as they point to the origin of innate immunity in vertebrates. We studied the phylogeny, biophysical and structural properties of IFN-γ and its receptors. Our phylogeny analysis suggests the existence of two groups of IFN-γ related proteins, one specific for Acanthomorpha , the other for Cypriniformes, Characiformes and Siluriformes. The analysis further shows an ancient duplication of the gene for IFN-γ receptor 1 (IFN- γR1) and the parallel existence of the duplicated genes in all current teleost fish species. In contrast, only one gene can be found for receptor 2, IFN- γR2. The specificity of the interaction between IFN- γ and both types of IFN- γR1 was determined by microscale thermophoresis measurements of the equilibrium dissociation constants for the proteins from three fish species. The measured preference of IFN- γ for one of the two forms of receptor 1agrees with the bioinformatic analysis of the coevolution between IFN- γ and receptor 1. To elucidate structural relationships between IFN-γ of fish and other vertebrate species, we determined the crystal structure of IFN-γ from olive flounder ( Paralichthys olivaceus , PoliIFN-γ) at crystallographic resolution of 2.3 Å and the low-resolution structures of Takifugu rubripes, Oreochromis niloticus, and Larimichthys crocea IFN-γ by small angle X-ray diffraction. The overall PoliIFN-γ fold is the same as the fold of the other known IFN- γ structures but there are some significant structural differences, namely the additional C-terminal helix G and a different angle between helices C and D in PoliIFN-γ. [ABSTRACT FROM AUTHOR]

Two non-pathogenic strains R89-1 and R90 T isolated from poppy seed ( Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes ( atpD , glnA , gyrB , recA , rpoB , 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261 T and Agrobacterium skierniewicense Ch11 T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G + C content is 56 mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90 T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum . The type strain of A. bohemicum is R90 T (=CCM 8736 T = DSM 104667 T ). [ABSTRACT FROM AUTHOR]

Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress. [ABSTRACT FROM AUTHOR]

Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π-cation motif of stacked residues KWRWRH, a NAG-W-NAG sandwich (where NAG stands for N-acetyl- d-glucosamine) and finally a helix formed by residues 78-85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2. [ABSTRACT FROM AUTHOR]

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity. [ABSTRACT FROM AUTHOR]

Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho -nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pham, Zahradník, Kolářová, Schneider and Fuertes.)