Thermostable WW-Domain Scaffold to Design Functional ß-Sheet Miniproteins.

There has been a recent surge in the design of miniproteins for medicinal chemistry, biomaterial design, or synthetic biology. In particular, there is an interest in peptide scaffolds that fold reliably, predictably, and with solid stability. In this article, we present the design of a highly thermostable WW domain, a three-stranded ß-sheet motif, with a superior melting temperature of about 90 °C to serve as a core scaffold onto which receptor-like properties can be grafted. We have performed specific rounds of sequence iteration on a WW-domain consensus sequence to decipher sequence positions that affect structural and, thus, thermal stability. We identified a sequence-structure relationship that yields a highly thermostable WW-domain scaffold. High-resolution NMR spectroscopy was applied, which enabled the identification of structural features at the atomic scale that contribute to this high thermostability. Finally, we grafted the binding motifs of the three WW-domain groups─Group I, Group II/III, and Group IV─and organophosphate and metal binding onto the highly thermostable WW-domain scaffold and obtained thermostable de novo WW domains that indeed display the different binding modes that were intended. The organophosphate-binding WW domains exhibit melting temperatures that are up to 34 K higher than previously reported top-down designs. These results impressively demonstrate that the highly thermostable WW-domain core scaffold is a solid platform for the design of discrete and reliably folding functional ß-sheet peptide miniproteins, providing an essential addition to the toolbox of peptide scaffolds previously used in synthetic biology and material design.

Rapid On-Resin N-Formylation of Peptides as One-Pot Reaction.

N-formylation is a common pre- and post-translational modification of the N-terminus or the lysine side chain of peptides and proteins that plays a role in the initiation of immune responses, gene expression, or epigenetics. Despite its high biological relevance, protocols for the chemical N-formylation of synthetic peptides are scarce. The few available methods are elaborate in their execution and the yields are highly sequence-dependent. We present a rapid, easy-to-use one-pot procedure that runs at room temperature and can be used to formylate protected peptides at both the N-terminus and the lysine side chain on the resin in near-quantitative yields. Only insensitive, storage-stable standard chemicals - formic acid, acetic anhydride, pyridine and DMF - are used. Formylation works for both short and long peptides of up to 34 amino acids and over the spectrum of canonical amino acids.

Inside-out Regulation of Ectodomain Cleavage of Cluster-of-Differentiation-44 (CD44) and of Neuregulin-1 Requires Substrate Dimerization.

Ectodomain shedding of transmembrane precursor proteins generates numerous life-essential molecules, such as epidermal growth factor receptor ligands. This cleavage not only releases the regulatory growth factor, but it is also the required first step for the subsequent processing by γ-secretase and the release of gene regulatory intracellular fragments. Signaling within the cell modifies the cytoplasmic tails of substrates, a step important in starting the specific and regulated cleavage of a large number of studied substrates. Ectodomain cleavage occurs, however, on the outside of the plasma membrane and is carried out by membrane-bound metalloproteases. How the intracellular domain modification communicates with the ectodomain of the substrate to allow for cleavage to occur is unknown. Here, we show that homodimerization of a cluster-of-differentiation-44 or of pro-neuregulin-1 monomers represents an essential pre-condition for their regulated ectodomain cleavage. Both substrates are associated with their respective metalloproteases under both basal or cleavage-stimulated conditions. These interactions only turn productive by specific intracellular signal-induced intracellular domain modifications of the substrates, which in turn regulate metalloprotease access to the substrates' ectodomain and cleavage. We propose that substrate intracellular domain modification induces a relative rotation or other positional change of the dimerization partners that allow metalloprotease cleavage in the extracellular space. Our findings fill an important gap in understanding substrate-specific inside-out signal transfer along cleaved transmembrane proteins and suggest that substrate dimerization (homo- or possibly heterodimerization) might represent a general principle in ectodomain shedding.

VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis.

BACKGROUND & AIMS: Senescence prevents cellular transformation. We investigated whether vascular endothelial growth factor (VEGF) signaling via its receptor, VEGFR2, regulates senescence and proliferation of tumor cells in mice with colitis-associated cancer (CAC). METHODS: CAC was induced in VEGFR2(ΔIEC) mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2(fl/fl) mice (controls) by administration of azoxymethane followed by dextran sodium sulfate. Tumor development and inflammation were determined by endoscopy. Colorectal tissues were collected for immunoblot, immunohistochemical, and quantitative polymerase chain reaction analyses. Findings from mouse tissues were confirmed in human HCT116 colorectal cancer cells. We analyzed colorectal tumor samples from patients before and after treatment with bevacizumab. RESULTS: After colitis induction, VEGFR2(ΔIEC) mice developed significantly fewer tumors than control mice. A greater number of intestinal tumor cells from VEGFR2(ΔIEC) mice were in senescence than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Tumor cell senescence promoted an anti-tumor immune response by CD8(+) T cells in mice. Patients whose tumor samples showed an increase in the proportion of senescent cells after treatment with bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment. CONCLUSIONS: Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with bevacizumab.

Identification of novel functional mini-receptors by combinatorial screening of split-WW domains.

ß-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications. Since the sequence-structure relationships of ß-sheet motifs are generally complex compared to the well-studied α-helical coiled coil (CC), other approaches such as combinatorial screening should be included to vary the function of the peptide. In this study, we present a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold, which takes advantage of the successful reconstitution of the fragmented WW domain of hPin1 (hPin1WW) by CC association. Fragmentation of hPin1WW was performed in both loop 1 (CC-hPin1WW-L1) and loop 2 (CC-hPin1WW-L2), and the respective fragments were linked to the strands of an antiparallel heterodimeric CC. Structural analysis by CD and NMR spectroscopy revealed structural reconstitution of the WW-domain scaffold only in CC-hPin1WW-L1, but not in CC-hPin1WW-L2. Furthermore, by using 1H-15N HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1WW in CC-hPin1WW-L1 were fully restored by CC association. To demonstrate the power of this approach as a combinatorial screening platform, we synthesized a four-by-six library of N- and C-terminal hPin1WW-CC peptide fragments that was screened for a WW domain that preferentially binds to ATP over cAMP, phophocholine, or IP6. Using this screening platform, we identified one WW domain, which specifically binds ATP, and a phosphorylcholine-specific WW-based mini-receptor, both having binding dissociation constants in the lower micromolar range.

Designed peptides as nanomolar cross-amyloid inhibitors acting via supramolecular nanofiber co-assembly.

Amyloid self-assembly is linked to numerous devastating cell-degenerative diseases. However, designing inhibitors of this pathogenic process remains a major challenge. Cross-interactions between amyloid-ß peptide (Aß) and islet amyloid polypeptide (IAPP), key polypeptides of Alzheimer's disease (AD) and type 2 diabetes (T2D), have been suggested to link AD with T2D pathogenesis. Here, we show that constrained peptides designed to mimic the Aß amyloid core (ACMs) are nanomolar cross-amyloid inhibitors of both IAPP and Aß42 and effectively suppress reciprocal cross-seeding. Remarkably, ACMs act by co-assembling with IAPP or Aß42 into amyloid fibril-resembling but non-toxic nanofibers and their highly ordered superstructures. Co-assembled nanofibers exhibit various potentially beneficial features including thermolability, proteolytic degradability, and effective cellular clearance which are reminiscent of labile/reversible functional amyloids. ACMs are thus promising leads for potent anti-amyloid drugs in both T2D and AD while the supramolecular nanofiber co-assemblies should inform the design of novel functional (hetero-)amyloid-based nanomaterials for biomedical/biotechnological applications.

Asc-1 regulates white versus beige adipocyte fate in a subcutaneous stromal cell population.

Adipose tissue expansion, as seen in obesity, is often metabolically detrimental causing insulin resistance and the metabolic syndrome. However, white adipose tissue expansion at early ages is essential to establish a functional metabolism. To understand the differences between adolescent and adult adipose tissue expansion, we studied the cellular composition of the stromal vascular fraction of subcutaneous adipose tissue of two and eight weeks old mice using single cell RNA sequencing. We identified a subset of adolescent preadipocytes expressing the mature white adipocyte marker Asc-1 that showed a low ability to differentiate into beige adipocytes compared to Asc-1 negative cells in vitro. Loss of Asc-1 in subcutaneous preadipocytes resulted in spontaneous differentiation of beige adipocytes in vitro and in vivo. Mechanistically, this was mediated by a function of the amino acid transporter ASC-1 specifically in proliferating preadipocytes involving the intracellular accumulation of the ASC-1 cargo D-serine.

Gamma secretase dependent release of the CD44 cytoplasmic tail upregulates IFI16 in cd44-/- tumor cells, MEFs and macrophages.

The adhesion molecule and co-receptor of receptor tyrosine kinases, CD44, is expressed in all cells of the immune system, but also in numerous non-immune cells. CD44 plays roles in the cellular response to different pathogens. The molecular actions of CD44 during these processes are by and large still unknown. The CD44 molecule undergoes a sequential proteolytic cleavage which leads to the release of a soluble intracellular domain (CD44-ICD). Previous reports had shown that the CD44-ICD is taken up into the nucleus where it enhances transcription of specific target genes. By RNA profiling we identified a CD44-dependent transcriptional increase of interferon-responsive genes, among them IFI16. IFI16 is important in the innate immune response. It senses and binds pathogenic DNA and, together with cGAS, activates the cGAS-cGAMP-STING pathway and induces the expression of genes relevant for the response, e.g. IFN-ß. Our results show that the enhancement of IFI16 expression depended on CD44 cleavage. A CD44-negative tumor cell line, embryonic fibroblasts and bone marrow-derived macrophages from cd44-/- mice were reduced in their response to IFN-γ, to viral DNA fragments and to Listeria monocytogenes infection. We could rescue the deficiency of CD44 negative RPM-MC cells and cd44-/- MEFs by expressing only the soluble CD44-ICD in the absence of any other CD44 domain. Expression of the CD44-ICD carrying a mutation that prevented the uptake into the nucleus, could not rescue the absence of CD44. This molecular aspect of regulation by CD44 may explain part of the immune phenotypes of mice with cd44 gene disruption.

[Treatment of patients with radioiodine refractory, differentiated thyroid carcinoma. A Consensus Statement]. / Therapie des Patienten mit Radioiod-refraktärem, differenziertem Schilddrüsenkarzinom. Ein Konsensusstatement.

There is no clear standard therapy for patients with radioactive iodine (131I)-refractory locally advanced or metastatic differentiated thyroid cancer. The therapeutic options for this indication have expanded with the recently approved multiple kinase inhibitor sorafenib. Recommendations for the definition and the management of iodine refractory patients were worked up by an interdisciplinary expert panel, consisting of endocrine surgeons, medical oncologists and nuclear medicine specialists.

["Is there a doctor on board?" - legal aspects of medical care in emergency situations during spare time]. / "Is there a doctor on board?" - Rechtliches zur ärztlichen Hilfeleistung in Notfallsituationen in der Freizeit.

Medical emergencies on international flights are not uncommon. In these situations the question often arises whether physicians are obliged to render first aid and whether omission leads to legal consequences. The general obligation to aid those in need applies to everyone, not only to physicians. Evading this duty makes liable to prosecution for omittance of defence of a third person in line with Art. 128 of the Swiss Penal Code, punishable by custodial sentence up to three years or an equivalent punitive fine. Vocational and professional law extend the duty to aid for physicians to urgent cases. Although resulting from the performance of a legal obligation, malpractice occurred in the course of first aid can lead to claims for compensation - even from foreign patients, and that according to their own domestic law.

Les urgences médicales dans les avions ne sont pas rares. Dans ces situations, la question se pose souvent de savoir si les médecins sont tenus d'effectuer les premiers soins et si le fait de s'y soustraire peut avoir des conséquences au niveau juridique. Le devoir d'assister toute personne dans le besoin concerne chacun de nous, et pas seulement les médecins. Quiconque ne respecte pas ce devoir d'assistance peut être condamné, au vu de l'art. 128 du code pénal, à une amende ou même une peine d'emprisonnement pouvant aller jusqu'à trois ans. Les obligations professionnelles et la déontologie prévoient pour les médecins un devoir d'assistance accru pour les cas d'urgence. Bien que ces devoirs soient prévus par la loi, il n'empêche pas que toute erreur professionnelle en cours de traitement peut entraîner des poursuites civiles en dommages et intérêts, de même pour des patients d'origine étrangère, selon la législation en vigueur dans leur pays.

A link between two tumorigenic proteins, CD44 and p21WAF1: CD44 increases phorbol ester-induced expression of p21WAF1 by stabilizing its mRNA and extending protein half-life.

The cell surface glycoprotein CD44 enhances phorbol-12-myristate 13-acetate (TPA)-induced expression of p21WAF1 by stabilizing its mRNA and enhancing the protein's half-life in several cell lines. Only the plasma membrane-anchored cytoplasmic tail of CD44 and its interacting ezrin, radixin, moesin (ERM) proteins are required for this effect. A mitogen activated kinase (MEK) inhibitor abolishes the action of CD44 on p21. Down-regulation of p21 dramatically decreased anchorage-independence of a cancer cell line, whereas CD44 expression in this background could partially rescue the phenotype.

Stochastic resonance training reduces musculoskeletal symptoms in metal manufacturing workers: a controlled preventive intervention study.

OBJECTIVES: This study examined the effects of stochastic resonance whole-body vibration training on work-related musculoskeletal symptoms and accidents. PARTICIPANTS: Participants were white and blue-collar employees of a Swiss metal manufacturer (N=38), and participation was voluntary. METHODS: The study was designed as a switching-replications longitudinal trial with randomized group allocation. The randomized controlled cross-over design consisted of two groups each given four weeks of exercise and no intervention during a second four-week period. Outcome was measured on a daily basis with questionnaires. Three components constituted musculoskeletal symptoms: musculoskeletal pain, related function limitations and musculoskeletal well-being. Accidents were assessed by ratings for balance and daily near-accidents. For statistical analysis, a mixed model was calculated. RESULTS: At the end of the training period musculoskeletal pain and related function limitation were significantly reduced, whereas musculoskeletal well-being had significantly increased. For function limitation and musculoskeletal well-being, change over time was linear. There was no effect on balance or near-accidents. CONCLUSIONS: Stochastic resonance whole-body vibration was found to be effective in the prevention of work-related musculoskeletal symptoms. It is well suited for the use in a work environment since it requires very little effort in terms of infrastructure, time and investment from participants.