Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.     

Impact of the Method of G6PD Deficiency Assessment on Genetic Association Studies of Malaria Susceptibility

Background

Clinical association studies have yielded varied results regarding the impact of glucose-6-phosphate dehydrogenase (G6PD) deficiency upon susceptibility to malaria. Analyses have been complicated by varied methods used to diagnose G6PD deficiency.

Methodology/Prinicipal Findings

We compared the association between uncomplicated malaria incidence and G6PD deficiency in a cohort of 601 Ugandan children using two different diagnostic methods, enzyme activity and G6PD genotype (G202A, the predominant East African allele). Although roughly the same percentage of males were identified as deficient using enzyme activity (12%) and genotype (14%), nearly 30% of males who were enzymatically deficient were wild-type at G202A. The number of deficient females was three-fold higher with assessment by genotype (21%) compared to enzyme activity (7%). Heterozygous females accounted for the majority (46/54) of children with a mutant genotype but normal enzyme activity. G6PD deficiency, as determined by G6PD enzyme activity, conferred a 52% (relative risk [RR] 0.48, 95% CI 0.31–0.75) reduced risk of uncomplicated malaria in females. In contrast, when G6PD deficiency was defined based on genotype, the protective association for females was no longer seen (RR = 0.99, 95% CI 0.70–1.39). Notably, restricting the analysis to those females who were both genotypically and enzymatically deficient, the association of deficiency and protection from uncomplicated malaria was again demonstrated in females, but not in males (RR = 0.57, 95% CI 0.37–0.88 for females).

Conclusions/Significance

This study underscores the impact that the method of identifying G6PD deficient individuals has upon association studies of G6PD deficiency and uncomplicated malaria. We found that G6PD-deficient females were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency is described using enzyme activity. These observations may help to explain the discrepancy in some published association studies involving G6PD deficiency and uncomplicated malaria.     

Pooling for improved screening of combinatorial libraries for directed evolution

Following diversity generation in combinatorial protein engineering, a significant amount of effort is expended in screening the library for improved variants. Pooling, or combining multiple cells into the same assay well when screening, is a means to increase throughput and screen a larger portion of the library with less time and effort. We have developed and validated a Monte Carlo simulation model of pooling and used it to screen a library of beta-galactosidase mutants randomized in the active site to increase their activity toward fucosides. Here, we show that our model can successfully predict the number of highly improved mutants obtained via pooling and that pooling does increase the number of good mutants obtained. In unpooled conditions, we found a total of three mutants with higher activity toward p-nitrophenyl-beta-D-fucoside than that of the wild-type beta-galactosidase, whereas when pooling 10 cells per well we found a total of approximately 10 improved mutants. In addition, the number of "supermutants", those with the highest activity increase, was also higher when pooling was used. Pooling is a useful tool for increasing the efficiency of screening combinatorial protein engineering libraries.     

Design and characterization of a modified T‐flask bioreactor for continuous monitoring of engineered tissue stiffness

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force‐displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T‐flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T‐flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R² > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF‐β, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Ultrasonographic soft markers of aneuploidy in second trimester: are we lost?

Chromosomal abnormalities occur in 0.1% to 0.2% of live births, and the most common clinically significant aneuploidy among live-born infants is Down syndrome (trisomy 21). Other sonographically detectable aneuploidies include trisomy 13, 18, monosomy X, and triploidy. Second-trimester ultrasound scan detects 2 types of sonographic markers suggestive of aneuploidy. Markers for major fetal structural abnormalities comprise the first type; the second type of markers are known as "soft markers" of aneuploidy. These latter markers are nonspecific, often transient, and can be readily detected during the second-trimester ultrasound. The most commonly studied soft markers of aneuploidy include a thickened nuchal fold, rhizomelic limb shortening, mild fetal pyelectasis, echogenic bowel, and echogenic intracardiac focus and choroid plexus cyst. There is a great deal of interest in the ultrasound detection of aneuploidy, as evidenced by the large number of publications in the literature on this topic. Unfortunately, studies evaluating the significance of the soft markers of aneuploidy vary widely and show contradictory results. In this article, we review the most common ultrasonographic soft markers used to screen aneuploidy and discuss ultrasonographic technique and measurement criteria for the detection of soft markers. We also review the clinical relevance of soft markers to aneuploidy risk assessment and evidence-based strategies for the management of affected pregnancies with each of these markers in light of current literature.     

The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax,modulate functional interactions with the anti-apoptotic protein Bcl-x<Subscript>L</Subscript>

Background  

Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-x_L.