Developing a recombinant model of the P2Y1 and P2Y11 receptor interactions mediating relaxation in gut smooth muscle

ATP and ADP mediate gut smooth muscle relaxation through two receptors, P2Y1 and P2Y11. This project aims to investigate the interaction between these two receptors by developing a recombinant model of the P2Y receptors expressed in gut smooth muscle cells (SMCs) by transfecting the human P2Y11 receptor cDNA into CHO-K1 cells, which express an endogenous P2Y1 receptor. Individual clonal cell lines expressing different densities of hP2Y11 were isolated from this stably-transfected CHO-K1:P2Y11 pool and characterized. A clone expressing a “high” density of hP2Y11 (13) and a clone expressing a “low” density of hP2Y11 (6) were selected for further study. Control 1321N1 cell lines expressing each receptor in isolation (1321N1-hP2Y1 and 1321N1-hP2Y11) were used for comparison purposes. The potency (EC50) of eight different nucleotide agonists was determined in calcium assays in the co-expressing cell lines. ADP and 2meSATP responses were biphasic in clone 13 but monophasic in clone 6. To investigate the nature of the two sites of the biphasic curves in clone 13, the effect of MRS 2179, NF 340 and Reactive Red on agonist responses was determined. MRS 2179 antagonized the high affinity site of the biphasic ADP and 2meSATP responses in clone 13 without affecting the low affinity site. NF 340 had no effect on agonist responses in clone 13. Reactive Red antagonized both sites of the biphasic curves in clone 13. These data suggest that the high-affinity site of the biphasic ADP and 2meSATP responses in clone 13 corresponds to P2Y1. The low-affinity site of the 2meSATP curve is most likely P2Y11. The low-affinity site the ADP response displays both P2Y1 and P2Y11-like. The novel ADP site, therefore, is elicited by differences in the expression level of P2Y11 and may correspond to a P2Y1:hP2Y11 receptor heteromer or a macromolecular complex containing both P2Y1 and P2Y11

Genetic contributions to visuospatial cognition in Williams syndrome: insights from two contrasting partial deletion patients

Background Williams syndrome (WS) is a rare neurodevelopmental disorder arising from a hemizygotic deletion of approximately 27 genes on chromosome 7, at locus 7q11.23. WS is characterised by an uneven cognitive profile, with serious deficits in visuospatial tasks in comparison to relatively proficient performance in some other cognitive domains such as language and face processing. Individuals with partial genetic deletions within the WS critical region (WSCR) have provided insights into the contribution of specific genes to this complex phenotype. However, the combinatorial effects of different genes remain elusive. Methods We report on visuospatial cognition in two individuals with contrasting partial deletions in the WSCR: one female (HR), aged 11 years 9 months, with haploinsufficiency for 24 of the WS genes (up to GTF2IRD1), and one male (JB), aged 14 years 2 months, with the three most telomeric genes within the WSCR deleted, or partially deleted. Results Our in-depth phenotyping of the visuospatial domain from table-top psychometric, and small- and large-scale experimental tasks reveal a profile in HR in line with typically developing controls, albeit with some atypical features. These data are contrasted with patient JB’s atypical profile of strengths and weaknesses across the visuospatial domain, as well as with more substantial visuospatial deficits in individuals with the full WS deletion. Conclusions Our findings point to the contribution of specific genes to spatial processing difficulties associated with WS, highlighting the multifaceted nature of spatial cognition and the divergent effects of genetic deletions within the WSCR on different components of visuospatial ability. The importance of general transcription factors at the telomeric end of the WSCR, and their combinatorial effects on the WS visuospatial phenotype are also discussed

Cooperativity-Dependent Folding of Single-Stranded DNA

The folding of biological macromolecules is a fundamental process of which we lack a full comprehension. Mostly studied in proteins and RNA, single-stranded DNA (ssDNA) also folds, at physiological salt conditions, by forming nonspecific secondary structures that are difficult to characterize with biophysical techniques. Here, we present a helix-coil model for secondary-structure formation, where ssDNA bases are organized in two different types of domains (compact and free). The model contains two parameters: the energy gain per base in a compact domain, ε , and the cooperativity related to the interfacial energy between different domains, γ . We test the ability of the model to quantify the formation of secondary structure in ssDNA molecules mechanically stretched with optical tweezers. The model reproduces the experimental force-extension curves in ssDNA of different molecular lengths and varying sodium and magnesium concentrations. Salt-correction effects for the energy of compact domains and the interfacial energy are found to be compatible with those of DNA hybridization. The model also predicts the folding free energy and the average size of domains at zero force, finding good agreement with secondary-structure predictions by mfold. We envision the model could be further extended to investigate native folding in RNA and protein

Validation of virtual water phantom software for pre-treatment verification of single-isocenter multiple-target stereotactic radiosurgery

Objectiu múltiple; SRS; Fantasma virtualObjetivo múltiple; SRS; Fantasma virtualMultiple‐target; SRS; Virtual phantomThe aim of this study was to benchmark the accuracy of the VIrtual Phantom Epid dose Reconstruction (VIPER) software for pre-treatment dosimetric verification of multiple-target stereotactic radiosurgery (SRS). VIPER is an EPID-based method to reconstruct a 3D dose distribution in a virtual phantom from in-air portal images. Validation of the VIPER dose calculation was assessed using several MLC-defined fields for a 6 MV photon beam. Central axis percent depth doses (PDDs) and output factors were measured with an ionization chamber in a water tank, while dose planes at a depth of 10 cm in a solid flat phantom were acquired with radiochromic films. The accuracy of VIPER for multiple-target SRS plan verification was benchmarked against Monte Carlo simulations. Eighteen multiple-target SRS plans designed with the Eclipse treatment planning system were mapped to a cylindrical water phantom. For each plan, the 3D dose distribution reconstructed by VIPER within the phantom was compared with the Monte Carlo simulation, using a 3D gamma analysis. Dose differences (VIPER vs. measurements) generally within 2% were found for the MLC-defined fields, while film dosimetry revealed gamma passing rates (GPRs) ≥95% for a 3%/1 mm criteria. For the 18 multiple-target SRS plans, average 3D GPRs greater than 93% and 98% for the 3%/2 mm and 5%/2 mm criteria, respectively. Our results validate the use of VIPER as a dosimetric verification tool for pre-treatment QA of single-isocenter multiple-target SRS plans. The method requires no setup time on the linac and results in an accurate 3D characterization of the delivered dose

Direct detection of molecular intermediates from first-passage times

All natural phenomena are governed by energy landscapes. However, the direct measurement of this fundamen-tal quantity remains challenging, particularly in complex systems involving intermediate states. Here, we uncover key details of the energy landscapes that underpin a range of experimental systems through quantitative analysis of first-passage time distributions. By combined study of colloidal dynamics in confinement, transport through a biological pore, and the folding kinetics of DNA hairpins, we demonstrate conclusively how a short-time, power-law regime of the first-passage time distribution reflects the number of intermediate states associated with each of these processes, despite their differing length scales, time scales, and interactions. We thereby establish a powerful method for investigating the underlying mechanisms of complex molecular processes

Neurodevelopmental disorders

Recent technological advances allow us to measure how the infant brain functions in ways that were not possible just a decade ago. Although methodological advances are exciting, we must also consider how theories guide research: what we look for and how we explain what we find. Indeed, the ways in which research findings are interpreted affects the design of policies, educational practices, and interventions. Thus, the theoretical approaches adopted by scientists have a real impact on the lives of children with neurodevelopmental disorders (NDDs) and their families, as well as on the wider community. Here, we introduce and compare two theoretical approaches that are used to understand NDDs: the neuropsychological account and neuroconstructivism. We show how the former, adult account, is inadequate for explaining NDDs and illustrate this using the examples of Williams syndrome and specific language impairment. Neuroconstructivism, by contrast, focuses on the developing organism and is helping to change the way in which NDDs are investigated. Whereas neuropsychological static approaches assume that one or more ‘modules’ (e.g., visuospatial ability in Williams syndrome) are impaired while the rest of the system is spared (e.g., language in Williams syndrome), neuroconstructivism proposes that basic‐level deficits have subtle cascading effects on numerous domains over development. Neuroconstructivism leads researchers to embrace complexity by establishing large research consortia to integrate findings at multiple levels (e.g., genetic, neural, cognitive, environmental) across developmental time

Route knowledge and configural knowledge in typical and atypical development: a comparison of sparse and rich environments

Background: Individuals with Down syndrome (DS) and individuals with Williams syndrome (WS) have poor navigation skills, which impact their potential to become independent. Two aspects of navigation were investigated in these groups, using virtual environments (VE): route knowledge (the ability to learn the way from A to B by following a fixed sequence of turns) and configural knowledge (knowledge of the spatial relationships between places within an environment). Methods: Typically developing (TD) children aged 5 to 11 years (N = 93), individuals with DS (N = 29) and individuals with WS (N = 20) were presented with a sparse and a rich VE grid maze. Within each maze, participants were asked to learn a route from A to B and a route from A to C before being asked to find a novel shortcut from B to C. Results: Performance was broadly similar across sparse and rich mazes. The majority of participants were able to learn novel routes, with poorest performance in the DS group, but the ability to find a shortcut, our measure of configural knowledge, was limited for all three groups. That is, 59 % TD participants successfully found a shortcut, compared to 10 % participants with DS and 35 % participants with WS. Differences in the underlying mechanisms associated with route knowledge and configural knowledge and in the developmental trajectories of performance across groups were observed. Only the TD participants walked a shorter distance in the last shortcut trial compared to the first, indicative of increased configural knowledge across trials. The DS group often used an alternative strategy to get from B to C, summing the two taught routes together. Conclusions: Our findings demonstrate impaired configural knowledge in DS and in WS, with the strongest deficit in DS. This suggests that these groups rely on a rigid route knowledge based method for navigating and as a result are likely to get lost easily. Route knowledge was also impaired in both DS and WS groups and was related to different underlying processes across all three groups. These are discussed with reference to limitations in attention and/or visuo-spatial processing in the atypical groups

Language and memory for object location

In three experiments, we investigated the influence of two types of language on memory for object location: demonstratives (this, that) and possessives (my, your). Participants first read instructions containing demonstratives/possessives to place objects at different locations, and then had to recall those object locations (following object removal). Experiments 1 and 2 tested contrasting predictions of two possible accounts of language on object location memory: the Expectation Model (Coventry, Griffiths, & Hamilton, 2014) and the congruence account (Bonfiglioli, Finocchiaro, Gesierich, Rositani, & Vescovi, 2009). In Experiment 3, the role of attention allocation as a possible mechanism was investigated. Results across all three experiments show striking effects of language on object location memory, with the pattern of data supporting the Expectation Model. In this model, the expected location cued by language and the actual location are concatenated leading to (mis)memory for object location, consistent with models of predictive coding (Bar, 2009; Friston, 2003)

Palaeoceanographic implications of current-controlled sedimentation in the Alboran Sea after the opening of the Strait of Gibraltar

This study focuses on the Alboran Sea area (Westernmost Mediterranean), where a seismic analysis of the Pliocene and Quaternary stratigraphy was conducted in the Alboran Sea (Westernmost Mediterranean) using ~2000 profiles consisting of single and multi-channel seismic records. The seismic facies and architectural analysis of the deposits evidence the presence of bottom-current deposits (plastered, sheeted, elongated-separated and confined monticular drifts) and associated erosive features (terraces, scarps, moats and channels). Many of these deposits were previously considered to be open slope turbidite deposits which have now been reinterpreted as contourites.The contourite features have developed under the continuous influence of Mediterranean water masses, after the opening of the Strait of Gibraltar (roughly divided into light and dense Mediterranean waters), with plastered drifts dominating on the Spanish and Moroccan continental slopes, and sheeted drifts infilling the subbasins. The location and growth of contourite features have been mainly controlled by two main factors: i) tectonics, which has governed the relocation of the main pathways of the water masses; and ii) climate, which has influenced both water mass conditions and the depth of interfaces, as well as hinterland sediment sources, conditioning the morphoseismic characteristics of the drifts (facies and geometry) and terrace formation (dimensions). The mapping of the contourite facies through time has allowed defining three main scenarios for deep water circulation since the opening of the Strait of Gibraltar, which are: i) Atlantic Zanclean flooding; ii) the Pliocene sea, with two different stages caused by the progressive relocation of flow pathways; and iii) the Quaternary sea, with well defined characteristics and mostly stable flow pathways for the AW, and light and dense Mediterranean waters.This work lead us to consider the geologic framework characterizing the Alboran Sea may have played an important role in the interaction of the Mediterranean Waters before entering the Strait of Gibraltar, and thus in forming the MOW. Additionally, the results of this work may help in understanding the sedimentation in other Mediterranean margins affected by the same water masses and other partly land-locked basins with exchanges of waters over a confining sill