Biomimetic spider silk and bioactive hydrogels formed by engineered recombinant spider silk proteins

Spider silk is a unique material and its properties have fascinated material scientists, biologists and physicians for decades. Spider silk display one of the highest toughness found among fibers in nature, is used by spiders for web-spinning, prey-wrapping and cocoon building, while scientists have explored its biomaterials properties for multiple purposes. Spider silk appears to be generally well tolerated when implanted and is biodegrade but it comes with a limited availability and variable quality. Artificial silk production by recombinant expression of spider silk proteins (spidroins) in heterologous hosts is a promising path to overcome drawbacks associated with natural silk. To recapitulate the elaborate structure of natural silk one must understand the nature of silk formation and mimic this process closely. Previously, an artificial spidroin, NT2RepCT, was developed that can be spun into fibers with impressive mechanical properties in a biomimetic setup. However, NT2RepCT fibers cannot match the properties of natural silk fibers which may be due to incomplete biomimicry of the spidroin, and/or spinning procedure. In paper I, we analyzed to what extent the spidroin solution (dope), from which the silk fiber are spun, recapitulates important features of natural dope and found that it has shear-thinning and viscoelastic behavior and undergoes pH-induced phase-separation and structural changes similar to native dope, but lacks the high viscosity typically seen for natural spinning dope. In Paper II, we took advantage of insights in the constraints that spidroins have evolved under and used rational protein engineering of the repeat region of NT2RepCT. More specifically, we increased the hydrophobicity of the b-sheet forming poly-Ala regions since hydrophobic amino acid residues side chains are generally more prone to form b-sheets and steric zippers. Such proteins are unlikely to be secreted since the translocon would inserts proteins with hydrophobic sections in the endoplasmic reticulum membrane. Since the NT2RepCT proteins accumulate intracellularly during expression in prokaryotic hosts, we are not confined by these restrictions. When spun into fibers in a biomimetic spinning device, the toughness of fibers spun from several of the engineered proteins improved significantly compared to fibers spun from NT2RepCT. Importantly, one of the fibers had an unprecedented toughness for an as-spun artificial silk fiber. Furthermore, expression of the engineered spidroin in a bioreactor resulted in protein yields that make large-scale production economically feasible. Paper III explores the surprising finding that the hyper-soluble and stable spidroin N-terminal domain (NT) forms hydrogels when incubated at 37°C, and that gel formation is associated with a conversion of NT into amyloid-like fibrils. The high structural flexibility of NT combined with the presence of amyloidogenic sequences in its a-helices are factors that are important for formation of the gel. Furthermore, by fusing NT to target proteins, we present a novel immobilization platform in which NT is used both as an expression tag for high yield production of soluble fusion proteins and as a fibrillar scaffold in the gel. As hydrogel formation occurred rapidly and under benign conditions also for NT2RepCT, Paper IV focuses on the potential application of NT2RepCT hydrogels as a drug release device and for cell encapsulation. Successful encapsulation and release of active green fluorescent protein suggest that the hydrogels could be suitable candidates for use as drug release devices. An encapsulated cell line released the bioactive molecule progranulin for 31 days to a similar extent as cells cultured under standard conditions. Human mesenchymal stem cells encapsulated in the hydrogels showed high survival but limited proliferation, likely due to restricted space in the dense fibrillar network that is characteristic of the gels. This thesis describes major steps forward in the development of novel spidroin-based materials

Instagram-Auftritte deutscher Hochschulbibliotheken

Untersuchungsziel der vorliegenden Arbeit ist herauszufinden, welche Chancen das soziale Netzwerk Instagram für deutsche Hochschulbibliotheken bietet und in welcher Art und Weise diese umgesetzt werden. Darüber hinaus wird untersucht, wie deutsche Hochschulbibliotheken organisiert sind, um ihre Auftritte und Profile auf sozialen Netzwerken zu pflegen. Die Ergebnisse werden mit Hilfe einer Analyse von Beiträgen auf Instagram, sowie Experteninterviews gewonnen. Die Arbeit richtet sich an Social-Media-Beauftragte deutscher Hochschulbibliotheken, aber auch anderer Bibliothekstypen. Ebenso richtet sie sich an das interessierte Fachpublikum. Die Arbeit zeigt auf, dass es sich nicht immer für jede Bibliothek lohnt auf dem sozialen Netzwerk Instagram aktiv zu sein. Benötigt werden gewisse Ressourcen, um erfolgreich Präsenz auf dem Social-Media-Kanal zu zeigen. Dennoch bietet Instagram gute Möglichkeiten, visuell den Bestand und die angebotenen Dienstleistungen zu bewerben. In die Analyse flossen 292 Beiträge von sieben Instagram-Profilen deutscher Hochschulbibliotheken in Form von Fotos und Videos ein. Die gewonnenen Daten wurden statistisch ausgewertet. Darüber hinaus wurden Experteninterviews mit Mitarbeitern acht deutscher Hochschulbibliotheken durchgeführt, die sich für die Auftritte ihrer Bibliotheken in sozialen Netzwerken verantwortlich zeigten

FACT: Functional annotation transfer between proteins with similar feature architectures

<p>Abstract</p> <p>Background</p> <p>The increasing number of sequenced genomes provides the basis for exploring the genetic and functional diversity within the tree of life. Only a tiny fraction of the encoded proteins undergoes a thorough experimental characterization. For the remainder, bioinformatics annotation tools are the only means to infer their function. Exploiting significant sequence similarities to already characterized proteins, commonly taken as evidence for homology, is the prevalent method to deduce functional equivalence. Such methods fail when homologs are too diverged, or when they have assumed a different function. Finally, due to convergent evolution, functional equivalence is not necessarily linked to common ancestry. Therefore complementary approaches are required to identify functional equivalents.</p> <p>Results</p> <p>We present the <b>F</b>eature <b>A</b>rchitecture <b>C</b>omparison <b>T</b>ool <url>http://www.cibiv.at/FACT</url> to search for functionally equivalent proteins. FACT uses the similarity between feature architectures of two proteins, i.e., the arrangements of functional domains, secondary structure elements and compositional properties, as a proxy for their functional equivalence. A scoring function measures feature architecture similarities, which enables searching for functional equivalents in entire proteomes. Our evaluation of 9,570 EC classified enzymes revealed that FACT, using the full feature, set outperformed the existing architecture-based approaches by identifying significantly more functional equivalents as highest scoring proteins. We show that FACT can identify functional equivalents that share no significant sequence similarity. However, when the highest scoring protein of FACT is also the protein with the highest local sequence similarity, it is in 99% of the cases functionally equivalent to the query. We demonstrate the versatility of FACT by identifying a missing link in the yeast glutathione metabolism and also by searching for the human GolgA5 equivalent in <it>Trypanosoma brucei</it>.</p> <p>Conclusions</p> <p>FACT facilitates a quick and sensitive search for functionally equivalent proteins in entire proteomes. FACT is complementary to approaches using sequence similarity to identify proteins with the same function. Thus, FACT is particularly useful when functional equivalents need to be identified in evolutionarily distant species, or when functional equivalents are not homologous. The most reliable annotation transfers, however, are achieved when feature architecture similarity and sequence similarity are jointly taken into account.</p

Cytosolic PrP can participate in prion-mediated toxicity.

Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. Importance: During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and prion formation is undefined. Here we generated Drosophila transgenic for cytosolic PrP expression in order to investigate its toxic potential in the absence or presence of exogenous prions. Cytosolic ovine PrP expressed in Drosophila was not overtly detrimental to the flies. However, cytosolic PrP transgenic Drosophila exposed to ovine scrapie showed a toxic phenotype absent from similar flies exposed to scrapie-free material. Significantly, the scrapie-induced toxic phenotype in cytosolic transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that cytosolic PrP can participate in the generation of transmissible prion-induced toxicity and highlight the utility of Drosophila as a model of mammalian prion disease.This work was supported by funds from the China Scholarship Council and the Cambridge Overseas Trust, which provided Ph.D. studentship support for C.Z. We acknowledge funding support from the NC3Rs.This is the accepted manuscript version. The final version is available from ASM at http://jvi.asm.org/content/88/14/8129.full

Gemeinsam lernen, gemeinsam handeln - Transferprozesse digitaler Hochschulbildungskonzepte

Kooperation und Transfer gewinnen angesichts der zahlreichen Förderprogramme und der großen Anzahl vorhandener Ansätze und Konzepte für digitale Hochschulbildung an Bedeutung. Wie und warum kooperieren konkurrierende Hochschuleinrichtungen im Bereich Digitalisierung? Wie kann Transfer angesichts der verschiedenen Akteur_innen auf unterschiedlichsten Ebenen der Hochschulbildung gelingen? Welche Bedeutung und welcher Nutzen - insbesondere hinsichtlich der Lehre - wird digitalen Medien zugeschrieben? Das aktuell stark quantitativ erforschte Themenfeld und diese Fragestellungen werden im Projekt BRIDGING durch ein mehrstufiges qualitatives Forschungsdesign erschlossen. Dort wird untersucht, wie digitale Hochschulbildungskonzepte von Akteur_innen auf Landes-, Fakultäts- sowie Fachlehrendenebene diskutiert und umgesetzt werden und welchen Einfluss Organisation und Fachkultur auf den Transfer haben. Im Sinne von Open Science gibt der Beitrag darüber hinaus Einblicke in ein laufendes Forschungsprojekt. (DIPF/Orig.

Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

International audienceInnate immune recognition of the major human-specific Gram-positive pathogen Strepto-coccus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2-and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon in-ternalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 −/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures

Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context

Testing for deficient mismatch repair and microsatellite instability : A focused update

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021). Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education. // Der Nachweis der Mismatch-Reparatur-Defizienz (dMMR) mit konsekutiver hochgradiger Mikrosatelliteninstabilität (MSI-H) ist inzwischen fester Bestandteil der Diagnostik des kolorektalen Karzinoms (KRK). Galt MSI anfänglich als möglicher Indikator einer erblichen Krebsdisposition (Lynch-Syndrom, LS) steht heute die Vorhersage des Therapieansprechens auf Immuncheckpoint-Inhibitoren (ICI) im Vordergrund. Entsprechende Empfehlungen und Testalgorithmen liegen für den Einsatz in der Primärdiagnostik vor (Übersicht in: Rüschoff et al. 2021). Aufgrund des damit verbundenen routinemäßigen Einsatzes und des sich erweiternden Indikationsspektrums von ICI-Therapien für Nicht-KRK wie Endometrium‑, Dünndarm‑, Magen- und Gallenwegskarzinome wird eine aktualisierte Übersicht zur dMMR/MSI-Testung vorgelegt. Fokus sind die Herausforderungen bei der Beurteilung immunhistochemischer Färbungen und die Wertigkeit PCR-basierter Verfahren unter Berücksichtigung des erweiterten ICI-Indikationsspektrums. Anhand neuer Daten zur Häufigkeit und Art von Diskordanzen zwischen dMMR- und MSI-Befund und der möglichen Rolle von Next Generation Sequencing zu deren Aufklärung wird ein praxisorientiertes Diagramm zur Entscheidungsfindung im diagnostischen Alltag vorgestellt. Wir weisen zudem auf die Bedeutung systematischer Qualitätssicherungsmaßnahmen (z. B. QuIP MSI-Portal und Ringversuche) einschließlich einer regelmäßigen Fortbildung hin

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform

Recombinant spider silks are of interest but the multimodal and aggregation-prone nature of them is a limitation. Here, the authors report on a miniature spidroin based on the N-terminal domain which forms a hydrogel at 37 degrees C which allows for ease of production and fusion protein modification to generate functional biomaterials.Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 degrees C. The gelation is caused by NT alpha-helix to beta-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density

Expression and role of the immune checkpoint regulator PD-L1 in the tumor-stroma interplay of pancreatic ductal adenocarcinoma

Introduction: Immune checkpoint inhibitors (ICI), e.g., targeting programmed cell death protein 1-ligand 1 (PD-L1) or its receptor PD-1, have markedly improved the therapy of many cancers but so far failed in pancreatic ductal adenocarcinoma (PDAC). Macrophages represent one of the most abundant immune cell populations within the tumor microenvironment (TME) of PDAC being able to either support or restrain tumor progression depending on their phenotype. To better understand treatment failure of PD-L1/PD-1 inhibitors in PDAC, this study examined PD-L1 expression in the context of a dynamic TME in PDAC with a particular focus on the impact of macrophages. Methods: Formalin-fixed and paraffin embedded tissue samples of primary PDAC tissues and corresponding liver metastases were used for immunohistochemical analyses. Serial sections were stained with antibodies detecting Pan-Cytokeratin, CD68, CD163, CD8, and PD-L1.To investigate whether the PD-1/PD-L1 axis and macrophages contribute to immune escape of PDAC cells, a stroma enriched 3D spheroid coculture model was established in vitro, using different PDAC cell lines and macrophages subtypes as well as CD8+ T cells. Functional and flow cytometry analyses were conducted to characterize cell populations. Results: Immunohistochemical analyses revealed that PD-L1 is mainly expressed by stroma cells, including macrophages and not PDAC cells in primary PDAC tissues and corresponding liver metastases. Notably, high local abundance of macrophages and strong PD-L1 staining were commonly found at invasion fronts of tumoral lesions between CD8+ T cells and tumor cells. In order to investigate whether PD-L1 expressing macrophages impact the response of PDAC cells to treatment with PD-L1/PD-1 inhibitors, we developed a spheroid model comprising two different PDAC cell lines and different ratios of in vitro differentiated primary M1- or M2-like polarized macrophages. In line with our in situ findings, high PD-L1 expression was observed in macrophages rather than PDAC cells, which was further increased by the presence of PDAC cells. The effector phenotype of co-cultured CD8+ T cells exemplified by expression of activation markers and release of effector molecules was rather enhanced by PDAC macrophage spheroids, particularly with M1-like macrophages compared to mono-culture spheroids. However, this was not associated with enhanced PDAC cell death. ICI treatment with either Durvalumab or Pembrolizumab alone or in combination with Gemcitabine hardly affected the effector phenotype of CD8+ T cells along with PDAC cell death. Thus, despite strong PD-L1 expression in macrophages, ICI treatment did not result in an enhanced activation and cytotoxic phenotype of CD8+ T cells. Conclusion: Overall, our study revealed novel insights into the interplay of PDAC cells and macrophages in the presence of ICI