Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRα1 ECD, we generated a model of the human GlyRα1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRα1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.     

Oral contraceptives and the risk of gallbladder disease: a comparative safety study

Background

Recent concerns have been raised about the risk of gallbladder disease associated with the use of drospirenone, a fourth-generation progestin used in oral contraceptives. We conducted a study to determine the magnitude of this risk compared with other formulations of oral contraceptives.

Methods

We conducted a retrospective cohort study using the IMS LifeLink Health Plan Claims Database. We included women who were using an oral contraceptive containing ethinyl estradiol combined with a progestin during 1997–2009. To be eligible, women had to have been taking the oral contraceptive continuously for at least six months. We computed adjusted rate ratios (RRs) for gallbladder disease using a Cox proportional hazards model. In the primary analysis, gallbladder disease was defined as cholecystectomy; in a secondary analysis, it was defined as hospital admission secondary to gallbladder disease.

Results

We included 2 721 014 women in the cohort, 27 087 of whom underwent surgical or laparoscopic cholecystectomy during the follow-up period. Compared with levonorgestrel, an older second-generation progestin, a small, statistically significant increase in the risk of gallbladder disease was associated with desogestrel (adjusted RR 1.05, 95% confidence interval [CI] 1.01–1.09), drospirenone (adjusted RR 1.20, 95% CI 1.16–1.26) and norethindrone (adjusted RR 1.10, 95% CI 1.06–1.14). No statistically significant increase in risk was associated with the other formulations of oral contraceptive (ethynodiol diacetate, norgestrel and norgestimate).

Interpretation

In a large cohort of women using oral contraceptives, we found a small, statistically significant increase in the risk of gallbladder disease associated with desogestrel, drospirenone and norethindrone compared with levonorgestrel. However, the small effect sizes compounded with the possibility of residual biases in this observational study make it unlikely that these differences are clinically significant.Oral contraceptives are the most popular mode of birth control among women and are used by about 100 million women worldwide.¹ Long-term use of these drugs has been associated with a variety of serious adverse events, including deep vein thrombosis, stroke and pulmonary embolism.² In addition, both estrogen and progesterone have been shown to play an important role in the formation of gallstones.^3–5 However, the relative risk of gallbladder disease associated with different formulations of oral contraceptives, including newer formulations, is unknown.Recently, there have been concerns expressed in the media about reports of gallbladder disease necessitating cholecystectomy associated with the use of drospirenone, a fourth-generation progestin.⁶ Drospirenone combined with ethinyl estradiol is primarily marketed as Yaz and Yasmin in Canada and the United States and is one of the most prescribed oral contraceptives in North America, with worldwide sales of $2 billion in 2009.⁷ The scientific evidence on the risk of gallbladder disease associated with drospirenone consists of only anecdotal or spontaneous reports in databases of adverse drug events.A possible link between drospirenone and gallbladder disease may lead to cholecystectomy and possible surgical complications.⁸ If there were a substantial risk of gallbladder disease with drospirenone, this might influence its overall risk–benefit ratio and could prompt physicians to prescribe safer alternatives. Given that women using oral contraceptives have been found to be at increased risk of gallbladder disease compared with women not using oral contraceptives,⁴ any excess risk associated with the use of drospirenone merits quantification within the context of a comparative safety study.     

Multivariate analysis of digital gene expression profiles identifies a xylem signature of the vascular tissue of white spruce (Picea glauca)

A collection of cDNA libraries from white spruce (Picea glauca) and interior spruce (P. glauca × engelmanii) vascular tissue were analyzed to identify a set of genes that could serve as tissue-related markers within the coniferous vascular system. Multivariate exploratory methods identified up to 128 genes co-expressed similarly among three xylem libraries. The majority (87) of these genes formed three distinctive meta-clusters, denoting putative gene cliques in xylem tissue. Of the selected genes, 33 (25%) exhibited no significant sequence homology in queries against any public databases, indicating the possibility of their unique expression in the xylem tissue of conifers. Another 38 genes (30%) had ambiguous annotation. Validation of the annotated genes with analog data, obtained from a wet-lab study utilizing microarray slides with 18,881 spots, resulted in a screened list of 29 genes as xylem-related markers. Response to stress was the predominant category to which the screened genes corresponded. Among the screened genes, elements of the phenolics biosynthesis, cinnamyl alcohol dehydrogenase and laccase, together with the fundamental enzyme of the cell wall biosynthesis, cellulose synthase, prominently delineated characteristics of the wood-forming tissue, xylem.     

Nonparametric inverse-probability-weighted estimators based on the highly adaptive lasso

Inverse-probability-weighted estimators are the oldest and potentially most commonly used class of procedures for the estimation of causal effects. By adjusting for selection biases via a weighting mechanism, these procedures estimate an effect of interest by constructing a pseudopopulation in which selection biases are eliminated. Despite their ease of use, these estimators require the correct specification of a model for the weighting mechanism, are known to be inefficient, and suffer from the curse of dimensionality. We propose a class of nonparametric inverse-probability-weighted estimators in which the weighting mechanism is estimated via undersmoothing of the highly adaptive lasso, a nonparametric regression function proven to converge at nearly $n^{-1/3}$-rate to the true weighting mechanism. We demonstrate that our estimators are asymptotically linear with variance converging to the nonparametric efficiency bound. Unlike doubly robust estimators, our procedures require neither derivation of the efficient influence function nor specification of the conditional outcome model. Our theoretical developments have broad implications for the construction of efficient inverse-probability-weighted estimators in large statistical models and a variety of problem settings. We assess the practical performance of our estimators in simulation studies and demonstrate use of our proposed methodology with data from a large-scale epidemiologic study.     

Turbidimetric analysis of growth kinetics of bacteria in the laboratory environment using smartphone

For different microbiological and pathological studies, it is often required to monitor the growth of bacteria in a cultured medium in the laboratory environment. UV‐VIS spectrophotometer is commonly used to estimate the growth of bacterial cell population by measuring the absorbance at 600 nm over a period of time. Colony‐forming unit (CFU) is another approach, which has been routinely performed to estimate the live bacterial cells on semisolid agar plates. Herein, we demonstrate an alternative yet highly reliable sensing platform on a smartphone using which growth kinetics of different bacteria can be reliably monitored. The performance of the proposed smartphone sensor has been compared with the data obtained from OD600 and CFU analysis. A good correlation of bacterial growth rates enumerated based on the proposed smartphone sensor, bench‐top spectrophotometer and CFU analysis have been observed under the experimental conditions.     

Decoding Cellular Mechanisms for Mechanosensory Discrimination

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Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure

Systematic studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo and ex vivo model systems. Thus, we set out to develop a reproducible cell culture system that can accurately replicate the realistic microstructure of native cardiac tissues. Using cell micropatterning techniques, we aligned cultured cardiomyocytes at micro- and macroscopic spatial scales to follow local directions of cardiac fibers in murine ventricular cross sections, as measured by high-resolution diffusion tensor magnetic resonance imaging. To elucidate the roles of ventricular tissue microstructure in macroscopic impulse conduction, we optically mapped membrane potentials in micropatterned cardiac cultures with realistic tissue boundaries and natural cell orientation, cardiac cultures with realistic tissue boundaries but random cell orientation, and standard isotropic monolayers. At 2 Hz pacing, both microscopic changes in cell orientation and ventricular tissue boundaries independently and synergistically increased the spatial dispersion of conduction velocity, but not the action potential duration. The realistic variations in intramural microstructure created unique spatial signatures in micro- and macroscopic impulse propagation within ventricular cross-section cultures. This novel in vitro model system is expected to help bridge the existing gap between experimental structure-function studies in standard cardiac monolayers and intact heart tissues.     

Design and utilization of epitope-based databases and predictive tools

In the last decade, significant progress has been made in expanding the scope and depth of publicly available immunological databases and online analysis resources, which have become an integral part of the repertoire of tools available to the scientific community for basic and applied research. Herein, we present a general overview of different resources and databases currently available. Because of our association with the Immune Epitope Database and Analysis Resource, this resource is reviewed in more detail. Our review includes aspects such as the development of formal ontologies and the type and breadth of analytical tools available to predict epitopes and analyze immune epitope data. A common feature of immunological databases is the requirement to host large amounts of data extracted from disparate sources. Accordingly, we discuss and review processes to curate the immunological literature, as well as examples of how the curated data can be used to generate a meta-analysis of the epitope knowledge currently available for diseases of worldwide concern, such as influenza and malaria. Finally, we review the impact of immunological databases, by analyzing their usage and citations, and by categorizing the type of citations. Taken together, the results highlight the growing impact and utility of immunological databases for the scientific community.