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Slotman B.J.,VU University Amsterdam | Faivre-Finn C.,University of Manchester | van Tinteren H.,NKI | Keijser A.,IKNL | And 11 more authors.
Lung Cancer | Year: 2017

Introduction In ES-SCLC patients with residual intrathoracic disease after first-line chemotherapy, the addition of thoracic radiotherapy reduces the risk of intrathoracic recurrence, and improves 2-year survival. To identify patient subgroups for future trials investigating higher dose (extra)thoracic radiotherapy, we investigated the prognostic importance of number and sites of metastases in patients included in the CREST trial. Materials/ methods Additional data on sites and numbers of metastases were collected from individual records of 260 patients from the top 9 recruiting centers in the randomized CREST trial (53% of 495 study patients), which compared thoracic radiotherapy (TRT) to no TRT in ES-SCLC patients after any response to chemotherapy. All patients received prophylactic cranial irradiation. Results The clinical characteristics and outcomes of the 260 patients analyzed here did not differ significantly from that of the other 235 patients included in the CREST trial, except that fewer patients had a WHO = 0 performance status (24% vs 45%), and a higher proportion had WHO = 2 (15% vs 5%; p < 0.0001). No distant metastases were recorded in 5%, 39% had metastases confined to one organ, 34% to two, and 22% to three or more organ sites. Metastases were present in the liver (47%), bone (40%), lung (28%), extrathoracic (non-supraclavicular) lymph nodes (19%), supraclavicular nodes (18%), adrenals (17%) and other sites (12%). The OS (p = 0.02) and PFS (p = 0.04) were significantly better in patients with 2 or fewer metastases, with OS significantly worse if liver (p = 0.03) and/or bone metastases (p = 0.04) were present. Discussion This analysis of patients recruited from the top 9 accruing centers in the CREST trial suggests that future studies evaluating more intensive thoracic and extra-thoracic radiotherapy in ES-SCLC should focus on patients with fewer than 3 distant metastases. © 2017 The Author(s)


Kryshtafovych A.,University of California at Davis | Moult J.,University of Maryland College Park | Bartual S.G.,CSIC - Institute of Physical Chemistry "Rocasolano" | Bartual S.G.,University of Santiago de Compostela | And 29 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2011

One goal of the CASP community wide experiment on the critical assessment of techniques for protein structure prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, that is, the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this article, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fiber protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iβ dimerization/docking domain, the ectodomain of the JTB (jumping translocation breakpoint) transmembrane receptor, Autotaxin in complex with an inhibitor, the DNA-binding J-binding protein 1 domain essential for biosynthesis and maintenance of DNA base-J (β-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the phycobilisome core-membrane linker phycobiliprotein ApcE from Synechocystis, the heat shock protein 90 activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae. © 2011 Wiley-Liss, Inc.


PubMed | University of Aarhus, PSI, Helmholtz Center for Heavy Ion Research, SLK Klinik Heilbronn and 12 more.
Type: Journal Article | Journal: Physica medica : PM : an international journal devoted to the applications of physics to medicine and biology : official journal of the Italian Association of Biomedical Physics (AIFB) | Year: 2014

This report, compiled by experts on the treatment of mobile targets with advanced radiotherapy, summarizes the main conclusions and innovations achieved during the 4D treatment planning workshop 2013. This annual workshop focuses on research aiming to advance 4D radiotherapy treatments, including all critical aspects of time resolved delivery, such as in-room imaging, motion detection, motion managing, beam application, and quality assurance techniques. The report aims to revise achievements in the field and to discuss remaining challenges and potential solutions. As main achievements advances in the development of a standardized 4D phantom and in the area of 4D-treatment plan optimization were identified. Furthermore, it was noticed that MR imaging gains importance and high interest for sequential 4DCT/MR data sets was expressed, which represents a general trend of the field towards data covering a longer time period of motion. A new point of attention was work related to dose reconstructions, which may play a major role in verification of 4D treatment deliveries. The experimental validation of results achieved by 4D treatment planning and the systematic evaluation of different deformable image registration methods especially for inter-modality fusions were identified as major remaining challenges. A challenge that was also suggested as focus for future 4D workshops was the adaptation of image guidance approaches from conventional radiotherapy into particle therapy. Besides summarizing the last workshop, the authors also want to point out new evolving demands and give an outlook on the focus of the next workshop.


No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Human HOIP and HOIL-1L cDNA were purchased from Open Biosystems (cloneIDs 4653017 and 3877587, respectively). HOIP RBR (residues 696–1,072), HOIP RING2L (residues 853–1,072) and full-length HOIL-1L were cloned into the pET-NKI-6xHis-3C-LIC vector30 coding for an N-terminal 6×His tag with a 3C protease cleavage site. HOIP UBA–RBR (residues 475–1,072) was cloned into a pET-NKI-6×His-eGFP-3C-LIC vector that codes for a 3C-cleavable His-tagged enhanced green fluorescent protein (eGFP) followed by the HOIP sequence. Human UbcH5B and Cdc34 DNA were a gift from M. Petroski. Coding sequences for UbcH13 and Uev1a were extracted out of a human cDNA library (Agilent Megaman). For crystallization, UbcH5B (residues 2–147) with the mutations S22R (to prevent backside ubiquitin binding31) and C85K (to enable covalent ubiquitin linkage21) was cloned into the pET-NKI-6×His-3C-LIC vector. UbcH5B without S22R and C85K mutations (used for enzymatic assays), Cdc34, UbcH13 and Uev1a were cloned into the same vector. Untagged mono-ubiquitin with native N and C termini, used for crystallization and linear ubiquitination assays, was cloned into the pET29 vector (Novagen) using NdeI/XhoI restriction sites. N-terminally blocked mono-ubiquitin used for thioester assays was cloned in the pET-NKI-6×His-3C-LIC vector. Untagged linear di-ubiquitin was cloned with overlap extension PCR and ligated into the pET29 vector (Novagen) using NdeI/XhoI restriction sites. N- and C-terminally blocked di-ubiquitin with a N-terminal His tag and a C-terminal Ala–Ser sequence was cloned into the pET-NKI-6×His-3C-LIC vector. Human ubiquitin-activating enzyme E1 (Ube1) was cloned into a pET28 vector resulting in an N-terminal His tag. For NF-κB assays full-length HOIP with an N-terminal Flag tag and HOIL-1L with an N-terminal myc tag were cloned into pcDNA3.1(+) (Invitrogen) using EcoRI/NotI restriction sites. Mutations in UbcH5B, ubiquitin and HOIP were introduced using standard site-directed mutagenesis techniques. All proteins were expressed in BL21(DE3) E. coli after induction with 0.5 mM IPTG overnight at 20 °C. For expression of HOIP and HOIL-1L constructs, 0.5 mM ZnCl was added to the cultures before induction. Bacteria were harvested by centrifugation, lysed by addition of lysozyme and sonication in the presence of protease inhibitors (PMSF and leupeptin) and DNase. Lysates were cleared by centrifugation and His-tagged proteins were initially purified using Ni-NTA agarose (Qiagen). For HOIP RBR used for crystallization, and UbcH5B, Cdc34, UbcH13, Uev1a, wild-type ubiquitin to generate K48-linked di-ubiquitin and HOIL-1L His tags were removed by addition of 3C protease overnight at 4 °C. HOIP RBR and HOIL-1L were further purified using Superdex 200 10/300 GL or HiLoad 16/600 Superdex 200 pg size-exclusion chromatography columns (GE Healthcare) equilibrated in protein buffer (10 mM HEPES pH 7.9, 100 mM NaCl). UbcH5B used for biochemical assays was further purified on a Superdex 75 10/300 GL size-exclusion chromatography column (GE Healthcare) equilibrated in protein buffer. HOIP mutants for activity assays, and Cdc34, UbcH13 and Uev1a were desalted into protein buffer directly after Ni-NTA purification using PD MidiTrap G-25 desalting columns (GE Healthcare). Ube1 for biochemical assays was further purified using ion-exchange chromatography (Source Q) in 10 mM HEPES pH 7.9, 10 mM NaCl and eluted with a gradient from 10–500 mM NaCl. N-terminally His-tagged (di-)ubiquitin was purified using Ni-NTA as described above followed by size-exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in protein buffer or buffer exchange into protein buffer using PD MidiTrap G-25 desalting columns. To purify untagged mono- or di-ubiquitin, 0.5 mM EDTA and 100 mM sodium acetate pH 4.5 were added to the bacterial lysates and lysates were cleared by centrifugation, diluted sevenfold with 50 mM sodium acetate pH 4.5 and applied to a Source S 10/100 ion exchange column (GE Healthcare) equilibrated in 50 mM sodium acetate pH 4.5. Ubiquitin was eluted with a 0–500 mM NaCl gradient and further purified by size-exclusion chromatography on a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in protein buffer. His-eGFP-HOIP was purified using size-exclusion chromatography as described for HOIP RBR, followed by 3C cleavage and removal of His-eGFP via a second round of size-exclusion chromatography. All proteins were generally flash frozen in liquid nitrogen in small aliquots and stored at −80 °C. UbcH5B~ubiquitin linkage was performed based on published methods21. Briefly, Ube1, UbcH5B(S22R/C85K) and ubiquitin were mixed and buffer exchanged into 50 mM Tris pH 10, 150 mM NaCl using PD-10 desalting columns (GE Healthcare). 10 mM MgCl , 5 mM ATP and 1 mM TCEP were added and the protein solution was incubated at 37 °C for 16 h. The completeness of the reaction was monitored using SDS–PAGE and covalently linked UbcH5B~ubiquitin was purified from unreacted proteins and Ube1 using a Superdex 75 10/300 GL size-exclusion chromatography column (GE Healthcare) equilibrated in protein buffer. HOIP RBR was mixed with a 1.3-fold molar excess of UbcH5B~ubiquitin and applied to a Superdex 200 10/300 GL size-exclusion chromatography column equilibrated in protein buffer. Complex formation and purity was confirmed using SDS–PAGE, and complex containing fractions were pooled and concentrated to ~12 mg ml−1 for crystallization. Crystallization was performed using the vapour diffusion technique in sitting drop MRC 96-well plates (Molecular Dimensions). Initial crystals were obtained mixing HOIP/UbcH5B~ubiquitin complex solution with an equimolar amount of free ubiquitin in the Morpheus Screen (Molecular Dimensions). Subsequently, 2 μl of the protein complex were mixed with 0.6 μl reservoir solution (0.1 M Morpheus Buffer 3 pH 8.5 (Tris/Bicine), 0.12 M Morpheus Alcohols Mix (0.02 M each of 1,6-hexanediol; 1-butanol; 1,2-propanediol (racemic); 2-propanol; 1,4-butanediol; 1,3-propanediol), 30% Morpheus P550MME_P20K mix (20% PEG550MME, 10% PEG20K) and 8% glycerol) in MRC 48-well plates (Molecular Dimensions). Crystals appeared after about one week at 12 °C and were cryo-cooled, and evaluated on a rotating anode X-ray generator (Rigaku FR-E superbright). Seeding and dehydration of the crystals was performed to improve crystal diffraction. For successful dehydration, reservoir was slowly added to the protein drop (3 × 0.5 μl within ~2 h) and subsequently equilibrated overnight at 12 °C against a reservoir solution with increased P550MME_P20K concentration by adding 11 μl 60% Morpheus P550MME_P20K stock solution to 50 μl reservoir solution. The new reservoir solution was then slowly added to the protein drop (3 × 0.5 μl, followed by 2 × 1 μl with removal of 1 μl each in the last steps). After further overnight equilibration, crystals were harvested from the drop and directly cryo-cooled in a cryogenic nitrogen stream at 100 K. Crystals diffracted in-house to 4–6 Å. Complete diffraction data were measured at 100 K at beamline 23ID-D of the General Medical Sciences and Cancer Institutes Structural Biology Facility at the Advanced Photon Source (GM/CA @ APS), Argonne National Laboratory. Despite their size (common dimensions of ~200 × 140 × 100 μm3) crystals exhibited substantial inhomogeneity resulting in split and smeared diffraction spots. Using raster scans32, a suitable region for data collection could be identified at the edge of the crystal. Using a small (20 μm diameter) beam, split spots could be separated to allow reliable indexing and integration. Utilization of a small beam necessitated higher flux to retain reliable diffraction. To mitigate the radiation damage, the total dose was distributed over a 100-μm stretch of the crystal by using the ‘helical’ mode of ‘vector’ data collection as implemented in JBluIce33. Data were measured at 1.282 Å wavelength with a Pilatus3 6M pixel array detector with a 1-mm-thick sensor (Dectris). Data were collected from a single crystal and indexed, integrated and scaled in XDS/XSCALE34. Data were further processed using AIMLESS35 from the CCP4 suite36 with a resolution cut-off of 3.48 Å, resulting in an and CC1/2 = 0.648 in the highest resolution shell. Phasing was carried out in Phaser37 using an MR-SAD protocol as implemented in PHENIX38. For this, independent molecular replacement searches were initially performed for the RING2L domain of HOIP (from PDB: 4LJP (ref. 14)), UbcH5B (from PDB: 3A33 (ref. 39)), and ubiquitin (from PDB: 4LJP (ref. 14)) with the four C-terminal residues deleted. Various ambiguous solutions were identified that could not be separated, and Zn2+ sites could not be identified using MR-SAD due to incompleteness of resultant models. However, manual inspection revealed that some MR solutions contained ubiquitin oriented near identically to the symmetry-related donor ubiquitin observed in the HOIP RING2L/ubiquitin-ubiquitin transfer complex (PDB: 4LJP (ref. 14)). Based on this observation, a trimmed search model was created that contained a complex of the core of HOIP RING2L (with residues 906–924 and 949–999 removed) and C-terminally truncated ubiquitin. An MR search using this model found a single solution for two copies of the complex. After successful iterative searches for two UbcH5B molecules and two further ubiquitin molecules, MR-SAD using Phaser identified 15 distinct Zn2+ sites including the known Zn2+ sites in the RING2L domain of HOIP. Further molecular replacement in Phaser using a single unit of the initial HOIP RING2L/UbcH5B~ubiquitin complex (without the additional second ubiquitin), and the NMR structure of HOIP IBR (zinc atoms removed, deposited in Protein Data Bank40 under PDB accession number 2CT7, unpublished) correctly placed a single HOIP IBR domain, which was then manually copied to the other NCS-related HOIP in the asymmetric unit. For molecular replacement of the HOIP RING1, Sculptor41 was used to generate a search model based on the structure of the RING1 domain of HHARI (PDB: 4KBL (ref. 11)). However, Phaser was not able to correctly place this domain, probably owing to the low sequence conservation of only 27% identity. However, since mutational analysis of HOIP suggested that the RING/E2 interaction is preserved between RING-type E3 ligases and RBR-type E3 ligases5, we overlaid the E2 of the published RNF4–RING/UbcH5A~ubiquitin structure (PDB: 4AP4 (ref. 21)) with the E2 in our structure and then used this overlay to add the RING1 model generated by Sculptor. This overlay placed the HOIP RING1 Zn2+-coordinating residues near the last remaining free Zn2+ ions found earlier by Phaser MR-SAD, indicating correct placement of the RING1 domain. In the final round of molecular replacement, the two additional ubiquitin (Ub ) molecules were reinstated at the RING1–IBR interface. At this stage, Refmac42 was used for refinement using settings optimized for low-resolution refinement43 including ‘jelly body refinement’ and Babinet scaling. ProSMART44 was used to generate external restraints against high-resolution structures (PDB: 4LJO (ref. 14) for HOIP RING2L and ubiquitin, and PDB: 2ESK (ref. 45) for UbcH5B). After this, clear extra electron density became visible for the unmodelled helical linker regions of the RING1–IBR and IBR–RING2L transitions and for other regions omitted in the initial search models. Further model building and refinement was manually performed in Coot46 and Refmac. During refinement additional clear positive difference map electron density became visible and Phaser was used to place one additional UbcH5B molecule (UbcH5B ) into this density. TLS restraints were generated using the TLSMD server47 and NCS restraints were used throughout refinement. One overall B-factor was refined in Refmac. In later rounds of refinement the PDB_REDO server48 was used for refinement optimization and MolProbity49 was used for structure validation. Data processing and refinement statistics are summarized in Extended Data Fig. 2b. Ramachandran statistics were calculated using MolProbity and 94.8% of all residues are in favoured regions, 4.9% in allowed regions and 0.3% are outliers. The final structure has a MolProbity score of 1.75 (100th percentile). In the final structure the two HOIP RBR molecules (see also Extended Data Fig. 3) are defined by electron density from residues 699 to 707, 711 to 948, 969 to 991, and 996 to 1,011 (chain A) and 699 to 754, 760 to 957, 967 to 1,015, 1,019 to 1,035 and 1,054 to 1,066 (chain B). The catalytic UbcH5B~ubiquitin conjugates are defined from UbcH5B residues 3 to 147 and ubiquitin residues 1 to 76 (chains C and E), and UbcH5B residues 2 to 147 and ubiquitin residues 1 to 76 (chains D and F). The allosteric ubiquitin chains (chains G and H) are defined from residues 1 to 76 and the additional UbcH5B (chain I) is defined from residues 2 to 146. PHENIX was used to calculate simulated annealing (SA) composite omit maps and feature enhanced maps (FEM). All molecular figures were prepared in PyMOL (Schrödinger, LLC). K48-linked and K63-linked ubiquitin chains were formed through a linkage-specific enzymatic reaction using Cdc34 and UbcH13/Uev1a E2 ubiquitin-conjugating enzymes, respectively, as described in the literature50. Ubiquitin chains were separated using ion-exchange chromatography as described above for purification of mono-ubiquitin. Purified K48-linked di-ubiquitin was directly desalted into protein buffer using PD-10 desalting columns, whereas K63-linked di-ubiquitin was further purified on a Superdex 75 10/300 GL size-exclusion chromatography column equilibrated in protein buffer. Native ubiquitin without additional residues was used to generate di-ubiquitin chains for ITC experiments, whereas N-terminally blocked ubiquitin was used to form K48-linked di-ubiquitin for testing allosteric activation of HOIP RBR. Linear ubiquitin formation assays were performed in 50 mM HEPES pH 7.9, 100 mM NaCl, 10 mM MgCl and 0.6 mM DTT using 200 nM E1, 1 μM UbcH5B, 1 μM HOIP RBR or HOIP RING2L and 40 μM untagged ubiquitin. Reactions were started by addition of 10 mM ATP and were incubated at 30 °C for 2 h. Samples were taken at the indicated time points and treated with 50 mM sodium acetate pH 4.5 as described previously6, mixed with SDS sample buffer and analysed by SDS–PAGE using 12% Bolt Bis-Tris gels (Life Technologies). Proteins were visualized with Coomassie Brilliant blue dye. To test the activating effect of linear di-ubiquitin on auto-inhibited HOIP UBA–RBR, 5 μM HOIP UBA–RBR was pre-incubated with N- and C-terminally blocked linear di-ubiquitin or HOIL-1L at the indicated concentrations before addition of the remaining assay components. Samples were taken after 60 min and subsequently treated as described above. To monitor HOIP~ubiquitin thioester ubiquitin transfer from UbcH5B to HOIP, Ube1 (100 nM), UbcH5B (4 μM) and N-terminally blocked ubiquitin (32 μM) were mixed in 50 mM HEPES pH 7.9, 100 mM NaCl, 10 mM MgCl and 5 mM ATP and incubated at 25 °C for 5 min when 2 μM HOIP RBR was added. Samples were taken 10 s after HOIP addition, quenched by addition of pre-heated SDS protein-loading buffer without DTT, and run on a 12% SDS–PAGE gel (Life Technologies). The 10-s time point used was empirically determined with a time-course experiment (Extended Data Fig. 9g). Gels were stained with Coomassie Brilliant blue dye and scanned on a Li-COR Odyssey scanner using the 700 nm (red) channel. For the thioester transfer assay shown in Fig. 3d, 200 nM Ube1, 2 μM UbcH5B, 8 μM HOIP RBR, 8 μM N-terminally blocked ubiquitin and 10 mM ATP were used and samples taken after 30 s. Furthermore, proteins were transferred to a PVDF membrane and ubiquitin was visualized on a LI-COR Odyssey scanner at 800 nm using an anti-ubiquitin antibody (P4D1, Santa Cruz, 1:200 dilution in TBST (50 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20)) followed by an IRDye 800CW secondary antibody (LI-COR, 1:10,000 dilution in TBST). All quantitative experiments shown in graphs were performed in triplicates and band intensities were quantified using the ImageStudio software (LI-COR). HOIP thioester transfer activity was calculated as the fraction of HOIP~ubiquitin to total HOIP for each mutant and normalized against thioester transfer activity of wild-type HOIP. Data were analysed in GraphPad Prism using two-tailed unpaired Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. To test the allosteric activation of HOIP RBR by linear di-ubiquitin, a modified ubiquitin transfer assay was performed. HOIP RBR was pre-incubated with N- and C-terminally blocked linear di-ubiquitin at the indicated final concentrations for 5 min at 25 °C. At the same time, Ube1, UbcH5B, ubiquitin and ATP were premixed and incubated for 5 min at 25 °C, resulting in fully loaded UbcH5B~ubiquitin. Both mixtures were subsequently mixed together, resulting in final concentrations of 100 nM Ube1, 2 μM UbcH5B, 8 μM N-terminally blocked ubiquitin and 2 μM HOIP RBR in the final buffer described for the standard ubiquitin transfer assay. Samples were taken after 30 s and further treated as described for the standard transfer assay. A 30-s time point was determined to give the best results in this assay, in which lower E2 and mono-ubiquitin concentrations were used, resulting in an overall slower reaction rate. The experiments comparing the effects of linear versus K48-linked di-ubiquitin (Extended Data Fig. 9e) were performed similarly, with the difference that all samples were incubated with apyrase (Sigma) for 5 min to deplete ATP before addition of HOIP/di-ubiquitin and prevent E2-loading of K48-linked di-ubiquitin, which features a free C terminus on one of the ubiquitin units. Sedimentation equilibrium experiments were performed in a ProteomeLab XL-I (Beckman Coulter) analytical ultracentrifuge. HOIP RBR/UbcH5B~ubiquitin as used for crystallization was loaded into a 6-channel equilibrium cell at 5.0, 2.5 and 1.25 μM concentration and centrifuged at 10,000 r.p.m., 20 °C in an An-50 Ti 8-place rotor until equilibrium was achieved. Data were analysed using HeteroAnalysis software (J. L. Cole and J. W. Lary, University of Connecticut; http://www.biotech.uconn.edu/auf/). ITC experiments were performed on an ITC200 calorimeter (Microcal). Aliquots (2 μl each) of 500–650 μM UbcH5B~ubiquitin or di-ubiquitin solution were injected into the cell containing 40–50 μM HOIP RBR or HOIP RBR/di-ubiquitin complexes. The experiments were performed at 23 °C in buffer containing 10 mM HEPES pH 7.9, 100 mM NaCl. For titrations of UbcH5B~ubiquitin into HOIP RBR/di-ubiquitin complexes, HOIP RBR was pre-incubated with an equimolar amount of di-ubiquitin before the ITC experiments. Data were analysed using the Origin software (Microcal). Human embryonic kidney (HEK) 293T cells (ATCC) were co-transfected with NF-κB-luc reporter plasmid that contains an NF-κB response element upstream of the promoter driving the luciferase reporter gene, pGL4.74[hRluc/TK] control vector (Promega) and epitope tagged Flag-HOIP or myc-HOIL-1L pcDNA3.1(+) plasmids in 6-well plates in triplicates using Lipofectamine 2000 transfection reagent. Since this assay could be carried out in a variety of cellular contexts, HEK293T cells were used because they are easy to transfect and suitable for the assay. The cells tested negative for mycoplasma contamination. Empty pcDNA3.1(+) vector was used as control. After 36 h, cells were lysed and 20 μl cell lysates were used to measure firefly luciferase and Renilla luciferase (transfection control) signals using the dual luciferase reporter assay system according to the manufacturer’s protocol (Promega). Data were analysed in GraphPad Prism and one-way ANOVA followed by Tukey’s post hoc tests were used for statistical analysis. Immunoblotting was performed with anti-Flag (clone M2, Sigma-Aldrich) and anti-myc (clone 9E10, Sigma-Aldrich) antibodies, to confirm equivalent wild-type and mutant protein expression levels.


News Article | November 29, 2016
Site: www.eurekalert.org

PITTSBURGH, PA (November 29, 2016) -- Neuro Kinetics, Inc. (NKI), the leader in clinical eye-tracking and non-invasive neuro-otologic diagnostic testing, announced today the publication of an important study in the field of concussion detection that illustrates the potential clinical utility of an integrated, multi-modal battery of oculomotor, vestibular, and reaction time (OVRT) tests. The paper, titled "Oculomotor, Vestibular, and Reaction Time Tests in Mild Traumatic Brain Injury," is jointly authored by investigators from the University of Pittsburgh, San Diego Naval Medical Center, Madigan Army Medical Center, NKI, and the University of Miami Miller School of Medicine. The results of this novel study indicate the value of a clinical tool that can aid doctors in the timely and objective detection of concussive symptoms. "Objective diagnosis is vital in the management of concussion. This study will pave the way for tools that can be used at the point of injury, as well as in the emergency room or a provider's office," says Michael E. Hoffer, M.D., co-lead author and Professor of Otolaryngology at the University of Miami Miller School of Medicine. Concussions and mild traumatic brain injuries (mTBI) are diagnosed following a head injury when the Glasgow Coma Scale is 13 or greater and the loss of consciousness and/or confusion is less than 30 minutes. Most TBIs that occur each year are mild, commonly called concussions. According to a 2016 report from the U.S. Centers for Disease Control (CDC), TBIs accounted for approximately 2.5 million emergency department (ED) visits, hospitalizations, or deaths in the U.S. in 2010. Concussions contributed to approximately 50,000 deaths in 2010. The number of mTBIs is believed to be higher than 2.5 million annually, however, given that ED visits for a TBI have trended up in recent years (CDC cites a 70% increase during the period 2001-2010), and many who are injured do not visit an ED or hospital for assessment or treatment. Individuals with mTBI can complain of short-term or long-term cognitive problems, headaches, attention deficits, sleeping issues, and/or light sensitivity. While any one concussion may not be debilitating, multiple concussions -- particularly if repeat concussions happen before the patient has recovered -- can "add up," and cumulative effects can be devastating. The 18-to-45-year-old male and female population on whom data were collected participated in a battery of OVRT tests on I-Portal® NOTC's (Neuro-Otologic Test Centers) at the University of Miami Miller School of Medicine, San Diego, and Madigan Army Medical Center. The researchers evaluated and compared one set of 100 controls to 50 concussions (mTBI), "Cohort 1", and a second set of 100 controls to 50 concussions, "Cohort 2". The subjects had all been diagnosed as concussed by an emergency room doctor. Testing occurred approximately 2.6 days post-concussive event on average. The study results reveal promising sensitivities and specificities exceeding 97% and 89%, respectively. "It is no surprise that no single test was able to generate results that clearly identify a concussion. Rather, successful separation of controls from concussions required a combination of tests," says Howison Schroeder, CEO of Neuro Kinetics. The study protocol included sixteen OVRT tests, ten of which are already cleared by the U.S. Food & Drug Administration (FDA) for vestibular and neuro-otologic evaluation. Lead author Dr. Carey Balaban, Professor of Otolaryngology, Neurobiology, Communication Sciences & Disorders, and Bioengineering at the University of Pittsburgh, states: "This study provides a basis for a new generation of objective diagnostic tools for concussion that uses traditional oculomotor and vestibular tests. It offers the considerable advantage of not requiring baseline testing." "We are excited by such highly sensitive and specific results, and thank the Department of Defense for supporting such a transformational study," says Schroeder. Majority funding for this study was provided by the Department of Defense's Army Medical Research and Material Command and its Hearing Center of Excellence under Contract No. W81XWH-12-C-0205. Additional funding was awarded by the Head Health Challenge II sponsors, which include the National Football League, Under Armour, Inc., and General Electric Company. To learn more about NKI, please visit http://www. . Jennifer Smith Director of Media Relations University of Miami Miller School of Medicine Office: 305-243-3018 jennifer.smith@med.miami.edu Neuro Kinetics, Inc. (NKI) is the leader in clinical eye-tracking and non-invasive neuro-otologic diagnostic testing. The eye is the portal to the brain and research has shown the detection of abnormal eye responses are used to diagnose more than 200 diseases and medical conditions. With over 140 I-Portal installations, NKI's FDA cleared I-Portal® devices are sold to audiologists, ENT's, neurotologists, neuro-ophthalmologists and neurologists around the globe. The company's cleared patented diagnostic platforms include the I-Portal® NOTC (Neuro-Otologic Test Center), I-Portal® VNG (Video Nystagmography) and I-Portal® VOG (Video Oculography), along with related accessories, software, training and support services.


Read R.J.,University of Cambridge | Adams P.D.,Lawrence Berkeley National Laboratory | Arendall III W.B.,Duke University | Brunger A.T.,Howard Hughes Medical Institute | And 16 more authors.
Structure | Year: 2011

This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators. © 2011 Elsevier Ltd.


PubMed | NKI, Elekta Inc and Netherlands Cancer Institute
Type: Journal Article | Journal: Medical physics | Year: 2016

The purpose of this planning study is to investigate the influence of the ERE on the day-to-day dose distribution in rectal cancer patients, where changes in gas-pockets frequently occur.Daily CT scans of 5 patients treated neo-adjuvant with 55Gy for rectal cancer were used. We optimized two plans on the planning CT (Monaco, 1 mm3 dosegrid), a conventional 7-field 6MV IMRT plan (Dconv) and a plan in the presence of a 1.5T field (Dmrl). We recalculated the plans on all repeat-CT scans and evaluated under/over-dosage of the daily CTVs. Changes of more than 1% were considered significant. In the bowel area, we investigated the relative dose changes due to the ERE, where the contribution of the ERE was separated from other effects such as attenuation.Both plans were comparable and compliant with ICRU 62 for all patients. For 2 fractions in one patient under-dosage in the CTV was significant, due to a disappearing gas-pocket. Here the V95 was 96.82 and 97.36% in in Dmrl compared to 98.85 and 98.66% in Dconv, respectively. For 3 fractions in another patient appearing gas-pockets resulted in significant over-dosage of the CTV. In these fractions the V107 was 1.88-2.68% in Dmrl compared to 0.33-1.27% in Dconv. In the bowel area the dose changes attributable to the ERE were approximately 5% in 1cc, at low dose levels.We were able to calculate acceptable treatment plans with and without a magnetic field. The ERE was present in the Dmrl, but the volumetric effect within the CTV was limited. Outside the CTV relative dose differences were similar, but on small volumes at lower, less relevant dose levels. This suggests that there is no clinical relevant ERE on dose distributions in rectal cancer patients on a 1.5T MR-Linac.


Suijkerbuijk S.J.E.,Universiteitsweg 100 | van Dam T.J.P.,University Utrecht | van Dam T.J.P.,Radboud University Nijmegen | Karagoz G.E.,University Utrecht | And 8 more authors.
Developmental Cell | Year: 2012

Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive. In one exception, vertebrate BUBR1-defined by the KEN box-preserved the kinase domain but allowed nonconserved degeneration of catalytic motifs. Although BUBR1 evolved to a typical pseudokinase in some vertebrates, it retained the catalytic triad in humans. However, we show that putative catalysis by human BUBR1 is dispensable for error-free chromosome segregation. Instead, residues that interact with ATP in conventional kinases are essential for conformational stability in BUBR1. We propose that parallel evolution of BUBR1 orthologs rendered its kinase function dispensable in vertebrates, producing an unusual, triad-containing pseudokinase.


Joosten R.P.,NKI | Joosten K.,NKI | Cohen S.X.,NKI | Vriend G.,Radboud University Nijmegen | Perrakis A.,NKI
Bioinformatics | Year: 2011

Motivation: Macromolecular crystal structures in the Protein Data Bank (PDB) are a key source of structural insight into biological processes. These structures, some >30 years old, were constructed with methods of their era. With PDB_REDO, we aim to automatically optimize these structures to better fit their corresponding experimental data, passing the benefits of new methods in crystallography on to a wide base of non-crystallographer structure users. Results: We developed new algorithms to allow automatic rebuilding and remodeling of main chain peptide bonds and side chains in crystallographic electron density maps, and incorporated these and further enhancements in the PDB_REDO procedure. Applying the updated PDB_REDO to the oldest, but also to some of the newest models in the PDB, corrects existing modeling errors and brings these models to a higher quality, as judged by standard validation methods. © The Author(s) 2011. Published by Oxford University Press.

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