Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells. Conclusions/Significance: Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.

Assessing an intuitive condition for stability under a range of traffic conditions via a generalised Lu-Kumar network

We argue the importance both of developing simple sufficientconditions for the stability of general multiclass queueing networks and also of assessing such conditions under a range of assumptions on the weight of the traffic flowing between service stations. To achieve the former, we review a peak-rate stability condition and extend its range of application and for the latter, we introduce a generalisation of the Lu-Kumar network on which the stability condition may be tested for a range of traffic configurations. The peak-rate condition is close to exact when the between-station traffic is light, but degrades as this traffic increases.

Evaluating the Effectiveness of Red Light Running Camera Enforcement in Cedar Rapids and Developing Guidelines For Selection and use of Red Light Running Countermeasures, Midwest Transportation Consortium, Final Report November 2011.

Red light running (RLR) is a problem in the US that has resulted in 165,000 injuries and 907 fatalities annually. In Iowa, RLR-related crashes make up 24.5 percent of all crashes and account for 31.7 percent of fatal and major injury crashes at signalized intersections. RLR crashes are a safety concern due to the increased likelihood of injury compared to other types of crashes. One tool used to combat red light running is automated enforcement in the form of RLR cameras. Automated enforcement, while effective, is often controversial. Cedar Rapids, Iowa installed RLR and speeding cameras at seven intersections across the city. The intersections were chosen based on crash rates and whether cameras could feasibly be placed at the intersection approaches. The cameras were placed starting in February 2010 with the last one becoming operational in December 2010. An analysis of the effect of the cameras on safety at these intersections was determined prudent in helping to justify the installation and effectiveness of the cameras. The objective of this research was to assess the safety effectiveness of the RLR program that has been implemented in Cedar Rapids. This was accomplished by analyzing data to determine changes in the following metrics:  Reductions in red light violation rates based on overall changes, time of day changes, and changes by lane  Effectiveness of the cameras over time  Time in which those running the red light enter the intersection  Changes in the average headway between vehicles entering the intersection

From Empirical Bayes to Full Bayes: Methods for Analyzubg Traffic Safey Data, October 25, 2004

Traffic safety engineers are among the early adopters of Bayesian statistical tools for analyzing crash data. As in many other areas of application, empirical Bayes methods were their first choice, perhaps because they represent an intuitively appealing, yet relatively easy to implement alternative to purely classical approaches. With the enormous progress in numerical methods made in recent years and with the availability of free, easy to use software that permits implementing a fully Bayesian approach, however, there is now ample justification to progress towards fully Bayesian analyses of crash data. The fully Bayesian approach, in particular as implemented via multi-level hierarchical models, has many advantages over the empirical Bayes approach. In a full Bayesian analysis, prior information and all available data are seamlessly integrated into posterior distributions on which practitioners can base their inferences. All uncertainties are thus accounted for in the analyses and there is no need to pre-process data to obtain Safety Performance Functions and other such prior estimates of the effect of covariates on the outcome of interest. In this light, fully Bayesian methods may well be less costly to implement and may result in safety estimates with more realistic standard errors. In this manuscript, we present the full Bayesian approach to analyzing traffic safety data and focus on highlighting the differences between the empirical Bayes and the full Bayes approaches. We use an illustrative example to discuss a step-by-step Bayesian analysis of the data and to show some of the types of inferences that are possible within the full Bayesian framework.

A Survey of Traffic Safety Culture Among Iowa Adults, TS-1101, 2012

Iowa’s traffic safety culture is influenced by laws and policies, enforcement methods, driver education, roadway engineering, and drivers’ behaviors. The Center for Social and Behavioral Research at the University of Northern Iowa was contracted by the Iowa Department of Transportation to conduct a general population survey of adult Iowans. Telephone interviews were conducted with 1,088 adult Iowans from October to December 2011. A dual-frame (cell phone and landline) sampling design was used. The interview covered a wide range of traffic safety topics (e.g., traffic safety policies, enforcement techniques, and distracted driving). Most Iowans said driving in Iowa is about as safe now as it was 5 years ago; however, one-fourth said driving in Iowa is less safe now. There are a number of driving-related behaviors many adult Iowans consider serious threats to traffic safety and never acceptable to do while driving. Yet, many Iowans report often seeing other drivers engaging in these behaviors and admit engaging in some themselves. For example, nearly 1 in 5 adult Iowa drivers said they have sent or read a text message or email while driving in the past 30 days despite this being prohibited since July of 2011. A slight majority said they support using cameras on highways, interstates, and city streets to automatically ticket drivers for speeding, with even stronger support for red light cameras. A comprehensive approach to traffic safety in Iowa is required to encourage protective factors that enhance traffic safety and reduce the impact of detrimental factors.

Evaluation of the Confirmation Light System at Signalized Intersections in Medium to Large Iowa Communities to Reduce Red Light Running Violations”, TR-657, 2015

Red light running continues to be a serious safety concern for many communities in the United States. The Federal Highway Administration reported that in 2011, red light running accounted for 676 fatalities nationwide. Red light running crashes at a signalized intersections are more serious, especially in high speed corridors where speeds are above 35 mph. Many communities have invested in red light countermeasures including low-cost strategies (e.g. signal backplates, targeted enforcement, signal timing adjustments and improvement with signage) to high-cost strategies (e.g. automated enforcement and intersection geometric improvements). This research study investigated intersection confirmation lights as a low-cost strategy to reduce red light running violations. Two intersections in Altoona and Waterloo, Iowa were equipped with confirmation lights which targeted the through and left turning movements. Confirmation lights enable a single police officer to monitor a specific lane of traffic downstream of the intersection. A before-after analysis was conducted in which a change in red light running violations prior to- and 1 and 3 months after installation were evaluated. A test of proportions was used to determine if the change in red light running violation rates were statistically significant at the 90 and 95 percent levels of confidence. The two treatment intersections were then compared to the changes of red light running violation rates at spillover intersections (directly adjacent to the treatment intersections) and control intersections. The results of the analysis indicated a 10 percent reduction of red light running violations in Altoona and a 299 percent increase in Waterloo at the treatment locations. Finally, the research team investigated the time into red for each observed red light running violation. The analysis indicated that many of the violations occurred less than one second into the red phase and that most of the violation occurred during or shortly after the all-red phase.

The Effectiveness of Iowa's Automated Red Light Running Enforcement Programs, 2007

The Federal Highway Administration estimates that red light running causes more than 100,000 crashes and 1,000 fatalities annually and results in an estimated economic loss of over $14 billion per year in the United States. In Iowa alone, a statewide analysis of red light running crashes, using crash data from 2001 to 2006, indicates that an average of 1,682 red light running crashes occur at signalized intersections every year. As a result, red light running poses a significant safety issue for communities. Communities rarely have the resources to place additional law enforcement in the field to combat the problem and they are increasingly using automated red light running camera-enforcement systems at signalized intersections. In Iowa, three communities currently use camera enforcement since 2004. These communities include Davenport, Council Bluffs, and Clive. As communities across the United States attempt to address red light running, a number of communities have implemented red light running camera enforcement programs. This report examines the red light running programs in Iowa and summarizes results of analyses to evaluate the effectiveness of such cameras.

A Beacon based Localization System for Autonomous Mobile Robots and its Applications in Traffic and Transport Control

ACCURATE sensing of vehicle position and attitude is still a very challenging problem in many mobile robot applications. The mobile robot vehicle applications must have some means of estimating where they are and in which direction they are heading. Many existing indoor positioning systems are limited in workspace and robustness because they require clear lines-of-sight or do not provide absolute, driftfree measurements.The research work presented in this dissertation provides a new approach to position and attitude sensing system designed specifically to meet the challenges of operation in a realistic, cluttered indoor environment, such as that of an office building, hospital, industrial or warehouse. This is accomplished by an innovative assembly of infrared LED source that restricts the spreading of the light intensity distribution confined to a sheet of light and is encoded with localization and traffic information. This Digital Infrared Sheet of Light Beacon (DISLiB) developed for mobile robot is a high resolution absolute localization system which is simple, fast, accurate and robust, without much of computational burden or significant processing. Most of the available beacon's performance in corridors and narrow passages are not satisfactory, whereas the performance of DISLiB is very encouraging in such situations. This research overcomes most of the inherent limitations of existing systems.The work further examines the odometric localization errors caused by over count readings of an optical encoder based odometric system in a mobile robot due to wheel-slippage and terrain irregularities. A simple and efficient method is investigated and realized using an FPGA for reducing the errors. The detection and correction is based on redundant encoder measurements. The method suggested relies on the fact that the wheel slippage or terrain irregularities cause more count readings from the encoder than what corresponds to the actual distance travelled by the vehicle.The application of encoded Digital Infrared Sheet of Light Beacon (DISLiB) system can be extended to intelligent control of the public transportation system. The system is capable of receiving traffic status input through a GSM (Global System Mobile) modem. The vehicles have infrared receivers and processors capable of decoding the information, and generating the audio and video messages to assist the driver. The thesis further examines the usefulness of the technique to assist the movement of differently-able (blind) persons in indoor or outdoor premises of his residence.The work addressed in this thesis suggests a new way forward in the development of autonomous robotics and guidance systems. However, this work can be easily extended to many other challenging domains, as well.

An Encoded Infrared Sheet Of Light Navigational Beacon System For Precise Localization Of Indoor Mobile Robot Vehicles

A new localization approach to increase the navigational capabilities and object manipulation of autonomous mobile robots, based on an encoded infrared sheet of light beacon system, which provides position errors smaller than 0.02m is presented in this paper. To achieve this minimal position error, a resolution enhancement technique has been developed by utilising an inbuilt odometric/optical flow sensor information. This system respects strong low cost constraints by using an innovative assembly for the digitally encoded infrared transmitter. For better guidance of mobile robot vehicles, an online traffic signalling capability is also incorporated. Other added features are its less computational complexity and online localization capability all these without any estimation uncertainty. The constructional details, experimental results and computational methodologies of the system are also described

Color detection and segmentation for road and traffic signs

This paper aims to present three new methods for color detection and segmentation of road signs. The images are taken by a digital camera mounted in a car. The RGB images are converted into IHLS color space, and new methods are applied to extract the colors of the road signs under consideration. The methods are tested on hundreds of outdoor images in different light conditions, and they show high robustness. This project is part of the research taking place in Dalarna University / Sweden in the field of the ITS.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

Simulation of visible light communications in vehicular networks

The rapid development in the field of lighting and illumination allows low energy consumption and a rapid growth in the use, and development of solid-state sources. As the efficiency of these devices increases and their cost decreases there are predictions that they will become the dominant source for general illumination in the short term. The objective of this thesis is to study, through extensive simulations in realistic scenarios, the feasibility and exploitation of visible light communication (VLC) for vehicular ad hoc networks (VANETs) applications. A brief introduction will introduce the new scenario of smart cities in which visible light communication will become a fundamental enabling technology for the future communication systems. Specifically, this thesis focus on the acquisition of several, frequent, and small data packets from vehicles, exploited as sensors of the environment. The use of vehicles as sensors is a new paradigm to enable an efficient environment monitoring and an improved traffic management. In most cases, the sensed information must be collected at a remote control centre and one of the most challenging aspects is the uplink acquisition of data from vehicles. My thesis discusses the opportunity to take advantage of short range vehicle-to-vehicle (V2V) and vehicle-to-roadside (V2R) communications to offload the cellular networks. More specifically, it discusses the system design and assesses the obtainable cellular resource saving, by considering the impact of the percentage of vehicles equipped with short range communication devices, of the number of deployed road side units, and of the adopted routing protocol. When short range communications are concerned, WAVE/IEEE 802.11p is considered as standard for VANETs. Its use together with VLC will be considered in urban vehicular scenarios to let vehicles communicate without involving the cellular network. The study is conducted by simulation, considering both a simulation platform (SHINE, simulation platform for heterogeneous interworking networks) developed within the Wireless communication Laboratory (Wilab) of the University of Bologna and CNR, and network simulator (NS3). trying to realistically represent all the wireless network communication aspects. Specifically, simulation of vehicular system was performed and introduced in ns-3, creating a new module for the simulator. This module will help to study VLC applications in VANETs. Final observations would enhance and encourage potential research in the area and optimize performance of VLC systems applications in the future.

Explaining light vehicle demand evolution in interurban toll roads: a dynamic panel data analysis in Spain

Tolls have increasingly become a common mechanism to fund road projects in recent decades. Therefore, improving knowledge of demand behavior constitutes a key aspect for stakeholders dealing with the management of toll roads. However, the literature concerning demand elasticity estimates for interurban toll roads is still limited due to their relatively scarce number in the international context. Furthermore, existing research has left some aspects to be investigated, among others, the choice of GDP as the most common socioeconomic variable to explain traffic growth over time. This paper intends to determine the variables that better explain the evolution of light vehicle demand in toll roads throughout the years. To that end, we establish a dynamic panel data methodology aimed at identifying the key socioeconomic variables explaining changes in light vehicle demand over time. The results show that, despite some usefulness, GDP does not constitute the most appropriate explanatory variable, while other parameters such as employment or GDP per capita lead to more stable and consistent results. The methodology is applied to Spanish toll roads for the 1990?2011 period, which constitutes a very interesting case on variations in toll road use, as road demand has experienced a significant decrease since the beginning of the economic crisis in 2008.

Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast MicroscopyV⃞

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20–30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5–7 min at 37°C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.

A computer program (VEHSIM) for vehicle fuel economy and performance simulation (automobiles and light trucks). Volume I: description and analysis. Final report.

National Highway Traffic Safety Administration, Office of Research and Development, Washington, D.C.