Ion-induced transient potential fluctuations facilitate pore formation and cation transport through lipid membranes

Unassisted ion transport through lipid membranes plays a crucial role in many cell functions without which life would not be possible, yet the precise mechanism behind the process remains unknown due to its molecular complexity. Here, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. High-throughput wide-field second harmonic (SH) microscopy shows that membrane potential fluctuations are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu$^{2+}$, Ca$^{2+}$, Ba$^{2+}$, Mg$^{2+}$) passing through GUV membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.Comment: 8 pages, 2 figure

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa

BackgroundThe awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent.MethodologyWe searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients.FindingsA total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections.ConclusionThe small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies

Water as a contrast agent for imaging interfacial structure and ion transport in giant vesicles

Hydrated lipid bilayer membranes and their asymmetry play a fundamental role in living cells by maintaining and regulating concentration gradients between cells, their environment, and their compartments. They achieve this not only through various channels and transporters, but also by being inherently semi-permeable to various molecules, including water and ions. However, molecular information about passive ion transport, as well as the complex structure of membrane hydration remain elusive, mainly due to a lack of experimental methods that can access this information, and relate it to micro- and macroscopic membrane properties. In this work, we study the molecular structure of lipid bilayer membranes and ion transport across them with high throughput wide-field second harmonic (SH) microscopy by utilizing water as the contrast agent. We show that by detecting small amounts of asymmetry in the structure of interfacial water, it is possible to measure the distribution of charged species across the membrane with high sensitivity. We apply this surface-selective technique to measure ion-lipid-water interactions, track ion transport, and quantify electro-chemical surface properties at the interfaces of lipid bilayer membranes in the form of giant unilamellar vesicles (GUVs). We start by improving the throughput of wide field SH microscopy in order to probe low-asymmetry interfaces with high contrast in a label-free manner. To do this, we design a custom optical parametric amplifier (OPA) with a tunable output in the 670-1000 nm range, up to a 1 MHz repetition rate, and an ultra-short 23 fs pulse duration. We then experimentally demonstrate the achieved throughput improvement. Next, we establish a way to probe interfacial hydration of GUVs. We quantify the surface properties of vesicles composed of different ratios of zwitterionic and anionic lipids, and show that only a few percent of anionic headgroups are ionized. We also observe spatial and temporal fluctuations in surface properties, and demonstrate that these fluctuations are universally found in lipid bilayer systems. Following that, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. Molecular dynamics simulations reveal that these fluctuations reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through GUV membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport. Then, we focus on unassisted Ca2+ translocation in more detail. We vary the hydrophobic core of bilayer membranes and observe different types of behavior in high throughput wide-field SH images. Ca2+ translocation is observed through mono-unsaturated membranes, significantly reduced upon adding cholesterol, and completely inhibited for branched and poly-unsaturated membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion induced transient pores, which require non-equilibrium membrane restructuring. This results in different transport rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought

Imaging Cu2+ binding to charged phospholipid membranes by high-throughput second harmonic wide-field microscopy

The interaction of divalent copper ions (Cu2+) with cell membranes is crucial for a variety of physiological processes of cells, such as hormone synthesis and cellular energy production. These interactions would not be possible without membrane hydration. However, the role of water has not received a lot of attention in membrane studies. Here, we use high-throughput wide-field second harmonic (SH) microscopy to study the interaction between Cu2+ and hydrated freestanding Montal-Muller lipid membranes. The symmetric lipid membranes are composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and either 1,2-diphytanoyl-sn-glycero-3-phosphate or 1,2-diphytanoyl-sn-glycero-3-phospho L-serine and are brought into contact with divalent Cu2+, which are added to one leaflet while maintaining the ionic strength balance. We observe transient domains of high SH intensity. In these areas, Cu2+ ions bind to the charged head groups, leading to charge neutralization on one side of the membrane. This exposes the ordered water at the non-interacting side of the membrane interface, which can be used to compute the interfacial membrane potential difference. We find that the domains of lipids with phosphatidic acid head groups display a higher interfacial membrane potential than those with phosphatidylserine head groups, which converts into higher dynamic electrostatic free energies and binding constants. (C) 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Estimating Pedestrian Accident Exposure: Approaches to a Statewide Pedestrian Exposure Database

This report discusses approaches to addressing the need for better and more widely available pedestrian volume data in the state of California. While a variety of approaches could be used, this report focuses on the strategy of a statewide pedestrian volume database.This database would meet a variety of data needs for different stakeholder groups. One of its principal purposes would be to allow safety professionals at the state and local levels to estimate pedestrian exposure to risk at specific sites.Since exposure data is essentially equivalent to facility usage data, a pedestrian exposure data would be used for many purposes beyond risk analysis. Facility usage data might be used by municipalities to pinpoint new infrastructure needs, or to determine whether new infrastructure encourages more pedestrian activity. Facility usage data might also be used by advocacy groups as a means to promote new facility investments.If the database includes information beyond pedestrian volumes, such as facility characteristics (e.g. the availability of sidewalks and intersection crossings) or planning variables (e.g. land uses and population densities), it may be used as a means to improve pedestrian demand modeling techniques or to investigate the relationship between pedestrian environmental quality and pedestrian demand. Furthermore, if facility funding data are included, the database may also be used as a means to track spending on pedestrian projects.In short, there is a wide range of usage for a pedestrian volume database. In designing the database, it is important to maximize its utility to pedestrian stakeholder groups while recognizing the costs associated with increased complexity.Creation of a pedestrian volume database for the state of California involves several major decision points. This report examines these decision points and provides a range of database approaches given different funding and institutional constraints, and describes the challenges that will need to be addressed in the database development process.Chapter 2 discusses the technical and institutional challenges inherent in creation of a pedestrian exposure database. Chapter 3 discusses the need for an inventory of the pedestrian network as a starting point for the database, and present two existing sources for the network. Chapter 4 presents a range of approaches to data collection process, and suggests data points that might be appropriate for inclusion in the data collection process. Chapter 5 discusses how pedestrian demand modeling might be used to estimate pedestrian volumes with limited data inputs. Chapter 6 summarizes the report and provides recommendations for future development of the database

Estimating Pedestrian Accident Exposure: Protocol Report

Walking is a healthful, environmentally benign form of travel, and is the most basicform of human mobility. Walking trips account for more than 8 percent of all tripstaken in California, making walking the second most commonly used mode of travelafter the personal automobile (Caltrans, 2002). In addition, many trips made byvehicle or public transit begin and end with walking.In spite of the importance and benefits of walking, pedestrians suffer adisproportionate share of the harm of traffic incidents in California. As noted above,walking trips make up just 8 percent of all trips in the state, but 17 percent of alltraffic fatalities are suffered by pedestrians. In 2004, 694 pedestrians were killed inthe state of California and 13,892 were injured (California Highway Patrol, 2004).To address this problem, significant resources are focused on countermeasures thataim to reduce the risk of pedestrian injury. Because resources are limited, riskanalysis is necessary to develop cost-effective countermeasures (Hoj and Kroger,2002).In the field of pedestrian safety, risk analysis involves assessing factors thatcontribute to the danger that a pedestrian is struck by a vehicle. These factors mayinclude physical characteristics of the street, such as lack of sidewalks; behavioralissues, such as pedestrian or driver alcohol use; as well as other environmentalvariables. In order to fully understand how these factors contribute to risk, it isnecessary to collect information on pedestrian exposure. Collection of pedestrianexposure information is an essential component of risk analysis.Pedestrian exposure is a concept that refers to the amount that people are exposedto the risk of being involved in a traffic collision. In principle, pedestrians are exposedto this risk whenever they are walking in the vicinity of automobiles. There are manymetrics that can be used to measure pedestrian exposure, but pedestrian volumesare the most frequently used.Although many state, regional, and local agencies have developed methodologies tocollect pedestrian volume data, there is no consensus on which method is best(Schneider et al., 2005; Schweizer, 2005). This is because there is no “one size fitsall” method of counting pedestrians. Rather, the choice of strategy depends on acomplex range of factors, including the characteristics of the area being studied; theresources available for data collection; and the specific purpose of data collection.This protocol aims to improve pedestrian data collection in the state of California byproviding information and guidance for each decision point in the data collectionprocess. Each chapter represents one of these decision points, and each will guidethe user through important considerations relevant to the data collection stage. Inaddition, each chapter provides a combination of real-world and hypotheticalexample scenarios to illustrate the issues discussed in the text

Estimating Pedestrian Accident Exposure

We are pleased to present the final report of Caltrans Task Orders 5211 and 6211, “Estimating Pedestrian Accident Exposure.” The project focused on defining pedestrian exposure and evaluating methods for measuring it within the State of California. The project was funded by the California Department of Transportation as part of the California Partners for Advanced Transit and Highways (PATH) Program of the University of California.Deliverables associated with the project include (I) a protocol report on assessing pedestrian exposure, which is accompanied by a training curriculum and an evaluation of manual pedestrian counting methods; (II) an evaluation and test of automated pedestrian counting methods; and (III) a report on strategies to create a statewide pedestrian exposure database and (IV) a protocol for Pedestrian Exposure Study in Alameda County. The deliverables are discussed in more detail below

Estimating Pedestrian Accident Exposure: Approaches to a Statewide Pedestrian Exposure Database

This report discusses approaches to addressing the need for better and more widely available pedestrian volume data in the state of California. While a variety of approaches could be used, this report focuses on the strategy of a statewide pedestrian volume database.This database would meet a variety of data needs for different stakeholder groups. One of its principal purposes would be to allow safety professionals at the state and local levels to estimate pedestrian exposure to risk at specific sites.Since exposure data is essentially equivalent to facility usage data, a pedestrian exposure data would be used for many purposes beyond risk analysis. Facility usage data might be used by municipalities to pinpoint new infrastructure needs, or to determine whether new infrastructure encourages more pedestrian activity. Facility usage data might also be used by advocacy groups as a means to promote new facility investments.If the database includes information beyond pedestrian volumes, such as facility characteristics (e.g. the availability of sidewalks and intersection crossings) or planning variables (e.g. land uses and population densities), it may be used as a means to improve pedestrian demand modeling techniques or to investigate the relationship between pedestrian environmental quality and pedestrian demand. Furthermore, if facility funding data are included, the database may also be used as a means to track spending on pedestrian projects.In short, there is a wide range of usage for a pedestrian volume database. In designing the database, it is important to maximize its utility to pedestrian stakeholder groups while recognizing the costs associated with increased complexity.Creation of a pedestrian volume database for the state of California involves several major decision points. This report examines these decision points and provides a range of database approaches given different funding and institutional constraints, and describes the challenges that will need to be addressed in the database development process.Chapter 2 discusses the technical and institutional challenges inherent in creation of a pedestrian exposure database. Chapter 3 discusses the need for an inventory of the pedestrian network as a starting point for the database, and present two existing sources for the network. Chapter 4 presents a range of approaches to data collection process, and suggests data points that might be appropriate for inclusion in the data collection process. Chapter 5 discusses how pedestrian demand modeling might be used to estimate pedestrian volumes with limited data inputs. Chapter 6 summarizes the report and provides recommendations for future development of the database

Estimating Pedestrian Accident Exposure: Automated Pedestrian Counting Devices Report

Automated methods are commonly used to count motorized vehicles, but are notfrequently used to count pedestrians. This is because the automated technologiesavailable to count pedestrians are not very developed, and their effectiveness hasnot been widely researched. Moreover, most automated methods are used primarilyfor the purpose of detecting, rather than counting, pedestrians (Dharmaraju et al.,2001; Noyce and Dharmaraju, 2002; Noyce et al., 2006).Automated pedestrian counting technologies are attractive because they have thepotential to reduce the labor costs associated with manual methods, and to recordpedestrian activity for long periods of time that are currently difficult to capturethrough traditional methods. Data input and storage may also be less timeconsuming than with manual methods.On the other hand, the capital costs of automated equipment may be high;specialized training may be required to operate it; and automated devices aregenerally not capable of collecting information on pedestrian characteristics andbehavior. For these reasons, automated devices are not appropriate for allpedestrian data collection efforts.The choice between which method is more appropriate to collect pedestrian datamust be based on the accuracy level desired, budget constraints, and data needsspecifications.Automated Counting TechnologiesMuch of the research on automated pedestrian tracking devices has focused onpedestrian detection, not pedestrian counting. Extensive reviews of pedestriandetection technologies were conducted by Noyce and Dharmaraju (2002) and byChan et al. (2006). Technologies include piezoelectric sensors, acoustic, active andpassive infrared, ultrasonic sensors, microwave radar, laser scanners, video imaging(computer vision).Of the technologies listed above, those most adaptable to the purpose of pedestriancounting are: infra-red beam counters; passive infrared counters; piezoelectric pads;laser scanners; and computer vision technology. None of these devices are widelyused for the purpose of counting pedestrians outdoors, but all have some potential tobe adapted for that purpose.This report describes each of these technologies in detail, and discusses some ofthe technical strengths and weaknesses of each method. It is important to be awarethat technical limitations are only one consideration among many when choosing anappropriate counting device. The device “packaging,” such as the method andlocation of installation may be equally important. For example, the location andaccessibility of the device may create liability issues or promote vandalism

Estimating Pedestrian Accident Exposure: Approaches to a Statewide Pedestrian Exposure Database

This report discusses approaches to addressing the need for better and more widely available pedestrian volume data in the state of California. While a variety of approaches could be used, this report focuses on the strategy of a statewide pedestrian volume database. This database would meet a variety of data needs for different stakeholder groups. One of its principal purposes would be to allow safety professionals at the state and local levels to estimate pedestrian exposure to risk at specific sites. Since exposure data is essentially equivalent to facility usage data, a pedestrian exposure data would be used for many purposes beyond risk analysis. Facility usage data might be used by municipalities to pinpoint new infrastructure needs, or to determine whether new infrastructure encourages more pedestrian activity. Facility usage data might also be used by advocacy groups as a means to promote new facility investments. If the database includes information beyond pedestrian volumes, such as facility characteristics (e.g. the availability of sidewalks and intersection crossings) or planning variables (e.g. land uses and population densities), it may be used as a means to improve pedestrian demand modeling techniques or to investigate the relationship between pedestrian environmental quality and pedestrian demand. Furthermore, if facility funding data are included, the database may also be used as a means to track spending on pedestrian projects. In short, there is a wide range of usage for a pedestrian volume database. In designing the database, it is important to maximize its utility to pedestrian stakeholder groups while recognizing the costs associated with increased complexity. Creation of a pedestrian volume database for the state of California involves several major decision points. This report examines these decision points and provides a range of database approaches given different funding and institutional constraints, and describes the challenges that will need to be addressed in the database development process. Chapter 2 discusses the technical and institutional challenges inherent in creation of a pedestrian exposure database. Chapter 3 discusses the need for an inventory of the pedestrian network as a starting point for the database, and present two existing sources for the network. Chapter 4 presents a range of approaches to data collection process, and suggests data points that might be appropriate for inclusion in the data collection process. Chapter 5 discusses how pedestrian demand modeling might be used to estimate pedestrian volumes with limited data inputs. Chapter 6 summarizes the report and provides recommendations for future development of the database.