Development of an open prospective observational multicentre cohort study to determine the impact of standardization of laparoscopic intraperitoneal onlay mesh repair (IPOM) for incisional hernia on clinical outcome and quality of life (LIPOM-Trial).

BACKGROUND: Incisional hernias are one of the most frequent complications in abdominal surgery. Laparoscopic repair has been widely used since its first description but has not been standardized. A panel of hernia experts with expertise on the subject "incisional hernia" was established to review existing literature and define a standard approach to laparoscopic IPOM-repair for incisional hernia. All involved surgeons agreed to perform further IPOM-repairs of incisional hernia according to the protocol. METHODS/DESIGN: This article summarizes the development of an open prospective observational multicentre cohort study to analyse the impact of a standardization of laparoscopic IPOM-repair for incisional hernia on clinical outcome and quality of life (health care research study). DISCUSSION: Our literature search found that there is a lack of standardization in the surgical approach to incisional hernia and the use of medical devices. The possibility of different surgical techniques, various meshes and a variety of mesh fixation techniques means that the results on outcome after incisional hernia repair are often not comparable between different studies. We believe there is a need for standardization of the surgical procedure and the use of medical devices in order to make the results more comparable and eliminate confounding factors in interpreting the results of surgical hernia repair. This approach, in our view, will also illustrate the influence of the operative technique on the general quality of surgical treatment of incisional hernias better than a "highly selective" study and will indicate the "reality" of surgical treatment not only in specialist centres. TRIAL REGISTRATION: The LIPOM-trial is registered at www.clinicaltrials.gov, with identifier: NCT02089958.

Small angle scattering in ribosomal structure research: localization of the messenger RNA within ribosomal elongation states.

Besides EM and biochemical studies small angle scattering (SAS) examinations have contributed significantly to our current knowledge about the ribosomal structure. SAS does not only allow the validation of competing models but permits independent model building. However, the major contribution of SAS to ribosomal structure research derived from its ability to reveal the spatial distribution of the individual ribosomal components (57 in the E. coli ribosome) within the ribosomal structure. More recently, an improved scattering method (proton-spin contrast variation) made it possible also to address the question of mapping functional ligands in defined ribosomal elongation states. Here, we review the contributions of SAS to the current understanding of the ribosome. Furthermore we present the direct localization of a small mRNA fragment within 70S elongation complexes and describe its movement upon the translocation reaction. The successful mapping of this fragment comprising only about 0.6% of the total mass of the complex proves that proton-spin contrast-variation is a powerful tool in modern ribosome research.

Escherichia coli 70 S ribosome at 15 A resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein.

Cryo-electron microscopy of the ribosome in different binding states with mRNA and tRNA helps unravel the different steps of protein synthesis. Using over 29,000 projections of a ribosome complex in single-particle form, a three-dimensional map of the Escherichia coli 70 S ribosome was obtained in which a single site, the P site, is occupied by fMet-tRNAfMet as directed by an AUG codon containing mRNA. The superior resolution of this three-dimensional map, 14.9 A, has made it possible to fit the tRNA X-ray crystal structure directly and unambiguously into the electron density, thus determining the locations of anticodon-codon interaction and peptidyltransferase center of the ribosome. Furthermore, at this resolution, one of the distinctly visible domains corresponding to a ribosomal protein, L1, closely matches with its X-ray structure.

Ribosomal tRNA binding sites: three-site models of translation.

The first models of translation described protein synthesis in terms of two operationally defined tRNA binding sites, the P-site for the donor substrate, the peptidyl-tRNA, and the A-site for the acceptor substrates, the aminoacyl-tRNAs. The discovery and analysis of the third tRNA binding site, the E-site specific for deacylated tRNAs, resulted in the allosteric three-site model, the two major features of which are (1) the reciprocal relationship of A-site and E-site occupation, and (2) simultaneous codon-anticodon interactions of both tRNAs present at the elongating ribosome. However, structural studies do not support the three operationally defined sites in a simple fashion as three topographically fixed entities, thus leading to new concepts of tRNA binding and movement: (1) the hybrid-site model describes the tRNAs' movement through the ribosome in terms of changing binding sites on the 30S and 50S subunits in an alternating fashion. The tRNAs thereby pass through hybrid binding states. (2) The alpha-epsilon model introduces the concept of a movable tRNA-binding domain comprising two binding sites, termed alpha and epsilon. The translocation movement is seen as a result of a conformational change of the ribosome rather than as a diffusion process between fixed binding sites. The alpha-epsilon model reconciles most of the experimental data currently available.

Structure of the elongating ribosome: arrangement of the two tRNAs before and after translocation.

The ribosome uses tRNAs to translate the genetic information into the amino acid sequence of proteins. The mass ratio of a tRNA to the ribosome is in the order of 1:100; because of this unfavorable value it was not possible until now to determine the location of tRNAs within the ribosome by neutron-scattering techniques. However, the new technique of proton-spin contrast-variation improves the signal-to-noise ratio by more than one order of magnitude, thus enabling the direct determination of protonated tRNAs within a deuterated ribosome for the first time. Here we analyze a pair of ribosomal complexes being either in the pre- or post-translocational states that represent the main states of the elongating ribosome. Both complexes were derived from one preparation. The orientation of both tRNAs within the ribosome and their mutual arrangement are determined by using an electron microscopy model for the Escherichia coli ribosome and the tRNA structure. The mass center of gravity of the (tRNA)2mRNA complex moves within the ribosome by 12 +/- 4 A in the course of translocation as previously reported. The main results of the present analysis are that the mutual arrangement of the two tRNAs does not change on translocation and that the angle between them is, depending on the model used, 110 degrees +/- 10 degrees before and after translocation. The translocational movement of the constant tRNA complex within the ribosome can be described as a displacement toward the head of the 30S subunit combined with a rotational movement by about 18 degrees.

Different consequences of incorporating chloroplast ribosomal proteins L12 and S18 into the bacterial ribosomes of Escherichia coli.

We have incorporated chloroplast ribosomal proteins (R-proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)-dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coil S18, 74 residues), containing a repeating, chloroplast-specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 30S subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coil ribosomes by a chloroplast R-protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12-lacking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)-dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a 'chloroplast loop' present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R-proteins concerned with specific functions are more divergent than their assembly properties. We have previously described methods suitable for overproduction and purification of chloroplast R-proteins that are encoded in organellar DNA (approximately 20), but that gave poor yield for those encoded in the nuclear DNA (approximately 45). Here we describe a method that overcomes this problem and allows the purification of nucleus-encoded chloroplast R-proteins in milligram quantities.

Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation).

A new technique for neutron scattering, the proton-spin contrast-variation, improves the signal-to-noise ratio more than one order of magnitude as compared to conventional techniques. The improved signal enables small RNA ligands within a large deuterated ribonucleic acid-protein complex to be measured. We used this technique to determine the positions of the two tRNAs within the elongating ribosome before and after translocation. Using a four-sphere model for each of the L-shaped tRNAs, unequivocal solutions were found for the localization of the mass centre of both tRNAs. The centre of gravity is located in the interface cavity separating the ribosomal subunits near the neck of the 30 S subunit. It moves during translocation by 12(+/-4) A towards the head of the 30 S subunit and slightly towards the L1 protuberance of the 50 S subunit.

[Phosphate sites of RNA-ligands interacting with ribosome at different stages of translation. Thiophosphate method of analysis]. / Fosfatnye uchastki RNK-ligandov, vzaimodeistvuiushchie s ribosomoi na razlichnykh stadiiakh transliatsii. Izuchenie s pomoshch'iu tiofosfatnogo metoda.

A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.

In vivo deuteration of transfer RNAs: overexpression and large-scale purification of deuterated specific tRNAs.

Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Large-scale preparation of fully deuterated cell components. Ribosomes from Escherichia coli with high biological activity.

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, mu < or = 0.35 h-1, for acetate/succinate mu < or = 0.1 h-1) which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source. The cells were first grown in fed-batch to high cell densities (above 50 g wet cells/l) under conditions of substrate limitations. A steady-flow fermentation followed keeping the growth rate at about mu of 0.1 h-1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.