252 research outputs found
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 Previously encountered limitations were superseded in two key respects.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
Calculation of a subject specific adaptive motion correction factor for improved real-time navigator echo gated MR coronary angiography
There
has
been conflicting
data
in the literature regarding the use
of
wide
navigator echo
(NE)
acceptance windows
in
combination with adaptive motion correction
for
magnetic resonance coronary
angiography
(MRCA).
This
in
part
may
be due
to
the use
of
a fixed
correction
factor
when applying
the adaptive motion-correction algorithm, which may potentially result
in
miscorrection
of
the imaging
slice
in
subjects
whose
correction
factor
differs widely
from
the mean.
We
have
addressed
this issue
by measuring the superior/inferior correction
factor
in 25 subjects and assessing the effect
of
using
a
subject-specific correction
factor
(CFss)
for
MRCA in comparison with
no
adaptive motion correction (CF,)
and
erroneous adaptive motion correction with
a
correction
factor
of
1.0
(CF,).
There
was
a
wide variation in the correction
factor
between subjects
(proximal
right coronary artery,
0.49
2
0.15,
range
0.20-0.70;
proximal left coronary artery, mean
0.59
?
0.I5,
range
0.20-0.85).
The subject-specific correction
factor
was accurately calculated
from
motion
of
the aortic root in
the coronal
plane
between expiratory
and
inspiratory breathhold
(correction
factor
calculated
from
coronal image versus correction factor calculated after localization
of
coronary arteries,
r
=
0.92,
p
<
0.001).
MRCA image quality was improved
using
a
subject-specific correction
factor,
for
both
a
6-mm
NE
acceptance window
(CF,
versus
CF,,
p
=
0.008;
CF,
versus CF,,
p
=
0.02)
and
a
16-
mm
NE window
(CFss
versus
CF,,
p
=
0.01;
CF,
versus
CF,,
p
=
0.007).
Furthermore, image quality
was maintained between the two
NE
windows
if
the subjects-specific
correction
factor
was
used
(6
versus
16
mm,
p
=
0.21),
with an improvement
in
scan
efficiency
(6
versus
16
mm,
49
5
17%
versus
81
&
22% respectively,
p
<
0.001).
Thus,
for
adaptive motion correction
to
be
implemented,
a
subject-
specific correction
factor
should be used
and calculated
from
simple coronal expiratory
and
inspiratory breathholds.
For
real-time NE-gated cardiac
MR
with adaptive motion correction, the
NE
window
can
be
widened to reduce the acquisition period without
loss
of
image quality
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
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