PUB22 and PUB23 U-box E3 ubiquitin ligases negatively regulate 26S proteasome activity under proteotoxic stress conditions.

The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear. Here, we showed that arsenite-induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U-box E3 ubiquitin ligases and that pub22pub23 double mutants displayed arsenite-insensitive seed germination and root growth phenotypes. PUB22/PUB23 downregulated 26S proteasome activity by promoting the dissociation of the 19S regulatory particle from the holo-proteasome complex, resulting in intracellular accumulation of UbG76V -GFP, an artificial substrate of the proteasome complex, and insoluble poly-ubiquitinated proteins. These results suggest that PUB22/PUB23 play a critical role in arsenite-induced proteotoxic stress response via negative regulation of 26S proteasome integrity.

The N-Terminal UND Motif of the Arabidopsis U-Box E3 Ligase PUB18 Is Critical for the Negative Regulation of ABA-Mediated Stomatal Movement and Determines Its Ubiquitination Specificity for Exocyst Subunit Exo70B1.

The Arabidopsis thaliana U-box E3 ligases PUB18/PUB19 and PUB22/PUB23 are negative regulators of drought stress responses. PUB18/PUB19 regulate the drought stress response in an abscisic acid (ABA)-dependent manner, whereas PUB22/PUB23 regulate this response in an ABA-independent manner. A major structural difference between PUB18/PUB19 and PUB22/PUB23 is the presence of the UND (U-box N-terminal domain). Here, we focused on elucidating the molecular mechanism that mediates the functional difference between PUB18 and PUB22 and found that the UNDPUB18 was critically involved in the negative regulation of ABA-mediated stomatal movements. Exo70B1, a subunit of the exocyst complex, was identified as a target of PUB18, whereas Exo70B2 was a substrate of PUB22. However, the ∆UND-PUB18 derivative failed to ubiquitinate Exo70B1, but ubiquitinated Exo70B2. By contrast, the UNDPUB18-PUB22 chimeric protein ubiquitinated Exo70B1 instead of Exo70B2, suggesting that the ubiquitination specificities of PUB18 and PUB22 to Exo70B1 and Exo70B2, respectively, are dependent on the presence or absence of the UNDPUB18 motif. The ABA-insensitive phenotypes of the pub18 pub19 exo70b1 triple mutant were reminiscent of those of exo70b1 rather than pub18 pub19, indicating that Exo70B1 functions downstream of PUB18. Overall, our results suggest that the UNDPUB18 motif is crucial for the negative regulation of ABA-dependent stomatal movement and for determination of its ubiquitination specificity to Exo70B1.

Comparison of sagittal values between lateral decubitus plain radiography and supine computed tomography in thoracolumbar fractures: a greater degree of kyphosis is observed in plain radiography than CT.

OBJECTIVE: Radiologic parameters are important factors for planning the treatment for thoracolumbar fracture. However, we noted that measurements of the degree of kyphosis by lateral decubitus plain radiography were greater than supine CT. The cause of this discrepancy is unclear. METHODS: We retrospectively reviewed the plain radiographs and CT scans of 90 patients with thoracolumbar fractures (fracture group). We measured the segmental sagittal angle (SSA) on lateral decubitus plain radiographs and in the median sagittal plane on CT scans obtained in the supine position. The method agreement (plain radiography versus CT) was determined by utilizing Bland-Altman plots. For the purpose of comparison, the same analyses were performed in a group of age and sex-matched controls (normal group). After establishing the method disagreement in the fracture group, the factors that contributed to the difference in the SSA between plain radiography and CT, as well as their threshold values, were determined. RESULTS: On Bland-Altman plots for the fracture group, the mean difference was 4.53° [95% confidence interval (CI) - 4.87° to 13.93°]. For the normal group, the mean difference was - 0.64° (95% CI - 5.87° to 4.58°). On univariate analysis, male sex, thoracolumbar level, and SSA(X) were significant factors associated with ∆SSA (P = 0.03, 0.002, and 0.000, respectively). Multivariable regression analysis showed that SSA(X) was the only significant factor. Receiver operating characteristic curve analysis indicated that the optimal threshold of SSA(X) was 17° with a sensitivity of 78% and a specificity of 75% (area under curve: 0.752). CONCLUSIONS: The mean SSA determined on lateral decubitus plain radiographs indicated significantly more kyphosis than that determined on CT images obtained in supine position. When the SSA on plain radiography is more than 17°, there might be a significant discrepancy between the two imaging modalities. This discrepancy seems to be mainly attributable to the difference in patient positioning (lateral decubitus position for plain radiography versus supine position for CT imaging).

Light sheet fluorescence microscopy using axi-symmetric binary phase filters.

Light sheet fluorescence microscopy (LSFM) has become an indispensable tool in biomedical studies owing to its depth-sectioning capability and low photo-bleaching. The axial resolution in LSFM is determined mainly by the thickness of the illumination sheet, and a high numerical-aperture lens is thus preferred in the illumination to increase the axial resolution. However, a rapid divergence of the illumination beam limits the effective field-of-view (FoV), that provides high-resolution images. Several strategies have been demonstrated for FoV enhancement, which involve the use of Bessel or Airy beams, for example. However, the generation of these beams requires complicated optical setup or phase filters with continuous phase distributions, which are difficult to manufacture. In contrast, a binary phase filter (BPF) comprising concentric rings with 0 or π phases produces a response similar to its continuous original and is easy to realize. Here, we present a novel form of LSFM that integrates BPFs derived from two representative axi-symmetric aberrations, including phase axicon and spherical aberrations, to improve the imaging performance. We demonstrate that these BPFs significantly increase the FoV, and those derived from axicon generate self-reconstructing beams, which are highly desirable in imaging through scattering specimens. We validate its high-contrast imaging capability over extended FoV by presenting three-dimensional images of microspheres, imaginal disc of Drosophila larva, and Arabidopsis.

Arabidopsis group XIV ubiquitin-conjugating enzymes AtUBC32, AtUBC33, and AtUBC34 play negative roles in drought stress response.

AtUBC32, AtUBC33, and AtUBC34 comprise Arabidopsis group XIV E2 ubiquitin-conjugating enzymes. Yeast two-hybrid, in vitro pull-down, and bimolecular fluorescence complementation assays revealed that group XIV E2s are interacting partners of the U-box-type E3 ligase PUB19, a negative regulator of drought stress response. These three AtUBCs are co-localized with PUB19 to the punctae-like structures, most of which reside on the endoplasmic reticulum membrane of tobacco leaf cells. Suppression of AtUBC32, AtUBC33, and AtUBC34 resulted in increased abscisic acid-mediated stomatal closure and tolerance to drought stress. These results indicate that Arabidopsis group XIV E2s play negative roles in drought stress response.