<i>In vitro</i> activity of MurT and GatD.

<p>(A) Purified recombinant GatD and MurT enzymes were incubated with purified lipid II. In the negative control (lane 1) the reaction was immediately stopped by the addition of BuOH/Pyr/Ac. <b>Time dependency of the GatD/MurT catalyzed reaction.</b> (B) Purified [¹⁴C]-lipid II was incubated in the presence of 2 µg MurT and GatD each, glutamine and ATP. The reaction was stopped by the addition of BuOH/PyrAc and the amount of amidated lipid II was quantified using phosphoimaging; mean values of three independent experiments are given.</p

Amidation of the cell wall precursor lipid II <i>in vitro</i>.

<p>(A) Amidation is catalyzed by the cooperative action of GatD and MurT (lane 1) and MurT cannot be replaced by MurE ligase (lane 2) or any other Mur-ligase (not shown). <b>Interdependency of GatD/MurT.</b> (B) Increasing concentrations of purified GatD (dots) or MurT (squares) proteins (0–2 µg) were incubated in the presence of glutamine, ATP and [¹⁴C]-lipid II in the presence of a fixed concentration of 2 µg MurT and GatD, respectively. A maximum conversion to amidated lipid II was observed only at equimolar ratio, considering the molecular masses of 49.2 kD (MurT) and 29.7 kD (GatD).</p

Perihematomal diffusion restriction as a common finding in large intracerebral hemorrhages in the hyperacute phase

<div><p>Purpose</p><p>There is growing evidence that a perihematomal area of restricted diffusion (PDR) exists in intraparenchymal hemorrhages (IPH) within 1 week of symptom onset (SO). Here, we study characteristics and the clinical impact of the PDR in patients with hyperacute (≤ 6 hours from SO) IPH by means of apparent diffusion coefficient (ADC).</p><p>Methods</p><p>This monocentric, retrospective study includes 83 patients with first-ever primary IPH from 09/2002-10/2015. 3D volumetric segmentation was performed for the IPH, PDR, and perihematomal edema (PHE) on fluid-attenuated inversion recovery, T2*/susceptibility weighted images, and ADC images.</p><p>Results</p><p>A PDR was seen in 56/83 patients (67.5%) presenting with hyperacute IPH. Multivariate logistic regression analysis revealed every 10-year increase of age (HR 1.929, 95% CI 1.047–3.552, <i>P</i> = .035) and male gender (HR 5.672, 95% CI 1.038–30.992, <i>P</i> = .045) as significant predictors of the presence of a PDR, but not IPH size, IPH location, nor National Institutes of Health Stroke Scale Score (NIHSS) at admission. We found no difference in NIHSS at discharge, hematoma removal, or mortality rate in PDR-positive patients. ADC values of the PDR show a step-wise normalization with increasing time from SO.</p><p>Conclusions</p><p>Occurrence of a PDR is a common finding in supratentorial hyperacute IPH, but shows no adverse short-term clinical impact. It may represent transient oligemic and metabolic changes.</p></div

Model for the GatD/MurT-catalyzed amidation during cell wall biosynthesis in <i>S. aureus</i>.

<p>The GatD/MurT bi-enzyme complex uses glutamine as the primary nitrogen donor and ammonia is shuttled from the GatD glutaminase active site to the MurT synthetase active-site. MurT finally catalyzes the amidation in an ATP-dependent fashion to the acceptor substrate which could be lipid II or, as depicted here, lipid II-Gly₅.</p

Multivariate regression analysis with gender, age, IPH location, time from symptom onset to MRI, NIHSS at admission, and log IPH volume as coefficients.

<p>Multivariate regression analysis with gender, age, IPH location, time from symptom onset to MRI, NIHSS at admission, and log IPH volume as coefficients.</p

Sequence parameters of FLAIR, DWI, and T2*/SWI of the different scanners.

<p>Sequence parameters of FLAIR, DWI, and T2*/SWI of the different scanners.</p

Mass spectrometry of non-amidated and amidated Lipid II.

<p>ESI-MS spectra were obtained with a micrOTOF-Q instrument running in negative mode. Peaks at m/z 936.52 correspond to lipid II (A) and at m/z 936.02 to amidated lipid II (B) for the doubly charged molecules, corresponding to a neutral mass of 1875 and 1874, respectively.</p

Iterative Reconstruction Improves Both Objective and Subjective Image Quality in Acute Stroke CTP

<div><p>Purpose</p><p>Computed tomography perfusion (CTP) imaging in acute ischemic stroke (AIS) suffers from measurement errors due to image noise. The purpose of this study was to investigate if iterative reconstruction (IR) algorithms can be used to improve the diagnostic value of standard-dose CTP in AIS.</p><p>Methods</p><p>Twenty-three patients with AIS underwent CTP with standardized protocol and dose. Raw data were reconstructed with filtered back projection (FBP) and IR with intensity levels 3, 4, 5. Image quality was objectively (quantitative perfusion values, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR)) and subjectively (overall image quality) assessed. Ischemic core and perfusion mismatch were visually rated. Discriminative power for tissue outcome prediction was determined by the area under the receiver operating characteristic curve (AUC) resulting from the overlap between follow-up infarct lesions and stepwise thresholded CTP maps.</p><p>Results</p><p>With increasing levels of IR, objective image quality (SNR and CNR in white matter and gray matter, elimination of error voxels) and subjective image quality improved. Using IR, mean transit time (MTT) was higher in ischemic lesions, while there was no significant change of cerebral blood volume (CBV) and cerebral blood flow (CBF). Visual assessments of perfusion mismatch changed in 4 patients, while the ischemic core remained constant in all cases. Discriminative power for infarct prediction as represented by AUC was not significantly changed in CBV, but increased in CBF and MTT (mean (95% CI)): 0.72 (0.67–0.76) vs. 0.74 (0.70–0.78) and 0.65 (0.62–0.67) vs 0.67 (0.64–0.70).</p><p>Conclusion</p><p>In acute stroke patients, IR improves objective and subjective image quality when applied to standard-dose CTP. This adds to the overall confidence of CTP in acute stroke triage.</p></div

Acceptor substrate specificity of the GatD/MurT catalyzed reaction.

<p>(A) Radiolabeled lipid I, lipid II and lipid II-Gly₅ were incubated together with glutamine and ATP in the presence (gray bars) and absence (white bars) of GatD/MurT. The addition of a 5- and 10-fold molar excess of UDP-MurNAc-pentapeptide (black bars) only resulted in minor reduction of amidated lipid II synthesized. <b>Amidation is not catalyzed in concert with MurA-F.</b> (B) Purified MurA-F enzymes were incubated in the presence and absence of GatD/MurT enzymes. After incubation the reaction product was added to a MraY-catalyzed lipid I synthesis assay using C₅₅-P. No change in migration behavior of the respective lipid I reaction products was observed.</p

Sequence parameters of FLAIR, DWI, and T2*/SWI of the different scanners.

<p>Sequence parameters of FLAIR, DWI, and T2*/SWI of the different scanners.</p