Specificity and Kinetics of Haloalkane Dehalogenase

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols. The active site is buried inside the protein and lined with hydrophobic residues. The reaction proceeds via a covalent substrate-enzyme complex. This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase. The kinetic mechanism for the “natural” substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given. In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs. The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds. However, the rate-determining step in the conversion (kcat) of 1,2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds. The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates.

Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state kcat with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Clα) from 1,2-dichloroethane interacts with Trp125 and the Clβ interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in kcat could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The kcat for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Clα or Brα) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.

Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the Clβ of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher kcat/Km for 1-chlorohexane and a 2-fold higher kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Blood group probabilities by next of kin

For rare blood groups the recruitment of donor relatives, for example siblings, is expected to be effective, since the probability of a similar rare blood group is likely. However, the likelihood differs between blood groups and is not commonly available. This paper provides a unified mathematical formulation to calculate such likelihoods. From a mathematical and probabilistic point of view, it is shown that these likelihoods can be obtained from the computation of a stationary genotype distribution. This, in turn, can be brought down to a system of quadratic stochastic operators. A generic mathematical approach is presented which directly leads to a stationary genotype distribution for arbitrary blood groups. The approach enables an exact computation for the effectiveness of recruiting next of kin for blood donorship. Next to an illustration of computations for ‘standard’ ABO and Rhesus-D blood groups, it is particularly illustrated for the extended Rhesus blood group system. Also other applications requiring next of kin blood group associations can be solved directly by using the unified mathematical formulation

Specificity and kinetics of haloalkane dehalogenase

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols, The active site is buried inside the protein and lined with hydrophobic residues, The reaction proceeds via a covalent substrate-enzyme complex, This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase, The kinetic mechanism for the ''natural'' substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given, In general, brominated substrates had a lower K-m, but a similar k(cat) than the chlorinated analogs, The rate of C-Br bond cleavage was higher than the rate of C-CL bond cleavage, which is in agreement with the leaving group abilities of these halogens, The lower K-m for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower K-m for bromo-compounds, However, the rate-determining step in the conversion (k(cat)) of 1,2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different K-m but similar k(cat) values for these compounds, The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates

The MICADO Atmospheric Dispersion Corrector:Optomechanical design, expected performance and calibration techniques

The differential refraction of light passing through the atmosphere can have a severe impact on image quality if no atmospheric dispersion corrector (ADC) is used. For the Extremely Large Telescope (ELT) this holds true well into the infrared. MICADO, the near-infrared imaging camera for the ELT, will employ a cryogenic ADC consisting of two counter-rotating Amici prisms with diameters of 125 mm. The mechanism will reduce the atmospheric dispersion to below 2.5 milli arcseconds (mas), with a set goal of 1 mas. In this report, we provide an overview of the current status of the ADC in development for MICADO. We summarise the optomechanical design and discuss how the cryogenic environment impacts the performance. We will also discuss our plan to use a diffraction mask in the cold pupil to calibrate and validate the performance once the instrument is fully integrated

Use of IBASPM atlas-based automatic segmentation toolbox in pathological brains: effect of template selection

Proceeding of: 2008 IEEE Nuclear Science Symposium Conference Record (NSS '08), Dresden, Germany, 19-25 Oct. 2008IBASPM software is an atlas-based method for automatic segmentation of brain structures, available as a freeware toolbox for the SPM package. To test the influence of the atlas when segmenting normal and pathologic brains, manual segmentation of the caudate nucleus head was compared to automatic segmentations using four different atlases: the default MNI AAL atlas; a customized atlas created from a combined sample of patients (n=20) and controls (n=18); and a customized atlas obtained separately for each group. Maximum average ratio of overlapping voxels (dice overlap) between manual and automatic segmentation was 71 o~ for controls and 52% for patients. In both groups, overlap ratios were better when using the customized atlases, instead of the standard MNI AAL atlas. Accuracy of the method was biased between left and right hemispheres, and also between groups, individual variability being higher in patients than in controls. Volumetric measurements using the customized atlases were also more accurate than using the MNI AAL atlas. Volume data were closer to manual segmentation values than dice overlap ratio (average differences ranging from 22.7°~ for MNI AAL atlas to 10.1 for customized atlas of patients and controls combined). Results suggests a low overaU performance of IBASPM as an automatic segmentation method for the head of the caudate nucleus. Because of the biases observed, the use of this method for analyzing caudate nucleus in patients presenting anatomical abnormalities should be cautiously carried out.This work is partially funded by the following projects: CD-TEAM Project, CENIT Program (Spanish Ministerio de Industria)~ FIS PI052271 (Spanish Ministerio de Sanidad y Consumo)~ and Fundación Mutua Madrilen

Surgical treatment for non-parasitic liver cysts improves quality of life

BACKGROUND&PURPOSE: Liver cysts occur frequently. Most are harmless, however some carry a significant patient burden. Optimizing treatment strategy is complicated as needs differ between patients. The current study assesses the effect of surgery on quality of life (QoL) of patients with non-parasitic liver cysts. METHODS: A retrospective cohort study of all patients who underwent surgery for non-parasitic liver cysts in three major Dutch medical centers from 1993 to 2017. Patient characteristics and surgery related variables were collected from the electronic patient file. QoL was measured before and after surgery using the EORTC QLQ-C30. Summary scores (SumSc) were calculated and compared to reference values of the general population. Multivariate analysis using logistic regression was performed for identifying outcome related factors. Increase of ≥ 10% in SumSc was defined as clinically relevant. MAIN FINDINGS: Eighty-eight of 132 eligible patients (67%) completed two QoL assessments. Respondents demonstrated significant improvement in the global health status, on all 5 functional scales (all p ≤ 0.005), on all 9 symptom scales after surgery (all p < 0.05), and on SumSc (p < 0.001) to levels similar or better than the general population. Patients with complications demonstrated a significant QoL gain (p < 0.05), and reported a similar postoperative status compared to patients without complications (p = 0.74). QoL gain for patients who underwent open and laparoscopic cyst fenestration were similar (p = 0.08). Multivariate analysis of SumSc found mechanical complaints as significant factor for ≥ 10% SumSc increase (OR 0.11, 95% CI (0.02-0.55). CONCLUSIONS: Surgery is a safe and effective strategy to significantly improve QoL in patients with symptomatic liver cysts

ABLE: Automated Brain Lines Extraction Based on Laplacian Surface Collapse.

The archetypical folded shape of the human cortex has been a long-standing topic for neuroscientific research. Nevertheless, the accurate neuroanatomical segmentation of sulci remains a challenge. Part of the problem is the uncertainty of where a sulcus transitions into a gyrus and vice versa. This problem can be avoided by focusing on sulcal fundi and gyral crowns, which represent the topological opposites of cortical folding. We present Automated Brain Lines Extraction (ABLE), a method based on Laplacian surface collapse to reliably segment sulcal fundi and gyral crown lines. ABLE is built to work on standard FreeSurfer outputs and eludes the delineation of anastomotic sulci while maintaining sulcal fundi lines that traverse the regions with the highest depth and curvature. First, it segments the cortex into gyral and sulcal surfaces; then, each surface is spatially filtered. A Laplacian-collapse-based algorithm is applied to obtain a thinned representation of the surfaces. This surface is then used for careful detection of the endpoints of the lines. Finally, sulcal fundi and gyral crown lines are obtained by eroding the surfaces while preserving the connectivity between the endpoints. The method is validated by comparing ABLE with three other sulcal extraction methods using the Human Connectome Project (HCP) test-retest database to assess the reproducibility of the different tools. The results confirm ABLE as a reliable method for obtaining sulcal lines with an accurate representation of the sulcal topology while ignoring anastomotic branches and the overestimation of the sulcal fundi lines. ABLE is publicly available via https://github.com/HGGM-LIM/ABLE .This work was supported by the project exAScale ProgramIng models for extreme Data procEssing (ASPIDE), that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 801091. This work has received funding from “la Caixa” Foundation under the project code LCF/PR/HR19/52160001. Susanna Carmona funded by Instituto de Salud Carlos III, co-funded by European Social Fund “Investing in your future” (Miguel Servet Type I research contract CP16/00096). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Yasser Alemán-Gómez is supported by the Swiss National Science Foundation (185897) and the National Center of Competence in Research (NCCR) SYNAPSY - The Synaptic Bases of Mental Diseases, funded as well by the Swiss National Science Foundation (51AU40-1257).S