Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds

For many protein families, the deluge of new sequence information together with new statistical protocols now allow the accurate prediction of contacting residues from sequence information alone. This offers the possibility of more accurate ab initio (non-homology-based) structure prediction. Such models can be used in structure solution by molecular replacement (MR) where the target fold is novel or is only distantly related to known structures. Here, AMPLE, an MR pipeline that assembles search-model ensembles from ab initio structure predictions (`decoys'), is employed to assess the value of contact-assisted ab initio models to the crystallographer. It is demonstrated that evolutionary covariance-derived residue–residue contact predictions improve the quality of ab initio models and, consequently, the success rate of MR using search models derived from them. For targets containing β-structure, decoy quality and MR performance were further improved by the use of a β-strand contact-filtering protocol. Such contact-guided decoys achieved 14 structure solutions from 21 attempted protein targets, compared with nine for simple Rosetta decoys. Previously encountered limitations were superseded in two key respects. Firstly, much larger targets of up to 221 residues in length were solved, which is far larger than the previously benchmarked threshold of 120 residues. Secondly, contact-guided decoys significantly improved success with β-sheet-rich proteins. Overall, the improved performance of contact-guided decoys suggests that MR is now applicable to a significantly wider range of protein targets than were previously tractable, and points to a direct benefit to structural biology from the recent remarkable advances in sequencing

Structure of the 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis

The 1.1 angstrom resolution structure of a periplasmic phosphate-binding protein from Stenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement using MOLREP in which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in the MOLREP peak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. A BLAST search then indicated that the molecule was most probably a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of the S. maltophilia protein has been refined to an R factor of 10.15% and an Rfree of 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles

Fast fully automatic segmentation of the severely abnormal human right ventricle from cardiovascular magnetic resonance images using a multi-scale 3D convolutional neural network

Cardiac magnetic resonance (CMR) is regarded as the reference examination for cardiac morphology in tetralogy of Fallot (ToF) patients allowing images of high spatial resolution and high contrast. The detailed knowledge of the right ventricular anatomy is critical in ToF management. The segmentation of the right ventricle (RV) in CMR images from ToF patients is a challenging task due to the high shape and image quality variability. In this paper we propose a fully automatic deep learning-based framework to segment the RV from CMR anatomical images of the whole heart. We adopt a 3D multi-scale deep convolutional neural network to identify pixels that belong to the RV. Our robust segmentation framework was tested on 26 ToF patients achieving a Dice similarity coefficient of 0.8281±0.1010 with reference to manual annotations performed by expert cardiologists. The proposed technique is also computationally efficient, which may further facilitate its adoption in the clinical routine

Use of the intravascular contrast agent NC100150 Injection in spin echo and gradient echo imaging of the heart

This is the first study of the intravascular iron oxide particle contrast agent, NC100150 Injection (Nycomed Imaging AS, Oslo, Norway, a part of Nycomed Amersham) in magnetic resonance imaging of the human heart. Eighteen healthy male volunteers were studied at both 0.5 and 1.5 T before and after the administration of NC100150 Injection. Transaxial spin-echo images were acquired at both field strengths, conventional gradient-echo cine images at 0.5 T, and breathhold Turbo-FLASH cine images at 1.5 T. Optimized cine imaging sequences were used postcontrast, with a high flip angle of 60-70”. In the spin-echo images there was a significant reduction in the blood pool flow artifact at the level of the right atrium (0.5 T, 57%, p < 0.01; 1.5 41%. p = 0.01) and the left ventricle (LV) (0.5 T, 45%, p = 0.01; 1.5 T, 45%, p < 0.01). In the conventional gradient-echo cines at 0.5 T, there was a significant increase in the LV blood pool and myocardial signal difference-to-noise ratio (SDNR) in the diastolic (56%, p = 0.01) and systolic (141%, p < 0.OOl)frames. There was also a significant increase in the signal intensity (SI) gradient at the LV blood pool-myocardial border in the diastolic and systolicframes (both p < 0.001). At higher doses of NClOO150 Injection (3 and 4 mg/kg), a rim of signal void around the LV blood pool was observed, perfectly defining the LV blood pool- myocardial border. In the Turbo-FLASH breathhold cines at 1.5 T, there was a significant increase in the LV blood pool-myocardial SDNR in the diastolic (221%, p < 0.001) and systolic (916%, p < 0.001) frames. Again, there was also a significant increase in the SI gradient at the LV blood pool- myocardial border in the diastolic and systolicframes (both p = 0.003). In conclusion, NC100150 Injection was given safely to 18 healthy subjects. Image quality and LV blood pool-myocardial definition were improved after the administration of NClOOI50 Injection. These improvements enable better spin-echo anatomical defiition, better definition of myocardial wall motion, and should improve the capability of automated edge detection algorithms

Yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) as zoomonitors of environmental contamination at a polluted area in Slovakia

<p>Abstract</p> <p>Background</p> <p>Free-living wild rodents are often used as zoomonitors of environmental contamination. In the present study, accumulation of cadmium (Cd), copper (Cu), iron (Fe), and zinc (Zn) in critical organs of yellow-necked mice (<it>Apodemus flavicollis</it>) and bank voles (<it>Myodes glareolus</it>) trapped in a polluted area in Nováky, Slovakia was investigated.</p> <p>Methods</p> <p>Yellow-necked mice (n = 8) and bank voles (n = 10) were collected using standard theriological methods for wood ecosystems. All animals were adult males in good physical condition. The concentrations of Cd, Cu, Fe, and Zn in the liver, kidney, and bone were determined by atomic absorption spectrophotometry.</p> <p>Results</p> <p>The highest concentrations of Cd and Zn were found in the bone of both species while Cu and Fe accumulated mainly in kidney or liver. Significant higher concentrations of Cd and Cu were detected in the liver of bank voles than in yellow-necked mice. Similar significant higher levels of Cd and Zn were found in the bone of bank voles. In contrast, significant higher concentrations of Cu and Fe were present in the kidney of yellow-necked mice.</p> <p>Conclusions</p> <p>In the yellow-necked mouse and bank vole, bone seems to accumulate Cd and Zn following prolonged exposure. On the contrary, kidney and liver store Cu and Fe after a long-term environmental exposure. In the present study, bank voles seemed to be more heavy metal loaded zoomonitors than yellow-necked mice.</p

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not infective to mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both

Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo: A Mouse Model

PURPOSE: Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure "transplant conditions" (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01. METHODS: Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts. RESULTS: Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001). CONCLUSION: Pre-confluent cells should be used to maximize graft cell viability

Results of NOPHO ALL2008 treatment for patients aged 1-45 years with acute lymphoblastic leukemia

Adults with acute lymphoblastic leukemia (ALL) do worse than children. From 7/2008 to 12/2014, Nordic and Baltic centers treated 1509 consecutive patients aged 1-45 years with Philadelphia chromosome-negative ALL according to the NOPHO ALL2008 without cranial irradiation. Overall, 1022 patients were of age 1-9 years (A), 266 were 10-17 years (B) and 221 were 18-45 years (C). Sixteen patients (three adults) died during induction. All others achieved remission after induction or 1-3 intensive blocks. Subsequently, 45 patients (12 adults) died, 122 patients relapsed (32 adults) with a median time to relapse of 1.6 years and 13 (no adult) developed a second malignancy. Median follow-up time was 4.6 years. Among the three age groups, older patients more often had higher risk ALL due to T-ALL (32%/25%/9%, PPeer reviewe

Three-dimensional late gadolinium enhancement cardiovascular magnetic resonance predicts inducibility of ventricular tachycardia in adults with repaired tetralogy of Fallot

Background - Adults with repaired tetralogy of Fallot (rTOF) die prematurely from ventricular tachycardia (VT) and sudden cardiac death. Inducible VT predicts mortality. Ventricular scar, the key substrate for VT, can be non-invasively defined with late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) but whether this relates to inducible VT is unknown. Methods - Sixty-nine consecutive rTOF patients (43 male, mean 40{plus minus}15 years) clinically scheduled for invasive programmed VT-stimulation were prospectively recruited for prior 3D LGE CMR. Ventricular LGE was segmented and merged with reconstructed cardiac chambers and LGE volume measured. Results - VT was induced in 22(31%) patients. Univariable predictors of inducible VT included increased RV LGE (OR 1.15;p=0.001 per cm3), increased non-apical vent LV LGE (OR 1.09;p=0.008 per cm3), older age (OR 1.6;p=0.01 per decile), QRS duration ≥180ms (OR 3.5;p=0.02), history of non-sustained VT (OR 3.5; p=0.02) and previous clinical sustained VT (OR 12.8;p=0.003); only prior sustained VT (OR 8.02;p=0.02) remained independent in bivariable analyses after controlling for RV LGE volume (OR 1.14;p=0.003). An RV LGE volume of 25cm3 had 72% sensitivity and 81% specificity for predicting inducible VT (AUC 0.81;p10cm3 was 100% sensitive and >36cm3 was 100% specific for predicting inducible VT. Conclusions - 3D LGE CMR-defined scar burden is independently associated with inducible VT and may help refine patient selection for programmed VT-stimulation when applied to an at least intermediate clinical risk cohort