Inheritance and fitness costs of resistance to Bacillus thuringiensis toxin Cry2Ad in laboratory strains of the diamondback moth, Plutella xylostella (L.).

The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the main pests of Brassica crops worldwide. Management of P. xylostella is particularly challenging, as different field populations have readily acquired resistance to a wide range of insecticides, including Bacillus thuringiensis (Bt) toxins. In this study, a novel strain of P. xyllostela (Fuzhou-R2Ad) with 120-fold resistance to Bt Cry2Ad was selected in the laboratory, after screening for 66 generations from the susceptible strain Fuzhou-S. In the absence of Bt Cry2Ad toxin, the Fuzhou-R2Ad had significantly lower fitness as compared to the susceptible strain, which might be related to induced genetic changes to Bt toxins. We used several models to measure the dominance levels of insecticide resistance among different strains and found an incompletely recessive inheritance pattern of the Fuzhou-R2Ad resistance, which might be controlled by multiple genes. This study constitutes the first report of laboratory-acquired resistance to Cry2Ad toxin in P. xylostella. Our work presents further insights into the mechanism of Bt resistance and has immediate implications for the integrated pest management of P. xylostella globally.

Arabidopsis Blue Light Receptor Phototropin 1 Undergoes Blue Light-Induced Activation in Membrane Microdomains.

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of phot1-GFP proteins we demonstrated that in the dark phot1 proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of phot1-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1D806N-GFP but did enhance its dimerization, suggesting that phot1 dimerization is independent of phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis revealed that the interaction between phot1-GFP and a marker of sterol-rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3-mCherry, indicating that BL facilitates the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as organizing platforms essential for the proper function of activated phot1 at the PM.

Exploring the Spatiotemporal Organization of Membrane Proteins in Living Plant Cells.

Plasma membrane proteins have important roles in transport and signal transduction. Deciphering the spatiotemporal organization of these proteins provides crucial information for elucidating the links between the behaviors of different molecules. However, monitoring membrane proteins without disrupting their membrane environment remains difficult. Over the past decade, many studies have developed single-molecule techniques, opening avenues for probing the stoichiometry and interactions of membrane proteins in their native environment by providing nanometer-scale spatial information and nanosecond-scale temporal information. In this review, we assess recent progress in the development of labeling and imaging technology for membrane protein analysis. We focus in particular on several single-molecule techniques for quantifying the dynamics and assembly of membrane proteins. Finally, we provide examples of how these new techniques are advancing our understanding of the complex biological functions of membrane proteins.

A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis.

Membrane proteins exert functions by forming oligomers or molecular complexes. Currently, step-wise photobleaching has been applied to count the fluorescently labelled subunits in plant cells, for which an accurate and reliable control is required to distinguish individual subunits and define the basal fluorescence. However, the common procedure using immobilized GFP molecules is obviously not applicable for analysis in living plant cells. Using the spatial intensity distribution analysis (SpIDA), we found that the A206K mutation reduced the dimerization of GFP molecules. Further ectopic expression of Myristoyl-GFPA206K driven by the endogenous AtCLC2 promoter allowed imaging of individual molecules at a low expression level. As a result, the percentage of dimers in the transgenic pCLC2::Myristoyl-mGFPA206K line was significantly reduced in comparison to that of the pCLC2::Myristoyl-GFP line, confirming its application in defining the basal fluorescence intensity of GFP. Taken together, our results demonstrated that pCLC2::Myristoyl-mGFPA206K can be used as a standard control for monomer GFP, facilitating the analysis of the step-wise photobleaching of membrane proteins in Arabidopsis thaliana.

Application of Variable Angle Total Internal Reflection Fluorescence Microscopy to Investigate Protein Dynamics in Intact Plant Cells.

Variable angle total internal reflection fluorescence microscopy (VA-TIRFM) is an optical method to observe the molecular events occurring in an extremely thin region near the plasma membrane. Recently, the VA-TIRFM technique has been widely used to study fluorescently labeled target molecules in living animal and plant cells. Here, we describe the optical principle of the VA-TIRFM technique and provide a detailed experimental procedure for the study of living plant cells.

Spatiotemporal Dynamics of the BRI1 Receptor and its Regulation by Membrane Microdomains in Living Arabidopsis Cells.

The major brassinosteroid (BR) receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRI1. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFlot1-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bri1-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.

Effects of flip angle uncertainty and noise on the accuracy of DCE-MRI metrics: comparison between standard concentration-based and signal difference methods.

Dynamic contrast-enhanced MRI is becoming an increasingly important tool to assess tumors and their response to treatment. In the most common method of computing tumor perfusion parameters, the concentration of the injected contrast agent is first computed in both tumor and blood which is subsequently fit to a perfusion model, typically the Tofts two compartment model. However, this strategy can be highly sensitive to errors in the excitation flip angle and noise. More recently, a simpler method of determining perfusion was developed in which the difference signal, obtained by subtracting the measured time course signal by the signal prior to bolus arrival, is utilized in lieu of the concentration values. The goal of this work is to compare the performance of these two strategies with simulation experiments in the presence of flip angle errors and different levels of image signal to noise ratios (SNRs). Results show that with the conventional method, if assumed pre-contrast T1 of blood is used, large errors in perfusion (exceeding 400% and 200% for K(trans) and ve, respectively) can occur in the presence of flip angle deviations typically observed in vivo. However, when baseline T1 values are measured for both tumor and blood, the errors become a function of flip angle difference between the two locations, with nearly no error if the flip angle errors are identical at both locations. The errors are substantially smaller with the signal difference strategy (less than 100% for both K(trans) and ve). The latter method also yields more consistent perfusion values at varying SNR levels. The results suggest that measuring the actual flip angle may be critical for obtaining absolute perfusion values, but in studies in which relative changes in perfusion is of primary interest or if true flip angles are not known, the signal difference strategy may be preferred over the standard concentration-based method.

Noncontrast enhanced four-dimensional dynamic MRA with golden angle radial acquisition and K-space weighted image contrast (KWIC) reconstruction.

PURPOSE: To explore the feasibility of 2D and 3D golden-angle radial acquisition strategies in conjunction with k-space weighted image contrast (KWIC) temporal filtering to achieve noncontrast enhanced dynamic MRA (dMRA) with high spatial resolution, low streaking artifacts and high temporal fidelity. METHODS: Simulations and in vivo examinations in eight normal volunteers and an arteriovenous malformation patient were carried out. Both 2D and 3D golden angle radial sequences, preceded by spin tagging, were used for dMRA of the brain. The radial dMRA data were temporally filtered using the KWIC strategy and compared with matched standard Cartesian techniques. RESULTS: The 2D and 3D dynamic MRA image series acquired with the proposed radial techniques demonstrated excellent image quality without discernible temporal blurring compared with standard Cartesian based approaches. The image quality of radial dMRA was equivalent to or higher than that of Cartesian dMRA by visual inspection. A reduction factor of up to 10 and 3 in scan time was achieved for 2D and 3D radial dMRA compared with the Cartesian-based counterparts. CONCLUSION: The proposed 2D and 3D radial dMRA techniques demonstrated image quality comparable or even superior to those obtained with standard Cartesian methods, but within a fraction of the scan time.

Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development.

Clathrin-mediated endocytosis, which depends on the AP2 complex, plays an essential role in many cellular and developmental processes in mammalian cells. However, the function of the AP2 complex in plants remains largely unexplored. Here, we show in Arabidopsis that the AP2 σ subunit mutant (ap2 σ) displays various developmental defects that are similar to those of mutants defective in auxin transport and/or signaling, including single, trumpet-shaped and triple cotyledons, impaired vascular pattern, reduced vegetative growth, defective silique development and drastically reduced fertility. We demonstrate that AP2 σ is closely associated and physically interacts with the clathrin light chain (CLC) in vivo using fluorescence cross-correlation spectroscopy (FCCS), protein proximity analyses and co-immunoprecipitation assays. Using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), we show that AP2 σ-mCherry spots colocalize with CLC-EGFP at the plasma membrane, and that AP2 σ-mCherry fluorescence appears and disappears before CLC-EGFP fluorescence. The density and turnover rate of the CLC-EGFP spots are significantly reduced in the ap2 σ mutant. The internalization and recycling of the endocytic tracer FM4-64 and the auxin efflux carrier protein PIN1 are also significantly reduced in the ap2 σ mutant. Further, the polar localization of PIN1-GFP is significantly disrupted during embryogenesis in the ap2 σ mutant. Taken together, our results support an essential role of AP2 σ in the assembly of a functional AP2 complex in plants, which is required for clathrin-mediated endocytosis, polar auxin transport and plant growth regulation.

Automatic coil selection for streak artifact reduction in radial MRI.

In radial MR imaging, streaking artifacts contaminating the entire field of view can arise from regions at the outer edges of the prescribed field of view. This can occur even when the Nyquist criterion is satisfied within the desired field of view. These artifacts become exacerbated when parts of the object lie in the superior/inferior regions of the scanner where the gradient strengths become weakened. When multiple coil arrays are used for signal reception, coils at the outer edges can be disabled before data acquisition to reduce the artifact levels. However, as the weakened gradient strengths near the edges often distort the object, causing the signal to become highly concentrated into a small region, the streaks are often not completely removed. Data from certain coils can also be excluded during reconstruction by visually inspecting the individual coil images, but this is impractical for routine use. In this work, a postprocessing method is proposed to automatically identify those coils whose images contain high levels of streaking for subsequent exclusion during reconstruction. The proposed method was demonstrated in vivo dynamic contrast enhanced MRI datasets acquired using a three-dimensional hybrid radial sequence. The results demonstrate that the proposed strategy substantially improves the image quality and show excellent agreement with images reconstructed with manually determined coil selection.

Neural differences between intrinsic reasons for doing versus extrinsic reasons for doing: an fMRI study.

The contemporary neural understanding of motivation is based almost exclusively on the neural mechanisms of incentive motivation. Recognizing this as a limitation, we used event-related functional magnetic resonance imaging (fMRI) to pursue the viability of expanding the neural understanding of motivation by initiating a pioneering study of intrinsic motivation by scanning participants' neural activity when they decided to act for intrinsic reasons versus when they decided to act for extrinsic reasons. As expected, intrinsic reasons for acting more recruited insular cortex activity while extrinsic reasons for acting more recruited posterior cingulate cortex (PCC) activity. The results demonstrate that engagement decisions based on intrinsic motivation are more determined by weighing the presence of spontaneous self-satisfactions such as interest and enjoyment while engagement decisions based on extrinsic motivation are more determined by weighing socially-acquired stored values as to whether the environmental incentive is attractive enough to warrant action.

Comparison of lung T2* during free-breathing at 1.5 T and 3.0 T with ultrashort echo time imaging.

Assessment of lung effective transverse relaxation time (T(2)*) may play an important role in the detection of structural and functional changes caused by lung diseases such as emphysema and chronic bronchitis. While T(2)* measurements have been conducted in both animals and humans at 1.5 T, studies on human lung at 3.0 T have not yet been reported. In this work, ultrashort echo time imaging technique was applied for the measurement and comparison of T(2)* values in normal human lungs at 1.5 T and 3.0 T. A 2D ultrashort echo time pulse sequence was implemented and evaluated in phantom experiments, in which an eraser served as a homogeneous short T(2)* sample. For the in vivo study, five normal human subjects were imaged at both field strengths and the results compared. The average T(2)* values measured during free-breathing were 2.11(±0.27) ms at 1.5 T and 0.74(±0.1) ms at 3.0 T, respectively, resulting in a 3.0 T/1.5 T ratio of 2.9. Furthermore, comparison of the relaxation values at end-expiration and end-inspiration, accomplished through self-gating, showed that during normal breathing, differences in T(2)* between the two phases may be negligible.

Direct MRI mapping of neuronal activity evoked by electrical stimulation of the median nerve at the right wrist.

Magnetic source MRI (msMRI) has being developed recently for direct detections of neuronal magnetic fields to map brain activity. However, controversial results have been reported by different research groups. In this study, more evidence was provided to demonstrate that the neuronal current signal could be detected by MRI using a rapid median nerve stimulation paradigm. The experiments were performed on six normal human participants to investigate the temporal specificity of the effect, as well as inter- and intrasubject reproducibility. Significant activation of contralateral primary sensory cortex (S1) was detected 80 ms after stimulation onset (corresponding to the P80 evoked potential peak). The 80-ms latency S1 activation was observed over three independent sessions for one subject and for all six participants. The magnitude of the signal change was 0.2-0.3%. Coinciding with our expectations, no S1 activation was found when MRI data acquisitions were targeted at the N20 and P30 peaks because of mutual cancellation of magnetic fields generated by those peaks. The results demonstrated good reproducibility of S1 activations and indicated that the S1 activations most likely originated from neuronal magnetic field rather than hemodynamic response.

Spaceflight induces both transient and heritable alterations in DNA methylation and gene expression in rice (Oryza sativa L.).

Spaceflight represents a complex environmental condition in which several interacting factors such as cosmic radiation, microgravity and space magnetic fields are involved, which may provoke stress responses and jeopardize genome integrity. Given the inherent property of epigenetic modifications to respond to intrinsic as well as external perturbations, it is conceivable that epigenetic markers like DNA methylation may undergo alterations in response to spaceflight. We report here that extensive alteration in both DNA methylation and gene expression occurred in rice plants subjected to a spaceflight, as revealed by a set of characterized sequences including 6 transposable elements (TEs) and 11 cellular genes. We found that several features characterize the alterations: (1) All detected alterations are hypermethylation events; (2) whereas alteration in both CG and CNG methylation occurred in the TEs, only alteration in CNG methylation occurred in the cellular genes; (3) alteration in expression includes both up- and down-regulations, which did not show a general correlation with alteration in methylation; (4) altered methylation patterns in both TEs and cellular genes are heritable to progenies at variable frequencies; however, stochastic reversion to wild-type patterns and further de novo changes in progenies are also apparent; and (5) the altered expression states in both TEs and cellular genes are also heritable to selfed progenies but with markedly lower transmission frequencies than altered DNA methylation states. Furthermore, we found that a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, the SWI/SNF chromatin remodeller (DDM1) and siRNA-related proteins are extremely sensitive to perturbation by spaceflight, which might be an underlying cause for the altered methylation patterns in the space-flown plants. We discuss implications of spaceflight-induced epigenetic variations with regard to health safety issues of spaceship crews and potentiality of spaceflight as a means for mutagenesis in crop breeding.

Direct MRI detection of neuronal magnetic fields in the brain: theoretical modeling.

Whether MRI can be used for direct detection of neuronal activity is a matter of debate. Controversial theoretical and experimental results have been reported. Here, we present an improved current-dipole model to compute magnetic field generated by neural firing and to calculate MRI signal changes resulting from the neuronal magnetic field (NMF). Each dendrite or each unmyelinated axon was modeled as a modified current-dipole. NMF were estimated based on a synchronized activity of multiple neurons. Sensitivity of using phase and magnitude MRI to measure effects of NMF was evaluated. Our results show that NMF can potentially generate up to a few percent changes in MRI magnitude signals. Phases of MRI signal tend to be destructively added and are insensitive to NMF in the activated region when the distribution of the activated dendrites is symmetrical. Phases could be detected when the distribution of the activated dendrites is asymmetrical and on some neighboring voxels. Our modeling implies that direct MRI detection of neuronal activity is possible.