High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

A Spectral Diagnostic for Density-Bounded HII Regions

The existence of density-bounded HII regions in spiral galaxies is supported by means of a spectral indicator based on the intensity of the [OI]6300A forbidden line. A grid of photoionization models providing spectral information of density-bounded nebulae is presented in order to test the validity of our indicator. The indicator is applicable to typical observed HII regions with solar or higher metallicities, and for the range of ages observed in normal HII regions. When applying the diagnostic to a large sample of HII regions of spiral galaxies taken from the literature, we find that a fraction of the selected HII regions with emission detected in the [OI]6300A line turn out to be consistent with the predictions of density-bounded photoionization models. Preliminary estimates of the fraction of Lyman continuum photons escaping ranges from 20% to 40%. The number of density-bounded HII regions could be even larger after confirmation of those regions for which the [OI] line was not detected, provided they are good density-bounded candidates. We stress the importance of having combined information on both good measurements or reliable upper limits for the intensity of this line, and good determinations of the Halpha luminosity for complete samples of HII regions in galaxies in order to make proper estimations as to whether UV photons escaping from HII regions are the main source of ionization of the diffuse ionized medium in galaxies.Comment: 15 pages, 5 figures, LaTeX, accepted for publication in M.N.R.A.

Early preparation during turn-taking:Listeners use content predictions to determine <i>what</i> to say but not <i>when</i> to say it

During conversation, there is often little gap between interlocutors' utterances. In two pairs of experiments, we manipulated the content predictability of yes/no questions to investigate whether listeners achieve such coordination by (i) preparing a response as early as possible or (ii) predicting the end of the speaker's turn. To assess these two mechanisms, we varied the participants' task: They either pressed a button when they thought the question was about to end (Experiments 1a and 2a), or verbally answered the questions with either yes or no (Experiments 1b and 2b). Predictability effects were present when participants had to prepare a verbal response, but not when they had to predict the turn-end. These findings suggest content prediction facilitates turn-taking because it allows listeners to prepare their own response early, rather than because it helps them predict when the speaker will reach the end of their turn

AINUR: Atlas of Images of NUclear Rings

We present the most complete atlas of nuclear rings to date. We include 113 rings found in 107 galaxies, six of which are elliptical galaxies, five are highly inclined disc galaxies, 18 are unbarred disc galaxies, and 78 are barred disc galaxies. Star-forming nuclear rings occur in 20% of disc galaxies with types between T=-3 and T=7. We aim to explore possible relationships between the size and morphology of the rings and various galactic parameters. We produce colour index and structure maps, as well as Halpha and Paalpha continuum-subtracted images from HST archival data. We derive ellipticity profiles from H-band 2MASS images in order to detect bars and find their metric parameters. We measure the non-axisymmetric torque parameter, Qg, and search for correlations between bar, ring metric parameters, and Qg. Our atlas of nuclear rings includes star-forming and dust rings. Nuclear rings span a range from a few tens of parsecs to a few kiloparsecs in radius. Star-forming nuclear rings can be found in a wide range of morphological types, from S0 to Sd, with a peak in the distribution between Sab and Sb, and without strong preference for barred galaxies. Dust nuclear rings are found in elliptical and S0 galaxies. For barred galaxies, the maximum radius that a nuclear ring can reach is a quarter of the bar radius. We found a nearly random distribution of PA offsets between nuclear rings and bars. There is some evidence that nuclear ring ellipticity is limited by bar ellipticity. We confirm that the maximum relative size of a star-forming nuclear ring is inversely proportional to the non-axisymmetric torque parameter, Qg, and that the origin of nuclear rings, even the ones in non-barred hosts, are closely linked to the existence of dynamical resonances.Comment: Accepted for publication in MNRAS. A full resolution version of the manuscript with high resolution figures can be found at http://www.iac.es/folleto/research/preprints

Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo's potential, affecting adult health

Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences

Comprehensive assessment of spotty calcifications on computed tomography angiography: Comparison to plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis

Vascular Biology and Interventio

Open and hidden agendas of "asymptomatic" patients who request check-up exams

BACKGROUND: Current guidelines for a check-up recommend routine screening not triggered by specific symptoms for some known risk factors and diseases in the general population. Patients' perceptions and expectations regarding a check-up exam may differ from these principles. However, quantitative and qualitative data about the discrepancy between patient- and provider expectations for this type of clinic consultation is lacking. METHODS: For a year, we prospectively enrolled 66 patients who explicitly requested a "check-up" at our medical outpatient division. All patients actively denied upon prompting having any symptoms or specific health concerns at the time they made their appointment. All consultations were videotaped and analysed for information about spontaneously mentioned symptoms and reasons for the clinic consultation ("open agendas") and for cues to hidden patient agendas using the Roter interaction analysis system (RIAS). RESULTS: All patients initially declared to be asymptomatic but this was ultimately the case in only 7 out of 66 patients. The remaining 59 patients spontaneously mentioned a mean of 4.2 ± 3.3 symptoms during their first consultation. In 23 patients a total of 31 hidden agendas were revealed. The primary categories for hidden agendas were health concerns, psychosocial concerns and the patient's concept of disease. CONCLUSIONS: The majority of patients requesting a general check-up tend to be motivated by specific symptoms and health concerns and are not "asymptomatic" patients who primarily come for preventive issues. Furthermore, physicians must be alert for possible hidden agendas, as one in three patients have one or more hidden reasons for requesting a check-up

N-acetylcysteine reduces oxidative stress in sickle cell patients

Oxidative stress is of importance in the pathophysiology of sickle cell disease (SCD). In this open label randomized pilot study the effects of oral N-acetylcysteine (NAC) on phosphatidylserine (PS) expression as marker of cellular oxidative damage (primary end point), and markers of hemolysis, coagulation and endothelial activation and NAC tolerability (secondary end points) were studied. Eleven consecutive patients (ten homozygous [HbSS] sickle cell patients, one HbSβ0-thalassemia patient) were randomly assigned to treatment with either 1,200 or 2,400 mg NAC daily during 6 weeks. The data indicate an increment in whole blood glutathione levels and a decrease in erythrocyte outer membrane phosphatidylserine exposure, plasma levels of advanced glycation end-products (AGEs) and cell-free hemoglobin after 6 weeks of NAC treatment in both dose groups. One patient did not tolerate the 2,400 mg dose and continued with the 1,200 mg dose. During the study period, none of the patients experienced painful crises or other significant SCD or NAC related complications. These data indicate that N-acetylcysteine treatment of sickle cell patients may reduce SCD related oxidative stress