A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.     

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.     

Impact of QTL properties on the accuracy of multi-breed genomic prediction

Background

Although simulation studies show that combining multiple breeds in one reference population increases accuracy of genomic prediction, this is not always confirmed in empirical studies. This discrepancy might be due to the assumptions on quantitative trait loci (QTL) properties applied in simulation studies, including number of QTL, spectrum of QTL allele frequencies across breeds, and distribution of allele substitution effects. We investigated the effects of QTL properties and of including a random across- and within-breed animal effect in a genomic best linear unbiased prediction (GBLUP) model on accuracy of multi-breed genomic prediction using genotypes of Holstein-Friesian and Jersey cows.

Methods

Genotypes of three classes of variants obtained from whole-genome sequence data, with moderately low, very low or extremely low average minor allele frequencies (MAF), were imputed in 3000 Holstein-Friesian and 3000 Jersey cows that had real high-density genotypes. Phenotypes of traits controlled by QTL with different properties were simulated by sampling 100 or 1000 QTL from one class of variants and their allele substitution effects either randomly from a gamma distribution, or computed such that each QTL explained the same variance, i.e. rare alleles had a large effect. Genomic breeding values for 1000 selection candidates per breed were estimated using GBLUP modelsincluding a random across- and a within-breed animal effect.

Results

For all three classes of QTL allele frequency spectra, accuracies of genomic prediction were not affected by the addition of 2000 individuals of the other breed to a reference population of the same breed as the selection candidates. Accuracies of both single- and multi-breed genomic prediction decreased as MAF of QTL decreased, especially when rare alleles had a large effect. Accuracies of genomic prediction were similar for the models with and without a random within-breed animal effect, probably because of insufficient power to separate across- and within-breed animal effects.

Conclusions

Accuracy of both single- and multi-breed genomic prediction depends on the properties of the QTL that underlie the trait. As QTL MAF decreased, accuracy decreased, especially when rare alleles had a large effect. This demonstrates that QTL properties are key parameters that determine the accuracy of genomic prediction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0124-6) contains supplementary material, which is available to authorized users.     

Bicuspid stenotic aortic valves: clinical characteristics and morphological assessment using MRI and echocardiography

Background

Bicuspid aortic valve (BAV) is one of the most common congenital heart defects with a population prevalence of 0.5% to 1.3%. Identifying patients with BAV is clinically relevant because BAV is associated with aortic stenosis, endocarditis and ascending aorta pathology.

Methods and Results

Patients with severe aortic stenosis necessitating aortic valve replacement surgery were included in this study. All dissected aortic valves were stored in the biobank of the University Medical Centre Utrecht. Additionally to the morphological assessment of the aortic valve by the surgeon and pathologist, echocardiographic and magnetic resonance imaging (MRI) images were evaluated. A total of 80 patients were included of whom 32 (40%) were diagnosed with BAV by the surgeon (gold standard). Patients with BAV were significantly younger (55 vs 71 years) and were more frequently male. Notably, a significant difference was found between the surgeon and pathologist in determining valve morphology. MRI was performed in 33% of patients. MRI could assess valve morphology in 96% vs 73% with echocardiography. The sensitivity of MRI for BAV in a population of patients with severe aortic stenosis was higher than echocardiography (75% vs 55%), whereas specificity was better with the latter (91% vs 79%). Typically, the ascending aorta was larger in patients with BAV.

Conclusion

Among unselected patients with severe aortic valve stenosis, a high percentage of patients with BAV were found. Imaging and assessment of the aortic valve morphology when stenotic is challenging.     

Charge modification of the endothelial surface layer modulates the permeability barrier of isolated rat mesenteric small arteries

We hypothesized that modulation of the effective charge density of the endothelial surface layer (ESL) results in altered arterial barrier properties to transport of anionic solutes. Rat mesenteric small arteries (diameter approximately 190 microm) were isolated, cannulated, perfused, and superfused with MOPS-buffered physiological salt solutions. MOPS-solutions were of normal ionic strength (162 mM, MOPS), low ionic strength (81 mM, LO-MOPS), or high ionic strength (323 mM, HI-MOPS), to modulate ESL charge density (normal, high, or low ESL charge, respectively). Osmolarity of MOPS, LO-MOPS, and HI-MOPS was kept constant at 297 mosmol/l, using additional glucose when necessary. Perfusate solutions were supplemented with 1% BSA. Arteries were cannulated with a double-barreled theta-pipet on the inlet side and a regular pipet on the outlet side. After infusion of FITC-labeled dextran of 50 kDa (FITC-Delta50) and the endothelial membrane dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, the dynamics of arterial dye filling were determined with confocal microscopy. ESL thickness, as determined from the initial exclusion zone for FITC-Delta50 on the luminal endothelial surface, was 6.3 +/- 1.4 microm for LO-MOPS, 2.7 +/- 1.0 microm for MOPS, and 1.1 +/- 1.3 microm for HI-MOPS. At low ionic strength, FITC-Delta50 permeated into the ESL with a total ESL permeation time (tauESL) of 26 min, and at normal ionic strength with a tauESL of 20 min. No apparent exclusion of FITC-Delta50 from the ESL could be observed at high ionic strength. In conclusion, we demonstrate that the modulation of solvent ionic strength influences the thickness and barrier properties of the ESL.     

Rapid production of recombinant human IgG With improved ADCC effector function in a transient expression system

Rapid production of recombinant human IgG with improved antibody dependent cell‐mediated cytotoxicity (ADCC) effector function is presented. The technique employs transient expression of IgG in suspension growing HEK‐293F cells in the presence of the glycosidase inhibitor kifunensine. The procedure takes ～7 days, provided that expression plasmids encoding the IgG of interest are available. Kifunensine inhibits the N‐linked glycosylation pathway of HEK‐293F cells in the endoplasmatic reticulum, resulting in IgG with oligomannose type glycans lacking core‐fucose. IgG1 transiently produced in kifunensine‐ treated HEK‐293F cells has improved affinity for the FcγRIIIA molecule as measured in an ELISA based assay, and almost eightfold enhanced ADCC using primary peripheral blood mononuclear effector cells. Biotechnol. Bioeng. 2010; 105: 350–357. © 2009 Wiley Periodicals, Inc.     

The essential fatty acid status in phenylketonuria patients under treatment

Phenylketonuric patients are on a special diet that lacks certain essential fatty acids. This study evaluates the essential fatty acid status of a group of phenylketonuric patients in the Netherlands undergoing dietary treatment. To this end, the essential fatty acid status of nine phenylketonuria patients was studied. On the basis of age and gender, two control subjects were selected for each patient. The essential fatty acid composition of duplicate food portions and the essential fatty acid status of plasma and erythrocytes were analyzed. Phenylketonuria subjects had a different essential fatty acid profile from their peers, especially concerning the n-3 fatty acids. N-6 and n-3 fatty long-chain polyenes were hardly consumed by phenylketonuria subjects, in contrast to the control subjects. Linoleic acid, on the other hand, was consumed in significantly higher amounts by phenylketonuria subjects and made up about 40% of their daily fat consumption. The essential fatty acid consumption pattern of the phenylketonuria subjects is mirrored by the essential fatty acid concentrations in blood. The essential fatty acid status of the phenylketonuric diet should be improved in order to prevent deficiency in n-3 fatty acids.     

sAPPα rescues deficits in amyloid precursor protein knockout mice following focal traumatic brain injury

The amyloid precursor protein (APP) is thought to be neuroprotective following traumatic brain injury (TBI), although definitive evidence at moderate to severe levels of injury is lacking. In the current study, we investigated histological and functional outcomes in APP-/- mice compared with APP+/+ mice following a moderate focal injury, and whether administration of sAPPα restored the outcomes in knockout animals back to the wildtype state. Following moderate controlled cortical impact injury, APP-/- mice demonstrated greater impairment in motor and cognitive outcome as determined by the ledged beam and Barnes Maze tests respectively (p < 0.05). This corresponded with the degree of neuronal damage, with APP-/- mice having significantly greater lesion volume (25.0 ± 1.6 vs. 20.3 ± 1.6%, p < 0.01) and hippocampal damage, with less remaining CA neurons (839 ± 245 vs. 1353 ± 142 and 1401 ± 263). This was also associated with an impaired neuroreparative response, with decreased GAP-43 immunoreactivity within the cortex around the lesion edge compared with APP+/+ mice. The deficits observed in the APP-/- mice related to a lack of sAPPα, as treatment with exogenously added sAPPα post-injury improved APP-/- mice histological and functional outcome to the point that they were no longer significantly different to APP+/+ mice (p < 0.05). This study shows that endogenous APP is potentially protective at moderate levels of TBI, and that this neuroprotective activity is related to the presence of sAPPα. Importantly, it indicates that the mechanism of action of exogenously added sAPPα is independent of the presence of endogenous APP.     

Anthrax toxin receptor 2 functions in ECM homeostasis of the murine reproductive tract and promotes MMP activity

Anthrax Toxin Receptor proteins function as receptors for anthrax toxin, however physiological activity remains unclear. To evaluate the biological role of Antxr2, we generated Antxr2-/- mice. Antxr2-/- mice were viable, however Antxr2 is required for parturition in young females and for preserving fertility in older female mice. Histological analysis of the uterus and cervix revealed aberrant deposition of extracellular matrix proteins such as type I collagen, type VI collagen and fibronectin. A marked disruption of both the circular and longitudinal myometrial cell layers was evident in Antxr2-/- mice. These changes progressed as the mice aged, resulting in a thickened, collagen dense, acellular stroma and the disappearance of normal uterine architecture. To investigate the molecular mechanism underlying the uterine fibrosis we performed immunoblotting for MMP2 using uterine lysates and zymography using conditioned medium from Antxr2-/- mouse embryonic fibroblasts and found reduced levels of activated MMP2 in both. This prompted us to investigate MT1-MMP status, as MMP2 processing is regulated by MT1-MMP. We found MT1-MMP activity, as measured by MMP2 processing and activation, was enhanced by expression of either ANTXR1 or ANTXR2. We identified an ANTXR2/MT1-MMP complex and demonstrated that MT1-MMP activity is dependent on ANTXR2 expression levels in cells. Thus, we have discovered that ANTXR1 and ANTXR2 function as positive regulators of MT1-MMP activity.