Quantitative changes in intracellular calcium and extracellular-regulated kinase activation measured in parallel in CHO cells stably expressing serotonin (5-HT) 5-HT2A or 5-HT2C receptors

<p>Abstract</p> <p>Background</p> <p>The serotonin (5-HT) 2A and 2C receptors (5-HT_2AR and 5-HT_2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (<it>Ca_i</it>⁺⁺) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands.</p> <p>Results</p> <p>We optimized the <it>Ca_i</it>⁺⁺assay for stable cell lines expressing either 5-HT_2AR or 5-HT_2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT_2AR- and 5-HT_2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT_2AR-expressing cells showed significant release of <it>Ca_i</it>⁺⁺in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT_2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.</p> <p>Conclusions</p> <p>These data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT_2AR and 5-HT_2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.</p

First-Year Spectroscopy for the SDSS-II Supernova Survey

This paper presents spectroscopy of supernovae discovered in the first season of the Sloan Digital Sky Survey-II Supernova Survey. This program searches for and measures multi-band light curves of supernovae in the redshift range z = 0.05 - 0.4, complementing existing surveys at lower and higher redshifts. Our goal is to better characterize the supernova population, with a particular focus on SNe Ia, improving their utility as cosmological distance indicators and as probes of dark energy. Our supernova spectroscopy program features rapid-response observations using telescopes of a range of apertures, and provides confirmation of the supernova and host-galaxy types as well as precise redshifts. We describe here the target identification and prioritization, data reduction, redshift measurement, and classification of 129 SNe Ia, 16 spectroscopically probable SNe Ia, 7 SNe Ib/c, and 11 SNe II from the first season. We also describe our efforts to measure and remove the substantial host galaxy contamination existing in the majority of our SN spectra.Comment: Accepted for publication in The Astronomical Journal(47pages, 9 figures

Alternative Splicing and Nonsense-Mediated RNA Decay Contribute to the Regulation of SHOX Expression

The human SHOX gene is composed of seven exons and encodes a paired-related homeodomain transcription factor. SHOX mutations or deletions have been associated with different short stature syndromes implying a role in growth and bone formation. During development, SHOX is expressed in a highly specific spatiotemporal expression pattern, the underlying regulatory mechanisms of which remain largely unknown. We have analysed SHOX expression in diverse embryonic, fetal and adult human tissues and detected expression in many tissues that were not known to express SHOX before, e.g. distinct brain regions. By using RT-PCR and comparing the results with RNA-Seq data, we have identified four novel exons (exon 2a, 7-1, 7-2 and 7-3) contributing to different SHOX isoforms, and also established an expression profile for the emerging new SHOX isoforms. Interestingly, we found the exon 7 variants to be exclusively expressed in fetal neural tissues, which could argue for a specific role of these variants during brain development. A bioinformatical analysis of the three novel 3′UTR exons yielded insights into the putative role of the different 3′UTRs as targets for miRNA binding. Functional analysis revealed that inclusion of exon 2a leads to nonsense-mediated RNA decay altering SHOX expression in a tissue and time specific manner. In conclusion, SHOX expression is regulated by different mechanisms and alternative splicing coupled with nonsense-mediated RNA decay constitutes a further component that can be used to fine tune the SHOX expression level

The Sloan Digital Sky Survey-II: Photometry and Supernova Ia Light Curves from the 2005 data

We present ugriz light curves for 146 spectroscopically confirmed or spectroscopically probable Type Ia supernovae from the 2005 season of the SDSS-II Supernova survey. The light curves have been constructed using a photometric technique that we call scene modelling, which is described in detail here; the major feature is that supernova brightnesses are extracted from a stack of images without spatial resampling or convolution of the image data. This procedure produces accurate photometry along with accurate estimates of the statistical uncertainty, and can be used to derive photometry taken with multiple telescopes. We discuss various tests of this technique that demonstrate its capabilities. We also describe the methodology used for the calibration of the photometry, and present calibrated magnitudes and fluxes for all of the spectroscopic SNe Ia from the 2005 season

The Sloan Digital Sky Survey-II: Photometry and Supernova Ia Light Curves from the 2005 Data

We present ugriz light curves for 146 spectroscopically confirmed or spectroscopically probable Type Ia supernovae from the 2005 season of the SDSS-II Supernova survey. The light curves have been constructed using a photometric technique that we call scene modeling, which is described in detail here; the major feature is that supernova brightnesses are extracted from a stack of images without spatial resampling or convolution of the image data. This procedure produces accurate photometry along with accurate estimates of the statistical uncertainty, and can be used to derive photometry taken with multiple telescopes. We discuss various tests of this technique that demonstrate its capabilities. We also describe the methodology used for the calibration of the photometry, and present calibrated magnitudes and fluxes for all of the spectroscopic SNe Ia from the 2005 season

The Sloan Digital Sky Survey-II Supernova Survey: Technical Summary

The Sloan Digital Sky Survey-II (SDSS-II) has embarked on a multi-year project to identify and measure light curves for intermediate-redshift (0.05 &lt; z &lt; 0.35) Type Ia supernovae (SNe Ia) using repeated five-band (ugriz) imaging over an area of 300 sq. deg. The survey region is a stripe 2.5 degrees wide centered on the celestial equator in the Southern Galactic Cap that has been imaged numerous times in earlier years, enabling construction of a deep reference image for discovery of new objects. Supernova imaging observations are being acquired between 1 September and 30 November of 2005-7. During the first two seasons, each region was imaged on average every five nights. Spectroscopic follow-up observations to determine supernova type and redshift are carried out on a large number of telescopes. In its first two three-month seasons, the survey has discovered and measured light curves for 327 spectroscopically confirmed SNe Ia, 30 probable SNe Ia, 14 confirmed SNe Ib/c, 32 confirmed SNe II, plus a large number of photometrically identified SNe Ia, 94 of which have host-galaxy spectra taken so far. This paper provides an overview of the project and briefly describes the observations completed during the first two seasons of operation

Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A

The major histocompatibility complex (MHC) on chromosome 6 is associated with susceptibility to more common diseases than any other region of the human genome, including almost all disorders classified as autoimmune. In type 1 diabetes the major genetic susceptibility determinants have been mapped to the MHC class II genes HLA-DQB1 and HLA-DRB1 (refs 1-3), but these genes cannot completely explain the association between type 1 diabetes and the MHC region. Owing to the region's extreme gene density, the multiplicity of disease-associated alleles, strong associations between alleles, limited genotyping capability, and inadequate statistical approaches and sample sizes, which, and how many, loci within the MHC determine susceptibility remains unclear. Here, in several large type 1 diabetes data sets, we analyse a combined total of 1,729 polymorphisms, and apply statistical methods - recursive partitioning and regression - to pinpoint disease susceptibility to the MHC class I genes HLA-B and HLA-A (risk ratios >1.5; Pcombined = 2.01 × 10-19 and 2.35 × 10-13, respectively) in addition to the established associations of the MHC class II genes. Other loci with smaller and/or rarer effects might also be involved, but to find these, future searches must take into account both the HLA class II and class I genes and use even larger samples. Taken together with previous studies, we conclude that MHC-class-I-mediated events, principally involving HLA-B*39, contribute to the aetiology of type 1 diabetes. ©2007 Nature Publishing Group

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points