A randomised, controlled trial evaluating a low cost, 3D-printed bronchoscopy simulator.

Low-fidelity, simulation-based psychomotor skills training is a valuable first step in the educational approach to mastering complex procedural skills. We developed a cost-effective bronchial tree simulator based on a human thorax computed tomography scan using rapid-prototyping (3D-print) technology. This randomised, single-blind study evaluated how realistic our 3D-printed simulator would mimic human anatomy compared with commercially available bronchial tree simulators (Laerdal® Airway Management Trainer with Bronchial Tree and AirSim Advance Bronchi, Stavanger, Norway). Thirty experienced anaesthetists and respiratory physicians used a fibreoptic bronchoscope to rate each simulator on a visual analogue scale (VAS) (0 mm = completely unrealistic anatomy, 100 mm = indistinguishable from real patient) for: localisation of the right upper lobe bronchial lumen; placement of a bronchial blocker in the left main bronchus; aspiration of fluid from the right lower lobe; and overall realism. The 3D-printed simulator was rated most realistic for the localisation of the right upper lobe bronchial lumen (p = 0.002), but no differences were found in placement of a bronchial blocker or for aspiration of fluid (p = 0.792 and p = 0.057) compared with using the commercially available simulators. Overall, the 3D-printed simulator was rated most realistic (p = 0.021). Given the substantially lower costs for the 3D-printed simulator (£85 (€100/US$110) compared with > ~ £2000 (€2350/US$2590) for the commercially available simulators), our 3D-printed simulator provides an inexpensive alternative for learning bronchoscopy skills, and offers the possibility of practising procedures on patient-specific models before attempting them in clinical practice.

NDRG1 prognosticates the natural course of disease in WHO grade II glioma.

There is a lack of relevant prognostic and predictive factors in neurooncology besides mutation of isocitrate dehydrogenase 1, codeletion of 1p/19q and promoter hypermethylation of O (6) -methylguanine-DNA-methyltransferase. More importantly, there is limited translation of these factors into clinical practice. The cancer genome atlas data and also clinical correlative analyses suggest a pivotal role for the epidermal growth factor receptor /protein kinase B/mammalian target of rapamycin (mTOR) pathway in both biology and the clinical course of gliomas. However, attempts to stratify gliomas by activating alterations in this pathway have failed thus far. The tumors of 40 patients with WHO grade II gliomas without immediate postoperative genotoxic treatment and known progression and survival status at a median follow-up of 12.2 years were analyzed for expression of the mTOR complex 2 downstream target N-myc downstream regulated gene (NDRG)1 using immunohistochemistry. Baseline characteristics for NDRG1 absent/low versus moderate/high patients were similar. Time to reintervention was significantly longer in the NDRG1 group (P = 0.026). NDRG1 may become a novel biomarker to guide the decision which WHO°II glioma patients may be followed without postsurgical intervention and which patients should receive genotoxic treatment early on. Validation of this hypothesis will be possible with the observational arm of the RTOG 9802 and the pretreatment step of the EORTC 22033/26032 trials.

Hypercalcemia mediated by parathyroid hormone-related protein as an early manifestation of pancreatic adenocarcinoma metastasis: a case report.

Humoral hypercalcemia of malignancy (HHM) is a paraneoplastic syndrome rarely associated with pancreatic adenocarcinoma. Parathyroid hormone-related peptide (PTHrP) is the central mediator of this condition. In our patient, hypercalcemia associated with elevated PTHrP was the initial manifestation of metastatic pancreatic adenocarcinoma. Successful palliation of HHM with bisphosphonates and loop diuretics has been previously reported and was effective in our patient. We report the first case of pancreatic adenocarcinoma metastasis after successful resection to present with hypercalcemia.

Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui.

Insertions were introduced by a two-step mutagenesis procedure into each of five double-helical regions of Escherichia coli 23 S rRNA, so as to extend the helix concerned by 17 bp. The helices chosen were at sites within the 23 S molecule (h9, h25, h45, h63 and h98) where significant length variations between different species are known to occur. At each of these positions, with the exception of h45, there are also significant differences between the 23 S rRNAs of E. coli and Haloarcula marismortui. Plasmids carrying the insertions were introduced into an E. coli strain lacking all seven rrn operons. In four of the five cases the cells were viable and 50 S subunits could be isolated; only the insertion in h63 was lethal. The modified subunits were examined by cryo-electron microscopy (cryo-EM), with a view to locating extra electron density corresponding to the insertion elements. The results were compared both with the recently determined atomic structure of H. marismortui 23 S rRNA in the 50 S subunit, and with previous 23 S rRNA modelling studies based on cryo-EM reconstructions of E. coli ribosomes. The insertion element in h45 was located by cryo-EM at a position corresponding precisely to that of the equivalent helix in H. marismortui. The insertion in h98 (which is entirely absent in H. marismortui) was similarly located at a position corresponding precisely to that predicted from the E. coli modelling studies. In the region of h9, the difference between the E. coli and H. marismortui secondary structures is ambiguous, and the extra electron density corresponding to the insertion was seen at a location intermediate between the position of the nearest helix in the atomic structure and that in the modelled structure. In the case of h25 (which is about 50 nucleotides longer in H. marismortui), no clear extra cryo-EM density corresponding to the insertion could be observed.

Aspirin (5 mmol/L) inhibits leukocyte attack and triggered reactive cell proliferation in a 3D human coronary in vitro model.

BACKGROUND: Leukocyte attack (LA) and the triggered reactive proliferation of smooth muscle cells (SMCs) are key events for the development of early atherosclerosis and restenosis. In the present study, we used a 3D human coronary in vitro model of LA (3DLA model) to examine the effect of high-dose aspirin on the adhesion and chemotaxis of leukocytes and the reactive proliferative response of SMCs. METHODS AND RESULTS: For dose-finding, the effect of aspirin (1, 2, 5, and 10 mmol/L) on the tumor necrosis factor-alpha-induced upregulation of intercellular adhesion molecule-1 was analyzed in monocultures of human coronary endothelial cells (HCAEC) and the SMCs of the human coronary media (HCMSMC). In cytoflow and Northern blot experiments, the expression of intercellular adhesion molecule-1 was slightly reduced after incubation with 5 mmol/L aspirin, and strong inhibition was found after incubation with 10 mmol/L. In 3DLA models, HCAECs and HCMSMCs were cultured on both sides of a porous filter. For LA, human monocytes or CD4(+) lymphocytes were seeded on the HCAEC side of the 3DLA unit. A dose of 5 mmol/L aspirin inhibited the adherence of monocytes or CD4(+) lymphocytes by 50% (P:<0.01) and the chemotaxis of monocytes by 90% (P:<0.01). The reactive proliferative response of cocultured HCMSMCs after LA, as measured by the uptake of bromodeoxyuridine, was significantly reduced by 83% after selective monocyte attack (P:<0.001) and by 42% after selective CD4(+) lymphocyte attack (P:<0.05). CONCLUSIONS: A local concentration of 5 mmol/L aspirin should be accepted as the lowest rational concentration for the beneficial in vitro effects of high-dose aspirin to be reproduced in clinical studies.

The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule.

Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple cross-links from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.

Benzofuro[3,2-b]pyridines as mixed ET(A)/ET(B) and selective ET(B) endothelin receptor antagonists.

The discovery, synthesis and structure-activity relationships of a series of novel benzofuro[3,2-b]pyridines as non-selective endothelin ET(A)/ET(B) as well as selective ET(B) receptor antagonists are described. The most potent non-selective inhibitor 7s displayed an IC50 of 21 nM and 41 nM for ET(A) and ET(B) receptors, respectively, whereas 7ee merely showed affinity for the ET(B) receptor (IC50 = 3.6 nM).

Expression of intercellular adhesion molecule-1 in human coronary endothelial and smooth muscle cells after stimulation with tumor necrosis factor-alpha.

BACKGROUND: The intercellular adhesion molecule-1 (ICAM-1) is one of several human cell adhesion molecules that play a critical role in the early stages of postangioplasty restenosis. In this study, the in-vitro expression of ICAM-1 in human coronary endothelial cells and human coronary smooth muscle cells (SMC) after stimulation with tumor necrosis factor-alpha (TNF-alpha) was investigated. METHODS AND RESULTS: SMC were isolated from the media of normal human coronary arteries (n = 26) up to 10 h post mortem (HCMSMC) and from human atherosclerotic coronary arteries (HCPSMC) that were extracted by thrombendarterectomy (n = 25). Endothelial cells of human coronary arteries (HCAEC) were purchased from Clonetics (Cell System, Remagen, Germany), and endothelial cells from human umbilical cord veins (HUVEC) were isolated after vaginal delivery. For investigations of the effect of TNF-alpha (2.5, 5, 10, and 20 ng/ml) on the proliferative activity of HUVEC, HCAEC, HCPSMC, and HCMSMC, serum-free media was used. After 24 h cell number and cell size distribution were measured in a cell analyzer system. The proliferation of HCPSMC and HCMSMC was increased by TNF-alpha; however, significant differences compared with controls were not reached. The proliferation of HUVEC and HCAEC was significantly reduced by TNF-alpha. For investigations of the effect of TNF-alpha (2.5, 5, 10, and 20 ng/ml) on the surface expression of ICAM-1, monoclonal anti-ICAM-1 antibodies (84H10) were used. The expression of ICAM-1 was analyzed using an immunofluorescence microscope. For flow cytometry analysis, 5 x 10(3) cells (100% gated) were analyzed using a fluorescence-activated cell sorter. In control cultures with no stimulation, the expression of ICAM-1 was positive in HCAEC, HCPSMC, HCMSMC, and HUVEC. TNF-alpha stimulated the expression of ICAM-1 in a time- and dose-dependent manner. After maximal stimulation with TNF-alpha (20 ng/ml for 24 h), the expression of ICAM-1 was stronger in HCMSMC than in HCPSMC. CONCLUSIONS: These results suggest that the cytokine TNF-alpha regulates the expression of ICAM-1 in both human coronary endothelial cells and SMC, and could therefore play an important role in the pathophysiology of inflammatory and immune processes in restenosis after angioplasty.

Endothelin antagonists: search for surrogates of methylendioxyphenyl by means of a Kohonen neural network.

The methylendioxyphenyl group, present in a number of potent endothelin receptor antagonists, could have undesirable metabolic interactions with cytochrome P450 in vivo. Using a self-organizing neural network we analysed the features of molecular electrostatic potentials of several endothelin receptor ligands. A library of small "fragments and functional groups" together with their corresponding Kohonen maps was generated. By means of this Kohonen map library we discovered the benzothiadiazole group as a surrogate for methylendioxyphenyl.

Endothelin antagonists: evaluation of 2,1,3-benzothiadiazole as a methylendioxyphenyl bioisoster.

The methylendioxyphenyl group is present in a number of endothelin receptor antagonists thus far reported. By means of a Kohonen neural network we discovered with a benzothiadiazole a bioisosteric replacement instead. This group should be devoid of the negative metabolic interactions with cytochrome P450 ascribed to methylendioxyphenyl in vivo. The synthesis of a potent benzothiadiazole analogue EMD 122801 together with in vitro studies of different methylendioxyphenyl, benzothiadiazole and benzofurazan derivatives is described.

Endothelin antagonists: discovery of EMD 122946, a highly potent and orally active ETA selective antagonist.

The discovery, in vitro and in vivo studies of the highly potent ETA antagonist EMD 122946 are presented. This compound displayed high binding affinity and functional antagonism [IC50 = 3.2 x 10(-11) M, pA2 = 9.5 (ETA)] and inhibited the ET-1 induced pressor response in pithed rats with an ED50 of 0.3 mg/kg. In conscious spontaneously hypertensive rats and in DOCA-salt hypertensive rats the compound lowered mean blood pressure with an ED50 of 0.06 mg/kg. EMD 122946 exhibited high bioavailability in rats and monkeys.

The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).

Getting closer to an understanding of the three-dimensional structure of ribosomal RNA.

Two experimentally unrelated approaches are converging to give a first low-resolution solution to the question of the three-dimensional organization of the ribosomal RNA from Escherichia coli. The first of these is the continued use of biochemical techniques, such as cross-linking, that provide information on the relative locations of different regions of the RNA. In particular, recent data identifying RNA regions that are juxtaposed to functional ligands such as mRNA or tRNA have been used to construct improved topographical models for the 16S and 23S RNA. The second approach is the application of high-resolution reconstruction techniques from electron micrographs of ribosomes in vitreous ice. These methods have reached a level of resolution at which individual helical elements of the ribosomal RNA begin to be discernible. The electron microscopic data are currently being used in our laboratory to refine the biochemically derived topographical RNA models.

The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.

The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.