P2Y₂ Nucleotide Receptors Expressed Heterologously in Sympathetic Neurons Inhibit Both N-Type Ca²⁺ and M-Type K⁺ Currents

The P2Y₂ receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y₂ receptor to neuronal N-type Ca²⁺ channels and to M-type K⁺ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y₂cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y₂nucleotide receptors of N-type calcium currents in rat sympathetic neurones. Br J Pharmacol 121:849–851], UTP inhibited the Ca²⁺ current (I_{Ca(N)} by up to 64%, with an IC₅₀ of ∼0.5 μm. We now find that UTP also inhibited the K⁺_{M} current (I_{K(M)} by up to 61%, with an IC₅₀ of ∼1.5 μm. UTP had no effect on either current in neurons not injected with P2Y₂ cRNA. Structure–activity relations for the inhibition of I_{Ca(N)} and I_{K(M)} in P2Y₂ cRNA-injected neurons were similar, with UTP ≥ ATP > ITP ≫ GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment withPertussis toxin (PTX) did not affect UTP-induced inhibition of I_{K(M)} but reduced inhibition of I_{Ca(N)} by ∼60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y₂ receptor can couple to at least two G-proteins to inhibit both Ca²⁺_{N} and K⁺_{M} channels with near-equal facility. This implies that the P2Y₂ receptor may induce a broad range of effector responses in the nervous system

Changes in undergraduate student alcohol consumption as they progress through university

BACKGROUND: Unhealthy alcohol use amongst university students is a major public health concern. Although previous studies suggest a raised level of consumption amongst the UK student population there is little consistent information available about the pattern of alcohol consumption as they progress through university. The aim of the current research was to describe drinking patterns of UK full-time undergraduate students as they progress through their degree course. METHOD: Data were collected over three years from 5895 undergraduate students who began their studies in either 2000 or 2001. Longitudinal data (i.e. Years 1–3) were available from 225 students. The remaining 5670 students all responded to at least one of the three surveys (Year 1 n = 2843; Year 2 n = 2219; Year 3 n = 1805). Results: Students reported consuming significantly more units of alcohol per week at Year 1 than at Years 2 or 3 of their degree. Male students reported a higher consumption of units of alcohol than their female peers. When alcohol intake was classified using the Royal College of Physicians guidelines [1] there was no difference between male and females students in terms of the percentage exceeding recommended limits. Compared to those who were low level consumers students who reported drinking above low levels at Year 1 had at least 10 times the odds of continuing to consume above low levels at year 3. Students who reported higher levels of drinking were more likely to report that alcohol had a negative impact on their studies, finances and physical health. Consistent with the reduction in units over time students reported lower levels of negative impact during Year 3 when compared to Year 1. CONCLUSION: The current findings suggest that student alcohol consumption declines over their undergraduate studies; however weekly levels of consumption at Year 3 remain high for a substantial number of students. The persistence of high levels of consumption in a large population of students suggests the need for effective preventative and treatment interventions for all year groups

Sporormiella as a tool for detecting the presence of large herbivores in the Neotropics

The reliability of using the abundance of Sporormiella spores as a proxy for the presence and abundance of megaherbivores was tested in southern Brazil. Mud-water interface samples from nine lakes, in which cattle-use was categorized as high, medium, or low, were assayed for Sporormiella representation. The sampling design allowed an analysis of both the influence of the number of animals using the shoreline and the distance of the sampling site from the nearest shoreline. Sporormiella was found to be a reliable proxy for the presence of large livestock. The concentration and abundance of spores declined from the edge of the lake toward the center, with the strongest response being in sites with high livestock use. Consistent with prior studies in temperate regions, we find that Sporormiella spores are a useful proxy to study the extinction of Pleistocene megafauna or the arrival of European livestock in Neotropical landscapes

Multilevel analysis in CSCL Research

Janssen, J., Erkens, G., Kirschner, P. A., & Kanselaar, G. (2011). Multilevel analysis in CSCL research. In S. Puntambekar, G. Erkens, & C. Hmelo-Silver (Eds.), Analyzing interactions in CSCL: Methods, approaches and issues (pp. 187-205). New York: Springer. doi:10.1007/978-1-4419-7710-6_9CSCL researchers are often interested in the processes that unfold between learners in online learning environments and the outcomes that stem from these interactions. However, studying collaborative learning processes is not an easy task. Researchers have to make quite a few methodological decisions such as how to study the collaborative process itself (e.g., develop a coding scheme or a questionnaire), on the appropriate unit of analysis (e.g., the individual or the group), and which statistical technique to use (e.g., descriptive statistics, analysis of variance, correlation analysis). Recently, several researchers have turned to multilevel analysis (MLA) to answer their research questions (e.g., Cress, 2008; De Wever, Van Keer, Schellens, & Valcke, 2007; Dewiyanti, Brand-Gruwel, Jochems, & Broers, 2007; Schellens, Van Keer, & Valcke, 2005; Strijbos, Martens, Jochems, & Broers, 2004; Stylianou-Georgiou, Papanastasiou, & Puntambekar, chapter #). However, CSCL studies that apply MLA analysis still remain relatively scarce. Instead, many CSCL researchers continue to use ‘traditional’ statistical techniques (e.g., analysis of variance, regression analysis), although these techniques may not be appropriate for what is being studied. An important aim of this chapter is therefore to explain why MLA is often necessary to correctly answer the questions CSCL researchers address. Furthermore, we wish to highlight the consequences of failing to use MLA when this is called for, using data from our own studies

RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.Fil: Merino, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Zamponi, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Vranych, Cecilia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

A primary care, multi-disciplinary disease management program for opioid-treated patients with chronic non-cancer pain and a high burden of psychiatric comorbidity

BACKGROUND: Chronic non-cancer pain is a common problem that is often accompanied by psychiatric comorbidity and disability. The effectiveness of a multi-disciplinary pain management program was tested in a 3 month before and after trial. METHODS: Providers in an academic general medicine clinic referred patients with chronic non-cancer pain for participation in a program that combined the skills of internists, clinical pharmacists, and a psychiatrist. Patients were either receiving opioids or being considered for opioid therapy. The intervention consisted of structured clinical assessments, monthly follow-up, pain contracts, medication titration, and psychiatric consultation. Pain, mood, and function were assessed at baseline and 3 months using the Brief Pain Inventory (BPI), the Center for Epidemiological Studies-Depression Scale scale (CESD) and the Pain Disability Index (PDI). Patients were monitored for substance misuse. RESULTS: Eighty-five patients were enrolled. Mean age was 51 years, 60% were male, 78% were Caucasian, and 93% were receiving opioids. Baseline average pain was 6.5 on an 11 point scale. The average CESD score was 24.0, and the mean PDI score was 47.0. Sixty-three patients (73%) completed 3 month follow-up. Fifteen withdrew from the program after identification of substance misuse. Among those completing 3 month follow-up, the average pain score improved to 5.5 (p = 0.003). The mean PDI score improved to 39.3 (p < 0.001). Mean CESD score was reduced to 18.0 (p < 0.001), and the proportion of depressed patients fell from 79% to 54% (p = 0.003). Substance misuse was identified in 27 patients (32%). CONCLUSIONS: A primary care disease management program improved pain, depression, and disability scores over three months in a cohort of opioid-treated patients with chronic non-cancer pain. Substance misuse and depression were common, and many patients who had substance misuse identified left the program when they were no longer prescribed opioids. Effective care of patients with chronic pain should include rigorous assessment and treatment of these comorbid disorders and intensive efforts to insure follow up

NEXT-100 Technical Design Report (TDR). Executive Summary

In this Technical Design Report (TDR) we describe the NEXT-100 detector that will search for neutrinoless double beta decay (bbonu) in Xe-136 at the Laboratorio Subterraneo de Canfranc (LSC), in Spain. The document formalizes the design presented in our Conceptual Design Report (CDR): an electroluminescence time projection chamber, with separate readout planes for calorimetry and tracking, located, respectively, behind cathode and anode. The detector is designed to hold a maximum of about 150 kg of xenon at 15 bar, or 100 kg at 10 bar. This option builds in the capability to increase the total isotope mass by 50% while keeping the operating pressure at a manageable level. The readout plane performing the energy measurement is composed of Hamamatsu R11410-10 photomultipliers, specially designed for operation in low-background, xenon-based detectors. Each individual PMT will be isolated from the gas by an individual, pressure resistant enclosure and will be coupled to the sensitive volume through a sapphire window. The tracking plane consists in an array of Hamamatsu S10362-11-050P MPPCs used as tracking pixels. They will be arranged in square boards holding 64 sensors (8 times8) with a 1-cm pitch. The inner walls of the TPC, the sapphire windows and the boards holding the MPPCs will be coated with tetraphenyl butadiene (TPB), a wavelength shifter, to improve the light collection.Comment: 32 pages, 22 figures, 5 table