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The purpose of this study is to provide an intravital noninvasive multiphoton microscopic platform for long-term ocular imaging in transgenic fluorescent mice with subcellular resolution. A multiphoton microscopic system with tunable laser output was employed. We designed a mouse holder incorporated with stereotaxic motorized stage for in vivo three-dimensional imaging of ocular surface in 3 transgenic mouse line with fluorescent protein (FP) expression to visualize distinct structures. With our imaging platform and the expression of FPs, we obtained the three-dimensional images across the whole cornea from epithelium to endothelium and in conjunctiva with subcellular resolution in vivo. Specified EGFP expression in corneal epithelium of K5-H2B-EGFP mice helped to identify both corneal and limbal epithelial cells while ubiquitous nuclear FP expression in R26R-GR mice allowed us to visualized nuclei of all cell types. Universal membrane-localized FP in mT/mG mice outlined all cell boundaries, nerve fibers, and capillaries. The simultaneously collected second harmonic generation signals from collagenous stroma provided architectural contrast. Time-lapsed recording enabled monitoring the mitotic activity of corneal epithelial cells and limbal epithelial cells. We developed an intravital multiphoton microscopic stereotaxic imaging platform and showed that, by incorporating FP-expressing transgenic mice, this platform enables in vivo 4-dimensional ophthalmic study at subcellular resolution.

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFβ1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFβ1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF., Pulmonary fibrosis is a major public health problem with unclear mechanism and limited therapeutic options. Here the authors show that a fibroblast-enriched endoplasmic reticulum protein, TXNDC5, promotes pulmonary fibrosis by stabilizing TGFBR1 and show the potential of TXNDC5 as a therapeutic target against pulmonary fibrosis.

Multiphoton microscopy allows long-term direct visualization of cells in live animals due to its low photodamage. When coupled with fluorescence protein targeting and second harmonic generation signals from natural collagen as contrast, multiphoton microscopy enables intravital tracing of cells while providing structural information from the extracellular matrix. Compared with conventional histological analysis, it can bring new insight into the cell dynamics in stem cell research. Here, we demonstrate cell imaging and tracing at a single cell resolution in the cornea, skin, and hair follicles using multiphoton microscopy in transgenic mice of which specific cell populations are tagged with fluorescent proteins.

Background Epigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. Methods H1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and β-actin, were performed by western blot analysis. Results Our data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. Conclusion Chidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.

Background and Objective Identification of methods to enhance anagen entry can be helpful for alopecia. Recently, nonablative laser has been proposed as a potential treatment for alopecia. However, how the laser parameters affect stem cell activity, hair cycles and the associated side effects have not been well characterized. Here we examine the effects of irradiation parameters of 1,550-nm fractional laser on hair cycles. Study Design/Materials and Methods The dorsal skin of eight-week-old female C57BL/6 mice with hair follicles in synchronized telogen was shaved and irradiated with a 1,550-nm fractional erbium-glass laser (Fraxel RE:STORE (SR1500) Laser System, Solta Medical, U.S.A.) with varied beam energies (5–35 mJ) and beam densities (500–3500 microthermal zones/cm2). The cutaneous changes were evaluated both grossly and histologically. Hair follicle stem cell activity was detected by BrdU incorporation and changes in gene expression were quantified by real-time PCR. Results Direct thermal injury to hair follicles could be observed early after irradiation, especially at higher beam energy. Anagen induction in the irradiated skin showed an all-or-non change. Anagen induction and ulcer formation were affected by the combination of beam energy and density. The lowest beam energy of 5 mJ failed to promote anagen entry at all beam densities tested. As beam energy increased from 10 mJ to 35 mJ, we found a decreasing trend of beam density that could induce anagen entry within 7–9 days with activation of hair follicle stem cells. Beam density above the pro-regeneration density could lead to ulcers and scarring followed by anagen entry in adjacent skin. Analysis of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, revealed that transient moderate inflammation was associated with anagen induction and intense prolonged inflammation preceded ulcer formation. Conclusion To avoid side effects of hair follicle injury and scarring, appropriate combination of beam energy and density is required. Parameters outside the therapeutic window can result in either no anagen promotion or ulcer formation. Lasers Surg. Med. 47:331–341, 2015. © 2015 Wiley Periodicals, Inc.

Transforming growth factor-β (TGF-β)-induced epithelial–mesenchymal transition is a critical process in the initiation of metastasis of various types of cancer. Chidamide is a class I histone deacetylase inhibitor with anti-tumor activity. This study investigated the effects of chidamide on TGF-β-mediated suppression of E-cadherin expression in adenocarcinomic lung epithelial cells and the molecular mechanisms involved in these effects. Western blot analysis, confocal microscopy, Quantitative methyl-specific PCR and bisulfite sequencing were used to evaluate the effects of different treatments on chidamide ameliorating TGF-β induced-E-cadherin loss. H3 acetylation binding to the promoter of E-cadherin was detected by chromatin immunoprecipitations (CHIP). We found that chidamide reduced the level of lung cancer cell migration observed using a Boyden chamber assay (as an indicator of metastatic potential). Chidamide inhibited TGF-β-induced SMAD2 phosphorylation and attenuated TGF-β-induced loss of E-cadherin expression in lung cancer cells by Western blotting and confocal microscopy, respectively. Quantitative methyl-specific PCR and bisulfite sequencing revealed that TGF-β-enhanced E-cadherin promoter methylation was ameliorated in cells treated with chidamide. We demonstrated that histone H3 deacetylation within the E-cadherin promoter was required for TGF-β-induced E-cadherin loss; cell treatment with chidamide increased the H3 acetylation detected by CHIP. Taken together, our results demonstrate that TGF-β suppressed E-cadherin expression by regulating promoter methylation and histone H3 acetylation. Chidamide significantly enhanced E-cadherin expression in TGF-β-treated cells and inhibited lung cancer cell migration. These findings indicate that chidamide has a potential therapeutic use due to its capacity to prevent cancer cell metastasis.