In vivo analysis of a salt bridge at the external gate of the Drosophila melanogaster serotonin transporter in response to amphetamines

Monoamine neurotransmitter transporters are membrane proteins responsible for the clearing of biogenic amines from a synapse. These transporters are targets for many important pharmaceuticals including antidepressants, as well as psychostimulant drugs such as cocaine and amphetamines. Amphetamines are believed to elicit their psychostimulant activity primarily by inducing a reversal of the transport cycle and increasing neurotransmitter release into the synapse, though the mechanism of this activity is incompletely understood. Previous in vitro research has suggested functional significance of a conserved salt bridge in the serotonin transporter (SERT) in amphetamine-induced 5-HT efflux. This salt bridge is disrupted in the Drosophila melanogaster SERT. Here, a mutant line of D. melanogaster expressing a SERT with a restored salt bridge (dSERT N484D) was studied. Changes in neurochemistry induced by methamphetamine (METH) or 3, 4-methylenedioxymethamphetamine (MDMA) were examined in vivo. HPLC/Mass spectrometry was used to quantify brain concentrations of neurotransmitters in fly tissue after drug treatment. N484D flies were found to have significantly depleted 5-HT in response to 0.05% MDMA relative to wild-type. This depletion of 5-HT was not observed after treatment with 0.6% MDMA or 0.6% METH. No significant drug-induced changes were observed in concentrations of other neurotransmitters examined. The results show that the presence of the salt bridge at the external gate of SERT may be important for amphetamine-induced 5-HT efflux, and helps explain pharmacological differences observed between hSERT and dSERT

Novel Introner-Like Elements in fungi are involved in parallel gains of spliceosomal introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom

Superconductivity in a two dimensional extended Hubbard model

The Roth's two-pole approximation has been used by the present authors to investigate the role of $d-p$ hybridization in the superconducting properties of an extended $d-p$ Hubbard model. Superconductivity with singlet $d_{x^2-y^2}$-wave pairing is treated by following Beenen and Edwards formalism. In this work, the Coulomb interaction, the temperature and the superconductivity have been considered in the calculation of some relevant correlation functions present in the Roth's band shift. The behavior of the order parameter associated with temperature, hybridization, Coulomb interaction and the Roth's band shift effects on superconductivity are studied.Comment: 14 pages, 8 figures, accepted for publication in European Physical Journal

Operator projection method applied to the single-particle Green's function in the Hubbard model

A new non-perturbative framework for many-body correlated systems is formulated by extending the operator projection method (OPM). This method offers a systematic expansion which enables us to project into the low-energy structure after extracting the higher-energy hierarchy. This method also opens a way to systematically take into account the effects of collective excitations. The Mott-Hubbard metal-insulator transition in the Hubbard model is studied by means of this projection beyond the second order by taking into account magnetic and charge fluctuations in the presence of the high-energy Mott-Hubbard structure. At half filling, the Mott-Hubbard gap is correctly eproduced between the separated two bands. Near half filling, a strongly renormalized low-energy single-particle excitations coexisting with the Mott-Hubbard bands are shown to appear. Signifcance of momentum-dependent self-energy in the results is stressed.Comment: 6 pages, final version to appear in J. Phys. Soc. Jp

Dyson Equation Approach to Many-Body Greens Functions and Self-Consistent RPA, First Application to the Hubbard Model

An approach for particle-hole correlation functions, based on the so-called SCRPA, is developed. This leads to a fully self-consistent RPA-like theory which satisfies the $f$-sum rule and several other theorems. As a first step, a simpler self-consistent approach, the renormalized RPA, is solved numerically in the one-dimensional Hubbard model. The charge and the longitudinal spin susceptibility, the momentum distribution and several ground state properties are calculated and compared with the exact results. Especially at half filling, our approach provides quite promising results and matches the exact behaviour apart from a general prefactor. The strong coupling limit of our approach can be described analytically.Comment: 35 pages, 18 Figures, Feynman diagrams as 10 additional eps-files, revised and enhanced version, accepted in Phys. Rev.

Manual hyperinflation partly prevents reductions of functional residual capacity in cardiac surgical patients - a randomized controlled trial

Cardiac surgery is associated with post-operative reductions of functional residual capacity (FRC). Manual hyperinflation (MH) aims to prevent airway plugging, and as such could prevent the reduction of FRC after surgery. The main purpose of this study was to determine the effect of MH on post-operative FRC of cardiac surgical patients. This was a randomized controlled trial of patients after elective coronary artery bypass graft and/or valve surgery admitted to the intensive care unit (ICU) of a university hospital. Patients were randomly assigned to a "routine MH group" (MH was performed within 30 minutes after admission to the ICU and every 6 hours thereafter, and before tracheal extubation), or a "control group" (MH was performed only if perceptible (audible) sputum was present in the larger airways causing problems with mechanical ventilation, or if oxygen saturation (SpO2) dropped below 92%). The primary endpoint was the reduction of FRC from the day before cardiac surgery to one, three, and five days after tracheal extubation. Secondary endpoints were SpO2 (at similar time points) and chest radiograph abnormalities, including atelectasis (at three days after tracheal extubation). A total of 100 patients were enrolled. Patients in the routine MH group showed a decrease of FRC on the first post-operative day to 71% of the pre-operative value, versus 57% in the control group (P = 0.002). Differences in FRC became less prominent over time; differences between the two study groups were no longer statistically significant at Day 5. There were no differences in SpO2 between the study groups. Chest radiographs showed more abnormalities (merely atelectasis) in the control group compared to patients in the routine MH group (P = 0.002). MH partly prevents the reduction of FRC in the first post-operative days after cardiac surgery. Netherlands Trial Register (NTR): NTR1384. http://www.trialregister.n

Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction

Background To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. Results In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation. In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Conclusion Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetecte

Comparison and transfer testing of multiplex ligation detection methods for GM plants

<p>Abstract</p> <p>Background</p> <p>With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.</p> <p>Results</p> <p>Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands).</p> <p>Conclusions</p> <p>From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.</p