The Influence of Disulfide, Thioacetal and Lanthionine-Bridges on the Conformation of a Macrocyclic Peptide.

Cyclisation of peptides by forming thioether (lanthionine), disulfide (cystine) or methylene thioacetal bridges between side chains is established as an important tool to stabilise a given structure, enhance metabolic stability and optimise both potency and selectivity. However, a systematic comparative study of the effects of differing bridging modalities on peptide conformation has not previously been carried out. In this paper, we have used the NMR deconvolution algorithm, NAMFIS, to determine the conformational ensembles, in aqueous solution, of three cyclic analogues of angiotensin(1-7), incorporating either disulfide, or non-reduceable thioether or methylene thioacetal bridges. We demonstrate that the major solution conformations are conserved between the different bridged peptides, but the distribution of conformations differs appreciably. This suggests that subtle differences in ring size and bridging structure can be exploited to fine-tune the conformational properties of cyclic peptides, which may modulate their bioactivities.

A network analysis of connected biophysical pathways to advice eelgrass (Zostera marina) restoration.

The North Sea and the Baltic Sea, including Danish coastal waters, have experienced a drastic decline in eelgrass Zostera marina coverage during the past century. Around 1900, eelgrass meadows covered about 6700 km2 of Danish coastal waters while the current potential distribution area is only about one third of this. In some areas, the potential distribution area is far from realized, and restoration efforts are needed to assist recovery. Such efforts are challenging, and resource-demanding and careful site selection is, therefore, important. In the present study, we aim to identify the connectivity of eelgrass populations as a basis for guiding site selection for restoration. We developed a coupled biophysical model to study eelgrass dispersal in the Kattegat. Partly submerged particles simulated the dispersal of reproductive eelgrass shoots containing seeds during the flowering season July-September. We then used network analysis to identify the potential connectivity between populations. We evaluated connectivity based on In-strength, Betweenness and Eigenvector centrality metrics and identified key areas in the Kattegat such as the central part of Aalborg Bay, to be considered to restore the network of Z. marina patches. The study proves the potentials of combining hydrodynamic models and network analysis to support marine conservation and planning, and highlights the importance of collaboration between ecologists, oceanographers, and practitioners in this endeavour.

The DnaA Tale.

More than 50 years have passed since the presentation of the Replicon Model which states that a positively acting initiator interacts with a specific site on a circular chromosome molecule to initiate DNA replication. Since then, the origin of chromosome replication, oriC, has been determined as a specific region that carries sequences required for binding of positively acting initiator proteins, DnaA-boxes and DnaA proteins, respectively. In this review we will give a historical overview of significant findings which have led to the very detailed knowledge we now possess about the initiation process in bacteria using Escherichia coli as the model organism, but emphasizing that virtually all bacteria have DnaA proteins that interacts with DnaA boxes to initiate chromosome replication. We will discuss the dnaA gene regulation, the special features of the dnaA gene expression, promoter strength, and translation efficiency, as well as, the DnaA protein, its concentration, its binding to DnaA-boxes, and its binding of ATP or ADP. Furthermore, we will discuss the different models for regulation of initiation which have been proposed over the years, with particular emphasis on the Initiator Titration Model.

Comparison of four MRI protocols for detection of extrahepatic colorectal cancer metastases.

PURPOSE: To compare three magnetic resonance imaging (MRI) protocols containing diffusion-weighted imaging with background suppression (DWIBS) and one traditional protocol for detecting extrahepatic colorectal cancer metastases. MATERIALS AND METHODS: Thirty patients with extrahepatic colorectal cancer metastases were scanned in three stations from the skull base to the upper thighs using a 1.5T MRI system with six different MRI sequences; transverse and coronal T2 -weighted (T2 W) turbo spin-echo (TSE), coronal short tau inversion recovery (STIR), 3D T1 W TSE, DWIBS, and a contrast-enhanced T1 W 3D gradient echo (GRE) sequence. The six sequences were used to build four hypothetical MRI interpretive sets which were read by two readers in consensus, blinded to prior imaging. Lesions were categorized into 13 anatomic regions. Fluorodeoxyglucose / positron emission tomography / computed tomography (FDG-PET/CT) read with full access to prior imaging and clinical records was used as the reference standard. Sensitivity, specificity, and false discovery rate (FDR) were calculated as appropriate and receiver operating characteristic (ROC) curves were constructed. RESULTS: In all, 177 malignant lesions were detected by FDG-PET/CT and distributed in 92 out of 390 scanned anatomic regions. The sensitivity was statistically higher in two out of three sets incorporating DWIBS on a per-lesion basis (66.7%, 63.3%, and 66.7% vs. 57.6%) (P = 0.01, P = 0.11, and P = 0.01, respectively) and in all sets incorporating DWIBS on a per-region basis (75.0%, 75.0%, and 77.2 vs. 66.3%) (P = 0.04, P = 0.04, and P = 0.01, respectively). There was no difference in specificity, FDR, or AUCROC . There was no difference between sets containing DWIBS irrespective of the use of a contrast-enhanced sequence. CONCLUSION: MRI sets containing DWIBS had superior sensitivity. This sensitivity was retained when omitting a contrast-enhanced sequence. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017;46:1619-1630.

miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells.

Oxaliplatin resistance in colorectal cancers (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, miR-625-3p was reported as a promising predictive marker. Herein, we show that miR-625-3p functionally induces oxaliplatin resistance in CRC cells, and identify the signalling networks affected by miR-625-3p. We show that the p38 MAPK activator MAP2K6 is a direct target of miR-625-3p, and, accordingly, is downregulated in non-responder patients of oxaliplatin therapy. miR-625-3p-mediated resistance is reversed by anti-miR-625-3p treatment and ectopic expression of a miR-625-3p insensitive MAP2K6 variant. In addition, reduction of p38 signalling by using siRNAs, chemical inhibitors or expression of a dominant-negative MAP2K6 protein induces resistance to oxaliplatin. Transcriptome, proteome and phosphoproteome profiles confirm inactivation of MAP2K6-p38 signalling as one likely mechanism of oxaliplatin resistance. Our study shows that miR-625-3p induces oxaliplatin resistance by abrogating MAP2K6-p38-regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p.

Segregation of chromosome arms in growing and non-growing Escherichia coli cells.

In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (O) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not according to the doughnut model.

The dislocating hip replacement - revision with a dual mobility cup in 56 consecutive patients.

INTRODUCTION: Recurrent dislocations of hip replacements are a difficult challenge. One treatment option for recurrent dislocations is the use of a dual mobility cup. The aim of this study was to retrospective investigate the effect of dual mobility cups as a treatment for recurrent dislocations in a consecutive series. Materials and. METHODS: 56 consecutive patients were revised in the period November 2000 to December 2010. The mean age at revision was 72 years (SD 11, range 37-92)) and median number of dislocations before revision surgery were 4 (IQR, 2-11). In all cases, revision was made with a Saturne dual mobility cup (Amplitude, Neyron, France). The mean follow-up period was 44 months (SD 30, range 0.1-119). RESULTS: One patient (1.8%) experienced a re-dislocation. Three patients (5.3%) had to be revised. One due to disintegration between the femoral head and inner shell, one due to loosening of the acetabular component, and one due to infection. Harris Hip Score improved from a mean of 76 before index surgery to 87 within one year after index surgery. CONCLUSION: This study advocates the use of a dual mobility cup for treatment of recurrent dislocations of THR. However, studies with a longer follow up are needed in order to evaluate implant survival.

Consumer attitudes toward new technique for preserving organic meat using herbs and berries.

This study aims to explore consumers' attitude toward a new preservation technique using herbs and berries in organic meat production, which enables to minimize the amount of chemical additives and to reduce the salt content in meat products. Consumer acceptance of the preservation technique using herbs and berries and intention to purchase products preserved with herbs and berries were investigated through a qualitative approach by means of three focus groups. In general, most participants were positive toward the preservation technique using herbs and berries and there were only few concerns related to the technique. Concerns were related not as much to the technique but more to the products. Four factors seem important in this relation: shelf life, taste, appearance and texture. The intention to purchase products preserved with herbs and berries is generally high, but is dependent on taste and appearance of the products, the price and information level.

High expression of microRNA-625-3p is associated with poor response to first-line oxaliplatin based treatment of metastatic colorectal cancer.

The backbone of current cytotoxic treatment of metastatic colorectal cancer (mCRC) consists of a fluoropyrimidine together with either oxaliplatin (XELOX/FOLFOX) or irinotecan (XELIRI/FOLFIRI). With an overall objective response rate of approximately 50% for either treatment combination, a major unsolved problem is that no predictors of response to these treatments are available. To address this issue, we profiled 742 microRNAs in laser-capture microdissected cancer cells from responding and non-responding patients receiving XELOX/FOLFOX as first-line treatment for mCRC, and identified, among others, high expression of miR-625-3p, miR-181b and miR-27b to be associated with poor clinical response. In a validation cohort of 94 mCRC patients treated first-line with XELOX, high expression of miR-625-3p was confirmed to be associated with poor response (OR = 6.25, 95%CI [1.8; 21.0]). Independent analyses showed that miR-625-3p was not dysregulated between normal and cancer samples, nor was its expression associated with recurrence of stage II or III disease, indicating that miR-625-3p solely is a response marker. Finally, we also found that these miRNAs were up-regulated in oxaliplatin resistant HCT116/oxPt (miR-625-3p, miR-181b and miR-27b) and LoVo/oxPt (miR-181b) colon cancer cell lines as compared with their isogenic parental cells. Altogether, our results suggest an association between miR-625-3p and response to first-line oxaliplatin based chemotherapy of mCRC.

A novel RNASEH2B splice site mutation responsible for Aicardi-Goutieres syndrome in the Faroe Islands.

AIM: The aim of the study was to identify the genetic background for Aicardi-Goutieres syndrome (AGS) in the Faroe Islands. METHODS: Four patients with AGS were identified. The patients had a variable phenotype, from a severe prenatal form with intrauterine foetal death to a milder phenotype, albeit still with an early onset, within the first 2-3 months. RESULTS: A genome-wide search for homozygosity revealed one single 15.6 Mb region of homozygosity on chromosome 13, which included RNASEH2B, where a splice site mutation c.322-3C>G was identified. Screening of 170 anonymous Faroese controls revealed a carrier frequency of approximately 1.8%, corresponding to an incidence of AGS in the Faroe Islands of around 1 in 12,300. CONCLUSION: The previously identified RNASEH2B mutations comprise altogether 20 mutations (missense, nonsense and splice site) with all patients harbouring at least one missense mutation. The severe phenotype of the Faroese patients compared with the previously reported patients with RNASEH2B mutations may be caused by the presence of two null alleles (although some residual normal splicing cannot be ruled out), whereas patients with one or two missense mutations may have some, albeit abnormal, RNASEH2B proteins, and hence some residual activity of RNASEH2B, explaining their milder phenotype.

Phase III trial of cetuximab with continuous or intermittent fluorouracil, leucovorin, and oxaliplatin (Nordic FLOX) versus FLOX alone in first-line treatment of metastatic colorectal cancer: the NORDIC-VII study.

PURPOSE: The NORDIC-VII multicenter phase III trial investigated the efficacy of cetuximab when added to bolus fluorouracil/folinic acid and oxaliplatin (Nordic FLOX), administered continuously or intermittently, in previously untreated metastatic colorectal cancer (mCRC). The influence of KRAS mutation status on treatment outcome was also investigated. PATIENTS AND METHODS: Patients were randomly assigned to receive either standard Nordic FLOX (arm A), cetuximab and FLOX (arm B), or cetuximab combined with intermittent FLOX (arm C). Primary end point was progression-free survival (PFS). Overall survival (OS), response rate, R0 resection rate, and safety were secondary end points. RESULTS: Of the 571 patients randomly assigned, 566 were evaluable in intention-to-treat (ITT) analyses. KRAS and BRAF mutation analyses were obtained in 498 (88%) and 457 patients (81%), respectively. KRAS mutations were present in 39% of the tumors; 12% of tumors had BRAF mutations. The presence of BRAF mutations was a strong negative prognostic factor. In the ITT population, median PFS was 7.9, 8.3, and 7.3 months for the three arms, respectively (not significantly different). OS was almost identical for the three groups (20.4, 19.7, 20.3 months, respectively), and confirmed response rates were 41%, 49%, and 47%, respectively. In patients with KRAS wild-type tumors, cetuximab did not provide any additional benefit compared with FLOX alone. In patients with KRAS mutations, no significant difference was detected, although a trend toward improved PFS was observed in arm B. The regimens were well tolerated. CONCLUSION: Cetuximab did not add significant benefit to the Nordic FLOX regimen in first-line treatment of mCRC.

Detection of Salmonella in meat: comparative and interlaboratory validation of a noncomplex and cost-effective pre-PCR protocol.

Cost-effective and rapid monitoring of Salmonella in the meat production chain can contribute to food safety. The objective of this study was to validate an easy-to-use pre-PCR sample preparation method based on a simple boiling protocol for screening of Salmonella in meat and carcass swab samples using a real-time PCR method. The protocol included incubation in buffered peptone water, centrifugation of an aliquot, and a boiling procedure. The validation study included comparative and interlaboratory trials recommended by the Nordic Organization for Validation of Alternative Microbiological Methods (NordVal). The comparative trial was performed against a reference method (NMKL 187, 2007) and a PCR method previously approved by NordVal with a semiautomated magnetic bead-based DNA extraction step using 122 artificially contaminated samples. The LOD was found to be 3.0, 3.2, and 3.4 CFU/sample for the boiling, magnetic bead-based, and NMKL 187 methods, respectively. When comparing the boiling method with the magnetic beads, the relative accuracy (AC), relative sensitivity (SE), and relative specificity (SP) were 98, 102, and 98%, respectively (Cohen's kappa index 0.95). When comparing results obtained by the boiling to the culture-based method, the AC, SE, and SP were found to be 98, 102, and 98%, respectively (kappa index 0.93). In the interlaboratory trial including valid results from 11 laboratories, apart from two false-positive samples by the boiling method combined with PCR, no deviating results were obtained (SP, SE, and AC were 100, 95, and 97%, respectively). This test is under implementation by the Danish meat industry, and can be useful for screening of large number of samples in the meat production, especially for fast release of minced meat with a short shelf life.

Cold atmospheric pressure plasma treatment of ready-to-eat meat: inactivation of Listeria innocua and changes in product quality.

The application of cold atmospheric pressure plasma for decontamination of a sliced ready-to-eat (RTE) meat product (bresaola) inoculated with Listeria innocua was investigated. Inoculated samples were treated at 15.5, 31, and 62 W for 2-60 s inside sealed linear-low-density-polyethylene bags containing 30% oxygen and 70% argon. Treatments resulted in a reduction of L. innocua ranging from 0.8 ± 0.4 to 1.6 ± 0.5 log cfu/g with no significant effects of time and intensity while multiple treatments at 15.5 and 62 W of 20 s with a 10 min interval increased reduction of L. innocua with increasing number of treatments. Concentrations of thiobarbituric acid reactive substances (TBARS) increased with power, treatments and storage time and were significantly higher than those of control samples after 1 and 14 days of storage at 5 °C. However, the levels were low (from 0.1 to 0.4 mg/kg) and beneath the sensory threshold level. Surface colour changes included loss of redness of ∼40% and 70% after 1 and 14 days of storage, respectively, regardless of plasma treatment. The results indicate that plasma may be applicable in surface decontamination of pre-packed RTE food products. However, oxidation may constitute an issue in some products.

YGFP: a spectral variant of GFP.

We describe YGFP, a slow bleaching green fluorescent protein (GFP) with unique spectral properties. YGFP is derived from an Escherichia coli codon-optimized synthetic gfp mutant 2 derivative. In addition to the GFP-mut 2 changes, it also carries S202F and T203I substitutions. YGFP can be used as a substitute for yellow fluorescent protein (YFP) in experiments in which two or more fluorescent proteins are fused to different cellular protein components, expanding the ability to study multiple labeled proteins in a cell at once.

Validation of a 20-h real-time PCR method for screening of Salmonella in poultry faecal samples.

Efficient and rapid monitoring of Salmonella in the poultry production chain is necessary to assure safe food. The objective was to validate an open-formula real-time PCR method for screening of Salmonella in poultry faeces (sock samples). The method consists of incubation in buffered peptone water for 18+/-2 h, centrifugation of a 1-ml subsample, DNA extraction on the pellet and PCR. The total analysis time is 20 h. The validation study included comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal). The comparative trial was performed against a reference method from the Nordic Committee on Food Analysis (NMKL187, 2007) using 132 artificially and naturally contaminated samples. The limit of detection (LOD50) was found to be 24 and 33 CFU/sample for the PCR and NMKL187 methods, respectively. The relative accuracy, relative sensitivity and relative specificity were all 100%, when including naturally contaminated samples and samples artificially contaminated with 10-100 CFU/sample. The collaborative trial included six laboratories and valid results were obtained from five of them. Apart from one of the samples that was artificially contaminated with 1-10 CFU/sample being a false negative with PCR for one of the laboratories, no false-positive or false-negative results were reported. This test supplies the growing demand for validated diagnostic PCR methods for screening of samples in the meat production chain to assure safe food.

The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid.

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.

Crystalline-to-plastic phase transitions in molecularly thin n-dotriacontane films adsorbed on solid surfaces.

Crystalline-to-rotator phase transitions have been widely studied in bulk hydrocarbons, in particular in normal alkanes. But few studies of these transitions deal with molecularly thin films of pure n-alkanes on solid substrates. In this work, we were able to grow dotriacontane (n-C(32)H(66)) films without coexisting bulk particles, which allows us to isolate the contribution to the ellipsometric signal from a monolayer of molecules oriented with their long axis perpendicular to the SiO(2) surface. For these submonolayer films, we found a step in the ellipsometer signal at approximately 331 K, which we identify with a solid-solid phase transition. At higher coverages, we observed additional steps in the ellipsometric signal that we identify with a solid-solid phase transition in multilayer islands ( approximately 333 K) and with the transition to the rotator phase in bulk crystallites ( approximately 337 K), respectively. After considering three alternative explanations, we propose that the step upward in the ellipsometric signal observed at approximately 331 K on heating the submonolayer film is the signature of a transition from a perpendicular monolayer phase to a denser phase in which the alkane chains contain on average one to two gauche defects per molecule.

Bileaflet blood cysts on the mitral valve in an adult.

Blood cysts within the heart are very rare entities in adults. The authors present possibly the first ever case in which blood cysts were found on both mitral valve leaflets. A 65-year-old woman was referred for transthoracic echocardiography because of vague chest discomfort. Transthoracic echocardiography displayed echo-free cysts on the tips of both mitral valve leaflets. Subsequent transesophageal echocardiography confirmed this finding. The blood cysts were successfully surgically removed.

Structure and growth of vapor-deposited n-dotriacontane films studied by X-ray reflectivity.

We have used synchrotron X-ray reflectivity measurements to investigate the structure of n-dotriacontane (n-C(32)H(66) or C32) films deposited from the vapor phase onto a SiO(2)-coated Si(100) surface. Our primary motivation was to determine whether the structure and growth mode of these films differ from those deposited from solution on the same substrate. The vapor-deposited films had a thickness of approximately 50 A thick as monitored in situ by high-resolution ellipsometry and were stable in air. Similar to the case of solution-deposited C32 films, we find that film growth in vacuum begins with a nearly complete bilayer adjacent to the SiO(2) surface formed by C32 molecules aligned with their long axis parallel to the interface followed by one or more partial layers of perpendicular molecules. These molecular layers coexist with bulk particles at higher coverages. Furthermore, after thermally cycling our vapor-deposited samples at atmospheric pressure above the bulk C32 melting point, we find the structure of our films as a function of temperature to be consistent with a phase diagram inferred previously for similarly treated solution-deposited films. Our results resolve some of the discrepancies that Basu and Satija (Basu, S.; Satija, S. K. Langmuir 2007, 23, 8331) found between the structure of vapor-deposited and solution-deposited films of intermediate-length alkanes at room temperature.

Validation of a same-day real-time PCR method for screening of meat and carcass swabs for Salmonella.

BACKGROUND: One of the major sources of human Salmonella infections is meat. Therefore, efficient and rapid monitoring of Salmonella in the meat production chain is necessary. Validation of alternative methods is needed to prove that the performance is equal to established methods. Very few of the published PCR methods for Salmonella have been validated in collaborative studies. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, non-commercial real-time PCR method for detection of Salmonella in meat and carcass swabs. RESULTS: The comparative trial was performed against a reference method (NMKL-71:5, 1999) using artificially and naturally contaminated samples (60 minced veal and pork meat samples, 60 poultry neck-skins, and 120 pig carcass swabs). The relative accuracy was 99%, relative detection level 100%, relative sensitivity 103% and relative specificity 100%. The collaborative trial included six laboratories testing minced meat, poultry neck-skins, and carcass swabs as un-inoculated samples and samples artificially contaminated with 1-10 CFU/25 g, and 10-100 CFU/25 g. Valid results were obtained from five of the laboratories and used for the statistical analysis. Apart from one of the non-inoculated samples being false positive with PCR for one of the laboratories, no false positive or false negative results were reported. Partly based on results obtained in this study, the method has obtained NordVal approval for analysis of Salmonella in meat and carcass swabs. The PCR method was transferred to a production laboratory and the performance was compared with the BAX Salmonella test on 39 pork samples artificially contaminated with Salmonella. There was no significant difference in the results obtained by the two methods. CONCLUSION: The real-time PCR method for detection of Salmonella in meat and carcass swabs was validated in comparative and collaborative trials according to NordVal recommendations. The PCR method was found to perform well. The test is currently being implemented for screening of several hundred thousand samples per year at a number of major Danish slaughterhouses to shorten the post-slaughter storage time and facilitate the swift export of fresh meat.