Ligand-induced conformational changes in a SMALP-encapsulated GPCR.

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs):their nature and potential

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR

G-protein-coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms

Protocol for the feasibility and implementation study of a model of best practice in primary care led postdiagnostic dementia care: PriDem

INTRODUCTION: Care is often inadequate and poorly integrated after a dementia diagnosis. Research and policy highlight the unaffordability and unsustainability of specialist-led support, and instead suggest a task-shared model, led by primary care. This study is part of the PriDem primary care led postdiagnostic dementia care research programme and will assess delivery of an evidence-informed, primary care based, person-centred intervention. The intervention involves Clinical Dementia Leads (CDLs) working in primary care to develop effective dementia care systems that build workforce capacity and support teams to deliver tailored support to people living with dementia and their carers. METHODS AND ANALYSIS: This is a 15-month mixed-methods feasibility and implementation study, situated in four National Health Service (NHS) primary care networks in England. The primary outcome is adoption of personalised care planning by participating general practices, assessed through a patient records audit. Feasibility outcomes include recruitment and retention; appropriateness and acceptability of outcome measures; acceptability, feasibility and fidelity of intervention components. People living with dementia (n=80) and carers (n=66) will be recruited through participating general practices and will complete standardised measures of health and well-being. Participant service use data will be extracted from electronic medical records. A process evaluation will explore implementation barriers and facilitators through methods including semistructured interviews with people living with dementia, carers and professionals; observation of CDL engagement with practice staff; and a practice fidelity log. Process evaluation data will be analysed qualitatively using codebook thematic analysis, and quantitatively using descriptive statistics. Economic analysis will determine intervention cost-effectiveness. ETHICS AND DISSEMINATION: The study has received favourable ethical opinion from Wales REC4. NHS Confidentiality Advisory Group support allows researchers preconsent access to patient data. Results will inform intervention adaptations and a future large-scale evaluation. Dissemination through peer-review journals, engagement with policy-makers and conferences will inform recommendations for dementia services commissioning. TRIAL REGISTRATION NUMBER: ISRCTN11677384

Scintillation-limited photometry with the 20-cm NGTS telescopes at Paranal Observatory

Ground-based photometry of bright stars is expected to be limited by atmospheric scintillation, although in practice observations are often limited by other sources of systematic noise. We analyse 122 nights of bright star (Gmag ≲ 11.5) photometry using the 20-cm telescopes of the Next-Generation Transit Survey (NGTS) at the Paranal Observatory in Chile. We compare the noise properties to theoretical noise models and we demonstrate that NGTS photometry of bright stars is indeed limited by atmospheric scintillation. We determine a median scintillation coefficient at the Paranal Observatory of CY=1.54⁠, which is in good agreement with previous results derived from turbulence profiling measurements at the observatory. We find that separate NGTS telescopes make consistent measurements of scintillation when simultaneously monitoring the same field. Using contemporaneous meteorological data, we find that higher wind speeds at the tropopause correlate with a decrease in long-exposure (t = 10 s) scintillation. Hence, the winter months between June and August provide the best conditions for high-precision photometry of bright stars at the Paranal Observatory. This work demonstrates that NGTS photometric data, collected for searching for exoplanets, contains within it a record of the scintillation conditions at Paranal

The relative emission from chromospheres and coronae: Dependence on spectral type and age

Extreme-ultraviolet and X-ray emission from stellar coronae drives mass loss from exoplanet atmospheres, and ultraviolet emission from stellar chromospheres drives photochemistry in exoplanet atmospheres. Comparisons of the spectral energy distributions of host stars are, therefore, essential for understanding the evolution and habitability of exoplanets. The large number of stars observed with the MUSCLES, Mega-MUSCLES, and other recent Hubble Space Telescope observing programs has provided for the first time a large sample (79 stars) of reconstructed Lyα fluxes that we compare with X-ray fluxes to identify significant patterns in the relative emission from these two atmospheric regions as a function of stellar age and effective temperature. We find that as stars age on the main sequence, the emissions from their chromospheres and coronae follow a pattern in response to the amount of magnetic heating in these atmospheric layers. A single trend-line slope describes the pattern of X-ray versus Lyα emission for G and K dwarfs, but the different trend lines for M dwarf stars show that the Lyα fluxes of M stars are significantly smaller than those of warmer stars with the same X-ray flux. The X-ray and Lyα luminosities divided by the stellar bolometric luminosities show different patterns depending on stellar age. The L (Lyα)/L(bol) ratios increase smoothly to cooler stars of all ages, but the L(X)/L(bol) ratios show different trends. For older stars, the increase in coronal emission with decreasing Teff is much steeper than that of chromospheric emission. We suggest a fundamental link between atmospheric properties and trend lines relating coronal and chromospheric heating.Fil: Linsky, Jeffrey L.. State University of Colorado at Boulder; Estados UnidosFil: Wood, Brian E.. Spece Sciences División. Naval Research Laboratory; Estados UnidosFil: Youngblood, Allison. State University of Colorado at Boulder; Estados UnidosFil: Brown, Alexander. State University of Colorado at Boulder; Estados UnidosFil: Froning, Cynthia S.. University of Texas at Austin; Estados UnidosFil: France, Kevin. State University of Colorado at Boulder; Estados UnidosFil: Buccino, Andrea Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Cranmer, Steven R.. State University of Colorado at Boulder; Estados UnidosFil: Mauas, Pablo Jacobo David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Miguel, Yamila. Leiden Observatory; Países BajosFil: Sebastian Pineda, J.. State University of Colorado at Boulder; Estados UnidosFil: Rugheimer, Sarah. University of Oxford; Reino UnidoFil: Vieytes, Mariela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Wheatley, Peter J.. University of Warwick; Reino UnidoFil: Wilson, David J.. University of Texas at Austin; Estados Unido

NGTS clusters survey -- II. White-light flares from the youngest stars in Orion

We present the detection of high energy white-light flares from pre-main sequence stars associated with the Orion complex, observed as part of the Next Generation Transit Survey (NGTS). With energies up to $5.2\times10^{35}$ erg these flares are some of the most energetic white-light flare events seen to date. We have used the NGTS observations of flaring and non-flaring stars to measure the average flare occurrence rate for 4 Myr M0-M3 stars. We have also combined our results with those from previous studies to predict average rates for flares above $1\times10^{35}$ ergs for early M stars in nearby young associations.STFC ST/M001962/1; ST/P000495/

Automatic vetting of planet candidates from ground based surveys : machine learning with NGTS

State of the art exoplanet transit surveys are producing ever increasing quantities of data. To make the best use of this resource, in detecting interesting planetary systems or in determining accurate planetary population statistics, requires new automated methods. Here we describe a machine learning algorithm that forms an integral part of the pipeline for the NGTS transit survey, demonstrating the efficacy of machine learning in selecting planetary candidates from multi-night ground based survey data. Our method uses a combination of random forests and self-organising-maps to rank planetary candidates, achieving an AUC score of 97.6% in ranking 12368 injected planets against 27496 false positives in the NGTS data. We build on past examples by using injected transit signals to form a training set, a necessary development for applying similar methods to upcoming surveys. We also make the autovet code used to implement the algorithm publicly accessible. autovet is designed to perform machine learned vetting of planetary candidates, and can utilise a variety of methods. The apparent robustness of machine learning techniques, whether on space-based or the qualitatively different ground-based data, highlights their importance to future surveys such as TESS and PLATO and the need to better understand their advantages and pitfalls in an exoplanetary context

TESS Duotransit Candidates from the Southern Ecliptic Hemisphere

Discovering transiting exoplanets with long orbital periods allows us to study warm and cool planetary systems with temperatures similar to the planets in our own Solar system. The TESS mission has photometrically surveyed the entire Southern Ecliptic Hemisphere in Cycle 1 (August 2018 - July 2019), Cycle 3 (July 2020 - June 2021) and Cycle 5 (September 2022 - September 2023). We use the observations from Cycle 1 and Cycle 3 to search for exoplanet systems that show a single transit event in each year - which we call duotransits. The periods of these planet candidates are typically in excess of 20 days, with the lower limit determined by the duration of individual TESS observations. We find 85 duotransit candidates, which span a range of host star brightnesses between 8 < $T_{mag}$ < 14, transit depths between 0.1 per cent and 1.8 per cent, and transit durations between 2 and 10 hours with the upper limit determined by our normalisation function. Of these candidates, 25 are already known, and 60 are new. We present these candidates along with the status of photometric and spectroscopic follow-up.Comment: 25 pages, 16 figures, submitted to Monthly Notices of the Royal Astronomical Societ

Phase II randomized preoperative window-of-opportunity study of the PI3K inhibitor pictilisib plus anastrozole compared with anastrozole alone in patients with estrogen receptor-positive breast cancer

Purpose: Preclinical data support a key role for the PI3K pathway in estrogen receptor-positive breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib (GDC-0941) can increase the antitumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. Patients and Methods: In this randomized, open-label phase II trial, postmenopausal women with newly diagnosed operable estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with anastrozole 1 mg once per day (n = 26) or the combination of anastrozole 1 mg with pictilisib 260 mg once per day (n = 49). The primary end point was inhibition of tumor cell proliferation as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. Results: There was significantly greater geometric mean Ki-67 suppression of 83.8% (one-sided 95% CI, ≥ 79.0%) for the combination and 66.0% (95% CI, ≤ 75.4%) for anastrozole (geometric mean ratio [combination: anastrozole], 0.48; 95% CI, ≤ 0.72; P = .004). PIK3CA mutations were not predictive of response to pictilisib, but there was significant interaction between response to treatment and molecular subtype (P =.03);for patients with luminal B tumors, the combination:anastrozole geometric mean ratio of Ki-67 suppression was 0.37 (95% CI, ≤ 0.67; P = .008), whereas no significant Ki-67 response was observed for pictilisib in luminal A tumors (1.01; P = .98). Multivariable analysis confirmed Ki-67 response to the combination treatment of patients with luminal B tumors irrespective of progesterone receptor status or baseline Ki-67 expression. Conclusion: Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer