Dynamic transport and localization of alpha-synuclein in primary hippocampal neurons.

BACKGROUND: Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. Abnormal accumulation of alpha-synuclein aggregates in dopaminergic neurons of the substantia nigra is diagnostic of sporadic Parkinson's disease, and mutations in the protein are linked to early onset forms of the disease. The folded conformation of the protein varies depending upon its environment and other factors that are poorly understood. When bound to phospholipid membranes, alpha-synuclein adopts a helical conformation that mediates specific interactions with other proteins. RESULTS: To investigate the role of the helical domain in transport and localization of alpha-synuclein, eGFP-tagged constructs were transfected into rat primary hippocampal neurons at 7 DIV. A series of constructs were analyzed in which each individual exon was deleted, for comparison to previous studies of lipid affinity and alpha-helix content. A53T and A30P substitutions, representing Parkinson's disease-associated variants, were analyzed as well. Single exon deletions within the lipid-binding N-terminal domain of alpha-synuclein (exons 2, 3, and 4) partially disrupted its presynaptic localization at 17-21 DIV, resulting in increased diffuse labeling of axons. Similar results were obtained for A30P, which exhibits decreased lipid binding, but not A53T. To examine whether differences in presynaptic enrichment were related to deficiencies in transport velocity, transport was visualized via live cell microscopy. Tagged alpha-synuclein migrated at a rate of 1.85 +/- 0.09 mum/s, consistent with previous reports, and single exon deletion mutants migrated at similar rates, as did A30P. Deletion of the entire N-terminal lipid-binding domain (Delta234GFP) did not significantly alter rates of particle movement, but decreased the number of moving particles. Only the A53TGFP mutant exhibited a significant decrease in transport velocity as compared to ASGFP. CONCLUSIONS: These results support the hypothesis that presynaptic localization involves a mechanism that requires helical conformation and lipid binding. Conversely, the rate of axonal transport is not determined by lipid affinity and is not sufficient to account for differences in presynaptic localization of alpha-synuclein-eGFP variants.This study was funded by the Branfman Family Foundation, including salary support for MLY, LH, and WSW

Evaluation of peak-picking algorithms for protein mass spectrometry

Peak picking is an early key step in MS data analysis. We compare three commonly used approaches to peak picking and discuss their merits by means of statistical analysis. Methods investigated encompass signal-to-noise ratio, continuous wavelet transform, and a correlation-based approach using a Gaussian template. Functionality of the three methods is illustrated and discussed in a practical context using a mass spectral data set created with MALDI-TOF technology. Sensitivity and specificity are investigated using a manually defined reference set of peaks. As an additional criterion, the robustness of the three methods is assessed by a perturbation analysis and illustrated using ROC curves

Cancer pain managment: experience of 702 consecutive cases in a teaching hospital in Hong Kong

Effective pain control is essential in the management of patients with cancer. We present here our experience in the management of 702 patients with cancer pain by the Pain Management Team, Department of Anaesthesiology, Queen Mary Hospital. Patients were discharged from the Pain Management Team with a visual analogue scale of pain (VAS) less than 3 in 87.7% of cases, and more than 90% of patients had improved appetite and sleep on discharge. These promising results were achieved through an emphasis on comfort and function, close liaison among clinicians from different specialties, and a variety of analgesic modalities. Oral drugs remained the mainstay of treatment, supplemented by alternative routes of drug administration such as subcutaneous, intravenous and transdermal delivery. Main side effects observed included nausea (16%) and constipation (8%). Neural blockade, including coeliac plexus blockade, and intercostal nerve blockade, and administration of opioids via subarachnoid or epidural routes were also employed in selected patients.published_or_final_versio

Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

<p>Abstract</p> <p>Background</p> <p>SXT is an integrating conjugative element (ICE) originally isolated from <it>Vibrio cholerae</it>, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.</p> <p>Results</p> <p>SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn²⁺or Mg²⁺ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in <it>E. coli </it>cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.</p> <p>Conclusions</p> <p>The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected <it>V. cholerae </it>cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.</p

Construction of online catalog topologies using decision trees

Organization of a Web site is important to help users get the most out of the site. A good Web site should help visitors find the information they want easily. Visitors typically find information by searching for selected terms of interest or by following links from one Web page to another. The first approach is more useful if the visitor knows exactly what he is seeking, while the second approach is useful when the visitor has less of a preconceived notion about what he wants. The organization of a Web site is especially important in the latter case. Traditionally, Web site organization is done by hand. In this paper, we introduce the problem of automatic Web site construction and propose a solution for solving a major step of the problem based on decision tree algorithms. The solution is found to be useful in automatic construction of product catalogs.published_or_final_versio

A network ridesharing experiment with sequential choice of transportation mode

Within the last decade, there has been a dramatic bloom in ridesharing businesses along with the emergence of new enabling technologies. A central issue in ridesharing, which is also important in the general domain of cost-sharing in economics and computer science, is that the sharing of cost implies positive externalities and hence coordination problems for the network users. We investigate these problems experimentally in the present study. In particular, we focus on how sequential observability of transportation mode choices can be a powerful facilitator of coordination in ridesharing. Our study abstracts the essential issues of coordination in ridesharing into a directed network game with experimentally testable predictions. In line with the theoretical analysis, our experimental evidence shows that even a limited extent of sequential choice observability might lead to efficient coordination. However, convergence to efficiency is slower with more limited observability, resulting in a significant increase in travel cost

Current status of urban wastewater treatment plants in China

© 2016 Elsevier Ltd. The study reported and analyzed the current state of wastewater treatment plants (WWTPs) in urban China from the perspective of treatment technologies, pollutant removals, operating load and effluent discharge standards. By the end of 2013, 3508 WWTPs have been built in 31 provinces and cities in China with a total treatment capacity of 1.48 × 108 m3/d. The uneven population distribution between China's east and west regions has resulted in notably different economic development outcomes. The technologies mostly used in WWTPs are AAO and oxidation ditch, which account for over 50% of the existing WWTPs. According to statistics, the efficiencies of COD and NH3-N removal are good in 656 WWTPs in 70 cities. The overall average COD removal is over 88% with few regional differences. The average removal efficiency of NH3-N is up to 80%. Large differences exist between the operating loads applied in different WWTPs. The average operating loading rate is approximately 83%, and 52% of WWTPs operate at loadings of <80%, treating up to 40% of the wastewater generated. The implementation of discharge standards has been low. Approximately 28% of WWTPs that achieved the Grade I-A Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant (GB 18918-2002) were constructed after 2010. The sludge treatment and recycling rates are only 25%, and approximately 15% of wastewater is inefficiently treated. Approximately 60% of WWTPs have capacities of 1 × 104 m3/d-5 × 104 m3/d. Relatively high energy consumption is required for small-scale processing, and the utilization rate of recycled wastewater is low. The challenges of WWTPs are discussed with the aim of developing rational criteria and appropriate technologies for water recycling. Suggestions regarding potential technical and administrative measures are provided

Split-Drain Magnetic Field-Effect Transistor Channel Charge Trapping and Stress Induced Sensitivity Deterioration

Session EB: Materials for ApplicationsThis paper proposed an analytical model on the deterioration of magnetic sensitivity of sectorial split-drain magnetic field-effect transistors (SD-MAGFETs). The deterioration is governed by the trap fill rate at the channel boundary traps, which is geometric dependent. Experimental results are presented which show good consistency with the analytical derivation. The deterioration is the most severe at a sector angle of 54.6°, which shows a design tradeoff with sensing hysteresis. Design guidelines for sectorial SD-MAGFET to obtain high sensitivity hysteresis and slow sensitivity deterioration are also presented which provide important information for efficient design. © 2013 IEEE.published_or_final_versio