New beginnings: A time-limited, group intervention for high-risk infants and mothers

‘New Beginnings’ (NB) is a structured, manualized program that addresses the mother–baby relationship within a group format. The program, developed at the Anna Freud Centre (Baradon, 2009, 2013), works with the nuanced, cross-modal emotional interactions between mother and infant, tracking attunement and communication errors and emphasizing interactive repair. To this purpose, open (non-defended) and genuine transactions are privileged, confounding intergenerational transference expectancies of rejection and shaming by a ‘bad’ world in which the individual is not seen as a worthwhile person. NB aims to increase mentalization in relation to self, baby, and the relationship between them (Baradon, with Biseo, Broughton, James, & Joyce, 2016). This takes place via the content of sessions, group processes (between the adults, adults and babies and the baby-group) and personalization of the program. A reopening of epistemic trust – trust in the authenticity and personal relevance of interpersonally transmitted knowledge (Fonagy, Luyten, & Allison, 2015) – is seen as the product of the above, and the vehicle for the socialization of the babies into a more benign social context. This chapter will begin by setting out the basic structure and aims of the program, before going on to explain how the program was developed, how the program runs and its theoretical foundations in attachment and mentalizing. We will then describe how NB has thus far been evaluated, and will finish with a more detailed account of the implementation of the program

Anion exchange chromatography - Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals

Assessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple quality attributes of EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and a pH gradient for elution. Baseline separation of charge variants and high-quality MS data were achieved using 30 mM ammonium formate pH 5.5 and 30 mM formic acid pH 2.5 as mobile phases. In a single experiment, assessment of critical quality attributes, such as charge heterogeneity, sialic acid content and number of N-ace-tyllactosamine units, was possible while providing additional information on other modifications such as O-acetylation and deamidation. In addition, good repeatability and robustness for the relative areas of the individual glycoforms and average number of Neu5Ac per EPO molecule were observed. The results were comparable to common pharmacopeia and standard methods with the advantage of requiring fewer analytical methods and less sample treatment saving time and costs. (C) 2020 The Authors. Published by Elsevier B.V.Proteomic

Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry

Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batche

Functional selectivity of adenosine receptor ligands

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as β-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the β-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins

Mechanism-based pharmacokinetic-pharmacodynamic modeling of antilipolytic effects of adenosine A(1) receptor agonists in rats: prediction of tissue-dependent efficacy in

ABSTRACT We have developed a pharmacokinetic-pharmacodynamic strategy based on the operational model of agonism to obtain estimates of apparent affinity and efficacy of N 6 -cyclopentyladenosine (CPA) analogs for the adenosine A 1 receptor-mediated in vivo effect on heart rate in the rat. All analogs investigated produced a significant decrease of the heart rate after intravenous infusion. Individual concentration-effect curves were fitted to the operational model of agonism with the values of E max and n constrained to the intrinsic activity (273 bpm) and Hill slope (1.18), respectively, obtained with the agonist that displayed the highest intrinsic activity, 5Ј-deoxy-CPA. In all cases, the model converged and estimates of apparent affinity and efficacy were obtained for each agonist. Affinity estimates correlated well with pK i values for the adenosine A 1 receptor in rat brain homogenates. In addition, a highly significant correlation was found between the estimates of the in vivo efficacy parameter and the GTP shift (the ratio between K i in the presence and absence of GTP). In conclusion, the operational model of agonism can provide meaningful measures of agonist affinity and efficacy at adenosine A 1 receptors in vivo. The model should be of use in the development of partial adenosine A 1 receptor agonists. Adenosine is believed to exert its physiological effects through interactions with at least four receptor subtypes: the A 1 , A 2a , A 2b and A 3 receptors (see In the search for partial agonists, several series of adenosine derivatives have been synthesized leading to the identification of analogs of CPA for which the ratio between apparent affinity in the presence and absence of GTP for the adenosine A 1 receptor in radioligand binding studies is lower than for CP

High-performance liquid chromatography of the adenosine A1 agonist N6-cyclopentyladenosine and the A1 antagonist 8-cyclopentyltheophylline and its application in a pharmacokinetic study in rats

This report describes a rapid and sensitive analysis for the simultaneous detection of the adenosine A1 receptor ligands N6-cyclopentyladenosine (CPA) and 8-cyclopentyltheophylline (CPT) in rat blood. The method involved alkaline extraction of the compounds and internal standard N6-cyclohexyladenosine (CHA) with ethyl acetate, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-microns MicroSphere C18 column with UV detection at 269 nm. The mobile phase consisted of a mixture of 10 mM acetate buffer (pH 4.0)-methanol-acetonitrile (56:40:4, v/v/v) with a flow-rate of 0.50 ml/min. The total run time was ca. 19 min. For CPA and CPT extraction yields were greater than 77 and 66% in the concentration range of 0.010-0.75 microgram/ml and 0.025-15 micrograms/ml, respectively, with intra- and inter-assay variations less than 9%. In 100 microliter blood samples the corresponding limits of detection were 3.3 and 6.2 ng/ml (signal-to-noise ratio = 3). CPA was found to be degraded in rat blood in vitro with a half-life of 24 min at 37 degrees C. The utility of the analytical method was established by analyzing blood samples from rats which had received an intravenous administration of 200 micrograms/kg CPA or 12 mg/kg CPT. Due to its rapidity and sensitivity this method is concluded to be particularly useful in pharmacokinetic studies with CPA and CP

Pharmacokinetic-pharmacodynamic relationship of the cardiovascular effects of adenosine A1 receptor agonist N6-cyclopentyladenosine in the rat

The purpose of the investigation was to develop a pharmacokinetic-pharmacodynamic model for the characterization of the cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in individual normotensive rats. After the i.v. administration of 200 micrograms/kg (0.60 mumol/kg) of CPA, the time course of heart rate and arterial blood pressure was monitored in conjunction with serial blood sampling. Potential interference of the concentration-cardiovascular effect relationship by the development of acute tolerance or the formation of (inter)active metabolites was investigated by infusion of CPA with different rates and by determination of blood concentrations both by high-performance liquid chromatography and radioreceptor assay. In the individual rats the concentration-hemodynamic effect relationships were satisfactorily modeled according to the sigmoidal Emax pharmacodynamic model. For the negative chronotropic effect, the pharmacodynamic parameters proved to be independent of the infusion rate, indicating the absence of development of acute tolerance during the experiment. Potency (EC50) and intrinsic efficacy (Emax) were 2.7 +/- 0.5 ng/ml and -209 +/- 10 bpm, respectively (mean +/- S.E., n = 17). The concentrations of CPA as determined by the radioreceptor assay were identical to those determined by high-performance liquid chromatography, thereby excluding the formation of (inter)active metabolites. It is concluded that on the basis of this integrated pharmacokinetic-pharmacodynamic model, with the negative chronotropic effect as a pharmacodynamic endpoint, estimates of the potency and the intrinsic efficacy of adenosine A1 receptor agonists in vivo can be obtained after the administration of a single dos