Insights into pediatric rhabdomyosarcoma research: Challenges and goals

Overall survival rates for pediatric patients with high‐risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.     

A randomised trial of two methods of issuing prenatal test results: the ARIA (Amniocentesis Results: Investigation of Anxiety) trial

BACKGROUND: Many pregnant women experience anxiety while waiting for the results of diagnostic tests. Policies and practices intended to reduce this anxiety require evaluation. OBJECTIVES: To test the following two hypotheses: * That giving amniocentesis results out on a fixed date alters maternal anxiety during the waiting period, compared with a policy of telling parents that the result will be issued "when available" (i.e. variable date). * That issuing early results from a rapid molecular test alters maternal anxiety during the waiting period, compared with not receiving any results prior to the karyotype. The effects of the two interventions on anxiety 1 month after receiving karyotype results were also examined. DESIGN: A multicentre, randomised, controlled, open fixed sample, 2 x 2 factorial design trial, with equal randomisation. SETTING: The prenatal diagnosis clinics in 12 hospitals in England offering amniocentesis as a diagnostic test for Down's syndrome. SAMPLE: Two hundred and twenty-six women who had had an amniocentesis were randomised between June 2002 and July 2004. Eight women with abnormal results or test failure were excluded post-randomisation. INTERVENTIONS: Issuing karyotype results on a prespecified fixed date, rather than issuing them as soon as they became available. Issuing karyotype results alone, or subsequent to issuing results from a rapid molecular test for the most common chromosomal abnormalities. MAIN OUTCOME MEASURES: Average anxiety during the waiting period, calculated using daily scores from the short version of the Spielberger State-Trait Anxiety Inventory (STAI). Anxiety 1 month after receiving karyotype results, measured using the short form STAI. RESULTS: Issuing early results from a partial but rapid test reduced maternal anxiety by a clinically significant amount during the waiting period (mean daily score 12.5 versus 14.8; scale score difference -2.36, 95% CI -1.2, -3.6), compared with receiving only the full karyotype results. There was no evidence that giving out karyotype results on a fixed or on a variable date altered maternal anxiety during the waiting period (mean daily score 13.2 versus 14.2; scale score difference -1.02, 95% CI -2.2, 0.2). One month after receiving normal karyotype results, anxiety was low in all groups, but women who had been given rapid test results tended to be more anxious than those who had not (mean single day score 9.2 versus 8.3; mean scale score difference 0.95, 95% CI -0.03, 1.9). This small to moderate effect did not reach conventional levels of statistical significance. CONCLUSIONS: Rapid testing was a beneficial addition to karyotyping, at least in the short term. This does not necessarily imply that early results would be preferred to comprehensive ones if women had to choose between them. Because there are no clear advantages in anxiety terms of issuing karyotype results as soon as they become available, or on a fixed date, women could be given a choice between them.     

ECK FISTULA LIVER SUBNORMAL IN PRODUCING HEMOGLOBIN AND PLASMA PROTEINS ON DIETS RICH IN LIVER AND IRON

The Eck fistula shunts the portal blood around the liver which receives its blood only by way of the hepatic artery. There are slight gross and histological changes in the Eck fistula liver of the dog. There is evidence at times of some functional abnormalities of the liver due to the Eck fistula but the dog can tolerate this fistula for 1 to 8 years and appear normal. Chloroform is tolerated by the Eck fistula dog, which may take twice a lethal dose for the control dog without evidence of significant liver injury. Acacia given by vein is deposited in the Eck fistula liver and impairs further its functional capacity to contribute to hemoglobin production. The stress of anemia brings out the fact that the anemic Eck fistula animal cannot utilize standard diet factors and iron as efficiently as the anemic non-Eck control dog. The output of new hemoglobin in some instances may drop to one-fourth of normal. When hypoproteinemia alone or combined with anemia is produced in the Eck fistula dog, we observe at times very low production of plasma protein—seven a drop to one-tenth of normal. This interrelation of liver abnormality, liver dysfunction, and lessened plasma protein and hemoglobin production is significant. It is generally accepted that the liver is concerned with the production of several plasma proteins—fibrinogen, prothrombin, and albumin. The experiments above indicate that the liver is concerned directly or indirectly with the production of new hemoglobin. Our belief is that the liver contributes to the fabrication of hemoglobin by means of the mobile plasma proteins which to a large extent derive from the liver.     

Synergistic effects of dopamine agonists and centrally administered neurotensin on feeding

Several lines of evidence indicate that neurotensin may modulate the activity of dopamine systems in the central nervous system. The present study investigated the possibility that intraperitoneal injections of the dopamine agonists l-dopa and bromocriptine would alter the aphagia produced by central administration of neurotensin. It was found that neurotensin suppressed feeding in food-deprived rats when injected into the lateral ventricle or the ventromedial hypothalamus. Food intake was not affected, however, when the peptide was placed in the lateral hypothalamus. A dose-dependent aphagia was also observed following peripheral injections of l-dopa and bromocriptine. Additionally, the anorectic effect of centrally administered neurotensin was potentiated by concurrent administration of doses of l-dopa or bromocriptine which, when given alone, had no effect on food intake. The data suggest that neurotensin aphagia may be mediated by the peptide's ability to increase the activity of dopamine systems in the central nervous system.     

Clarifying Relations Between Thought-Action Fusion, Religiosity, and Obsessive–Compulsive Symptoms Through Consideration of Intent

Conflicting findings regarding the relations between thought-action fusion (TAF), religiosity, and obsessive compulsive disorder (OCD) may be due to a lack of clarity regarding the intent associated with the negative thoughts under consideration. In Study 1, we examined the perceptions of the immorality of intentional and unwanted morally-relevant thoughts (Moral TAF) and their relations with OCD symptoms, religiosity, and obsessive beliefs in a non-clinical sample. In Study 2, we randomly assigned participants to complete one of two versions of a previously used sentence neutralization task that was varied in terms of intent. Perception of the immorality of intentional negative thoughts but not unwanted negative thoughts was associated with Protestant/Catholic affiliation and greater prayer frequency, and perception of the immorality of unwanted thoughts was consistently associated with obsessive beliefs. Neither form of Moral TAF was associated with OCD symptoms. Further, reaction to the modified non-intentional neutralization task was associated with OCD symptoms, thought-action fusion, and scrupulosity, while reaction to the original intentional task was only associated with Moral TAF. Overall, the findings suggest that individuals differ in their perceptions of intentional versus unintentional thoughts. Perceptions of intentional morally-relevant thoughts appear related to religiosity, while perceptions of unintentional thoughts are likely to be of greater relevance to our understanding of OCD.     

Translumbar catheter redirection using a tip-deflector technique

A new technique is described for reversing the direction of the catheter tip during translumbar aortography, without the need for partial withdrawal of the catheter from the aortic lumen. The method ensures optimal delivery of contrast medium at the desired level, while avoiding the risk of retroperitoneal bleeding or dislodgement during catheter manipulation.     

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Galectin-3 is a glycan-binding protein (GBP) that binds β-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells and regulation of immune responses. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the three-dimensional (3D) arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, have significant advantages over static and artificial systems. Here we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live human hepatic stellate cells and peripheral blood mononuclear cells. Understanding the identity of the glycoproteins and defining the structures of the glycans will empower efforts to design and develop selective therapeutics to mitigate galectin-3–mediated biological events.

The noncovalent interactions between glycan-binding proteins (GBPs) and glycans dictate many important biological events. Among such GBPs is galectin-3, a 26-kDa β-galactoside GBP that plays key roles in many physiological and pathological events (1). In hepatic fibrosis, a disease that manifests as the excessive buildup of scar tissue, liver-resident macrophages secrete galectin-3 (2, 3), which then binds cell surface glycans on quiescent hepatic stellate cells (HSCs), activating them to transdifferentiate into a muscle-like phenotype. Galectin-3–null mice exhibit attenuated liver fibrosis even after induced injury, highlighting its critical role (3). Galectin-3 is also known to interact with cells of the innate immune system (4, 5) to regulate apoptosis (6) or control dendritic cell differentiation (7). In these cases, as well as in other cases in which galectin-3 is involved, the full complement of interacting glycoprotein receptors remains unknown.Despite significant advances in glycoscience, the study of GBP–glycan interactions and the identification of glycan-mediated counter-receptors remains a recurring challenge. Capturing these binding events often requires some form of artificial reconstitution to amplify individually weak interactions into high-avidity binding. Indeed, glycan microarrays with defined mixtures of homogenous glycans or recombinant GBPs have significantly propelled our understanding of glycan-mediated function (8). Conventional immunoprecipitation and lectin affinity techniques using cell lysates have similarly been used to reveal an initial catalog of 100 to 185 galectin-3–associated proteins (9–14). However, these manipulations alter the cell’s native and three-dimensional (3D) configuration and multivalent arrangement, both of which are critically important in the study of GBP–glycan interactions (15, 16).Another key issue involves the underlying glycoprotein ligand. Although many glycoproteins carry the glycan epitope for binding a GBP, only a limited set should be recognized as physiologically relevant receptors, owing to the physical constraints imposed by the living cell (17). While often overlooked, the glycoprotein carrying the glycan can impart specific biological functions to a GBP–glycan binding event (17). Recent work has put forth the concept of “professional glycoprotein ligands,” in which a specific set of glycoproteins (instead of a broadly defined glycome) can exhibit exquisite binding and functional roles (18). Thus, determining the identity of the underlying core protein that anchors the glycan can be greatly empowering. Not only can it provide an understanding of the 3D arrangement of the glycan (if the 3D structure of the core protein is known), but it can also provide additional insight into its expression levels in different cell types and tissues, further informing strategies for selective drug development.Thus, comprehensive approaches that permit the study of GBP–glycan interactions in live cells while simultaneously facilitating identification of the physiological glycoprotein receptors have great potential to impact glycoscience. We hypothesize that proximity labeling strategies (19) using an engineered ascorbate peroxidase, APEX2 (20), could be compatible for elucidating glycan-mediated GBP–glycoprotein interactions. In this approach (Fig. 1), APEX2 is fused to a protein of interest, followed by the treatment of cells with biotin-phenol and subsequently with hydrogen peroxide (H₂O₂). Under these conditions, APEX2 catalyzes the formation of highly reactive, short-lived (<1 ms), and proximally restricted (<20 nm) biotin-phenoxyl radicals that covalently tag nearby electron-rich residues. The biotinylated proteins can then be enriched and identified using quantitative mass spectrometry (MS)-based proteomics. Because the (glyco)proteins adjacent to the APEX2 fusion protein are preferentially biotinylated, the resulting MS data provide a readout of its immediate environment.Open in a separate windowFig. 1.Schematic illustration of the identification of galectin-3 (Gal-3) interacting proteins by in situ proximity labeling. Recombinant APEX2 and galectin-3 fusion proteins are applied to living cells where galectin-3 can freely diffuse to bind its cognate ligands. On addition of biotin phenol (yellow circle with “B”; 30 min) and hydrogen peroxide (H₂O₂; 1 min), APEX2 catalyzes the formation of highly-reactive biotin-phenoxyl radicals that react within a short range (<20 nm) of the galectin-3 complex within a short time frame (<1 ms). The biotin-tagged protein interactors can then be identified using MS-based proteomics.We reasoned that proximity labeling could offer significant advantages over other approaches to determining GBP–glycan interactions, including the opportunity to perform the labeling in live cells and the ability to tag weakly bound glycan-mediated interactors, as the covalent biotinylation reaction ensures that the enrichment step no longer relies on weak GBP–glycan interactions alone. When coupled with inhibitors, the proximity labeling strategy can also distinguish between glycan-mediated and non–glycan-mediated interactors. Integration of this approach with quantitative MS-based proteomics would also expedite the assignment of the interacting proteins and provide calculable measures to distinguish interactors from nonspecific binders.Here we report that the use of an APEX2 and galectin-3 fusion protein (PX-Gal3) provides a sensitive and comprehensive approach to mapping the proteome-wide glycan-mediated galectin-3 interactome in live human HSCs and peripheral blood mononuclear cells (PBMCs). We found that the exogenous incubation of cells with PX-Gal3 in HSCs leads to glycan-dependent interactions, whereas its cellular overexpression does not. We further validated the interactions between galectin-3 and candidate proteins in vitro and discovered that some proteins are direct glycan-mediated receptors. Using MS-based glycomics, we also examined the glycomes of HSC surfaces, PX-Gal3 tagged glycoproteins, and an individual glycoprotein receptor for galectin-3. Our results highlight the utility of the in situ proximity labeling approach in discovering physiologically relevant GBP interactors in living cells.