The Nicotine Metabolite, Cotinine, Alters the Assembly and Trafficking of a Subset of Nicotinic Acetylcholine Receptors

Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs), leading to their up-regulation on the plasma membrane. Although the mechanism is not fully understood, nicotine-induced up-regulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH-sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry

Organelle-Specific Single-Molecule Imaging of \u3cem\u3eα\u3c/em\u3e4\u3cem\u3eβ\u3c/em\u3e2 Nicotinic Receptors Reveals the Effect of Nicotine on Receptor Assembly and Cell-Surface Trafficking

Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER versus those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure

Utilizing pHluorin-Tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins

Mammalian Cell-Derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly

A Cross-Correlation Analysis of Mg II Absorption Line Systems and Luminous Red Galaxies from the SDSS DR5

We analyze the cross-correlation of 2,705 unambiguously intervening Mg II (2796,2803A) quasar absorption line systems with 1,495,604 luminous red galaxies (LRGs) from the Fifth Data Release of the Sloan Digital Sky Survey within the redshift range 0.36<=z<=0.8. We confirm with high precision a previously reported weak anti-correlation of equivalent width and dark matter halo mass, measuring the average masses to be log M_h(M_[solar]h^-1)=11.29 [+0.36,-0.62] and log M_h(M_[solar]h^-1)=12.70 [+0.53,-1.16] for systems with W[2796A]>=1.4A and 0.8A<=W[2796A]<1.4A, respectively. Additionally, we investigate the significance of a number of potential sources of bias inherent in absorber-LRG cross-correlation measurements, including absorber velocity distributions and the weak lensing of background quasars, which we determine is capable of producing a 20-30% bias in angular cross-correlation measurements on scales less than 2'. We measure the Mg II - LRG cross-correlation for 719 absorption systems with v<60,000 km s^-1 in the quasar rest frame and find that these associated absorbers typically reside in dark matter haloes that are ~10-100 times more massive than those hosting unambiguously intervening Mg II absorbers. Furthermore, we find evidence for evolution of the redshift number density, dN/dz, with 2-sigma significance for the strongest (W>2.0A) absorbers in the DR5 sample. This width-dependent dN/dz evolution does not significantly affect the recovered equivalent width-halo mass anti-correlation and adds to existing evidence that the strongest Mg II absorption systems are correlated with an evolving population of field galaxies at z<0.8, while the non-evolving dN/dz of the weakest absorbers more closely resembles that of the LRG population.Comment: 21 pages, 19 figures; Published in Astrophysical Journa

Environmental effects on water intake and water intake prediction in growing beef cattle

Water is an essential nutrient, but there are few recent studies that evaluate how much water individual beef cattle consume and how environmental factors affect an individual’s water intake (WI). Most studies have focused on WI of whole pens rather than WI of individual animals. Thus, the objective of this study was to evaluate the impact of environmental parameters on individual-animal WI across different seasons and develop prediction equations to estimate WI, including within different environments and management protocols. Individual daily feed intake and WI records were collected on 579 crossbred steers for a 70-d period following a 21-d acclimation period for feed and water bunk training. Steers were fed in 5 separate groups over a 3-yr period from May 2014 to March 2017. Individual weights were collected every 14 d and weather data were retrieved from the Oklahoma Mesonet’s Stillwater station. Differences in WI as a percent of body weight (WI%) were analyzed accounting for average temperature (TAVG), relative humidity (HAVG), solar radiation (SRAD), and wind speed (WSPD). Seasonal (summer vs. winter) and management differences (ad libitum vs. slick bunk) were examined. Regression analysis was utilized to generate 5 WI prediction equations (overall, summer, winter, slick, and ad libitum). There were significant (P \u3c 0.05) differences in WI between all groups when no environmental parameters were included in the model. Although performance was more similar after accounting for all differences in weather variables, significant (P \u3c 0.05) seasonal and feed management differences were still observed for WI%, but were less than 0.75% of steer body weight. The best linear predictors of daily WI (DWI) were dry mater intake (DMI), metabolic body weights (MWTS), TAVG, SRAD, HAVG, and WSPD. Slight differences in the coefficient of determinations for the various models were observed for the summer (0.34), winter (0.39), ad libitum (0.385), slick bunk (0.41), and overall models (0.40). Based on the moderate R2 values for the WI prediction equations, individual DWI can be predicted with reasonable accuracy based on the environmental conditions that are present, MWTS, and DMI consumed, but substantial variation exists in individual animal WI that is not accounted for by these models

Recruitment and follow-up of adolescent and young adult cancer survivors: the AYA HOPE Study

IntroductionCancer is rare in adolescents and young adults (AYA), but these patients have seen little improvement in survival in contrast to most other age groups. Furthermore, participation in research by AYAs is typically low. We conducted a study to examine the feasibility of recruiting a population-based sample of AYA survivors to examine issues of treatment and health outcomes.MethodsIndividuals diagnosed in 2007-08 and age 15-39 at the time of diagnosis with acute lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, germ cell cancer or sarcoma were identified by 7 Surveillance, Epidemiology, and End-Results (SEER) cancer registries, mailed surveys within 14 months after diagnosis and again a year later, and had medical records reviewed.Results525 (43%) of the eligible patients responded, 39% refused and 17% were lost to follow-up. Extensive efforts were required for most potential respondents (87%). 76% of respondents completed the paper rather than online survey version. In a multivariate model, age, cancer site, education and months from diagnosis to the first mailing of the survey were not associated with participation, although males (p  &lt;  0.01), Hispanics and non-Hispanic blacks (p  &lt;  0.001) were less likely to participate. 91% of survivors completing the initial survey completed the subsequent survey.DiscussionDespite the response rate, those who participated adequately reflected the population of AYA cancer survivors. The study demonstrates that cancer registries are valuable foundations for conducting observational, longitudinal population-based research on AYA cancer survivors.Implications for cancer survivorsAchieving a reasonable response rate in this population is possible, but requires extensive resources

Genome-wide association study identifies 30 loci associated with bipolar disorder.

Bipolar disorder is a highly heritable psychiatric disorder. We performed a genome-wide association study (GWAS) including 20,352 cases and 31,358 controls of European descent, with follow-up analysis of 822 variants with P &lt; 1 × 10-4 in an additional 9,412 cases and 137,760 controls. Eight of the 19 variants that were genome-wide significant (P &lt; 5 × 10-8) in the discovery GWAS were not genome-wide significant in the combined analysis, consistent with small effect sizes and limited power but also with genetic heterogeneity. In the combined analysis, 30 loci were genome-wide significant, including 20 newly identified loci. The significant loci contain genes encoding ion channels, neurotransmitter transporters and synaptic components. Pathway analysis revealed nine significantly enriched gene sets, including regulation of insulin secretion and endocannabinoid signaling. Bipolar I disorder is strongly genetically correlated with schizophrenia, driven by psychosis, whereas bipolar II disorder is more strongly correlated with major depressive disorder. These findings address key clinical questions and provide potential biological mechanisms for bipolar disorder