Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity

Analysis of Mycobacterium tuberculosis in the state of Texas for rifampin resistance using molecular beacons

Mycobacterium tuberculosis, a bacillus known to cause disease in humans since ancient times, is the etiological agent of tuberculosis (TB). The infection is primarily pulmonary, although other organs may also be affected. The prevalence of pulmonary TB disease in the US is highest along the US-Mexico border, and of the four US states bordering Mexico, Texas had the second highest percentage of cases of TB disease among Mexico-born individuals in 1999 (CDC, 2001). Between the years of 1993 and 1998, the prevalence of drug-resistant (DR) TB was 9.1% among Mexican-born individuals and 4.4% among US-born individuals (CDC, 2001). In the same time period, the prevalence of multi-drug resistant (MDR) TB was 1.4% among Mexican-born individuals and 0.6% among US-born individuals (CDC, 2001). There is a renewed urgency in the quest for faster and more effective screening, diagnosis, and treatment methods for TB due to the resurgence of tuberculosis in the US during the mid-1980s and early 1990s (CDC, 2007a), and the emergence of drug-resistant, multidrug-resistant, and extremely drug-resistant tuberculosis worldwide. Failure to identify DR and MDR-TB quickly leads to poorer treatment outcomes (CDC, 2007b). The recent rise in TB/HIV comorbidity further complicates TB control efforts. The gold standard for identification of DR-TB requires mycobacterial growth in culture, a technique taking up to three weeks, during which time DR/MDR-TB individuals harboring resistant organisms may be receiving inappropriate treatment. The goal of this study was to determine the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) using molecular beacons in the Texas population. qPCR using molecular beacons is a novel approach to detect mycobacterial mutations conferring drug resistance. This technique is time-efficient and has been shown to have high sensitivity and specificity in several populations worldwide. Rifampin (RIF) susceptibility was chosen as the test parameter because strains of M. tuberculosis which are resistant to RIF are likely to also be MDR. Due to its status as a point of entry for many immigrants into the US, control efforts against TB and drug-resistant TB in Texas is a vital component of prevention efforts in the US as a whole. We show that qPCR using molecular beacons has high sensitivity and specificity when compared with culture (94% and 87%, respectively) and DNA sequencing (90% and 96%, respectively). We also used receiver operator curve analysis to calculate cutoff values for the objective determination of results obtained by qPCR using molecular beacons. ^

Multiplex detection of four pathogenic retroviruses using molecular beacons

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100,000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.

Development of guidelines for installation of intersection control beacons. Final report.

Federal Highway Administration, Washington, D.C.

Bonfires to beacons : Federal civil aviation policy under the Air commerce act, 1926-1938 /

Mode of access: Internet.

The American coast pilot : containing the courses and distances between the principal harbours, capes, and headlands, on the coast of North and South America; with directions for sailing into the same; describing the soundings, bearings of the light-houses and beacons from the rocks, shoals, ledges, &c. With the prevailing winds, settings of the currents, &c. and the latitudes and longitudes of the principal harbours and capes. Together with a tide table / By Edmund M. Blunt. To which is annexed, the Shipmaster's assistant; containing disbursements and other ship accounts, &c. /

Issued without the Shipmaster's assistant which was published separately in 1822 under the title: The merchant's and shipmaster's assistant.

Global reconfiguration of a team of networked mobile robots among obstacles

This paper presents a full system demonstration of dynamic sensorbased reconfiguration of a networked robot team. Robots sense obstacles in their environment locally and dynamically adapt their global geometric configuration to conform to an abstract goal shape. We present a novel two-layer planning and control algorithm for team reconfiguration that is decentralised and assumes local (neighbour-to-neighbour) communication only. The approach is designed to be resource-efficient and we show experiments using a team of nine mobile robots with modest computation, communication, and sensing. The robots use acoustic beacons for localisation and can sense obstacles in their local neighbourhood using IR sensors. Our results demonstrate globally-specified reconfiguration from local information in a real robot network, and highlight limitations of standard mesh networks in implementing decentralised algorithms.

Redundant sensing for localisation in outdoor industrial environments

We describe our experiences with automating a large fork-lift type vehicle that operates outdoors and in all weather. In particular, we focus on the use of independent and robust localisation systems for reliable navigation around the worksite. Two localisation systems are briefly described. The first is based on laser range finders and retro-reflective beacons, and the second uses a two camera vision system to estimate the vehicle’s pose relative to a known model of the surrounding buildings. We show the results from an experiment where the 20 tonne experimental vehicle, an autonomous Hot Metal Carrier, was conducting autonomous operations and one of the localisation systems was deliberately made to fail.

Driver’s behavioural changes with new Intelligent Transport System interventions at railway level crossings: A driving simulator study

Improving safety at railway level crossings is an important issue for the Australian transport system. Governments, the rail industry and road organisations have tried a variety of countermeasures for many years to improve railway level crossing safety. New types of Intelligent Transport System (ITS) interventions are now emerging due to the availability and the affordability of technology. These interventions target both actively and passively protected railway level crossings and attempt to address drivers’ errors at railway crossings, which are mainly a failure to detect the crossing or the train and misjudgement of the train approach speed and distance. This study aims to assess the effectiveness of three emerging ITS that the rail industry considers implementing in Australia: a visual in-vehicle ITS, an audio in-vehicle ITS, as well as an on-road flashing beacons intervention. The evaluation was conducted on an advanced driving simulator with 20 participants per trialled technology, each participant driving once without any technology and once with one of the ITS interventions. Every participant drove through a range of active and passive crossings with and without trains approaching. Their speed approach of the crossing, head movements and stopping compliance were measured. Results showed that driver behaviour was changed with the three ITS interventions at passive crossings, while limited effects were found at active crossings, even with reduced visibility. The on-road intervention trialled was unsuccessful in improving driver behaviour; the audio and visual ITS improved driver behaviour when a train was approaching. A trend toward worsening driver behaviour with the visual ITS was observed when no trains were approaching. This trend was not observed for the audio ITS intervention, which appears to be the ITS intervention with the highest potential for improving safety at passive crossings.

U.S. Caribbean fish trap fishery costs and earnings study

Executive Summary: This study describes the socio-economic characteristics of the U.S. Caribbean trap fishery that encompasses the Commonwealth of Puerto Rico and Territory of the U.S. Virgin Islands. In-person interviews were administered to one hundred randomly selected trap fishermen, constituting nearly 25% of the estimated population. The sample was stratified by geographic area and trap tier. The number of traps owned or fished to qualify for a given tier varied by island. In Puerto Rico, tier I consisted of fishermen who had between 1-40 fish traps, tier II was made up of fishermen who possessed between 41 and 100 fish traps, and tier III consisted of fishermen who held in excess of 100 fish traps. In St. Thomas and St. John, tier I was composed of fishermen who held between 1 and 50 fish traps, tier II consisted of fishermen who had between 51-150 fish traps and tier III was made up of fishermen who had in excess of 150 fish traps. Lastly, in St. Croix, tier I was made up of fishermen who had less than 20 fish traps and tier II consisted of fishermen who had 20 or more fish traps. The survey elicited information on household demographics, annual catch and revenue, trap usage, capital investment on vessels and equipment, fixed and variable costs, behavioral response to a hypothetical trap reduction program and the spatial distribution of traps. The study found that 79% of the sampled population was 40 years or older. The typical Crucian trap fisherman was older than their Puerto Rican and St. Thomian and St. Johnian counterparts. Crucian fishermen’s average age was 57 years whereas Puerto Rican fishermen’s average age was 51 years, and St. Thomian and St. Johnian fishermen’s average age was 48 years. As a group, St. Thomian and St. Johnian fishermen had 25 years of fishing experience, and Puerto Rican and Crucian fishermen had 30, and 29 years, respectively. Overall, 90% of the households had at least one dependent. The average number of dependents across islands was even, ranging between 2.8 in the district of St. Thomas and St. John and 3.4 in the district of St. Croix. The percentage utilization of catch for personal or family use was relatively low. Regionally, percentage use of catch for personal or family uses ranged from 2.5% in St. Croix to 3.8% in the St. Thomas and St. John. About 47% of the respondents had a high school degree. The majority of the respondents were highly dependent on commercial fishing for their household income. In St. Croix, commercial fishing made up 83% of the fishermen’s total household income, whereas in St. Thomas and St. John and Puerto Rico it contributed 74% and 68%, respectively. The contribution of fish traps to commercial fishing income ranged from 51% in the lowest trap tier in St. Thomas and St. John to 99% in the highest trap tier in St. Croix. On an island basis, the contribution of fish traps to fishing income was 75% in St. Croix, 61% in St. Thomas and St. John, and 59% in Puerto Rico. The value of fully rigged vessels ranged from $400 to $250,000. Over half of the fleet was worth $10,000 or less. The St. Thomas and St. John fleet reported the highest mean value, averaging $58,518. The Crucian and Puerto Rican fleets were considerably less valuable, averaging $19,831 and $8,652, respectively. The length of the vessels ranged from 14 to 40 feet. Fifty-nine percent of the sampled vessels were at least 23 feet in length. The average length of the St. Thomas and St. John fleet was 28 feet, whereas the fleets based in St. Croix and Puerto Rico averaged 21 feet. The engine’s propulsion ranged from 8 to 400 horsepower (hp). The mean engine power was 208 hp in St. Thomas and St. John, 108 hp in St. Croix, and 77 hp in Puerto Rico. Mechanical trap haulers and depth recorders were the most commonly used on-board equipment. About 55% of the sampled population reported owning mechanical trap haulers. In St. Thomas and St. John, 100% of the respondents had trap haulers compared to 52% in Puerto Rico and 20% in St. Croix. Forty-seven percent of the fishermen surveyed stated having depth recorders. Depth recorders were most common in the St. Thomas and St. John fleet (80%) and least common in the Puerto Rican fleet (37%). The limited presence of emergency position indication radio beacons (EPIRBS) and radar was the norm among the fish trap fleet. Only 8% of the respondents had EPIRBS and only 1% had radar. Interviewees stated that they fished between 1 and 350 fish traps. Puerto Rican respondents fished on average 39 fish traps, in contrast to St. Thomian and St. Johnian and Crucian respondents, who fished 94 and 27 fish traps, respectively. On average, Puerto Rican respondents fished 11 lobster traps, and St. Thomian and St. Johnian respondents fished 46 lobster traps. None of the Crucian respondents fished lobster traps. The number of fish traps built or purchased ranged between 0 and 175, and the number of lobster traps built or bought ranged between 0 and 200. Puerto Rican fishermen on average built or purchased 30 fish traps and 14 lobster traps, and St. Thomian and St. Johnian fishermen built or bought 30 fish traps and 11 lobster traps. Crucian fishermen built or bought 25 fish traps and no lobster traps. As a group, fish trap average life ranged between 1.3 and 5 years, and lobster traps lasted slightly longer, between 1.5 and 6 years. The study found that the chevron or arrowhead style was the most common trap design. Puerto Rican fishermen owned an average of 20 arrowhead traps. St. Thomian and St. Johnian and Crucian fishermen owned an average of 44 and 15 arrowhead fish traps, respectively. The second most popular trap design was the square trap style. Puerto Rican fishermen had an average of 9 square traps, whereas St. Thomian and St. Johnian fishermen had 33 traps and Crucian fishermen had 2 traps. Antillean Z (or S) -traps, rectangular and star traps were also used. Although Z (or S) -traps are considered the most productive trap design, fishermen prefer the smaller-sized arrowhead and square traps because they are easier and less expensive to build, and larger numbers of them can be safely deployed. The cost of a fish trap, complete with rope and buoys, varied significantly due to the wide range of construction materials utilized. On average, arrowhead traps commanded $94 in Puerto Rico, $251 in St. Thomas and St. John, and $119 in St. Croix. The number of trips per week ranged between 1 and 6. However, 72% of the respondents mentioned that they took two trips per week. On average, Puerto Rican fishermen took 2.1 trips per week, St. Thomian and St. Johnian fishermen took 1.4 trips per week, and Crucian fishermen took 2.5 trips per week. Most fishing trips started at dawn and finished early in the afternoon. Over 82% of the trips lasted 8 hours or less. On average, Puerto Rican fishermen hauled 27 fish traps per trip whereas St. Thomian and St. Johnian fishermen and Crucian fishermen hauled 68 and 26 fish traps per trip, respectively. The number of traps per string and soak time varied considerably across islands. In St. Croix, 84% of the respondents had a single trap per line, whereas in St. Thomas and St. John only 10% of the respondents had a single trap per line. Approximately, 43% of Puerto Rican fishermen used a single trap line. St. Thomian and St. Johnian fishermen soaked their traps for 6.9 days while Puerto Rican and Crucian fishermen soaked their traps for 5.7 and 3.6 days, respectively. The heterogeneity of the industry was also evidenced by the various economic surpluses generated. The survey illustrated that higher gross revenues did not necessarily translate into higher net revenues. Our analysis also showed that, on average, vessels in the trap fishery were able to cover their cash outlays, resulting in positive vessel income (i.e., financial profits). In Puerto Rico, annual financial profits ranged from $4,760 in the lowest trap tier to $32,467 in the highest tier, whereas in St. Thomas and St. John annual financial profits ranged from $3,744 in the lowest tier to $13,652 in the highest tier. In St. Croix, annual financial profits ranged between $9,229 and $15,781. The survey also showed that economic profits varied significantly across tiers. Economic profits measure residual income after deducting the remuneration required to keep the various factors of production in their existing employment. In Puerto Rico, annual economic profits ranged from ($9,339) in the lowest trap tier to $ 8,711 in the highest trap tier. In St. Thomas and St. John, annual economic profits ranged from ($7,920) in the highest tier to ($18,486) in the second highest tier. In St. Croix, annual economic profits ranged between ($7,453) to $10,674. The presence of positive financial profits and negative economic profits suggests that higher economic returns could be earned from a societal perspective by redirecting some of these scarce capital and human resources elsewhere in the economy. Furthermore, the presence of negative economic earnings is evidence that the fishery is overcapitalized and that steps need to be taken to ensure the long-run economic viability of the industry. The presence of positive financial returns provides managers with a window of opportunity to adopt policies that will strengthen the biological and economic performance of the fishery while minimizing any adverse impacts on local fishing communities. Finally, the document concludes by detailing how the costs and earnings information could be used to develop economic models that evaluate management proposals. (PDF contains 147 pages)

Deep tissue fluorescence imaging with time-reversed light

Advances in optical techniques have enabled many breakthroughs in biology and medicine. However, light scattering by biological tissues remains a great obstacle, restricting the use of optical methods to thin ex vivo sections or superficial layers in vivo. In this thesis, we present two related methods that overcome the optical depth limit—digital time reversal of ultrasound encoded light (digital TRUE) and time reversal of variance-encoded light (TROVE). These two techniques share the same principle of using acousto-optic beacons for time reversal optical focusing within highly scattering media, like biological tissues. Ultrasound, unlike light, is not significantly scattered in soft biological tissues, allowing for ultrasound focusing. In addition, a fraction of the scattered optical wavefront that passes through the ultrasound focus gets frequency-shifted via the acousto-optic effect, essentially creating a virtual source of frequency-shifted light within the tissue. The scattered ultrasound-tagged wavefront can be selectively measured outside the tissue and time-reversed to converge at the location of the ultrasound focus, enabling optical focusing within deep tissues. In digital TRUE, we time reverse ultrasound-tagged light with an optoelectronic time reversal device (the digital optical phase conjugate mirror, DOPC). The use of the DOPC enables high optical gain, allowing for high intensity optical focusing and focal fluorescence imaging in thick tissues at a lateral resolution of 36 µm by 52 µm. The resolution of the TRUE approach is fundamentally limited to that of the wavelength of ultrasound. The ultrasound focus (~ tens of microns wide) usually contains hundreds to thousands of optical modes, such that the scattered wavefront measured is a linear combination of the contributions of all these optical modes. In TROVE, we make use of our ability to digitally record, analyze and manipulate the scattered wavefront to demix the contributions of these spatial modes using variance encoding. In essence, we encode each spatial mode inside the scattering sample with a unique variance, allowing us to computationally derive the time reversal wavefront that corresponds to a single optical mode. In doing so, we uncouple the system resolution from the size of the ultrasound focus, demonstrating optical focusing and imaging between highly diffusing samples at an unprecedented, speckle-scale lateral resolution of ~ 5 µm. Our methods open up the possibility of fully exploiting the prowess and versatility of biomedical optics in deep tissues.