Antifibrotic effects of CXCR4 antagonist in bleomycin-induced pulmonary fibrosis in mice

Circulating fibrocytes had been reported to migrate into the injured lungs, and contribute to fibrogenesis via chemokine-chemokine receptor systems including CXCL12-CXCR4 axis. Here we hypothesized that blockade of CXCR4 might inhibit the migration of fibrocytes to the injured lungs and the subsequent pulmonary fibrosis. To explore the antifibrotic effects of blockade of CXCR4, we used a specific antagonist for CXCR4, AMD3100, in bleomycin-induced pulmonary fibrosis model in mice. Administration of AMD3100 significantly improved the loss of body weight of mice treated with bleomycin, and inhibited the fibrotic lesion in subpleural areas of the lungs. The quantitative analysis demonstrated that treatment with AMD3100 reduced the collagen content and fibrotic score (Aschcroft score) in the lungs. Although AMD3100 did not affect cell classification in bronchoalveolar lavage fluid on day 7, the percentage of lymphocytes was reduced by AMD3100 on day 14. AMD3100 directly inhibited the migration of human fibrocytes in response to CXCL12 in vitro, and reduced the trafficking of fibrocytes into the lungs treated with bleocmycin in vivo. These results suggest that the blockade of CXCR4 might be useful strategy for therapy of patients with pulmonary fibrosis via inhibiting the migration of circulating fibrocytes

Fluorescence Imaging-Based High-Throughput Screening of Fast- and Slow-Cycling LOV Proteins

<div><p>Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from <i>Avena sativa</i> phototropin 1 (AsLOV2). The gene library was introduced into <i>Escherichia coli</i> cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3×10³ s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.</p></div

Direct imaging the thermal reversion of wild-type AsLOV2.

<p>(A) Bacterial colonies expressing wild-type AsLOV2 on an agar plate were irradiated with blue light, and fluorescence recovery of its flavin cofactor was visualized with a stereoscopic fluorescence microscope. (B) Time-lapse imaging of the fluorescence recovery of wild-type AsLOV2 after irradiation with blue light. The region boxed with a white square in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082693#pone-0082693-g003" target="_blank">Fig. 3A</a> are magnified. (C) Time course of the fluorescence recovery of three independent bacterial colonies shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082693#pone-0082693-g003" target="_blank">Fig. 3B</a> with white dashed circles. The fluorescence recovery was fit with single exponential curves (solid lines).</p

A LOV domain and its photocycle.

<p>(A) The second light-oxygen-voltage (LOV) domain derived from <i>Avena sativa</i> phototropin 1 (AsLOV2) binds a flavin cofactor (FMN) to sense blue light. In the dark state, the C-terminal Jα helix of AsLOV2 is tightly bound to its core domain (switch-off, left panel). Upon irradiation with blue light, the Jα helix is released from the core domain of AsLOV2 (switch-on, right panel). When the blue light is turned off, the open conformation of AsLOV2 in the light state is returned back to its closed conformation in the dark state (right to left). The blue light-dependent conformational change of AsLOV2 switches the activity of an effector domain, such as a protein with enzymatic activity and a peptide, connected at the C-terminus of AsLOV2. (B) A photochemical reaction, known as a photocycle, occurring between a LOV domain and a flavin cofactor. Blue light irradiation induces the formation of a covalent bond between the thiol group of a cysteine within a LOV domain and the C4a position of the isoalloxazine ring of flavin (left to right). The photoadduct spontaneously breaks when the LOV domain is returned back to the dark condition (right to left). The photoadduct formation and its break lead to loss of fluorescence from the flavin cofactor and its recovery, respectively.</p

Mutagenesis and screening of AsLOV2 variants with improved thermal reversion kinetics.

<p>(A) Fluorescence recovery of wild-type AsLOV2 after irradiation with blue light at room temperature (closed circle), at 50°C (closed triangle), and on ice (closed diamond). The results are means ± S.D. of three independent measurements. (B) Time course of fluorescence recovery of wild-type AsLOV2 (red) and that of AsLOV2 variants with fast thermal reversion kinetics (black). Twelve different fast variants obtained in the present partial screening are shown. Fluorescence intensity was recorded every 1.7 s at room temperature. (C) Time course of fluorescence recovery of wild-type AsLOV2 (red) and that of AsLOV2 variants with slow thermal reversion kinetics (black). Eight different slow variants obtained in the present partial screening are shown. Fluorescence intensity was recorded every 6.8 s at room temperature. Wild-type AsLOV2 and its variants were expressed in bacterial cells and imaged with a stereoscopic fluorescence microscope on an agar plate (A, B and C). The fluorescence recovery was fit with single exponential curves (A, B and C).</p

Crystal structure of wilt-type AsLOV2 in the light state.

<p>Structural analysis of wild-type AsLOV2 (PDB: 2V1B) has previously revealed that approximately 20 amino acid residues surround the isoallexazine ring of FMN. Among them, seven amino acid residues, Val416, Thr418, Asn425, Ile427, Ile466, Phe494 and Leu496, represented by stick model, were selected for the present mutagenesis study. The crystal structure shows the formation of the covalent bond between Cys450 and FMN in the light state. Two alternative side chain conformations, dark conformation and light conformation, of Cys450 are shown in the structure because dark conformation is present in the light state structure at 10% occupancy. Inset shows an overview of AsLOV2 structure.</p

Structural analysis of Val416 of AsLO2 for thermal reversion.

<p>(A) A crystal structure of wild-type AsLOV2 in the dark state (PDB: 2V1A) shows the side chain of Val416 directed toward Cys450 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082693#pone.0082693-Halavaty1" target="_blank">[10]</a>. The valine residue is located within 4 of Cys450, but has no steric interaction with its side chain. (B) A crystal structure of wild-type VVD in the dark state (PDB: 2PD7) shows that Ile74, which is equivalent to Val416 of AsLOV2, positions its side chain in van der Waals contact with Cys108, which is equivalent to Cys450 of AsLOV2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082693#pone.0082693-Zoltowski1" target="_blank">[6]</a>. Zoltowski <i>et al.</i> explain that the steric interaction of the methyl group may affect the conformation stability of Cys108 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082693#pone.0082693-Zoltowski2" target="_blank">[13]</a>. (C) A crystal structure of VVD-I74V in the dark state (PDB: 3HJK) shows that a valine substitution of Ile74 removes the methyl group of Ile74 from van der Waals area of Cys108 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082693#pone.0082693-Zoltowski2" target="_blank">[13]</a>. Key amino acid residues described above are represented by stick model and sphere model with a radius equal to the van del Waals surface. Other key amino acid residues, which are Thr418, Asn425 and Ile427 of wild-type AsLOV2 (A), and Cys76, Thr83 and Ile85 of wild-type VVD (B), and the VVD-I74V variant (C), for thermal reversion in LOV domains are represented by line model. Crystal structures of wild-type AsLOV2 (A), wild-type VVD (B) and the VVD-I74V variant (C) in the dark state show the amino acid residues located near the solvent channel (blue sphere).</p

Parameters for recovery kinetics in AsLOV2 variants.

<p> NA, not available.</p

Association between Irregular Meal Timing and the Mental Health of Japanese Workers

Breakfast skipping and nighttime snacking have been identified as risk factors for obesity, diabetes, and cardiovascular diseases. However, the effects of irregularity of meal timing on health and daily quality of life are still unclear. In this study, a web-based self-administered questionnaire survey was conducted involving 4490 workers (73.3% males; average age = 47.4 ± 0.1 years) in Japan to investigate the association between meal habits, health, and social relationships. This study identified that irregular meal timing was correlated with higher neuroticism (one of the Big Five personality traits), lower physical activity levels, and higher productivity loss. Irregular meal timing was also associated with a higher incidence of sleep problems and lower subjective health conditions. Among health outcomes, a high correlation of irregular meal timing with mental health factors was observed. This study showed that irregularity of meal timing can be explained by unbalanced diets, frequent breakfast skipping, increased snacking frequency, and insufficient latency from the last meal to sleep onset. Finally, logistic regression analysis was conducted, and a significant contribution of meal timing irregularity to subjective mental health was found under adjustment for other confounding factors. These results suggest that irregular meal timing is a good marker of subjective mental health issues