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The innate immune recognition of the malaria-causing pathogen Plasmodium falciparum (P. falciparum) is not fully explored. Here, we identify the nucleoside 5'-methylthioinosine (MTI), a Plasmodium-specific intermediate of the purine salvage pathway, as a pathogen-derived Toll-like receptor 8 (TLR8) agonist. Co-incubation of MTI with the TLR8 enhancer poly(dT) as well as synthetic or P. falciparum-derived RNA strongly increase its stimulatory activity. Of note, MTI generated from methylthioadenosine (MTA) by P. falciparum lysates activates TLR8 when MTI metabolism is inhibited by immucillin targeting the purine nucleoside phosphorylase (PfPNP). Importantly, P. falciparum-infected red blood cells incubated with MTI or cultivated with MTA and immucillin lead to TLR8-dependent interleukin-6 (IL-6) production in human monocytes. Our data demonstrate that the nucleoside MTI is a natural human TLR8 ligand with possible in vivo relevance for innate sensing of P. falciparum., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cytokeratins (CKs) are intermediate filaments of epithelial cells. In humans, different types of epithelia as well as their neoplasms show distinct CK expression profiles. The aim of this study was to establish a panel of CKs for the identification of specialized canine epithelia that can be integrated in a routine diagnostic setting. Immunohistochemistry was performed on 42 formalin-fixed paraffin-embedded (FFPE) canine unaltered tissues including all epithelial tissues by using an antibody panel detecting CKs 7, 8, 13, 14, 17, 19 and 20 and the pancytokeratin marker AE1/AE3. Using this antibody panel, a differentiation scheme for the identification of canine tissues was developed. This allowed the identification of 23 out of the 42 examined canine tissues and the distinction of 9 groups of specialized epithelia. The statistical validation revealed high variations in the immunoreactivity for CKs 7, 8, 14, 17 and 20 between the donor dogs. The antibody detecting CK 7 (OV-TL 12/13) showed a decrease in immunostaining after a fixation time of 3 and 4 days. To the best of the authors' knowledge this is the first study that characterizes all canine epithelial tissues for their expression of CKs 7, 8, 13, 14, 17, 19 and 20 and the pancytokeratin marker AE1/AE3. Results of this study are an important prerequisite for comparative histology and for the investigation into similarities/differences of the cytokeratin expression between normal and neoplastic epithelia. Since this study was performed on FFPE tissue, it can be included in the workflow of a routine diagnostic laboratory.

Members of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) family are known auxin receptors. To analyze the possible receptor function of AUXIN BINDING PROTEIN1 (ABP1), an auxin receptor currently under debate, we performed different approaches. We performed a pharmacological approach using α-(2,4-dimethylphenylethyl-2-oxo)-indole-3-acetic acid (auxinole), α-(phenylethyl-2-oxo)-indole-3-acetic acid (PEO-IAA), and 5-fluoroindole-3-acetic acid (5-F-IAA) to discriminate between ABP1- and TIR1/AFB-mediated processes in Arabidopsis ( Arabidopsis thaliana ). We used a peptide of the carboxyl-terminal region of AtABP1 as a tool. We performed mutant analysis with the null alleles of ABP1 , abp1-c1 and abp1-TD1 , and the TILLING mutant abp1-5 We employed Coimbra, an accession that exhibits an amino acid exchange in the auxin-binding domain of ABP1. We measured either volume changes of single hypocotyl protoplasts or hypocotyl growth, both at high temporal resolution. 5-F-IAA selectively activated the TIR1/AFB pathway but did not induce protoplast swelling; instead, it showed auxin activity in the hypocotyl growth test. In contrast, PEO-IAA induced an auxin-like swelling response but no hypocotyl growth. The carboxyl-terminal peptide of AtABP1 induced an auxin-like swelling response. In the ABP1 -related mutants and Coimbra, no auxin-induced protoplast swelling occurred. ABP1 seems to be involved in mediating rapid auxin-induced protoplast swelling, but it is not involved in the control of rapid auxin-induced growth., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Crystallography is frequently used as follow-up method to validate hits identified by biophysical screening cascades. The capacity of crystallography to directly screen fragment libraries is often underestimated, due to its supposed low-throughput and need for high-quality crystals. We applied crystallographic fragment screening to map the protein-binding site of the aspartic protease endothiapepsin by individual soaking experiments. Here, we report on 41 fragments binding to the catalytic dyad and adjacent specificity pockets. The analysis identifies already known warheads but also reveals hydrazide, pyrazole, or carboxylic acid fragments as novel functional groups binding to the dyad. A remarkable swapping of the S1 and S1' pocket between structurally related fragments is explained by either steric demand, required displacement of a well-bound water molecule, or changes of trigonal-planar to tetrahedral geometry of an oxygen functional group in a side chain. Some warheads simultaneously occupying both S1 and S1' are promising starting points for fragment-growing strategies.

Methods for comparing protein binding sites are frequently validated on data sets of pockets that were obtained simply by extracting the protein area next to the bound ligands. With this strategy, any unoccupied pocket will remain unconsidered. Furthermore, a large amount of ligand-biased intrinsic shape information is predefined, inclining the subsequent comparisons as rather trivial even in data sets that hardly contain redundancies in sequence information. In this study, we present the results of a very simplistic and shape-biased comparison approach, which stress that unrestricted cavity extraction is essential to enable unexpected cross-reactivity predictions among proteins and function annotations of orphan proteins.