Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure.

PURPOSE: The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. METHODS: We assessed cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. RESULTS: Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-ß-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. CONCLUSIONS: In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.

Ocular cicatricial pemphigoid following Dipeptidyl Peptidase-4 inhibitor use: A case report.

Purpose: To report a rare Ocular Cicatricial Pemphigoid (OCP) case in a patient taking a Dipeptidyl Peptidase-4 Inhibitor (DPP-4 inhibitor), a medication used for the management of type 2 diabetes, for at least six years. Observations: A 64-year-old male presented with refractory bilateral conjunctival inflammation and ocular discharge that had persisted for two months, despite multiple prior therapies for presumed bacterial conjunctivitis. Upon initial examination, clinical findings strongly suggested OCP, and he had elevated levels of anti-BP180 antibodies. Despite receiving systemic treatments such as steroid pulse therapy and therapeutic plasma exchange after discontinuing DPP-4 inhibitors, his condition progressively worsened, with manifestations such as forniceal shortening in his left eye. Consequently, the patient required keratoepithelioplasty, amniotic membrane transplantation in his left eye, and bilateral eyelid entropion surgery. His condition initially worsened for a time after discontinuing the DPP-4 inhibitor, but it gradually improved over time, and ocular surface surgical intervention was not required in the right eye. Conclusions and Importance: The findings in this study demonstrate that severe refractory OCP may occur while taking the DPP-4 inhibitor, thus indicating that a detailed interview regarding medications is essential for patients with ocular pemphigoid, especially those with type 2 diabetes.

Gene expression signatures of human senescent corneal and conjunctival epithelial cells.

PURPOSE: This study aimed to investigate the senescent phenotypes of human corneal and conjunctival epithelial cells. METHODS: We examined cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation, and expression of senescence markers (p16 and p21). RNA sequencing analysis was conducted to compare gene expression profiles between senescent and non-senescent cells. Finally, the potential involvement of senescent cells in the pathogenesis of ocular surface diseases was investigated. RESULTS: X-irradiated corneal and conjunctival epithelial cells exhibited typical senescence phenotypes, i.e., flattened morphologies, increased SA-ß-gal activity, decreased cell proliferation, and increased expression of senescence markers, p16 and p21. RNA-seq analysis revealed substantial differences in gene expression profiles between senescent corneal (SCo) and conjunctival epithelial cells (SCj). Moreover, SCj were detected in pathological conjunctival tissues associated with limbal stem cell deficiency (LSCD) due to Stevens-Johnson syndrome or chemical burns, potentially being involved in abnormal differentiation. CONCLUSION: This study highlights the cellular and molecular characteristics of senescent ocular surface cells, particularly in SCj that show abnormal keratin expression, and their potential roles in severe ocular surface diseases and pathology.

Recent insights into the SWI/SNF complex and the molecular mechanism of hSNF5 deficiency in rhabdoid tumors.

Genetic information encoded by DNA is packaged in the nucleus using the chromatin structure. The accessibility of transcriptional elements in DNA is controlled by the dynamic structural changes of chromatin for the appropriate regulation of gene transcription. Chromatin structure is regulated by two general mechanisms, one is histone modification and the other is chromatin remodeling in an ATP-dependent manner. Switch/sucrose nonfermentable (SWI/SNF) complexes utilize the energy from ATP hydrolysis to mobilize nucleosomes and remodel the chromatin structure, contributing to conformational changes in chromatin. Recently, the inactivation of encoding genes for subunits of the SWI/SNF complexes has been documented in a series of human cancers, accounting for up to almost 20% of all human cancers. For example, human SNF5 (hSNF5), the gene that encodes a subunit of the SWI/SNF complexes, is the sole mutation target that drives malignant rhabdoid tumors (MRT). Despite remarkably simple genomes, the MRT has highly malignant characteristics. As a key to understanding MRT tumorigenesis, it is necessary to fully examine the mechanism of chromatin remodeling by the SWI/SNF complexes. Herein, we review the current understanding of chromatin remodeling by focusing on SWI/SNF complexes. In addition, we describe the molecular mechanisms and influences of hSNF5 deficiency in rhabdoid tumors and the prospects for developing new therapeutic targets to overcome the epigenetic drive of cancer that is caused by abnormal chromatin remodeling.

Reply to Marino et al. Does Subretinal Fluid Influence Choroidal Thickness (ChT) and Structure in Preeclampsia with Serous Retinal Detachment? Comment on "Fukui et al. Changes in Choroidal Thickness and Structure in Preeclampsia with Serous Retinal Detachment. J. Clin. Med. 2023, 12, 609".

We thank the authors for the interesting comments [...].

Changes in Choroidal Thickness and Structure in Preeclampsia with Serous Retinal Detachment.

Preeclampsia is a pregnancy-specific syndrome characterized by hypertension and proteinuria. We retrospectively investigated the clinical features, including choroidal layer thickness and luminal area to stromal area ratio, in a case series of preeclampsia with serous retinal detachment (SRD). The subjects were pregnant women with SRD during hospitalization for preeclampsia from October 2014 to June 2021. Based on medical records, affected eyes, time of onset, fundus examination findings, and subfoveal choroidal thickness (SCT), the choroidal layer thickness and choroidal vascular index (CVI) in each patient was examined. Thirteen eyes from seven patients (mean age 30.7 ± 4.7 years) were included in the study. In all cases, SRD improved without topical ocular treatment. The mean SCT at the initial visit was 424.4 ± 70.5 µm, and all patients had choroidal thickening, which significantly decreased to 286.0 ± 57.9 µm (p < 0.01) at the last visit. The mean choroidal inner layer was 162.7 ± 69.4 µm at the initial visit and 122.3 ± 35.5 µm at the final follow-up visit (p = 0.06), showing no significant difference; however, the mean choroidal outer layer was 261.7 ± 47.6 µm at the initial visit and 163.7 ± 37.1 µm at the final follow-up visit (p < 0.01), thus showing a significant decrease. The mean CVI was 67.2 ± 1.3% at the initial visit, yet it had significantly decreased to 65.4 ± 1.1% (p < 0.01) at the final follow-up visit. The findings of this study show that SRD with preeclampsia is associated with increased thickening of the choroidal outer layer, especially in the choroidal luminal area.

Tumor necrosis factor­related apoptosis­inducing ligand is a novel transcriptional target of runt­related transcription factor 1.

Runt­related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13­acetate­treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co­transfected with RUNX1 and core­binding factor ß (CBFß) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFß proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription­quantitative PCR (RT­qPCR). TRAIL and RUNX1­ETO expression was also measured in Kasumi­1 cells transfected with RUNX1­ETO siRNA and in KG­1 cells transfected with RUNX1­ETO expression plasmid, both by RT­qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi­1, KG­1, SKNO­1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1­dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1­ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1­ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.

C11orf21, a novel RUNX1 target gene, is down-regulated by RUNX1-ETO.

The fusion protein RUNX1-ETO is an oncogenic transcription factor generated by t(8;21) chromosome translocation, which is found in FAB-M2-type acute myeloid leukemia (AML). RUNX1-ETO is known to dysregulate the normal RUNX1 transcriptional network, which should involve essential factors for the onset of AML with t(8;21). In this study, we screened for possible transcriptional targets of RUNX1 by reanalysis of public data in silico, and identified C11orf21 as a novel RUNX1 target gene because its expression was down-regulated in the presence of RUNX1-ETO. The expression level of C11orf21 was low in AML patient samples with t(8;21) and in Kasumi-1 cells, which carry RUNX1-ETO. Knockdown of RUNX1-ETO in Kasumi-1 cells restored C11orf21 expression, whereas overexpression of RUNX1 up-regulated C11orf21 expression. In addition, knockdown of RUNX1 in other human leukemia cells without RUNX-ETO, such as K562, led to a decrease in C11orf21 expression. Of note, the C11orf21 promoter sequence contains a consensus sequence for RUNX1 binding and it was activated by exogenously expressed RUNX1 based on our luciferase reporter assay. This luciferase signal was trans-dominantly suppressed by RUNX1-ETO and site-directed mutagenesis of the consensus site abrogated the reporter activity. This study demonstrated that C11orf21 is a novel transcriptional target of RUNX1 and RUNX1-ETO suppressed C11orf21 transcription in t(8;21) AML. Thus, through this in silico approach, we identified a novel transcriptional target of RUNX1, and the depletion of C11orf21, the target gene, may be associated with the onset of t(8;21) AML.

Ultrafast Dynamics of a Solvatochromic Dye, Phenol Blue: Tautomerization and Coherent Wavepacket Oscillations.

Femtosecond time-resolved transient absorption spectroscopy was performed for a nonfluorescent solvatochromic dye, phenol blue, N-(4-dimethylaminophenyl)-1,4-benzoquinoneimine, which exhibits ultrafast nonradiative decay due to its flexible molecular structure. By exciting the molecule in ethanol (EtOH) solution with two excitation wavelengths located at shorter- and longer-wavelength sides of the visible absorption band, we observed ultrafast nonradiative decay from the excited state, followed by spectral evolution in the ground state. The nonradiative decay in the subpicosecond range creates a vibrationally hot ground state with the lifetime in the picosecond range. Subsequently, a tautomer that absorbs at shorter wavelengths is produced from the hot state, which causes a red shift of the ground-state bleach (GSB). The tautomerization presumably involves twisting of the benzoquinoneimine moiety induced by the breaking of the hydrogen bond (H-bond) between the solute and the solvent molecules. The recombination of the H-bond occurs with a time constant of ∼30 ps, and the system returns to its original state. We also observed low-frequency coherent wavepacket oscillations that modulate the GSB with dephasing times similar to the excited-state lifetime.

Lipopolysaccharide (LPS) inhibits ectopic bone formation induced by bone morphogenetic protein-2 and TGF-ß1 through IL-1ß production.

OBJECTIVES: In order to gain new insight into bacterial infection during bone-regenerative treatment using bone morphogenetic proteins (BMPs), we examined the effects of lipopolysaccharide (LPS) on ectopic bone formation induced by BMP-2 and transforming growth factor (TGF)-ß1 in mice. METHODS: We implanted collagen sponges containing BMP-2, TGF-ß1, and various amounts of LPS into mouse muscle tissues. Lump-like masses in which ectopic bones developed in mice were processed for microcomputed tomography, DNA microarray, reverse-transcription PCR, and histological analyses. RESULTS: LPS treatment caused a dose-dependent reduction in the volume of ectopic bone. The total volume of ectopic bone induced by BMP-2 + TGF-ß1 treatment was reduced by more than 75% in the presence of LPS. Histological analysis of the ectopic bone tissues revealed a significant reduction in total bone volume and bone volume/total volume in response to LPS. LPS treatment significantly increased the osteoblast number and osteoid volume, while the osteoclast number did not change. Since LPS induced production of TNF-α and IL-1ß in lump-like masses, we implanted collagen sponges containing BMP-2 and TGF-ß1 with or without LPS into TNF-α- or IL-1α/ß-deficient mice. LPS treatment reduced the volume of ectopic bones in TNF-α-deficient mice but not in IL-1α/ß-deficient mice. Furthermore, collagen sponges containing IL-1ß reduced ectopic bone formation by BMP-2 and TGF-ß1 in wild-type mice to the same extent as LPS treatment did. CONCLUSIONS: LPS suppresses the ectopic bone formation induced by BMP-2 and TGF-ß1 through IL-1ß production.

Induction of osteoblastic differentiation of neural crest-derived stem cells from hair follicles.

The neural crest (NC) arises near the neural tube during embryo development. NC cells migrate throughout the embryo and have potential to differentiate into multiple cell types, such as peripheral nerves, glial, cardiac smooth muscle, endocrine, and pigment cells, and craniofacial bone. In the present study, we induced osteoblast-like cells using whisker follicles obtained from the NC of mice. Hair follicle cells derived from the NC labeled with enhanced green fluorescent protein (EGFP) were collected from protein zero-Cre/floxed-EGFP double transgenic mice and cultured, then treated and cultured in stem cell growth medium. After growth for 14 days, results of flow cytometry analysis showed that 95% of the EGFP-positive (EGFP+) hair follicle cells derived from the NC had proliferated and 76.2% of those expressed mesenchymal stem cells markers, such as platelet-derived growth factor α and stem cell antigen-1, and also showed constitutive expression of Runx2 mRNA. Cells stimulated with bone morphogenetic protein-2 expressed osteocalcin, osterix, and alkaline phosphatase mRNA, resulting in production of mineralized matrices, which were detected by von Kossa and alizarin red staining. Moreover, EGFP+ hair follicle cells consistently expressed macrophage colony-stimulating factor and osteoprotegerin (OPG). Addition of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] (10-8 M) to the cultures suppressed OPG expression and induced RANKL production in the cells. Furthermore, multinucleated osteoclasts appeared within 6 days after starting co-cultures of bone marrow cells with EGFP+ cells in the presence of 1,25(OH)2D3 and PGE2. These results suggest that NC-derived hair follicle cells possess a capacity for osteoblastic differentiation and may be useful for developing new bone regenerative medicine therapies.

First-principles molecular dynamics study for average structure and oxygen diffusivity at high temperature in cubic Bi2O3.

Bismuth oxide, Bi(2)O(3), has a cubic structure (δ-phase) at high temperature. High oxygen conductivity of δ-Bi(2)O(3) should be closely related to disordering of the oxygen sublattice. In order to reconstruct the disordered structure in the crystal using first-principles molecular dynamics (FPMD), a sufficiently long simulation time is essentially required. In this study, the FPMD simulation up to 1 ns is performed with special interest given to the convergence of the average structure and the oxygen diffusivity with respect to the simulation time. The obtained average structure and the oxygen diffusivity are in good agreement with those obtained by experimental analysis.

R848, a toll-like receptor 7 agonist, inhibits osteoclast differentiation but not survival or bone-resorbing function of mature osteoclasts.

R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-ß was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-ß to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7.