Identification and functional characterization of small non-coding RNAs in Xanthomonas oryzae pathovar oryzae

<p>Abstract</p> <p>Background</p> <p>Small non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. <it>Xanthomonas oryzae </it>pathovar <it>oryzae </it>(<it>Xoo</it>) is an important plant pathogenic bacterium which causes serious bacterial blight of rice. However, little is known about the number, genomic distribution and biological functions of sRNAs in <it>Xoo</it>.</p> <p>Results</p> <p>Here, we performed a systematic screen to identify sRNAs in the <it>Xoo </it>strain PXO99. A total of 850 putative non-coding RNA sequences originated from intergenic and gene antisense regions were identified by cloning, of which 63 were also identified as sRNA candidates by computational prediction, thus were considered as <it>Xoo </it>sRNA candidates. Northern blot hybridization confirmed the size and expression of 6 sRNA candidates and other 2 cloned small RNA sequences, which were then added to the sRNA candidate list. We further examined the expression profiles of the eight sRNAs in an <it>hfq </it>deletion mutant and found that two of them showed drastically decreased expression levels, and another exhibited an Hfq-dependent transcript processing pattern. Deletion mutants were obtained for seven of the Northern confirmed sRNAs, but none of them exhibited obvious phenotypes. Comparison of the proteomic differences between three of the ΔsRNA mutants and the wild-type strain by two-dimensional gel electrophoresis (2-DE) analysis showed that these sRNAs are involved in multiple physiological and biochemical processes.</p> <p>Conclusions</p> <p>We experimentally verified eight sRNAs in a genome-wide screen and uncovered three Hfq-dependent sRNAs in <it>Xoo</it>. Proteomics analysis revealed <it>Xoo </it>sRNAs may take part in various metabolic processes. Taken together, this work represents the first comprehensive screen and functional analysis of sRNAs in rice pathogenic bacteria and facilitates future studies on sRNA-mediated regulatory networks in this important phytopathogen.</p

Minimizing the effects of magnetization transfer asymmetry on inhomogeneous magnetization transfer (ihMT) at ultra-high magnetic field (11.75 T)

International audienceOBJECTIVES: The recently reported inhomogeneous magnetization transfer technique (ihMT) has been proposed for specific imaging of inhomogeneously broadened lines, and has shown great promise for characterizing myelinated tissues. The ihMT contrast is obtained by subtracting magnetization transfer images obtained with simultaneous saturation at positive and negative frequency offsets (dual frequency saturation experiment, MT (+/-)) from those obtained with single frequency saturation (MT (+)) at the same total power. Hence, ihMT may be biased by MT-asymmetry, especially at ultra-high magnetic field. Use of the average of single positive and negative frequency offset saturation MT images, i.e., (MT (+)+MT (-)) has been proposed to correct the ihMT signal from MT-asymmetry signal. MATERIALS AND METHODS: The efficiency of this correction method was experimentally assessed in this study, performed at 11.75 T on mice. Quantitative corrected ihMT and MT-asymmetry ratios (ihMTR and MTRasym) were measured in mouse brain structures for several MT-asymmetry magnitudes and different saturation parameter sets. RESULTS: Our results indicated a "safe" range of magnitudes (/MTRasym/\textless4 %) for which MT-asymmetry signal did not bias the corrected ihMT signal. Moreover, experimental evidence of the different natures of both MT-asymmetry and inhomogeneous MT contrasts were provided. In particular, non-zero ihMT ratios were obtained at zero MTRasym values. CONCLUSION: MTRasym is not a confounding factor for ihMT quantification, even at ultra-high field, as long as MTRasym is restricted to ±4 %

Fast imaging strategies for mouse kidney perfusion measurement with pseudocontinuous arterial spin labeling (pCASL) at ultra high magnetic field (11.75 tesla)

International audienceBackground To derive an adapted protocol at ultra high magnetic field for mouse kidney perfusion measurements using pCASL in combination with three widely available fast imaging readouts: segmented SE EPI (sSE EPI), RARE, and TrueFISP. Methods pCASL sSE EPI, pCASL RARE, and pCASL TrueFISP were used for the acquisition of mouse kidney perfusion images in the axial and coronal planes at 11.75T. Results were compared in terms of perfusion sensitivity, signal-to-noise ratio (SNR), blood flow values, intrasession and intersession repeatability, and image quality (subjectively classified into three grades: good, satisfactory, and unacceptable). Results Renal cortex perfusion measurements were performed within 2 min with pCASL RARE/pCASL TrueFISP and 4 min with pCASL sSE EPI. In an axial direction, SNR values of 6.6/5.6/2.8, perfusion sensitivity values of 16.1 ± 3.7/13.6 ± 2.4/13.4 ± 1.0 %, blood flow values of 679 ± 149/466 ± 111/572 ± 46 mL/100 g/min and in-ROI variations values of 192/161/181 mL/100 g/min were obtained with pCASL sSE EPI/pCASL RARE/pCASL TrueFISP. Highest SNR per unit of time (1.8) and highest intra/intersession reliability (92.9% and 95.1%) were obtained with pCASL RARE, which additionally presented highly reproducible satisfactory image quality. In coronal plane, significantly lower SNR, perfusion sensitivity and perfusion values were obtained for all techniques compared with that in the axial plane (P \textless 0.05) due to magnetization saturation effects. Conclusion pCASL RARE demonstrated more advantages for longitudinal preclinical kidney perfusion studies at ultra high magnetic field. J. Magn. Reson. Imaging 2015;42:999–1008

On the boost effect of inhomogeneous Magnetization Transfer (ihMT)

International audienceA new implementation of inhomogeneous magnetization transfer (ihMT) has recently been introduced, consisting of concentrating the RF energy deposition within the saturation period and demonstrating a significant boost of the ihMT sensitivity. The boost effect has been characterized in this study among different ihMT sequences, species and field strengths and reveals common features. The optimal sequence settings vary with the number of consecutive MT pulses and are presumably related to the timescale of the underlying T_\textrm1D components and magnetization exchange rates

Anisotropy of inhomogeneous Magnetization Transfer (ihMT) in White Matter

International audienceInhomogeneous magnetization transfer (ihMT) is a new endogenous contrast mechanism that has been proposed for imaging myelinated tissues. The dipolar interaction underlying the ihMT effect is intrinsically anisotropic, exhibiting the well-known (3cos&sup2;&theta; -1) angle dependency. Here we report experimental evidence of the anisotropy of ihMT in white matter and we derive a realistic theoretical model combining the angular dependency of the myelin lineshape and dipolar-order RF saturation theory

The inhomogeneous MT (ihMT) technique: Achievements and perspectives

International audienceThe inhomogeneous (ihMT) technique represents a relatively simple addition to MT, but provides a different contrast that is sensitive to the dipolar relaxation time T_\textrm1D. A review is provided of: method(s) for its application; considerations for optimizing the ihMT signal; the use of models to obtain quantitative parameters and guide acquisition; and its application in vivo

The inhomogeneous MT (ihMT) technique: Achievements and perspectives

International audienceThe inhomogeneous (ihMT) technique represents a relatively simple addition to MT, but provides a different contrast that is sensitive to the dipolar relaxation time T_\textrm1D. A review is provided of: method(s) for its application; considerations for optimizing the ihMT signal; the use of models to obtain quantitative parameters and guide acquisition; and its application in vivo

Anisotropy of inhomogeneous Magnetization Transfer (ihMT) in White Matter

International audienceInhomogeneous magnetization transfer (ihMT) is a new endogenous contrast mechanism that has been proposed for imaging myelinated tissues. The dipolar interaction underlying the ihMT effect is intrinsically anisotropic, exhibiting the well-known (3cos&sup2;&theta; -1) angle dependency. Here we report experimental evidence of the anisotropy of ihMT in white matter and we derive a realistic theoretical model combining the angular dependency of the myelin lineshape and dipolar-order RF saturation theory

Magnetization transfer from inhomogeneously broadened lines (ihMT): Experimental optimization of saturation parameters for human brain imaging at 1.5 Tesla

International audiencePURPOSE: Recently a new MR endogenous contrast mechanism was reported. It allows specifically imaging the magnetization transfer (MT) effect arising from inhomogeneously broadened components of the NMR spectrum, and was hence dubbed ihMT. Such unique NMR lineshape properties are presumably occurring in myelin because of its specifically ordered, multilayered sheath structure. Here, optimization of a pulsed ihMT preparation module is presented to provide guidance for future studies and improve the understanding of underlying contrast mechanisms. METHODS: This study was performed at 1.5 Tesla on healthy volunteers. A pulsed ihMT preparation was implemented in combination with a HASTE readout module. The pulse width, interpulse repetition time, total saturation duration and RF saturation power were considered for optimization of the ihMT sensitivity and contrast. RESULTS: An optimal configuration of the preparation module was derived, leading to 10% ihMT signal in internal capsule (relative to unsaturated data) and around 200% signal increase relative to gray matter, i.e., approximately 10-fold superior contrast compared with conventional MT ratios, measured under similar experimental conditions. CONCLUSION: Overall the ihMT sequence was robust, sensitive and very specific for white matter. These findings suggest great potential for assessing brain myelination and for better characterization of myelin related disorders. Magn Reson Med 73:2111-2121, 2015. © 2014 Wiley Periodicals, Inc

High Resolution Magic Angle Spinning (HR-MAS) NMR characterization of inhomogeneous magnetization transfer (ihMT) responsive samples

International audienceInhomogeneous magnetization transfer (ihMT) is a promising technique for central nervous system imaging. Although ihMT signal is mostly observed in myelinated tissues, weaker ihMT signal may be revealed in other biological tissue. The fundamental relationship between the underlying NMR lineshape and the ihMT signal is still an open question. Here we investigate high resolution magic-angle spinning (HR-MAS) NMR to provide insight into the mechanisms underpinning line broadening of ihMT responsive samples. The resulting spectra evidence different dipolar Hamiltonian components contributing to line broadening and support ihMT as being dominated by inhomogeneous interaction in myelinated tissues