105 research outputs found
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Fluorescent reporters for functional analysis in rice leaves.
Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed the analysis of processes ranging from gene expression and protein localization to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualize fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins (mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato) that are robustly expressed and detectable in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolor imaging of different subcellular compartments. Overall, we conclude that mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato are suitable for high-resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice
Pharmacological Validation of Trypanosoma brucei Phosphodiesterases as Novel Drug Targets
The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug targets
Gene Conversion Transfers the GAF-A Domain of Phosphodiesterase TbrPDEB1 to One Allele of TbrPDEB2 of Trypanosoma brucei
Cyclic nucleotide specific phosphodiesterases are important regulators of cyclic nucleotide signalling in eukaryotes. In many organisms, including humans and trypanosomes, some of these enzymes contain specific domains (GAF domains) that bind cyclic nucleotides, and that are involved in the regulation of the catalytic domain. In the parasitic protozoon that causes human sleeping sickness, Trypanosoma brucei, two closely related phosphodiesterases each contain two such GAF domains, GAF-A and GAF-B. Their genes are tandemly located on chromosome 9, spaced by only a few thousand nucleotides. We here show that a gene conversion event has exchanged the region that codes for the GAF-A domain of the downstream gene by the closely similar corresponding sequence of the upstream gene. This domain exchange has no effect on intracellular localization of the two enzymes. The gene conversion event has occurred in one particular strain of trypanosomes (Lister427) and is found in all its derivatives, but not in any other strain or isolate. The presence or absence of this gene conversion represents a useful analytical marker for the stringent discrimination of Lister427 derivatives from other trypanosome strains
A Model of Brain Circulation and Metabolism: NIRS Signal Changes during Physiological Challenges
We construct a model of brain circulation and energy metabolism. The model is
designed to explain experimental data and predict the response of the
circulation and metabolism to a variety of stimuli, in particular, changes in
arterial blood pressure, CO2 levels, O2 levels, and
functional activation. Significant model outputs are predictions about blood
flow, metabolic rate, and quantities measurable noninvasively using
near-infrared spectroscopy (NIRS), including cerebral blood volume and
oxygenation and the redox state of the CuA centre in cytochrome
c oxidase. These quantities are now frequently measured in
clinical settings; however the relationship between the measurements and the
underlying physiological events is in general complex. We anticipate that the
model will play an important role in helping to understand the NIRS signals, in
particular, the cytochrome signal, which has been hard to interpret. A range of
model simulations are presented, and model outputs are compared to published
data obtained from both in vivo and in vitro
settings. The comparisons are encouraging, showing that the model is able to
reproduce observed behaviour in response to various stimuli
Recommended from our members
Fluorescent reporters for functional analysis in rice leaves.
Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed the analysis of processes ranging from gene expression and protein localization to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualize fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins (mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato) that are robustly expressed and detectable in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolor imaging of different subcellular compartments. Overall, we conclude that mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato are suitable for high-resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice
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