Pelvic organ prolapse surgery with and without tension-free vaginal tape in women with occult or asymptomatic urodynamic stress incontinence: a randomised controlled trial.

INTRODUCTION: We set out to determine if insertion of a retropubic tension-free vaginal tape (TVT) sling at the time of pelvic organ prolapse surgery improves continence outcomes in women with pre-operative occult stress incontinence (OSI) or asymptomatic urodynamic stress incontinence (USI). METHODS: We conducted a randomised controlled study of prolapse surgery with or without a TVT midurethral sling. The pre- and post-operative assessment at 6 months included history, physical examination and urodynamic testing. Quality of life (QOL) and treatment success was assessed with the UDI-6 SF, IIQ-7 SF and a numerical success score. The primary outcome was symptomatic stress urinary incontinence (SUI) requiring continence surgery (TVT) at 6 months. Long-term follow-up continued to a minimum of 24 months. Secondary outcomes were quality of life parameters. RESULTS: Eighty women received prolapse surgery alone (n = 43) or prolapse surgery with concurrent TVT (n = 37). Six months following prolapse surgery 3 out of 43 (7 %) patients in the no TVT group requested sling surgery compared with 0 out of 37 (0 %) in the TVT group (ARR 7 % [95 %CI: 3 to 19 %], p = 0.11). After 24 months there was one further participant in the no TVT group who received a TVT for treatment of SUI compared with none in the TVT group (4 out of 43, 9.3 % versus 0 out of 37; ARR 9.3 % [95 %CI: -1 to 22 %], p = 0.06). Both groups showed improvement in QOL difference scores for within-group analysis, without difference between groups. CONCLUSION: These results support a policy that routine insertion of a sling in women with OSI at the time of prolapse repair is questionable and should be subject to shared decision-making between clinician and patient.

Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex.

We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism.

Double-oblique free-breathing high resolution three-dimensional coronary magnetic resonance angiography.

OBJECTIVES: The goal of the present study was to develop a strategy for three-dimensional (3D) volume acquisition along the major axes of the coronary arteries. BACKGROUND: For high-resolution 3D free-breathing coronary magnetic resonance angiography (MRA), coverage of the coronary artery tree may be limited due to excessive measurement times associated with large volume acquisitions. Planning the 3D volume along the major axis of the coronary vessels may help to overcome such limitations. METHODS: Fifteen healthy adult volunteers and seven patients with X-ray angiographically confirmed coronary artery disease underwent free-breathing navigator-gated and corrected 3D coronary MRA. For an accurate volume targeting of the high resolution scans, a three-point planscan software tool was applied. RESULTS: The average length of contiguously visualized left main and left anterior descending coronary artery was 81.8 +/- 13.9 mm in the healthy volunteers and 76.2 +/- 16.5 mm in the patients (p = NS). For the right coronary artery, a total length of 111.7 +/- 27.7 mm was found in the healthy volunteers and 79.3 +/- 4.6 mm in the patients (p = NS). Comparing coronary MRA and X-ray angiography, a good agreement of anatomy and pathology was found in the patients. CONCLUSIONS: Double-oblique submillimeter free-breathing coronary MRA allows depiction of extensive parts of the native coronary arteries. The results obtained in patients suggest that the method has the potential to be applied in broader prospective multicenter studies where coronary MRA is compared with X-ray angiography.

Diagnostic value of lipopolysaccharide-binding protein and procalcitonin for sepsis diagnosis in forensic pathology.

The aims of this study were twofold. The first was to investigate the diagnostic performance of two biochemical markers, procalcitonin (PCT) and lipopolysaccharide-binding protein (LBP), considering each individually and then combined, for the postmortem diagnosis of sepsis. We also tested the usefulness of pericardial fluid for postmortem LBP determination. Two study groups were formed, a sepsis-related fatalities group of 12 cases and a control group of 30 cases. Postmortem native CT scans, autopsy, histology, neuropathology, and toxicology as well as other postmortem biochemical investigations were performed in all cases. Microbiological investigations were also carried out in the septic group. Postmortem serum PCT and LBP levels differed between the two groups. Both biomarkers, individually considered, allowed septic states to be diagnosed, whereas increases in both postmortem serum PCT and LBP levels were only observed in cases of sepsis. Similarly, normal PCT and LBP values in postmortem serum were identified only in non-septic cases. Pericardial fluid LBP levels do not correlate with the presence of underlying septic states. No relationship was observed between postmortem serum and pericardial fluid LBP levels in either septic or non-septic groups, or between pericardial fluid PCT and LBP levels.

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions.

Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal specific sequence motifs; however, CCG and CGC sequences seem to be overrepresented. Nonrandom association (multiple correspondence analysis, p < 0.0001) between silent genes, DNA methylation and MBD2 binding was observed. The association between MBD2 binding and transcriptional repression weakened as the distance between binding site and TSS increased, suggesting that MBD2 represses transcriptional initiation. This hypothesis may represent a functional explanation for the preferential binding of MBD2 at methyl-CpG in TSS regions.

Selective and powerful stress gene expression in Arabidopsis in response to malondialdehyde.

The provenance, half-life and biological activity of malondialdehyde (MDA) were investigated in Arabidopsis thaliana. We provide genetic confirmation of the hypothesis that MDA originates from fatty acids containing more than two methylene-linked double bonds, showing that tri-unsaturated fatty acids are the in vivo source of up to 75% of MDA. The abundance of the combined pool of free and reversibly bound MDA did not change dramatically in stress, although a significant increase in the free MDA pool under oxidative conditions was observed. The half-life of infiltrated MDA indicated rapid metabolic turnover/sequestration. Exposure of plants to low levels of MDA using a recently developed protocol powerfully upregulated many genes on a cDNA microarray with a bias towards those implicated in abiotic/environmental stress (e.g. ROF1 and XERO2). Remarkably, and in contrast to the activities of other reactive electrophile species (i.e. small vinyl ketones), none of the pathogenesis-related (PR) genes tested responded to MDA. The use of structural mimics of MDA isomers suggested that the propensity of the molecule to act as a cross-linking/modifying reagent might contribute to the activation of gene expression. Changes in the concentration/localisation of unbound MDA in vivo could strongly affect stress-related transcription.

Drug uptake in a rodent sarcoma model after intravenous injection or isolated lung perfusion of free/liposomal doxorubicin.

The distribution of free and liposomal doxorubicin (Liporubicin) administered by intravenous injection (IV) or isolated lung perfusion (ILP) was compared in normal and tumor tissues of sarcoma bearing rodent lungs. A single sarcomatous tumor was generated in the left lung of 35 Fischer rats, followed 10 days later by left-sided ILP (n=20) or IV drug administration (n=12), using 100 microg and 400 microg free or liposomal doxorubicin, respectively. The tumor and lung tissue drug concentration was measured by HPLC. Free doxorubicin administered by ILP resulted in a three-fold (100 microg) and 10-fold (400 microg) increase of the drug concentration in the tumor and normal lung tissue compared to IV administration. In contrast, ILP with Liporubicin resulted in a similar drug uptake in the tumor and lung tissue compared to IV injection. For both drug formulations and dosages, ILP resulted in a higher tumor to lung tissue drug ratio but also in a higher spatial heterogeneity of drug distribution within the lung compared to IV administration. ILP resulted in a higher tumor to lung tissue drug ratio and in a more heterogeneous drug distribution within the lung compared to IV drug administration.

Cell-free reconstitution of microautophagy in yeast.

Microautophagy is the direct uptake of soluble or particulate cellular constituents into lysosomes. Here, I describe methods to reconstitute and study this process in vitro, using vacuoles (lysosomes) from the yeast Saccharomyces cerevisiae as model organelles. Protocols to grow the cells, isolate vacuoles from them, and to induce microautophagy of soluble tracers are presented.

An infrared reflection-absorption spectroscopic (IRRAS) study of the interaction of lipid A and lipopolysaccharide Re with endotoxin-binding proteins.

Lipopolysaccharides (LPS, endotoxins) are main constituents of the outer membranes of Gram-negative bacteria, with the 'endotoxic principle' lipid A anchoring LPS into the membrane. When LPS is removed from the bacteria by the action of the immune system or simply by cell dividing, it may interact strongly with immunocompetent cells such as mononuclear cells. This interaction may lead, depending on the LPS concentration, to beneficial (at low) or pathophysiological (at high concentrations) reactions, the latter frequently causing the septic shock syndrome. There is a variety of endogenous LPS-binding proteins. To this class belong lactoferrin (LF) and hemoglobin (Hb), which have been shown to suppress and enhance the LPS-induced cytokine secretion in mononuclear cells, respectively. To elucidate the interaction mechanisms of endotoxins with these proteins, we have investigated in an infrared reflection-absorption spectroscopy (IRRAS) study the interaction of LPS or lipid A monolayers at the air/water interface with LF and Hb proteins, injected into the aqueous subphase. The data are clearly indicative of completely different interaction mechanisms of the endotoxins with the proteins, with the LF acting only at the LPS backbone, whereas Hb incorporates into the lipid monolayer. These data allow an understanding of the different reactivities in the biomedicinal systems.

Transcriptional Activation and Receptor Dimerization is Affected by Mutations in the NR2E3 Ligand-Binding Domain

Purpose: Mutations in the ligand-binding domain (LBD) of NR2E3 cause recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS), Goldmann-Favre syndrome (GFS) and clumped pigmentary retinal degeneration (CPRD). In addition to ligand binding, the LBD contains also essential amino acid sequences for the oligomerization of nuclear receptors. The aim of our studies is to characterize the impact of mutations in the LBD on receptor oligomerization and transcriptional activity of NR2E3. Methods: The different NR2E3 mutants were generated by QuickChange mutagenesis and analyzed in 293T-based transactivation studies and BRET2 (bioluminescence resonance electron transfer) assays. In silico homology modeling of mutant proteins was also performed using available crystallographic data of related nuclear receptors. Results: The mutants p.W234S, p.A256V, p.A256E, p.L263P, p.R309G, p.R311Q, p.R334G, p.L336P, p.L353V, p.R385P and p.M407K, all located in the LBD, showed impaired receptor dimerization at various degrees. Impaired repressor dimerization as assessed by BRET2 assays did not always correlate with impaired repressor function of NR2E3 as assessed by cell-based reporter assays. There were minor differences of transcriptional activity of mutant proteins on mouse S-opsin (opn1sw), mouse cone arrestin (arr3) and human cone arrestin, suggesting that the effect of LBD mutations was independent of the promoter context. Conclusions: Mutational analysis and homology modeling allowed the characterization of potential oligomerization interfaces of the NR2E3 LBD. Additionally, mutations in NR2E3 LBD may cause recessive retinal degenerations by different molecular mechanisms.

A 35.7 kb DNA fragment from the Bacillus subtilis chromosome containing a putative 12.3 kb operon involved in hexuronate catabolism and a perfectly symmetrical hypothetical catabolite-responsive element.

The Bacillus subtilis strain 168 chromosomal region extending from 109 degrees to 112 degrees has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and beta-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase alpha-subunit (FdhA), protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.

Spin-labeling coronary MR angiography with steady-state free precession and radial k-space sampling: initial results in healthy volunteers.

The purpose of this study was to prospectively compare free-breathing navigator-gated cardiac-triggered three-dimensional steady-state free precession (SSFP) spin-labeling coronary magnetic resonance (MR) angiography performed by using Cartesian k-space sampling with that performed by using radial k-space sampling. A new dedicated placement of the two-dimensional selective labeling pulse and an individually adjusted labeling delay time approved by the institutional review board were used. In 14 volunteers (eight men, six women; mean age, 28.8 years) who gave informed consent, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), vessel sharpness, vessel length, and subjective image quality were investigated. Differences between groups were analyzed with nonparametric tests (Wilcoxon, Pearson chi2). Radial imaging, as compared with Cartesian imaging, resulted in a significant reduction in the severity of motion artifacts, as well as an increase in SNR (26.9 vs 12.0, P < .05) in the coronary arteries and CNR (23.1 vs 8.8, P < .05) between the coronary arteries and the myocardium. A tendency toward improved vessel sharpness and vessel length was also found with radial imaging. Radial SSFP imaging is a promising technique for spin-labeling coronary MR angiography.

Free-breathing inner-volume black-blood imaging of the human heart using two-dimensionally selective local excitation at 3 T.

Black-blood fast spin-echo imaging is a powerful technique for the evaluation of cardiac anatomy. To avoid fold-over artifacts, using a sufficiently large field of view in phase-encoding direction is mandatory. The related oversampling affects scanning time and respiratory chest motion artifacts are commonly observed. The excitation of a volume that exclusively includes the heart without its surrounding structures may help to improve scan efficiency and minimize motion artifacts. Therefore, and by building on previously reported inner-volume approach, the combination of a black-blood fast spin-echo sequence with a two-dimensionally selective radiofrequency pulse is proposed for selective "local excitation" small field of view imaging of the heart. This local excitation technique has been developed, implemented, and tested in phantoms and in vivo. With this method, small field of view imaging of a user-specified region in the human thorax is feasible, scanning becomes more time efficient, motion artifacts can be minimized, and additional flexibility in the choice of imaging parameters can be exploited.