Wip1 confers G2 checkpoint recovery competence by counteracting p53‐dependent transcriptional repression

Activation of the DNA damage checkpoint causes a cell‐cycle arrest through inhibition of cyclin‐dependent kinases (cdks). To successfully recover from the arrest, a cell should somehow be maintained in its proper cell‐cycle phase. This problem is particularly eminent when a cell arrests in G2, as cdk activity is important to establish a G2 state. Here, we identify the phosphatase Wip1 (PPM1D) as a factor that maintains a cell competent for cell‐cycle re‐entry during an ongoing DNA damage response in G2. We show that Wip1 function is required throughout the arrest, and that Wip1 acts by antagonizing p53‐dependent repression of crucial mitotic inducers, such as Cyclin B and Plk1. Our data show that the primary function of Wip1 is to retain cellular competence to divide, rather than to silence the checkpoint to promote recovery. Our findings uncover Wip1 as a first in class recovery competence gene, and suggest that the principal function of Wip1 in cellular transformation is to retain proliferative capacity in the face of oncogene‐induced stress.     

The Structure of the Cytochrome P450cam–Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3 Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16 Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.     

Interactive learning and action: realizing the promise of synthetic biology for global health

The emerging field of synthetic biology has the potential to improve global health. For example, synthetic biology could contribute to efforts at vaccine development in a context in which vaccines and immunization have been identified by the international community as being crucial to international development efforts and, in particular, the millennium development goals. However, past experience with innovations shows that realizing a technology’s potential can be difficult and complex. To achieve better societal embedding of synthetic biology and to make sure it reaches its potential, science and technology development should be made more inclusive and interactive. Responsible research and innovation is based on the premise that a broad range of stakeholders with different views, needs and ideas should have a voice in the technological development and deployment process. The interactive learning and action (ILA) approach has been developed as a methodology to bring societal stakeholders into a science and technology development process. This paper proposes an ILA in five phases for an international effort, with national case studies, to develop socially robust applications of synthetic biology for global health, based on the example of vaccine development. The design is based on results of a recently initiated ILA project on synthetic biology; results from other interactive initiatives described in the literature; and examples of possible applications of synthetic biology for global health that are currently being developed.     

Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph-Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.     

Methyl group reorientation under ligand binding probed by pseudocontact shifts

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1–3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.     

Liver Fibrosis in Type I Gaucher Disease: Magnetic Resonance Imaging,Transient Elastography and Parameters of Iron Storage

Long term liver-related complications of type-1 Gaucher disease (GD), a lysosomal storage disorder, include fibrosis and an increased incidence of hepatocellular carcinoma. Splenectomy has been implicated as a risk factor for the development of liver pathology in GD. High ferritin concentrations are a feature of GD and iron storage in Gaucher cells has been described, but iron storage in the liver in relation to liver fibrosis has not been studied. Alternatively, iron storage in GD may be the result of iron supplementation therapy or regular blood transfusions in patients with severe cytopenia. In this pilot study, comprising 14 type-1 GD patients (7 splenectomized, 7 non-splenectomized) and 7 healthy controls, we demonstrate that liver stiffness values, measured by Transient Elastography and MR-Elastography, are significantly higher in splenectomized GD patients when compared with non-splenectomized GD patients (p = 0.03 and p = 0.01, respectively). Liver iron concentration was elevated (>60±30 µmol/g) in 4 GD patients of whom 3 were splenectomized. No relationship was found between liver stiffness and liver iron concentration. HFE gene mutations were more frequent in splenectomized (6/7) than in non-splenectomized (2/7) participants (p = 0.10). Liver disease appeared more advanced in splenectomized than in non-splenectomized patients. We hypothesize a relationship with excessive hepatic iron accumulation in splenectomized patients. We recommend that all splenectomized patients, especially those with evidence of substantial liver fibrosis undergo regular screening for HCC, according to current guidelines.     

Socio-Cultural Determinants of Health-Seeking Behaviour on the Kenyan Coast: A Qualitative Study

Background

Severe childhood illnesses present a major public health challenge for Africa, which is aggravated by a suboptimal response to the child''s health problems with reference to the health-seeking behaviour of the parents or guardians. We examined the health-seeking behaviour of parents at the Kenyan coast because understanding impediments to optimal health-seeking behaviour could greatly contribute to reducing the impact of severe illness on children''s growth and development.

Methods and Results

Health-seeking behaviour, and the factors influencing this behaviour, were examined in two traditional communities. We held in-depth interviews with 53 mothers, fathers and caregivers from two rural clinics at the Kenyan Coast. Biomedical medicine (from health facilities and purchased over the counter) was found to be the most popular first point of treatment. However, traditional healing still plays a salient role in the health care within these two communities. Traditional healers were consulted for various reasons: a) attribution of causation of ill-health to supernatural sources, b) chronic illness (inability of modern medicine to cure the problem) and c) as prevention against possible ill-health. In developing an explanatory model of decision-making, we observed that this was a complex process involving consultation at various levels, with elders, but also between both parents, depending on the perceived nature and chronicity of the illness. However, it was reported that fathers were the ultimate decision makers in relation to decisions concerning where the child would be taken for treatment.

Conclusions

Health systems need to see traditional healing as a complementary system in order to ensure adequate access to health care. Importantly, fathers also need to be addressed in intervention and education programs.     

Immobilization of thermostable β-galactosidase on epoxy support and its use for lactose hydrolysis and galactooligosaccharides biosynthesis

Thermoresistant, recombinant β-galactosidase from Thermotoga maritima was purified and immobilized on the surface of epoxy-coated magnetic beads. The enzyme, which has hexameric quaternary structure as shown by gel filtration chromatography, attaches to the resin through multiple covalent linkages that involve different subunits. The bound enzyme shows higher stability than the free form. The immobilized enzyme showed to be efficient for the hydrolysis of lactose and the biosynthesis of galactooligosaccharides (GOS). The chemical structure of synthesized GOS has been determined by NMR revealing that the main product was β-3′-galactosyl lactose. Although β-galactosidases from different sources have been used for the same purposes, the distinct advantage of the methodology described in this communication is that the enzyme can be easily produced, purified and immobilized in large quantities.     

Performance of Dutch Children on the Bayley III: A Comparison Study of US and Dutch Norms

Background

The Bayley Scales of Infant and Toddler Development-third edition (Bayley-III) are frequently used to assess early child development worldwide. However, the original standardization only included US children, and it is still unclear whether or not these norms are adequate for use in other populations. Recently, norms for the Dutch version of the Bayley-III (The Bayley-III-NL) were made. Scores based on Dutch and US norms were compared to study the need for population-specific norms.

Methods

Scaled scores based on Dutch and US norms were compared for 1912 children between 14 days and 42 months 14 days. Next, the proportions of children scoring < 1-SD and < -2 SD based on the two norms were compared, to identify over- or under-referral for developmental delay resulting from non-population-based norms.

Results

Scaled scores based on Dutch norms fluctuated around values based on US norms on all subtests. The extent of the deviations differed across ages and subtests. Differences in means were significant across all five subtests (p < .01) with small to large effect sizes (η _p ²) ranging from .03 to .26). Using the US instead of Dutch norms resulted in over-referral regarding gross motor skills, and under-referral regarding cognitive, receptive communication, expressive communication, and fine motor skills.

Conclusions

The Dutch norms differ from the US norms for all subtests and these differences are clinically relevant. Population specific norms are needed to identify children with low scores for referral and intervention, and to facilitate international comparisons of population data.