Mutational analysis of the aggregation-prone and disaggregation-prone regions of acylphosphatase

We have performed an extensive mutational analysis of aggregation and disaggregation of amyloid-like protofibrils of human muscle acylphosphatase. Our findings indicate that the regions that promote aggregation in 25% (v/v) 2,2,2 trifluoroethanol (TFE) are different from those that promote disaggregation under milder conditions (5% TFE). Significant changes in the rate of disaggregation of protofibrils in 5% TFE result not only from mutations situated in the regions of the sequence that play a key role in the mechanism of aggregation in 25% TFE, but also from mutations located in other regions. In order to rationalise these results, we have used a modified version of the Zyggregator aggregation propensity prediction algorithm to take into account structural rearrangements of the protofibrils that may be induced by changes in solution conditions. Our results suggest that a wider range of residues contributes to the stability of the aggregates in addition to those that play an important kinetic role in the aggregation process. The mutational approach described here is capable of providing residue-specific information on the structure and dynamics of amyloid protofibrils under conditions close to physiological and should be widely applicable to other systems.     

Counteracting Effects Operating on Src Homology 2 Domain-containing Protein-tyrosine Phosphatase 2 (SHP2) Function Drive Selection of the Recurrent Y62D and Y63C Substitutions in Noonan Syndrome

Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2.     

SIMPLE: implementation of recommendations from international evidence-based guidelines on caesarean sections in the Netherlands. Protocol for a controlled before and after study

Background

Caesarean section (CS) rates are rising worldwide. In the Netherlands, the most significant rise is observed in healthy women with a singleton in vertex position between 37 and 42 weeks gestation, whereas it is doubtful whether an improved outcome for the mother or her child was obtained. It can be hypothesized that evidence-based guidelines on CS are not implemented sufficiently. Therefore, the present study has the following objectives: to develop quality indicators on the decision to perform a CS based on key recommendations from national and international guidelines; to use the quality indicators in order to gain insight into actual adherence of Dutch gynaecologists to guideline recommendations on the performance of a CS; to explore barriers and facilitators that have a direct effect on guideline application regarding CS; and to develop, execute, and evaluate a strategy in order to reduce the CS incidence for a similar neonatal outcome (based on the information gathered in the second and third objectives).

Methods

An independent expert panel of Dutch gynaecologists and midwives will develop a set of quality indicators on the decision to perform a CS. These indicators will be used to measure current care in 20 hospitals with a population of 1,000 women who delivered by CS, and a random selection of 1,000 women who delivered vaginally in the same period. Furthermore, by interviewing healthcare professionals and patients, the barriers and facilitators that may influence the decision to perform a CS will be measured. Based on the results, a tailor-made implementation strategy will be developed and tested in a controlled before-and-after study in 12 hospitals (six intervention, six control hospitals) with regard to effectiveness, experiences, and costs.

Discussion

This study will offer insight into the current CS care and into the hindering and facilitating factors influencing obstetrical policy on CS. Furthermore, it will allow definition of patient categories or situations in which a tailor-made implementation strategy will most likely be meaningful and cost effective, without negatively affecting the outcome for mother and child.

Trial registration

http://www.clinicaltrials.gov: NCT01261676     

Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIVmac251 at Challenge Exposure

We used the simian immunodeficiency virus mac251 (SIV_mac251) macaque model to study the effect of the dose of mucosal exposure on vaccine efficacy. We immunized macaques with a DNA prime followed by SIV gp120 protein immunization with ALVAC-SIV and gp120 in alum, and we challenged them with SIV_mac251 at either a single high dose or at two repeated low-dose exposures to a 10-fold-lower dose. Infection was neither prevented nor modified following a single high-dose challenge of the immunized macaques. However, two exposures to a 10-fold-lower dose resulted in protection from SIV_mac251 acquisition in 3 out of 12 macaques. The remaining animals that were infected had a modulated pathogenesis, significant downregulation of interferon responsive genes, and upregulation of genes involved in B- and T-cell responses. Thus, the choice of the experimental model greatly influences the vaccine efficacy of vaccines for human immunodeficiency virus (HIV).     

Higher number of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> CagA EPIYA C phosphorylation sites increases the risk of gastric cancer,but not duodenal ulcer

Background  

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric cancer and peptic ulcer. Bacterial virulence factors such as CagA have been shown to increase the risk of both diseases. Studies have suggested a causal role for CagA EPIYA polymorphisms in gastric carcinogenesis, and it has been shown to be geographically diverse. We studied associations between H. pylori CagA EPIYA patterns and gastric cancer and duodenal ulcer, in an ethnically admixed Western population from Brazil. CagA EPIYA was determined by PCR and confirmed by sequencing. A total of 436 patients were included, being 188 with gastric cancer, 112 with duodenal ulcer and 136 with gastritis.     

Macromolecular composition of phloem exudate from white lupin (Lupinus albus L.)

Background  

Members of the legume genus Lupinus exude phloem 'spontaneously' from incisions made to the vasculature. This feature was exploited to document macromolecules present in exudate of white lupin (Lupinus albus [L.] cv Kiev mutant), in particular to identify proteins and RNA molecules, including microRNA (miRNA).