Ethanol/Naltrexone Interactions at the mu-Opioid Receptor. CLSM/FCS Study in Live Cells

BACKGROUND: Alcoholism is a widespread chronic disorder of complex aetiology with a significant negative impact on the individual and the society. Mechanisms of ethanol action are not sufficiently well understood at the molecular level and the pharmacotherapy of alcoholism is still in its infancy. Our study focuses at the cellular and molecular level on ethanol-induced effects that are mediated through the micro-opioid receptor (MOP) and on the effects of naltrexone, a well-known antagonist at MOP that is used clinically to prevent relapse in alcoholism. METHODOLOGY/PRINCIPAL FINDINGS: Advanced fluorescence imaging by Confocal Laser Scanning Microscopy (CLSM) and Fluorescence Correlation Spectroscopy (FCS) are used to study ethanol effects on MOP and plasma membrane lipid dynamics in live PC12 cells. We observed that relevant concentrations of ethanol (10-40 mM) alter MOP mobility and surface density, and affect the dynamics of plasma membrane lipids. Compared to the action of specific ligands at MOP, ethanol-induced effects show complex kinetics and point to a biphasic underlying mechanism. Pretreatment with naloxone or naltrexone considerably mitigates the effects of ethanol. CONCLUSIONS/SIGNIFICANCE: We suggest that ethanol acts by affecting the sorting of MOP at the plasma membrane of PC12 cells. Naltrexone exerts opposite effects on MOP sorting at the plasma membrane, thereby countering the effects of ethanol. Our experimental findings give new insight on MOP-mediated ethanol action at the cellular and molecular level. We suggest a new hypothesis to explain the well established ethanol-induced increase in the activity of the endogenous opioid system

Impact of a modified data capture period on Liu comorbidity index scores in Medicare enrollees initiating chronic dialysis

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author’s publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Background: The Liu Comorbidity Index uses the United States Renal Data System (USRDS) to quantify comorbidity in chronic dialysis patients, capturing baseline comorbidities from days 91 through 270 after dialysis initiation. The 270 day survival requirement results in sample size reductions and potential survivor bias. An earlier and shorter time-frame for data capture could be beneficial, if sufficiently similar comorbidity information could be ascertained. Methods: USRDS data were used in a retrospective observational study of 70,114 Medicare- and Medicaid-eligible persons who initiated chronic dialysis during the years 2000–2005. The Liu index was modified by changing the baseline comorbidity capture period to days 1–90 after dialysis initiation for persons continuously enrolled in Medicare. The scores resulting from the original and the modified comorbidity indices were compared, and the impact on sample size was calculated. Results: The original Liu comorbidity index could be calculated for 75% of the sample, but the remaining 25% did not survive to 270 days. Among 52,937 individuals for whom both scores could be calculated, the mean scores for the original and the modified index were 7.4 ± 4.0 and 6.4 ± 3.6 points, respectively, on a 24-point scale. The most commonly calculated difference between scores was zero, occurring in 44% of patients. Greater comorbidity was found in those who died before 270 days. Conclusions: A modified version of the Liu comorbidity index captures the majority of comorbidity in persons who are Medicare-enrolled at the time of chronic dialysis initiation. This modification reduces sample size losses and facilitates inclusion of a sicker portion of the population in whom early mortality is common. Keywords: Comorbidity, Kidney failure, Chronic, Renal dialysis, Epidemiologic research desig

Cladoceran birth and death rates estimates

I. Birth and death rates of natural cladoceran populations cannot be measured directly. Estimates of these population parameters must be calculated using methods that make assumptions about the form of population growth. These methods generally assume that the population has a stable age distribution. 2. To assess the effect of variable age distributions, we tested six egg ratio methods for estimating birth and death rates with data from thirty-seven laboratory populations of Daphnia pulicaria. The populations were grown under constant conditions, but the initial age distributions and egg ratios of the populations varied. Actual death rates were virtually zero, so the difference between the estimated and actual death rates measured the error in both birth and death rate estimates. 3. The results demonstrate that unstable population structures may produce large errors in the birth and death rates estimated by any of these methods. Among the methods tested, Taylor and Slatkin's formula and Paloheimo's formula were most reliable for the experimental data. 4. Further analyses of three of the methods were made using computer simulations of growth of age-structured populations with initially unstable age distributions. These analyses show that the time interval between sampling strongly influences the reliability of birth and death rate estimates. At a sampling interval of 2.5 days (equal to the duration of the egg stage), Paloheimo's formula was most accurate. At longer intervals (7.5–10 days), Taylor and Slatkin's formula which includes information on population structure was most accurate

Studying Flow Close to an Interface by Total Internal Reflection Fluorescence Cross Correlation Spectroscopy: Quantitative Data Analysis

Total Internal Reflection Fluorescence Cross Correlation Spectroscopy (TIR-FCCS) has recently (S. Yordanov et al., Optics Express 17, 21149 (2009)) been established as an experimental method to probe hydrodynamic flows near surfaces, on length scales of tens of nanometers. Its main advantage is that fluorescence only occurs for tracer particles close to the surface, thus resulting in high sensitivity. However, the measured correlation functions only provide rather indirect information about the flow parameters of interest, such as the shear rate and the slip length. In the present paper, we show how to combine detailed and fairly realistic theoretical modeling of the phenomena by Brownian Dynamics simulations with accurate measurements of the correlation functions, in order to establish a quantitative method to retrieve the flow properties from the experiments. Firstly, Brownian Dynamics is used to sample highly accurate correlation functions for a fixed set of model parameters. Secondly, these parameters are varied systematically by means of an importance-sampling Monte Carlo procedure in order to fit the experiments. This provides the optimum parameter values together with their statistical error bars. The approach is well suited for massively parallel computers, which allows us to do the data analysis within moderate computing times. The method is applied to flow near a hydrophilic surface, where the slip length is observed to be smaller than 10nm, and, within the limitations of the experiments and the model, indistinguishable from zero.Comment: 18 pages, 12 figure

Solitary caecal diverticulitis as an unusual cause of a right iliac fossa mass: a case report

Inflammation of a solitary caecal diverticulum is an uncommon pathological condition. Preoperatively the condition is almost indistinguishable from appendicitis, and is often confused with carcinoma of the caecum during operation. The typical patient with this condition is male, Asian, and in the fourth decade of life. This case is unusual in that the patient was a 26-year-old Caucasian man

High resolution array-CGH analysis of single cells

Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex(®) Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader–Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics

Proinsulin C-peptide elicits disaggregation of insulin resulting in enhanced physiological insulin effects

Using surface plasmon resonance (SPR) and electrospray mass spectrometry (ESI-MS), proinsulin C-peptide was found to influence insulin-insulin interactions. In SPR with chip-bound insulin, C-peptide mixed with analyte insulin increased the binding, while alone C-peptide did not. A control peptide with the same residues in random sequence had little effect. In ESI-MS, C-peptide lowered the presence of insulin hexamer. The data suggest that C-peptide promotes insulin disaggregation. Insulin/insulin oligomer μM dissociation constants were determined. Compatible with these findings, type 1 diabetic patients receiving insulin and C-peptide developed 66% more stimulation of glucose metabolism than when given insulin alone. A role of C-peptide in promoting insulin disaggregation may be important physiologically during exocytosis of pancreatic β-cell secretory granulae and pharmacologically at insulin injection sites. It is compatible with the normal co-release of C-peptide and insulin and may contribute to the beneficial effect of C-peptide and insulin replacement in type 1 diabetics

High-redshift quasar host galaxies with adaptive optics

We present K band adaptive optics observations of three high-redshift (z ~ 2.2) high-luminosity quasars, all of which were studied for the first time. We also bserved several point spread function (PSF) calibrators, non-simultaneously because of the small field of view. The significant temporal PSF variations on timescales of minutes inhibited a straightforward scaled PSF removal from the quasar images. Characterising the degree of PSF concentration by the radii encircling 20% and 80% of the total flux, respectively, we found that even under very different observing conditions the r_20 vs. r_80 relation varied coherently between individual short exposure images, delineating a well-defined relation for point sources. Placing the quasar images on this relation, we see indications that all three objects were resolved. We designed a procedure to estimate the significance of this result, and to estimate host galaxy parameters, by reproducing the statistical distribution of the individual short exposure images. We find in all three cases evidence for a luminous host galaxy, with a mean absolute magnitude of M_R = -27.0 and scale lengths around ~ 4-12 kpc. Together with a rough estimate of the central black hole masses obtained from C_iv line widths, the location of the objects on the bulge luminosity vs. black hole mass relation is not significantly dfferent from the low-redshift regime, assuming only passive evolution of the host galaxy. Corresponding Eddington luminosities are L_nuc/L_Edd ~ 0.1-0.6.Comment: 17 pages, 16 figures; submitted to Astronomy&Astrophysics. v2: Absolute magnitudes corrected and as a consequence updated two figures, black hole masses and stellar population age estimates in Discussion, but main conclusions remain the same; accepted by A&

C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells

Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC) in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86)Rb(+)) uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM). DNA binding activity was determined by electrical mobility shift assay (EMSA). Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1)-subunit protein expression, accompanied with increase in (86)Rb(+) uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1)-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6), concomitant with Na,K-ATPase α(1)-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1)-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C-peptide-mediated signaling. Importantly, only physiological concentrations of C-peptide elicit this effect

How does community context influence coalitions in the formation stage? a multiple case study based on the Community Coalition Action Theory

<p>Abstract</p> <p>Background</p> <p>Community coalitions are rooted in complex and dynamic community systems. Despite recognition that environmental factors affect coalition behavior, few studies have examined how community context impacts coalition formation. Using the Community Coalition Action theory as an organizing framework, the current study employs multiple case study methodology to examine how five domains of community context affect coalitions in the formation stage of coalition development. Domains are history of collaboration, geography, community demographics and economic conditions, community politics and history, and community norms and values.</p> <p>Methods</p> <p>Data were from 8 sites that participated in an evaluation of a healthy cities and communities initiative in California. Twenty-three focus groups were conducted with coalition members, and 76 semi-structured interviews were conducted with local coordinators and coalition leaders. Cross-site analyses were conducted to identify the ways contextual domains influenced selection of the lead agency, coalition membership, staffing and leadership, and coalition processes and structures.</p> <p>Results</p> <p>History of collaboration influenced all four coalition factors examined, from lead agency selection to coalition structure. Geography influenced coalition formation largely through membership and staffing, whereas the demographic and economic makeup of the community had an impact on coalition membership, staffing, and infrastructure for coalition processes. The influence of community politics, history, norms and values was most noticeable on coalition membership.</p> <p>Conclusions</p> <p>Findings contribute to an ecologic and theory-based understanding of the range of ways community context influences coalitions in their formative stage.</p