Living organisms detect changes in temperature using thermosensory molecules. However, these molecules and/or their mechanisms for sensing temperature differ among organisms. To identify thermosensory molecules in plants, we investigated chloroplast positioning in response to temperature changes and identified a blue-light photoreceptor, phototropin, that is an essential regulator of chloroplast positioning. Based on the biochemical properties of phototropin during the cellular response to light and temperature changes, we found that phototropin perceives temperature based on the temperature-dependent lifetime of the photoactivated chromophore. Our findings indicate that phototropin perceives both blue light and temperature and uses this information to arrange the chloroplasts for optimal photosynthesis. Because the photoactivated chromophore of many photoreceptors has a temperature-dependent lifetime, a similar temperature-sensing mechanism likely exists in other organisms. Thus, photoreceptors may have the potential to function as thermoreceptors.iving organisms perceive temperature using thermosensory molecules. Various types of thermosensory molecules have been identified in microorganisms, animals, and plants, and these molecules perceive temperature via temperature-dependent intraand intermolecular interactions (1-3). However, these thermosensory molecules and/or their mechanisms for temperature sensing differ among organisms (1).As photosynthetic organelles, chloroplasts change their intracellular position in response to light and temperature (4-7). For example, under low light conditions at 22°C, chloroplasts accumulate at the cell surface (along the periclinal cell wall) to maximize photosynthetic efficiency, in a phenomenon termed the accumulation response (4, 6). However, under the same light conditions, but at a temperature of 5°C, the chloroplasts move to the cell periphery (along the anticlinal cell wall), possibly to avoid the light, in a phenomenon termed the cold-avoidance response or the cold-positioning response (5, 6). Because the cold-avoidance response is temperature dependent (5), the response is likely controlled by a thermosensory molecule. In a previous study, we found that the blue-light (BL) photoreceptor phototropin (phot) is responsible for the cold-avoidance response in the fern Adiantum capillus-veneris, which has three types of phot molecules (phot1, phot2, and neochrome1) (5,8). In this fern, phot2 mediates the cold-avoidance response (5). Therefore, in the present study, we hypothesized that phot is involved in temperature perception and we further analyzed the cold-avoidance response in the liverwort Marchantia polymorpha, which has only a single copy of the PHOT gene (MpPHOT) (9). Results and DiscussionFirst, we examined whether MpPHOT is essential for the coldavoidance response in M. polymorpha. When the wild type (WT), the knockout mutant (Mpphot KO ), and the complementation line (Mpphot/Mpphot KO ) (9) (Fig. S1) were treated with low white light at 5°C for 24 h to induce ...

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Green fluorescent protein (GFP) was discovered from the jellyfish Aequorea victoria, and several improvements have been carried out to change its physicochemical properties. The resulting improved GFP variants have been used as reporter proteins for bioimaging techniques in various research fields including plant science. Almost all GFP variants were developed using Escherichia coli to improve fluorescence properties in mammalian cells, but the impact in other organisms such as plant cells remains to be determined. In this study, we performed comparative analysis of four improved GFP variants, GFP-S65T, eGFP, frGFP and sfGFP, with reference to the fluorescence intensity in Arabidopsis protoplasts, and found that sfGFP is the brightest. Using non-fluorescent fragments from the GFP variants, we also conducted bimolecular fluorescence complementation (BiFC) assays to find appropriate fragment pairs of GFP-based BiFC for visualization of protein-protein interactions in living plant cells. Our observations revealed that the brightest is the sfGFP-based BiFC. Further, as an evaluation method for the sfGFP-based BiFC, a BiFC competition assay was successfully completed for the first time in planta. The present study provides useful information for selection and improvement of the GFP molecule and its application to BiFC technology in plants.Key words: Arabidopsis, BiFC, BiFC competition, GFP, protein-protein interaction.Since the first attempt to employ wild-type green fluorescent protein (wtGFP) as a reporter protein, it has been developed for bioimaging techniques in various research fields including plant science (Chalfie et al. 1994;Heim et al. 1995). wtGFP is a typical β-barrel structure harboring a chromophore spontaneously formed by the three residues (S65-T66-G67), and thereby exhibits green fluorescence without any cofactor (Tsien 1998). To date, wtGFP has been improved by targeted or random mutations to increase its fluorescence intensity, and almost all of the improvements were performed using Escherichia coli for suitable expression in mammalian cells (Shaner et al. 2005). The first improvement was a single mutation of serine to threonine residue at position 65 (S65T) for the chromophore (Figure 1); the mutation shifted the excitation peak to 490 nm from 395 nm and 470 nm of wtGFP (Heim et al. 1995). Compared with wtGFP, this peak-shifted GFP (psGFP) gives 6-fold brighter Abbreviations: BiFC, bimolecular fluorescence complementation; eGC, the C-terminal fragment of eGFP; eGFP, enhanced GFP; eGN, the N-terminal fragment of eGFP; EYFP, enhanced yellow fluorescent protein; frGC, the C-terminal fragment of frGFP; frGFP, folding reporter GFP; frGN, the N-terminal fragment of frGFP; GFP, green fluorescent protein; MXMT, 7-methylxanthine methyltransferase; psGC, the C-terminal fragment of psGFP; psGFP, peak-shifted GFP; psGN, the N-terminal fragment of psGFP; PEG, polyethylene glycol; RC, the C-terminal fragment of DsRED monomer; sfGC, the C-terminal fragment of sfGFP; sfGFP, superfolder GFP; sfGN, the N-...

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Chronic obstructive pulmonary disease (COPD) has been recently characterized as a disease of accelerated lung aging, but the mechanism remains unclear. Tetraspanins have emerged as key players in malignancy and inflammatory diseases. Here, we found that CD9/CD81 double knockout (DKO) mice with a COPD-like phenotype progressively developed a syndrome resembling human aging, including cataracts, hair loss, and atrophy of various organs, including thymus, muscle, and testis, resulting in shorter survival than wild-type (WT) mice. Consistent with this, DNA microarray analysis of DKO mouse lungs revealed differential expression of genes involved in cell death, inflammation, and the sirtuin-1 (SIRT1) pathway. Accordingly, expression of SIRT1 was reduced in DKO mouse lungs. Importantly, siRNA knockdown of CD9 and CD81 in lung epithelial cells additively decreased SIRT1 and Foxo3a expression, but reciprocally upregulated the expression of p21 and p53, leading to reduced cell proliferation and elevated apoptosis. Furthermore, deletion of these tetraspanins increased the expression of pro-inflammatory genes and IL-8. Hence, CD9 and CD81 might coordinately prevent senescence and inflammation, partly by maintaining SIRT1 expression. Altogether, CD9/CD81 DKO mice represent a novel model for both COPD and accelerated senescence.

The aim of this study was to evaluate the usefulness of simultaneous measurement of plasma steroids, including precursors, for the evaluation of drug effects on adrenal steroidogenesis in vivo. Plasma concentrations of corticosterone and its precursors were examined in rats dosed with compounds that affect adrenal steroidogenesis via different modes of action as well as the relationships of the changes with blood chemistry and adrenal histopathology. Male rats were dosed with tricresyl phosphate, aminoglutethimide, trilostane (TRL), metyrapone (MET), ketoconazole (KET), or mifepristone for 7 days. In the TRL, MET, and KET groups, precursor levels were markedly increased, while there were no significant changes in the corticosterone level, suggesting that the precursors are more sensitive biomarkers to detect the effect on adrenal steroidogenesis. Also, the precursors with increased levels were those that are normally metabolized by the inhibited enzymes, reflecting the modes of action of the compounds. In addition, different patterns of changes were observed in blood chemistry and histopathology, supporting the mechanism suggested by the steroid changes. These results show that simultaneous measurement of plasma steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

To evaluate retrospectively any association between the degree of deformity correction by medial open-wedge high tibial osteotomy (HTO) and patellofemoral joint degeneration. We hypothesized that development of patellofemoral joint degeneration depended on the degree of intraoperative deformity correction. Fifty-seven patients who underwent medial open-wedge HTO for treatment of osteoarthritis in one knee were included in this study. Knees were classified into degeneration (D) and non-degeneration (ND) groups according to worsening of the patellar and/or femoral trochlear cartilage at the time of hardware removal (D group, 27 knees) and no degeneration or improvement (ND group, 30 knees). We compared pre- to post-surgery change in hip-knee-ankle angle (HKA) and medial-proximal-tibial angle (MPTA), open-wedge HTO correction angle, and arthroscopic findings between groups. Mean age, height, weight, and body mass index were 54.1 ± 9.9 years, 160.4 ± 8.7 cm, 66.4 ± 12.1 kg, and 25.7 ± 3.3 kg/m 2 , respectively. Change in both HKA and MPTA differed significantly between groups. The MPTA cut-off values to predict patellofemoral degeneration were determined to be 10°, associated with an AUC of 0.75 (95% confidence interval [CI] 0.62–0.87). This study evaluated retrospectively the effect of the correction angle during medial open-wedge HTO on patellofemoral joint degeneration. If deformity correction exceeds an MPTA of 10° during open-wedge HTO, degeneration of patellofemoral joint needs to be considered. Level of evidence: Level IV.

Neurite orientation dispersion and density imaging (NODDI) is a novel diffusion method for evaluating tissue microstructure, and may provide additional information over conventional diffusion tensor imaging (DTI). We evaluated NODDI and DTI parameters in cases of tuberous sclerosis (TS) to assess microstructural changes in the white matter. Eleven cases of tuberous sclerosis and eight age-matched controls underwent NODDI and DTI. We performed qualitative analysis and tract-based spatial statistics (TBSS) analysis of the NODDI parameters (Ficv: intracellular volume fraction, Fiso: isotropic fraction, ODI: orientation dispersion index) as well as DTI parameters (MD: mean diffusivity, FA: fractional anisotropy). We also performed a correlation analysis between clinical symptoms and parameters. The qualitative analysis indicated that the Ficv had a lower value in TS cases particularly in the tubers adjacent to the white matter. The TBSS analysis showed that the TS cases had decreased Ficv in a greater area compared to the other parameters including MD. In particular, the Ficv was decreased in deep white matter, such as the superior longitudinal fascicles (SLF). The application of NODDI to TS cases revealed tissue microstructural changes, and particularly the Ficv could detect more widespread abnormalities in white matter structure compared to DTI parameters.