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"Mason, Michael"
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Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics
A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA's performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment.
Journal Article
Sugar demand, not auxin, is the initial regulator of apical dominance
For almost a century the plant hormone auxin has been central to theories on apical dominance, whereby the growing shoot tip suppresses the growth of the axillary buds below. According to the classic model, the auxin indole-3-acetic acid is produced in the shoot tip and transported down the stem, where it inhibits bud growth. We report here that the initiation of bud growth after shoot tip loss cannot be dependent on apical auxin supply because we observe bud release up to 24 h before changes in auxin content in the adjacent stem. After the loss of the shoot tip, sugars are rapidly redistributed over large distances and accumulate in axillary buds within a timeframe that correlates with bud release. Moreover, artificially increasing sucrose levels in plants represses the expression of BRANCHED1 (BRC1), the key transcriptional regulator responsible for maintaining bud dormancy, and results in rapid bud release. An enhancement in sugar supply is both necessary and sufficient for suppressed buds to be released from apical dominance. Our data support a theory of apical dominance whereby the shoot tip’s strong demand for sugars inhibits axillary bud outgrowth by limiting the amount of sugar translocated to those buds.
Journal Article
Juno
Faced with an unplanned pregnancy, an offbeat young woman makes an unusual decision regarding her unborn child.
DVD
Nucleic acid purification from plants, animals and microbes in under 30 seconds
Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.
Journal Article
Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks
The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.
The authors describe how to easily prepare a large number of dipsticks from cellulose-based filter paper and use them to rapidly purify nucleic acids from a variety of sources.
Journal Article
Risky Substance Use Environments and Addiction: A New Frontier for Environmental Justice Research
Substance use disorders are widely recognized as one of the most pressing global public health problems, and recent research indicates that environmental factors, including access and exposure to substances of abuse, neighborhood disadvantage and disorder, and environmental barriers to treatment, influence substance use behaviors. Racial and socioeconomic inequities in the factors that create risky substance use environments may engender disparities in rates of substance use disorders and treatment outcomes. Environmental justice researchers, with substantial experience in addressing racial and ethnic inequities in environmental risk from technological and other hazards, should consider similar inequities in risky substance use environments as an environmental justice issue. Research should aim at illustrating where, why, and how such inequities in risky substance use environments occur, the implications of such inequities for disparities in substance use disorders and treatment outcomes, and the implications for tobacco, alcohol, and drug policies and prevention and treatment programs.
Journal Article
Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy
Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only ∼100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features ∼86
±
4
nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.
Journal Article