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

Intra-session test-retest reliability of pelvic floor muscle electromyography during running

Introduction and hypothesis: The prevalence of female stress urinary incontinence is high, and young adults are also affected, including athletes, especially those involved in "high-impact” sports. To date there have been almost no studies testing pelvic floor muscle (PFM) activity during dynamic functional whole body movements. The aim of this study was the description and reliability test of PFM activity and time variables during running. Methods: A prospective cross-sectional study including ten healthy female subjects was designed with the focus on the intra-session test-retest reliability of PFM activity and time variables during running derived from electromyography (EMG) and accelerometry. Results: Thirteen variables were identified based on ten steps of each subject: Six EMG variables showed good reliability (ICC 0.906-0.942) and seven time variables did not show good reliability (ICC 0.113-0.731). Time variables (e.g. time difference between heel strike and maximal acceleration of vaginal accelerator) showed low reliability. However, relevant PFM EMG variables during running (e.g., pre-activation, minimal and maximal activity) could be identified and showed good reliability. Conclusion: Further adaptations regarding measurement methods should be tested to gain better control of the kinetics and kinematics of the EMG probe and accelerometers. To our knowledge this is the first study to test the reliability of PFM activity and time variables during dynamic functional whole body movements. More knowledge of PFM activity and time variables may help to provide a deeper insight into physical strain with high force impacts and important functional reflexive contraction patterns of PFM to maintain or to restore continenc

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

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

Chloroplast development in green plant tissues: the interplay between light, hormone, and transcriptional regulation.

Chloroplasts are best known for their role in photosynthesis, but they also allow nitrogen and sulphur assimilation, amino acid, fatty acid, nucleotide and hormone synthesis. How chloroplasts develop is therefore relevant to these diverse and fundamental biological processes, but also to attempts at their rational redesign. Light is strictly required for chloroplast formation in all angiosperms and directly regulates the expression of hundreds of chloroplast-related genes. Light also modulates the levels of several hormones including brassinosteriods, cytokinins, auxins and gibberellins, which themselves control chloroplast development particularly during early stages of plant development. Transcription factors such as GOLDENLIKE1&2 (GLK1&2), GATA NITRATE-INDUCIBLE CARBON METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1) act downstream of both light and phytohormone signalling to regulate chloroplast development. Thus, in green tissues transcription factors, light signalling and hormone signalling form a complex network regulating the transcription of chloroplast- and photosynthesis-related genes to control the development and number of chloroplasts per cell. We use this conceptual framework to identify points of regulation that could be harnessed to modulate chloroplast abundance and increase photosynthetic efficiency of crops, and to highlight future avenues to overcome gaps in current knowledge

Continuous versus intermittent stochastic resonance whole body vibration and its effect on pelvic floor muscle activity

To determine the optimal stochastic whole body vibration (SR-WBV) load modality regarding pelvic floor muscle (PFM) activity in order to complete the SR-WBV training methodology for future PFM training with SR-WBV