pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst

A Second-Generation Device for Automated Training and Quantitative Behavior Analyses of Molecularly-Tractable Model Organisms

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science

A search of the Orion spur for continuous gravitational waves using a "loosely coherent" algorithm on data from LIGO interferometers

We report results of a wideband search for periodic gravitational waves from isolated neutron stars within the Orion spur towards both the inner and outer regions of our Galaxy. As gravitational waves interact very weakly with matter, the search is unimpeded by dust and concentrations of stars. One search disk (A) is $6.87^\circ$ in diameter and centered on $20^\textrm{h}10^\textrm{m}54.71^\textrm{s}+33^\circ33'25.29"$, and the other (B) is $7.45^\circ$ in diameter and centered on $8^\textrm{h}35^\textrm{m}20.61^\textrm{s}-46^\circ49'25.151"$. We explored the frequency range of 50-1500 Hz and frequency derivative from $0$ to $-5\times 10^{-9}$ Hz/s. A multi-stage, loosely coherent search program allowed probing more deeply than before in these two regions, while increasing coherence length with every stage. Rigorous followup parameters have winnowed initial coincidence set to only 70 candidates, to be examined manually. None of those 70 candidates proved to be consistent with an isolated gravitational wave emitter, and 95% confidence level upper limits were placed on continuous-wave strain amplitudes. Near $169$ Hz we achieve our lowest 95% CL upper limit on worst-case linearly polarized strain amplitude $h_0$ of $6.3\times 10^{-25}$, while at the high end of our frequency range we achieve a worst-case upper limit of $3.4\times 10^{-24}$ for all polarizations and sky locations.Comment: Fixed minor typo - duplicate name in the author lis

Cellular pharmacology studies of anticancer agents: recommendations from the EORTC-PAMM group

An increasing number of manuscripts focus on the in vitro evaluation of established and novel anti-tumour agents in experimental models. Whilst the design of such in vitro assays is inherently flexible, some of these studies lack the minimum information necessary to critically evaluate their relevance or have been carried out under unsuitable conditions. The use of appropriate and robust methods and experimental design has important implications for generating results that are reliable, relevant, and reproducible. The Pharmacology and Molecular Mechanisms (PAMM) group of the European Organization for Research and Treatment of Cancer (EORTC) is the largest group of academic scientists working on drug development and bundle decades of expertise in this field. This position paper addresses all researchers with an interest in the preclinical and cellular pharmacology of anti-tumour agents and aims at generating basic recommendations for the correct use of compounds to be tested for anti-tumour activity using a range of preclinical cellular models of cancer

Effective components of feedback from Routine Outcome Monitoring (ROM) in youth mental health care: study protocol of a three-arm parallel-group randomized controlled trial

Contains fulltext : 126077.pdf (publisher's version ) (Open Access)Background: Routine Outcome Monitoring refers to regular measurements of clients' progress in clinical practice, aiming to evaluate and, if necessary, adapt treatment. Clients fill out questionnaires and clinicians receive feedback about the results. Studies concerning feedback in youth mental health care are rare. The effects of feedback, the importance of specific aspects of feedback, and the mechanisms underlying the effects of feedback are unknown. In the present study, several potentially effective components of feedback from Routine Outcome Monitoring in youth mental health care in the Netherlands are investigated. Methods/Design: We will examine three different forms of feedback through a three-arm parallel-group randomized controlled trial. 432 children and adolescents (aged 4 to 17 years) and their parents, who have been referred to mental health care institution Pro Persona, will be randomly assigned to one of three feedback conditions (144 participants per condition). Randomization will be stratified by age of the child or adolescent and by department. All participants fill out questionnaires at the start of treatment, one and a half months after the start of treatment, every three months during treatment, and at the end of treatment. Participants in the second and third feedback conditions fill out an additional questionnaire. In condition 1, clinicians receive basic feedback regarding clients' symptoms and quality of life. In condition 2, the feedback of condition 1 is extended with feedback regarding possible obstacles to a good outcome and with practical suggestions. In condition 3, the feedback of condition 2 is discussed with a colleague while following a standardized format for case consultation. The primary outcome measure is symptom severity and secondary outcome measures are quality of life, satisfaction with treatment, number of sessions, length of treatment, and rates of dropout. We will also examine the role of being not on track (not responding to treatment). Discussion: This study contributes to the identification of effective components of feedback and a better understanding of how feedback functions in real-world clinical practice. If the different feedback components prove to be effective, this can help to support and improve the care for youth.11 p