Translation elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an elongation factor that enhances translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, KD of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (KD of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation.

Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation.

Key Points• Sterile inflammation inducing venous thrombosis is coordinated by the damageassociated molecular pattern HMGB1 delivered by platelets.• The effect of HMGB1 depends on the redox form, and disulfide HMGB1 induces NET formation, platelet aggregation, and monocyte activation.Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new antiinflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1 2/2 chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis. (Blood. 2016; 128(20):2435-2449

Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-l-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-l-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development.

SummaryThe present guideline (S2k) on allergen-specific immunotherapy (AIT) was established by the German, Austrian and Swiss professional associations for allergy in consensus with the scientific specialist societies and professional associations in the fields of otolaryngology, dermatology and venereology, pediatric and adolescent medicine, pneumology as well as a German patient organization (German Allergy and Asthma Association; Deutscher Allergie- und Asthmabund, DAAB) according to the criteria of the Association of the Scientific Medical Societies in Germany (Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften, AWMF).AIT is a therapy with disease-modifying effects. By administering allergen extracts, specific blocking antibodies, toler-ance-inducing cells and mediators are activated. These prevent further exacerbation of the allergen-triggered immune response, block the specific immune response and attenuate the inflammatory response in tissue.Products for SCIT or SLIT cannot be compared at present due to their heterogeneous composition, nor can allergen concentrations given by different manufacturers be compared meaningfully due to the varying methods used to measure their active ingredients. Non-modified allergens are used for SCIT in the form of aqueous or physically adsorbed (depot) extracts, as well as chemically modified allergens (allergoids) as depot extracts. Allergen extracts for SLIT are used in the form of aqueous solutions or tablets.The clinical efficacy of AIT is measured using various scores as primary and secondary study endpoints. The EMA stipulates combined symptom and medication scores as primary endpoint. A harmonization of clinical endpoints, e. g., by using the combined symptom and medication scores (CSMS) recommended by the EAACI, is desirable in the future in order to permit the comparison of results from different studies. The current CONSORT recommendations from the ARIA/GA2LEN group specify standards for the evaluation, presentation and publication of study results.According to the Therapy allergen ordinance (TAV), preparations containing common allergen sources (pollen from grasses, birch, alder, hazel, house dust mites, as well as bee and wasp venom) need a marketing authorization in Germany. During the marketing authorization process, these preparations are examined regarding quality, safety and efficacy. In the opinion of the authors, authorized allergen preparations with documented efficacy and safety, or preparations tradeable under the TAV for which efficacy and safety have already been documented in clinical trials meeting WAO or EMA standards, should be preferentially used. Individual formulations (NPP) enable the prescription of rare allergen sources (e.g., pollen from ash, mugwort or ambrosia, mold Alternaria, animal allergens) for specific immunotherapy. Mixing these allergens with TAV allergens is not permitted.Allergic rhinitis and its associated co-morbidities (e. g., bronchial asthma) generate substantial direct and indirect costs. Treatmen...

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BackroundVisual analogue scales (VAS) are psychometric measuring instruments designed to document the characteristics of disease-related symptom severity in individual patients and use this to achieve a rapid (statistically measurable and reproducible) classification of symptom severity and disease control. VAS can also be used in routine patient history taking and to monitor the course of a chronic disease such as allergic rhinitis (AR). More specifically, the VAS has been used to assess effectiveness of AR therapy in real life, both in intermittent and persistent disease.MethodsThis position paper takes a detailed look at the historical development of VAS and its method-specific principles. Particular focus is put on aspects of practical application in daily routine and on a critical discussion of the advantages and disadvantages of the individual methods.ResultsVAS are well validated for the measurement of AR symptoms and correlate well with the ARIA (allergic rhinitis and its impact on asthma) severity classification and also correlated well with rTNSS and RQLQ. Moreover, several treatment studies on AR have used VAS as an evaluation parameter. Thanks to the use of new (real-life and real-time) communication technologies, such as smartphone apps, Discussion: VAS can be used relatively simply and highly effectively to assess disease control. The VAS lends itself very well to digitization and has now been incorporated into a smartphone app (called Allergy Diary) to assess AR control and direct treatment decisions as part of an AR clinical decision support system (CDSS). MASK Rhinitis has developed this app, which is currently available in 15 different languages.