Mechanical limits of viral capsids

We study the elastic properties and mechanical stability of viral capsids under external force-loading with computer simulations. Our approach allows the implementation of specific geometries corresponding to specific phages such as $\phi$29 and CCMV. We demonstrate how in a combined numerical and experimental approach the elastic parameters can be determined with high precision. The experimentally observed bimodality of elastic spring constants is shown to be of geometrical origin, namely the presence of pentavalent units in the viral shell. A criterion for capsid breakage is defined, which explains well the experimentally observed rupture. From our numerics we find for the dependence of the rupture force on the F\"oppl-von K\'arm\'an (FvK) number a crossover from $\gamma^{2/3}$ to $\gamma^{1/2}$. For filled capsids high internal pressures lead to a stronger destabilization of viruses with a buckled ground state than unbuckled ones. Finally, we show how our numerically calculated energy maps can be used to extract information about the strength of protein-protein interactions from rupture experiments.Comment: 6 pages, 9 figure

Molecular structure refinement by direct fitting of atomic coordinates to experimental ESR spectra

An attempt is made to bypass spectral analysis and fit internal coordinates of radicals directly to experimental liquid- and solid-state electron spin resonance (ESR) spectra. We take advantage of the recently introduced large-scale spin dynamics simulation algorithms and of the fact that the accuracy of quantum mechanical calculations of ESR parameters has improved to the point of quantitative correctness. Partial solutions are offered to the local minimum problem in spectral fitting and to the problem of spin interaction parameters (hyperfine couplings, chemical shifts, etc.) being very sensitive to distortions in molecular geometry.Comment: Submitted for publicatio

Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G

The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription–translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition

Two-dimensional NMR lineshape analysis

NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions

EuraHS: the development of an international online platform for registration and outcome measurement of ventral abdominal wall hernia repair

Background Although the repair of ventral abdominal wall hernias is one of the most commonly performed operations, many aspects of their treatment are still under debate or poorly studied. In addition, there is a lack of good definitions and classifications that make the evaluation of studies and meta-analyses in this field of surgery difficult. Materials and methods Under the auspices of the board of the European Hernia Society and following the previously published classifications on inguinal and on ventral hernias, a working group was formed to create an online platform for registration and outcome measurement of operations for ventral abdominal wall hernias. Development of such a registry involved reaching agreement about clear definitions and classifications on patient variables, surgical procedures and mesh materials used, as well as outcome parameters. The EuraHS working group (European registry for abdominal wall hernias) comprised of a multinational European expert panel with specific interest in abdominal wall hernias. Over five working group meetings, consensus was reached on definitions for the data to be recorded in the registry. Results A set of well-described definitions was made. The previously reported EHS classifications of hernias will be used. Risk factors for recurrences and co-morbidities of patients were listed. A new severity of comorbidity score was defined. Post-operative complications were classified according to existing classifications as described for other fields of surgery. A new 3-dimensional numerical quality-of-life score, EuraHS-QoL score, was defined. An online platform is created based on the definitions and classifications, which can be used by individual surgeons, surgical teams or for multicentre studies. A EuraHS website is constructed with easy access to all the definitions, classifications and results from the database. Conclusion An online platform for registration and outcome measurement of abdominal wall hernia repairs with clear definitions and classifications is offered to the surgical community. It is hoped that this registry could lead to better evidence-based guidelines for treatment of abdominal wall hernias based on hernia variables, patient variables, available hernia repair materials and techniques

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates

Objective: To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. Methods: EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Results: Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins.Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate.By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation.ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Conclusions: Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates. Keywords: EndoC-βH1, Pseudoislets, Glucose stimulated insulin secretion, Somatostatin signaling, Proliferatio

Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins

Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A1 chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A1 chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A1 variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A1 chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A1 chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A1 chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs

Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM

The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNAPhe, Trp-tRNATrp, or Leu-tRNALeuI. The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same ‘loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection

2017 update of the WSES guidelines for emergency repair of complicated abdominal wall hernias

Emergency repair of complicated abdominal wall hernias may be associated with worsen outcome and a significant rate of postoperative complications. There is no consensus on management of complicated abdominal hernias. The main matter of debate is about the use of mesh in case of intestinal resection and the type of mesh to be used. Wound infection is the most common complication encountered and represents an immense burden especially in the presence of a mesh. The recurrence rate is an important topic that influences the final outcome. A World Society of Emergency Surgery (WSES) Consensus Conference was held in Bergamo in July 2013 with the aim to define recommendations for emergency repair of abdominal wall hernias in adults. This document represents the executive summary of the consensus conference approved by a WSES expert panel. In 2016, the guidelines have been revised and updated according to the most recent available literature.Peer reviewe