Rapid Corneal Calcification Following Endothelial Keratoplasty: A Case Report.

Corneal calcification typically progresses slowly but can occasionally advance rapidly. This report details severe stromal calcification following repeat Descemet's stripping automated endothelial keratoplasty (DSAEK) in a 75-year-old patient with diabetes, hypertension, and prior ocular surgeries, including cataract surgery, intraocular lens extraction with suturing, and trabeculectomy. Persistent epithelial defects after the surgery led to rapid central stromal calcification within four weeks, significantly reducing visual acuity. Management included switching from betamethasone sodium phosphate to fluorometholone, facilitating complete epithelial recovery within two months. However, persistent stromal opacity necessitated a subsequent penetrating keratoplasty. Infrared absorption spectrophotometry identified calcium phosphate as the primary component of the calcification. This case highlights the importance of vigilant monitoring and proactive management of epithelial defects to prevent rapid calcification following endothelial keratoplasty.

Refractory Fungal Keratitis Caused by Acrophialophora fusispora: A Case Report and Literature Review.

Acrophialophora fusispora is a filamentous fungus that is found in soil and rarely infects humans. We herein report the first case of fungal keratitis caused by A. fusispora in Japan and present a review of the literature on human infections with Acrophialophora species. A 62-year-old Japanese male on immunosuppressive therapy developed fungal keratitis after the removal of a corneal foreign body from his left eye. Voriconazole eye drops and systemic therapy for post-traumatic fungal keratitis did not resolve the infection, and the patient required a therapeutic corneal transplant. The isolate was identified as A. fusispora based on the nucleotide sequence of the internal transcribed spacer region. In a drug susceptibility test, the minimum inhibitory concentration of voriconazole was 0.5 µg/mL. Based on this case and previous cases from the literature review, fungal keratitis caused by A. fusispora is often refractory.

Effect of graphene-based nanomaterials on corneal wound healing in vitro.

Graphene-based nanomaterials (GBNs) are widely used due to their chemical and physical properties for multiple commercial and environmental applications. From an occupational health perspective, there is concern regarding the effects of inhalation on the respiratory system, and many studies have been conducted to study inhalation impacts on lung. Similar to the respiratory system, the eyes may also be exposed to GBNs and thus impacted. In this study, immortalized human corneal epithelial (hTCEpi) cells and rabbit corneal fibroblasts (RCFs) were used to investigate the toxicity of eight types of GBN: graphene oxide (GO; 400 nm), GO (1 µm), partially reduced graphene oxide (PRGO; 400 nm), reduced graphene oxide (RGO; 400 nm), RGO (2 µm), graphene (110 nm), graphene (140 nm), and graphene (1 µm). We next examined the effects of these GBNs on hTCEpi cell migration. We also determined whether the expression of α-smooth muscle actin (αSMA), a myofibroblast marker, is altered by the GBNs using RCFs. We found that RGO (400 nm) and RGO (2 µm) were highly toxic to hTCEPi cells and RCFs meanwhile, PRGO (400 nm) was toxic only to hTCEpi cells. In addition, PRGO (400 nm), RGO (400 nm), and RGO (2 µm) inhibited hTCEpi cell migration and significantly increased αSMA mRNA expression. Further study in vivo is required to determine if RGO nanomaterials delay corneal epithelial healing and induce scar formation.

Corneal Copper Deposition Secondary to Monoclonal Gammopathy in a Patient With Chronic Lymphocytic Leukemia: A Case Report.

Hypercupremia-induced corneal copper deposition secondary to monoclonal gammopathy is rare and shows a characteristic corneal opacity quite different from other causes of hypercupremia, such as Wilson's disease. This report describes a case of corneal copper deposition in a patient with monoclonal gammopathy associated with chronic lymphocytic leukemia. An 84-year-old man with slowly progressive corneal opacity was referred to our hospital. The corneal opacity was present at least five years ago. The patient's best-corrected visual acuity was 20/25 OU (in both eyes) at the initial visit to our hospital. Slit-lamp examination and anterior segment optical coherence tomography revealed bilateral brown-colored opacity localized to deep layers of the central cornea. In vivo confocal microscopy (IVCM) showed indistinct corneal stromal cells in the deep layer and endothelial cells. The possible differential diagnoses were corneal dystrophy and Wilson's disease, but the color, shape, or site of corneal opacity was inconsistent with the disease. As the patient had a history of chronic lymphocytic leukemia, which is often associated with monoclonal gammopathy, we suspected that the corneal opacity was copper deposition in association with the hematologic diseases. Laboratory examinations showed elevated serum copper and normal ceruloplasmin. Serum protein electrophoresis revealed significantly high IgG levels with depression of IgA, IgE, and IgM. These results supported our diagnosis. Followingly, we consulted the patient's attending hematologist, and the doctor initiated treatment for hypercupremia. In conclusion, hypercupremia secondary to monoclonal gammopathy should be considered a possible cause of central brown-colored corneal opacity.

Cytotoxicity of 2D engineered nanomaterials in pulmonary and corneal epithelium.

Two-dimensional (2D) engineered nanomaterials are widely used in consumer and industrial goods due to their unique chemical and physical characteristics. Engineered nanomaterials are incredibly small and capable of being aerosolized during manufacturing, with the potential for biological interaction at first-contact sites such as the eye and lung. The unique properties of 2D nanomaterials that make them of interest to many industries may also cause toxicity towards epithelial cells. Using murine and human respiratory epithelial cell culture models, we tested the cytotoxicity of eight 2D engineered nanomaterials: graphene (110 nm), graphene oxide (2 um), graphene oxide (400 nm), reduced graphene oxide (2 um), reduced graphene oxide (400 nm), partially reduced graphene oxide (400 nm), molybdenum disulfide (400 nm), and hexagonal boron nitride (150 nm). Non-graphene nanomaterials were also tested in human corneal epithelial cells for ocular epithelial cytotoxicity. Hexagonal boron nitride was found to be cytotoxic in mouse tracheal, human alveolar, and human corneal epithelial cells. Hexagonal boron nitride was also tested for inhibition of wound healing in alveolar epithelial cells; no inhibition was seen at sub-cytotoxic doses. Nanomaterials should be considered with care before use, due to specific regional cytotoxicity that also varies by cell type. Supported by U01ES027288 and T32HL007013 and T32ES007059.

Metal Oxide Engineered Nanomaterials Modulate Rabbit Corneal Fibroblast to Myofibroblast Transformation.

Purpose: Corneal keratocyte-fibroblast-myofibroblast (KFM) transformation plays a critical role in corneal stromal wound healing. However, the impact of engineered nanomaterials (ENMs), found in an increasing number of commercial products, on this process is poorly studied. This study investigates the effects of metal oxide ENMs on KFM transformation in vitro and in vivo. Methods: Cell viability of rabbit corneal fibroblasts (RCFs) was tested following treatment with 11 metal oxide ENMs at concentrations of 0.5 to 250 µg/ml for 24 hours. Messenger RNA (mRNA) and protein expression of αSMA, a marker of myofibroblast transformation, were measured using RCFs after exposure to 11 metal oxide ENMs at a concentration that did not affect cell viability, in media containing either 0 or 10 ng/ml of TGF-ß1. Additionally, the effect of topical Fe2O3 nanoparticles (NPs) (50 ng/ml) on corneal stromal wound healing following phototherapeutic keratectomy (PTK) was determined. Results: V2O5, Fe2O3, CuO, and ZnO ENMs were found to significantly reduce cell viability as compared to vehicle control and the other seven metal oxide ENMs tested. V2O5 nanoflakes significantly reduced mRNA and protein αSMA concentrations in the presence of TGF-ß1. Fe2O3 NPs significantly increased αSMA mRNA expression in the presence of TGF-ß1 but did not alter αSMA protein expression. Topically applied Fe2O3 NPs in an in vivo rabbit corneal stromal wound healing model did not delay healing. Conclusions: Fe2O3 NPs promote corneal myofibroblast induction in vitro but do not impair corneal stromal wound healing in vivo. Translational Relevance: These experimental results can apply to human nanomedical research.

Foveal structure in nanophthalmos and visual acuity.

PURPOSE: To evaluate the fovea in nanophthalmic eyes using spectral domain optical coherence tomography (SD-OCT) and OCT angiography (OCTA), and to investigate the relationship between the macular microstructure and visual acuity. METHODS: This is a retrospective case series of five nanophthalmic patients. The foveal avascular zone (FAZ) area was measured in superficial and deep vascular layers with OCTA. The thickness of the inner retinal layer (IRL) was measured with SD-OCT. The ratio of the foveal and parafoveal IRL thickness (fIRL/pIRL ratio) was calculated. The relationship between these parameters and visual acuity was then investigated. RESULTS: Eight eyes were identified as nanophthalmic with a mean axial length of 17.19 ± 1.44 mm (range: 15.71 to 19.88 mm). The mean best-corrected visual acuity (BCVA) in the logarithm of the minimum angle of resolution (logMAR) was 0.12 ± 0.18 (range: - 0.18 to 0.40). OCTA showed that FAZs were either absent or undeveloped in the superficial and deep capillary plexuses. Two patients did not show any visual impairments despite small FAZ and a shallow foveal depression. Although the BCVA was significantly correlated with the deep FAZ size, it did not correlate with the superficial FAZ size, axial length, or fIRL/pIRL ratio. However, the refractive error, axial length, and deep FAZ size were all significantly correlated with the fIRL/pIRL ratio. CONCLUSIONS: The FAZs were commonly found to be small in the superficial and deep capillary plexuses. Although the deep FAZ size correlated with visual acuity, it is unclear whether the retinal microstructure and the FAZ size are responsible for the visual impairments observed in the same individuals.

Matrin3 promotes homologous recombinational repair by regulation of RAD51.

Matrin3 is a highly conserved inner nuclear matrix protein involved in multiple stages of RNA metabolism. Although Matrin3 may also play a role in DNA repair, its precise roles have remained unclear. In this study, we showed that the depletion of Matrin3 led to decreased homologous recombination (HR) efficiency and increased radiation sensitivity of cells. Matrin3-depleted cells showed impaired DNA damage-dependent focus formation of RAD51, a key protein in HR. These findings suggest that Matrin3 promotes HR by regulating RAD51.

Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations.

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities.

SUMO modification system facilitates the exchange of histone variant H2A.Z-2 at DNA damage sites.

Histone exchange and histone post-translational modifications play important roles in the regulation of DNA metabolism, by re-organizing the chromatin configuration. We previously demonstrated that the histone variant H2A.Z-2 is rapidly exchanged at damaged sites after DNA double strand break induction in human cells. In yeast, the small ubiquitin-like modifier (SUMO) modification of H2A.Z is involved in the DNA damage response. However, whether the SUMO modification regulates the exchange of human H2A.Z-2 at DNA damage sites remains unclear. Here, we show that H2A.Z-2 is SUMOylated in a damage-dependent manner, and the SUMOylation of H2A.Z-2 is suppressed by the depletion of the SUMO E3 ligase, PIAS4. Moreover, PIAS4 depletion represses the incorporation and eviction of H2A.Z-2 at damaged sites. These findings demonstrate that the PIAS4-mediated SUMOylation regulates the exchange of H2A.Z-2 at DNA damage sites.

Coordinated Regulation of TIP60 and Poly(ADP-Ribose) Polymerase 1 in Damaged-Chromatin Dynamics.

The dynamic exchange of histones alleviates the nucleosome barrier and simultaneously facilitates various aspects of cellular DNA metabolism, such as DNA repair and transcription. In response to DNA damage, the acetylation of Lys5 in the histone variant H2AX, catalyzed by TIP60, plays a key role in promoting histone exchange; however, the detailed molecular mechanism still is unclear. Here, we show that the TIP60 complex includes poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is required for the rapid exchange of H2AX on chromatin at DNA damage sites. It is known that PARP-1 binds dynamically to damaged chromatin and is crucial for the subsequent recruitment of other repair factors, and its auto-poly(ADP-ribosyl)ation is required for the dynamics. We also show that the acetylation of histone H2AX at Lys5 by TIP60, but not the phosphorylation of H2AX, is required for the ADP-ribosylation activity of PARP-1 and its dynamic binding to damaged chromatin. Our results indicate the reciprocal regulation of K5 acetylation of H2AX and PARP-1, which could modulate the chromatin structure to facilitate DNA metabolism at damage sites. This could explain the rather undefined roles of PARP-1 in various DNA damage responses.

Relaxed Chromatin Formation and Weak Suppression of Homologous Pairing by the Testis-Specific Linker Histone H1T.

Linker histones bind to nucleosomes and compact polynucleosomes into a higher-order chromatin configuration. Somatic and germ cell-specific linker histone subtypes have been identified and may have distinct functions. In this study, we reconstituted polynucleosomes containing human histones H1.2 and H1T, as representative somatic and germ cell-specific linker histones, respectively, and found that H1T forms less compacted chromatin, as compared to H1.2. An in vitro homologous pairing assay revealed that H1T weakly inhibited RAD51/RAD54-mediated homologous pairing in chromatin, although the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, substantially suppressed it. An in vivo recombination assay revealed that H1T overproduction minimally affected the recombination frequency, but significant suppression was observed when H1.2 was overproduced in human cells. These results suggested that the testis-specific linker histone, H1T, possesses a specific function to produce the chromatin architecture required for proper chromosome regulation, such as homologous recombination.

hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.

Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.

Structural basis for ubiquitin recognition by ubiquitin-binding zinc finger of FAAP20.

Several ubiquitin-binding zinc fingers (UBZs) have been reported to preferentially bind K63-linked ubiquitin chains. In particular, the UBZ domain of FAAP20 (FAAP20-UBZ), a member of the Fanconi anemia core complex, seems to recognize K63-linked ubiquitin chains, in order to recruit the complex to DNA interstrand crosslinks and mediate DNA repair. By contrast, it is reported that the attachment of a single ubiquitin to Rev1, a translesion DNA polymerase, increases binding of Rev1 to FAAP20. To clarify the specificity of FAAP20-UBZ, we determined the crystal structure of FAAP20-UBZ in complex with K63-linked diubiquitin at 1.9 Å resolution. In this structure, FAAP20-UBZ interacts only with one of the two ubiquitin moieties. Consistently, binding assays using surface plasmon resonance spectrometry showed that FAAP20-UBZ binds ubiquitin and M1-, K48- and K63-linked diubiquitin chains with similar affinities. Residues in the vicinity of Ala168 within the α-helix and the C-terminal Trp180 interact with the canonical Ile44-centered hydrophobic patch of ubiquitin. Asp164 within the α-helix and the C-terminal loop mediate a hydrogen bond network, which reinforces ubiquitin-binding of FAAP20-UBZ. Mutations of the ubiquitin-interacting residues disrupted binding to ubiquitin in vitro and abolished the accumulation of FAAP20 to DNA damage sites in vivo. Finally, structural comparison among FAAP20-UBZ, WRNIP1-UBZ and RAD18-UBZ revealed distinct modes of ubiquitin binding. UBZ family proteins could be divided into at least three classes, according to their ubiquitin-binding modes.

Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.