Asynchronous globin chain synthesis in rabbit reticulocyte lystates induced by hemin-controlled repressor

To define further the role of hemin-controlled repressor (HCR) in globin synthesis, we studied its effect on the synthesis of individual globin chains in a rabbit reticulocyte lysate cell-free system. In the presence of HCR there was a marked globin chain imbalance, resulting in a lowered alpha/beta ratio. These findings in vitro may have relevance to certain clinical heme deficiency states in which a similar globin chain imbalance has been observed.     

Activation of coagulation factor XI, without detectable contact activation in dengue haemorrhagic fever

A prospective cohort study was performed in 50 patients with dengue haemorrhagic fever (DHF) to determine the potential role of the contact activation system and factor XI activation (intrinsic pathway) in the coagulation disorders in DHF. To establish whether TAFI (thrombin-activatable fibrinolysis inhibitor) was involved in the severity of the coagulation disorders, the TAFI antigen and activity levels were also determined. Markers of contact activation (kallikrein--C1-inhibitor complexes), the intrinsic pathway of coagulation (factor XIa--C1-inhibitor complexes) and TAFI were measured and correlated to thrombin generation markers (thrombin--anti-thrombin complexes (TAT), prothrombin fragment 1+2 (F1+2)) and a marker for fibrinolysis [plasmin--alpha 2--anti-plasmin complexes (PAP)]. Activation of the intrinsic pathway of coagulation was clearly demonstrated by elevated levels of factor XIa--C1-inhibitor complexes, without evidence of contact activation, reflected by undetectable kallikrein--C1-inhibitor complexes. Both TAFI antigen and activity levels were decreased in all patients, which may contribute to the severity of bleeding complications in DHF because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis. These findings in a human viral infection model are in accordance with earlier findings in bacterial sepsis.     

Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor (Kd=120 nM,MW=734 Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

Metastasis is a complex multistep process (1, 2) employing, among others, mechanisms governing actin cytoskeleton dynamics involving integrin signaling and actin regulatory proteins (3–5). So far, all approved antimetastatic drugs antagonize integrins (6) or inhibit downstream kinases (7, 8) (SI Appendix, Fig. S1). In the metastatic setting however, these drugs appear to have only limited success (9–13) and 5-y survival is not increasing satisfactorily (14, 15), making new approaches in antimetastatic drug development essential to meet this urgent medical need.The enabled/vasodilator stimulated phosphoprotein protein family (Ena/VASP) acts as a crucial hub in cell migration by linking actin filaments to invadopodia and focal adhesions (16–22). Due to their role in the transformation of benign lesions into invasive and metastatic cancer, Ena/VASP proteins are discussed as part of the invasive signature and as a marker of breast carcinogenesis (23–25). At the advanced tumor stage, the protein family is overexpressed (26–28), which has been shown to increase migration speed in vivo (29) and to potentiate invasiveness (30). Yet, no sufficiently potent probes to interfere with Ena/VASP in vivo have been reported.The three vertebrate Ena/VASP family members, enabled homolog (ENAH), VASP, and Ena-VASP-like (EVL), share a tripartite structural organization in which two Ena/VASP homology domains (EVH1 and EVH2) are separated by a more divergent proline-rich central part. Interactions of the EVH2 domain are involved in the elongation and protection of barbed-end actin filaments from capping proteins and tetramerization (31, 32). EVH1 folds into a structured globular domain that interacts with proteins at focal adhesions (33), the leading edge (34, 35), and invadopodia (36, 37) by recognizing the motif [F/W/L/Y]PxϕP (35, 38) (ϕ hydrophobic, x any; SI Appendix, Fig. S3) in poly-L-proline type II helix (PPII) conformation.In the course of our research into small molecules as potential inhibitors of protein–protein interactions (39) we recently in silico designed and stereo-selectively synthesized scaffolds, coined ProMs, which mimic pairs of prolines in PPII conformation (40). The modular combination of different ProMs thereby allowed us to generate nonpeptidic secondary-structure mimetics that fulfill the steric requirements of the addressed proline-rich motif-recognizing domain (41–47). For the EVH1 domain, our proof-of-concept study yielded a canonically binding, nontoxic, cell-membrane-permeable, 706-Da inhibitor 1 (Fig. 1A) composed of two different ProM scaffolds and 2-chloro-(L)-phenylalanine (2-Cl-Phe) (40). While the synthetic inhibitor 1 represents the compound with the highest reported affinity toward Ena/VASP EVH1 domains, a further improvement was required for in vivo experiments. Here we report successful structure-based optimization of inhibitor 1 based on 22 high-resolution crystal structures of ENAH EVH1 in complex with different inhibitors (SI Appendix, Tables S1–S6), including the well-resolved C-terminal binding epitope TEDEL of ActA from Listeria monocytogenes (48). Newly identified interaction sites adjacent to the C terminus of 1 were addressed by in silico designed and stereo-selectively synthesized modifications of the ProM-1 scaffold (Fig. 1A). While drastically increasing the affinity against a rather flat protein surface we conserved structural simplicity, low molecular weight, nontoxicity, and cell-membrane permeability. Potent compounds against Ena/VASP were shown to also act in vivo, i.e., by inhibiting cancer cell extravasation in zebrafish at only 1 μM, thereby paving the way for future preclinical studies.Open in a separate windowFig. 1.(A) Structure of the first-generation Ena/VASP EVH1 inhibitor 1. All compositions share the N-acetylated 2-chloro-phenylalanine unit (blue) attached to a central ProM-2 scaffold (red). Esterification of the C terminus renders the inhibitors cell-membrane permeable (40). (B) General (modular) architecture of nonpeptidic, conformationally preorganized inhibitors used in this study. Structural variation (pink) was achieved by replacing the C-terminal ProM-1 unit (green) by ProM-9, ProM-13, ProM-12, ProM-15, or ProM-17 (Table 1).     

Evaluation of collateral blood flow by myocardial contrast enhanced echocardiography

Contrast enhanced cross sectional echocardiography is a new method for the real-time evaluation of regional myocardial perfusion. Two patients with a history of anteroseptal myocardial infarction and echocardiographically detected septal dyskinesia were examined by this new method. The first patient had two severe stenoses of the left anterior descending coronary artery and normal echocontrast opacification of the interventricular septum caused by collaterals from the right coronary artery. The second patient had good patency of left anterior descending coronary artery and no septal opacification. Thus contrast enhanced cross sectional echocardiography can be used to assess the importance of collateral blood flow in the myocardium.     

Factor IXa-factor VIIIa-cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo.

We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.     

Agreement on Child Maltreatment Decisions: A Nonrandomized Study on the Effects of Structured Decision-Making

Background

Practitioners investigating cases of suspected child maltreatment often disagree whether a child is subject to or at risk of abuse or neglect in the family and, if so, what to do about such abuse or neglect. Structured decision-making is considered to be a solution to the problem of subjective judgments and decisions.

Objective

This study investigates the effects of ORBA, a method for structured decision-making in Advice and Reporting Centres for Child Abuse and Neglect (ARCCAN), on interrater agreement of judgments and decisions.

Methods

Two groups of ARCCAN practitioners, one trained in using ORBA and one untrained, used a questionnaire to make judgments and decisions on the same case vignettes. Interrater agreement on the judgments was obtained by calculating the percentage of agreement, intra class correlation, and the Kappa coefficient.

Results

Both ORBA trained and untrained practitioners showed little agreement on judgments and decisions, except for the judgment on child maltreatment substantiation, for which trained practitioners showed fair agreement. Agreement among trained and untrained practitioners only differed for some judgments and decisions, and differences were not always in the same direction.

Conclusions

This result indicates no convincing evidence that structured decision-making leads to better agreement on decisions concerning child abuse and neglect. Recommendations for improvements in uniform decision-making and further research are given.     

Interpretation of radiological images: towards a framework of knowledge and skills

The knowledge and skills that are required for radiological image interpretation are not well documented, even though medical imaging is gaining importance. This study aims to develop a comprehensive framework of knowledge and skills, required for two-dimensional and multiplanar image interpretation in radiology. A mixed-method study approach was applied. First, a literature search was performed to identify knowledge and skills that are important for image interpretation. Three databases, PubMed, PsycINFO and Embase, were searched for studies using synonyms of image interpretation skills or visual expertise combined with synonyms of radiology. Empirical or review studies concerning knowledge and skills for medical image interpretation were included and relevant knowledge and skill items were extracted. Second, a preliminary framework was built and discussed with nine selective experts in individual semi-structured interviews. The expert team consisted of four radiologists, one radiology resident, two education scientists, one cognitive psychologist and one neuropsychologist. The framework was optimised based on the experts comments. Finally, the framework was applied to empirical data, derived from verbal protocols of ten clerks interpreting two-dimensional and multiplanar radiological images. In consensus meetings adjustments were made to resolve discrepancies of the framework with the verbal protocol data. We designed a framework with three main components of image interpretation: perception, analysis and synthesis. The literature study provided four knowledge and twelve skill items. As a result of the expert interviews, one skill item was added and formulations of existing items were adjusted. The think-aloud experiment showed that all knowledge items and three of the skill items were applied within all three main components of the image interpretation process. The remaining framework items were apparent only within one of the main components. After combining two knowledge items, we finally identified three knowledge items and thirteen skills, essential for image interpretation by trainees. The framework can serve as a guideline for education and assessment of two- and three-dimensional image interpretation. Further validation of the framework in larger study groups with different levels of expertise is needed.     

Varying sun protection of young children by migrant and Australian‐born mothers

Objective: To compare sun protection by Australian‐born and migrant mothers of three‐year‐old children. Methods: Australian‐born and migrant mothers taking part in the Environments for Healthy Living prospective birth‐cohort study were asked standard questions about their child's sun protection. Children were given a skin cancer susceptibility score based on grandparents' ethnic origin. Logistic regression was used to estimate odds ratios (ORs) to measure the association of sun protection of children according to mothers' migrant status adjusted for socio‐demographic characteristics. Results: A total of 613 Australian‐born and 224 migrant mothers of three‐year‐old children were studied. Mothers who had migrated less than four years ago were more likely to allow their three‐year‐old to spend more than two hours outdoors between 10 am and 3 pm compared to Australian‐born mothers (OR=2.80, 95%CI 1.20–6.57). Mothers from high latitude countries (>45 degrees) were more likely to apply sunscreen to their child than those from lower latitude countries (OR=3.15, 95%CI 1.03–9.61). Conclusions and implications: Strategies should aim to increase general awareness about the need for sun protection of young children, and recent migrants should be alerted to the harms of excessive sun exposure.