SARS-CoV-2 infection status in corneal preservation solution and COVID-19 prevalence after corneal transplantation.

The potential risks associated with organs from COVID-19-infected donors were unclear. To determine the SARS-CoV-2 infection status of corneas transplanted during the COVID-19 pandemic, we performed a polymerase chain reaction (PCR) using the corneal preservation solution that was used for corneal transplantation. We also examined the postoperative health status of the recipients. This study included 144 transplants in 143 eyes. Ninety-nine eyes of imported corneas and 10 of the 14 corneas donated in the prefecture were PCR tested at our hospital, and all were SARS-CoV-2 negative. All corneal transplants were performed after confirming their SARS-CoV-2 negativity by a PCR using a corneal preservation solution at our hospital or a nasopharyngeal swab at a previous facility. Despite postoperative steroid administration, no patient developed COVID-19 infection until discharge. Hence, if the donor's nasopharyngeal swab test is SARS-CoV-2 negative, COVID-19 infection in the recipient due to corneal transplantation may be prevented. Since corneal transplant recipients are susceptible to infection due to prolonged steroid administration and are at high risk for severe diseases if infection occurs, SARS-CoV-2 detection testing using nasopharyngeal swabs in donors should be performed.

A Novel Method for Creating Heterologous Lethal Antibiotic Producers by Screening from Combi-OGAB Library with Various Promoters in a Biosynthetic Gene Cluster.

In this study, we devised a novel method to create heterologous producers of lethal antibiotics against host bacteria. Heterologous producers cannot be created when antibiotics are toxic to host bacteria. To overcome this challenge, we developed a novel method involving construction of a combinatorial library with various promoters and screening based on the production. To realize this, we utilized Combi-OGAB (Combinatorial Ordered Gene Assembly in Bacillus subtilis), which technology can effectively construct diverse combinatorial library and accelerate screening procedures. B. subtilis and Gramicidin S were selected as the host bacterium and the targeted antibiotic, respectively. The screened producer from Combi-OGAB screening cycles achieved >30-fold productivity over the lethal level. These results suggest that our strategy has the potential to maximize the phenotypic resistance of host bacteria to create heterologous lethal antibiotic producers.

Novel compound heterozygous mutations in the PLEC gene in a neonate with epidermolysis bullosa simplex with pyloric atresia.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited disorders characterized by the blistering of the skin and mucous membranes. Although the molecular basis of EB has been significantly elucidated, the precise phenotypes of the lethal types of EB have not been completely characterized. Herein, we report a severe case of EB with pyloric atresia (PA). The patient was a Japanese boy who not only had skin lesions but also various complications such as PA, dysphagia, hypotonia, infectious keratitis with corneal ulcer, obstructive uropathy and protein-losing enteropathy. Genetic analysis led to the identification of two novel compound heterozygous mutations in the last exon of the plectin (PLEC) gene. Based on this finding, EB simplex with PA was diagnosed. Immunostaining with anti-plectin antibodies revealed truncated plectin proteins lacking the C-terminus in the patient's skin. We also conducted a prenatal diagnosis in subsequent pregnancy. Our report further highlights the crucial role of plectin in many organs and provides valuable information regarding the phenotypes resulting from mutations in the PLEC gene.

Demonstration of a multicolor metasurface holographic movie based on a cinematographic approach.

This study uses a dielectric metasurface to demonstrates a multicolor holographic movie. Overlapping of multiple-wavelength images at 445 nm, 532 nm, and 633 nm was achieved by maintaining the ratio between the wavelengths and the pixel periods constant. Polarization-independent pillar waveguides made of single-crystal silicon are used as meta-atoms. A movie of the rotating earth was designed by the iterative Fourier transform algorithm and fabricated using electron beam lithography to a silicon-on-sapphire substrate. The multicolor movie consists of 20 frames was successfully reproduced at the maximum speed of 30 frames per second.

Simultaneous Vitreoretinal Surgery and Sclerokeratoplasty for Keratoglobus with Intraocular Hemorrhage and Extensive Corneal Rupture.

We reported a case of simultaneous vitrectomy and sclerokeratoplasty (SKP) performed for keratoglobus with extensive corneal rupture and intraocular hemorrhage caused by trauma. A 73-year-old woman was treated for keratoglobus and glaucoma. She was punched in both eyes, her right eye showed corneal rupture and the left eye showed prolapse of the ocular contents due to eyeball rupture. She immediately underwent corneal sutures in the right eye and resection of the prolapsed ocular contents in the left eye at a nearby ophthalmological clinic. Three days after the injury, the patient was referred to our clinic for vision recovery. The best corrected visual acuity of the right eye was measured by counting fingers. Her right eye presented severe corneal edema with a sutured corneal wound in the upper periphery, which was positive in the Seidel test. B-mode ultrasound revealed choroidal detachment and subchoroidal hemorrhage. Fourteen days after injury, simultaneous corneal suture and posterior sclerotomy were performed in the right eye, but corneal fragility and corneal opacity were prominent, and B-mode examination revealed prolonged vitreous hemorrhage and retinal detachment. Twenty-one days after injury, we performed simultaneous SKP and 25-G pars plana vitrectomy (PPV). In this procedure, we initially performed SKP followed by 25-G PPV without a keratoprosthesis or endoscope. The visibility of the fundus through the corneoscleral graft was good during vitrectomy. Three months after surgery, her corrected visual acuity improved to 10/1,000. Although there was mild corneal stromal edema and khodadoust line, there were no obvious fundus complications. Simultaneous SKP and PPV for keratoglobus with extensive corneal rupture and vitreous diseases may be a good option.

Role of the Neurokinin-1 Receptor in the Promotion of Corneal Epithelial Wound Healing by the Peptides FGLM-NH2 and SSSR in Neurotrophic Keratopathy.

Purpose: Neurotrophic keratopathy is a corneal epitheliopathy induced by trigeminal denervation that can be treated with eyedrops containing the neuropeptide substance P (or the peptide FGLM-NH2 derived therefrom) and insulin-like growth factor 1 (or the peptide SSSR derived therefrom). Here, we examine the mechanism by which substance P (or FGLM-NH2) promotes corneal epithelial wound healing in a mouse model of neurotrophic keratopathy. Methods: The left eye of mice subjected to trigeminal nerve axotomy in the right eye served as a model of neurotrophic keratopathy. Corneal epithelial wound healing was monitored by fluorescein staining and slit-lamp examination. The distribution of substance P, neurokinin-1 receptor (NK-1R), and phosphorylated Akt was examined by immunohistofluorescence analysis. Cytokine and chemokine concentrations in intraocular fluid were measured with a multiplex assay. Results: Topical administration of FGLM-NH2 and SSSR promoted corneal epithelial wound healing in the neurotrophic keratopathy model in a manner sensitive to the NK-1R antagonist L-733,060. Expression of substance P and NK-1R in the superficial layer of the corneal epithelium decreased and increased, respectively, in model mice compared with healthy mice. FGLM-NH2 and SSSR treatment suppressed the production of interleukin-1α, macrophage inflammatory protein 1α (MIP-1α) and MIP-1ß induced by corneal epithelial injury in the model mice. It also increased the amount of phosphorylated Akt in the corneal epithelium during wound healing in a manner sensitive to prior L-733,060 administration. Conclusions: The substance P-NK-1R axis promotes corneal epithelial wound healing in a neurotrophic keratopathy model in association with upregulation of Akt signaling and attenuation of changes in the cytokine-chemokine network.

Simultaneous Vitreoretinal Surgery and Penetrating Keratoplasty without a Keratoprosthesis or Endoscopy for Vitreoretinal Disease Associated with Corneal Opacity.

We evaluated the visual outcome of combined penetrating keratoplasty (PKP) and 25G pars plana vitrectomy (PPV) performed without a temporary keratoprosthesis or endoscopy in a patient with vitreoretinal disease complicated by severe corneal opacity. The patient was a 68-year-old woman who had severe corneal opacity and silicone oil in her left eye after several previous intraocular surgeries for rhegmatogenous retinal detachment and proliferative vitreoretinopathy. We successfully performed a combined surgery of conventional PKP followed by 25G PPV without the use of a keratoprosthesis. At 6 months after surgery, visual acuity had not improved, and the density of corneal endothelial cells of the donor cornea had declined from 3,205 to 1,969 cells/mm2. However, corneal transparency remained good, and additional surgery for vitreoretinal disease was not necessary. The combined surgical procedure designed to minimize the number of open-sky steps and to limit vitreoretinal complications thus proved to be safe and achieved stable corneal clarity in a patient with vitreoretinal disease and severe corneal opacity.

Biaryl axially chiral derivatizing agent for simultaneous separation and sensitive detection of proteinogenic amino acid enantiomers using liquid chromatography-tandem mass spectrometry.

Novel sophisticated derivatizing agents for the efficient enantioselective separation and mass spectrometric detection of d- and l-amino acids have been developed. Two new axially chiral reagents derived from 6,6'-dimethyl-2,2'-biphenyldiamine were synthesized. Their chiral separation and detection abilities were evaluated by derivatizing proteinogenic amino acid standards in reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting diastereomers derived from the developed derivatizing agents and amino acids could be completely separated, because of the effective chiral environment constructed by the axially chiral biphenyl moiety. After optimizing the reactive group, (R)-4-nitrophenyl N-[2'-(diethylamino)-6,6'-dimethyl-[1,1'-biphenyl]-2-yl]carbamate hydrochloride ((R)-BiAC) was found to be the best reagent for highly sensitive simultaneous d,l-amino acid analysis. Using (R)-BiAC, the complete chiral separation of all derivatized proteinogenic amino acids was achieved within 11.5 min with Rs greater than 1.9, except for certain allo-isomers. An exceptional feature of this reagent was its control of elution order, i.e., it afforded elution of the diastereomers derived from d-amino acids before their l-amino acid counterparts for all 19 proteinogenic amino acids. Sensitive detection was also achieved by introducing a dialkyl amino group and selectively cleaving it at the binding site between the reagent and amino acid. Attomole (amol) detection limits (signal-to-noise ratio = 3) were obtained for the tested d,l-amino acids, in the range 7.0-127 amol. As an example of application, the method was applied to food sample analysis, and detected several d-amino acids. Consequently, the developed method seems likely to facilitate simultaneous determination of enantiomers, including the tiny amounts of d-amino acids found in nature.

Highly sensitive quantification for human plasma-targeted metabolomics using an amine derivatization reagent.

Amino acids and their related metabolites play important roles in various physiological processes and have consequently become biomarkers for diseases. However, accurate quantification methods have only been established for major compounds, such as amino acids and a limited number of target metabolites. We previously reported a highly sensitive high-throughput method for the simultaneous quantification of amines using 3-aminopyridyl-N-succinimidyl carbamate as a derivatization reagent combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Herein, we report the successful development of a practical and accurate LC-MS/MS method to analyze low concentrations of 40 physiological amines in 19 min. Thirty-five of these amines showed good linearity, limits of quantification, accuracy, precision, and recovery characteristics in plasma, with scheduled selected reaction monitoring acquisitions. Plasma samples from 10 healthy volunteers were evaluated using our newly developed method. The results revealed that 27 amines were detected in one of the samples, and that 24 of these compounds could be quantified. Notably, this new method successfully quantified metabolites with high accuracy across three orders of magnitude, with lowest and highest averaged concentrations of 31.7 nM (for spermine) and 18.3 µM (for α-aminobutyric acid), respectively.

Differential Changes in Intraocular Pressure and Corneal Manifestations in Individuals With Viral Endotheliitis After Keratoplasty.

PURPOSE: To investigate the clinical characteristics of viral endotheliitis after keratoplasty, we evaluated clinical parameters in individuals with anterior chamber inflammation after keratoplasty classified according to the absence or presence of DNA from various viruses in aqueous humor. METHODS: A total of 29 eyes of 27 subjects with anterior chamber inflammation after keratoplasty were enrolled in the study. The subjects were classified into the herpes simplex virus (HSV) group (7 patients, 8 eyes), cytomegalovirus (CMV) group (6 patients, 6 eyes), varicella zoster virus group (2 patients, 2 eyes), and nonviral group (12 patients, 13 eyes) on the basis of detection of viral DNA in aqueous humor. The interval between keratoplasty and the onset of inflammation, changes in intraocular pressure (IOP), and corneal manifestations of inflammation were retrospectively reviewed. RESULTS: Sixteen of the 29 eyes developed anterior chamber inflammation within 2 years after keratoplasty. IOP during active inflammation was significantly increased in the HSV and CMV groups compared with preinflammation values, and the increased IOP was significantly ameliorated in association with resolution of inflammation. Linear-type keratoprecipitates were frequently detected in the HSV (5 of 8 eyes), CMV (3 of 6 eyes), and nonviral (6 of 13 eyes) groups. Corneal edema was also commonly observed in the HSV (7 of 8 eyes) and nonviral (13 of 13 eyes) groups. CONCLUSIONS: Changes in IOP and corneal manifestations may provide a basis for differentiation of viral endotheliitis from allograft rejection and among viral pathogens in individuals with anterior chamber inflammation after keratoplasty, and they may thus allow initiation of appropriate treatment before viral DNA is identified.

[Evaluation of Iris Morphology Viewed through Stromal Edematous Corneas by Infrared Camera].

BACKGROUND: We reported that the application of infrared camera enables us to observe iris morphology in Peters' anomaly through edematous corneas. PURPOSE: To observe the iris morphology in bullous keratopathy or failure grafts with an infrared camera. SUBJECTS AND METHODS: Eleven bullous keratopathy or failure grafts subjects (6 men and 5 women, mean age ± SD; 72.7 ± 13.0 years old) were enrolled in this study. The iris morphology was observed by applying visible light mode and near infrared light mode of infrared camera (MeibomPen). The detectability of pupil shapes, iris patterns and presence of iridectomy was evaluated. RESULTS: Infrared mode observation enabled us to detect the pupil shapes in 11 out of 11 cases, iris patterns in 3 out of 11 cases, and presence of iridetomy in 9 out of 11 cases although visible light mode observation could not detect any iris morphological changes. CONCLUSION: Applying infrared optics was valuable for observation of the iris morphology through stromal edematous corneas.

Potential New Modes of Treatment of Neurotrophic Keratopathy.

The cornea focuses external light onto the retina, a function for which it must be transparent and possess a smooth surface. Homeostasis of the corneal epithelium is regulated by various humoral factors present in the tear fluid and by neural factors derived from the trigeminal nerve. Neurotrophic keratopathy (NK) is characterized by corneal epithelial disorders that result from impairment of trigeminal nerve function and a consequent deficiency of neural factors. The ideal mode of treatment for this condition is the regeneration of damaged trigeminal nerve fibers, but such therapy is not currently available. In this review, we describe established and potential new treatments of NK. Our research demonstrated that a combination of the neurotransmitter substance P and insulin-like growth factor 1 (IGF-1) has a synergistic stimulatory effect on corneal epithelial migration in vitro and on corneal wound closure in vivo. Furthermore, we identified the minimal amino acid sequences of substance P and IGF-1 required for this synergistic action based on the assumption that the clinical application of peptides corresponding to these sequences would have fewer side effects compared with the full-length molecules. Combination of the substance P-derived peptide FGLM-amide and the IGF-1-derived peptide SSSR promoted corneal epithelial wound healing in patients with NK.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01756456.

Recovery of the Corneal Stroma Associated With Rapid Reepithelialization Induced by the Fibronectin-Derived Peptide PHSRN in 2 Cases of Corneal Perforation Due to a Persistent Epithelial Defect.

PURPOSE: To report 2 cases of corneal perforation associated with a persistent epithelial defect (PED), which were treated with eye drops containing the fibronectin-derived peptide PHSRN (Pro-His-Ser-Arg-Asn). METHODS: A 67-year-old man and a 58-year-old man presented with corneal perforation associated with a PED caused by lagophthalmos. PHSRN eye drops were administered 4 times a day to both patients. RESULTS: Both patients experienced healing of the epithelial defect and closure of corneal perforation within 3 or 4 days after the onset of PHSRN treatment. Anterior segment optical coherence tomography also revealed recovery of corneal stromal thickness at the lesion site. CONCLUSIONS: PHSRN eye drops were effective for the treatment of corneal perforation due to the PED, with rapid reepithelialization being followed by full restoration of stromal thickness.

Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples.

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells.

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amid from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1 pM, respectively. Meanwhile, the ratios of GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amide) to total GLP-1 peptides were similar (6 ± 3, 26 ± 3, and 78 ± 5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells.

Quantitative analysis of collagen lamellae in the normal and keratoconic human cornea by second harmonic generation imaging microscopy.

PURPOSE: To characterize the structural properties of collagen lamellae in the normal and keratoconic human corneal stroma, we measured their width and angle relative to Bowman's layer (BL). METHODS: Thirteen normal and four keratoconic corneas were examined. Collagen lamellae in tissue blocks from the central cornea were visualized by second harmonic generation imaging microscopy. Images obtained in 1-µm steps from BL to Descemet's membrane (DM) were subjected to three-dimensional reconstruction. The reconstructed data sets were divided into 10 layers of equal depth (L1-L10) for analysis. The width of lamellae adherent to BL (L0) was also determined. RESULTS: For the normal cornea, the width (mean ± SD) of collagen lamellae was 6.5 ± 1.7 µm at L0, decreased to 4.3 ± 1.3 µm at L1, and then increased gradually with progression toward DM to 122.2 ± 34.5 µm at L10, whereas the angle of lamellae was 20.9° ± 5.4° at L1 and decreased initially to 10.6° ± 3.2° at L2 before declining gradually to 2.7° ± 2.2° at L10. The width and angle of collagen lamellae in the keratoconic cornea were significantly larger and smaller, respectively, relative to those in the normal cornea. CONCLUSIONS: In the normal human cornea, collagen lamellae adjacent to BL are narrow and form a steep angle with BL, whereas they increase in width and their angle relative to BL flattens with progression toward DM. These properties of collagen lamellae are altered in keratoconus and are likely related to abnormalities of corneal shape.

Congenital hypoplastic trigeminal nerve revealed by manifestation of corneal disorders likely caused by neural factor deficiency.

PURPOSE: To report a case of hypoplastic trigeminal nerve associated with corneal epithelial disorders that were successfully treated with peptides derived from substance P and insulin-like growth factor-1 (IGF-1). CASE REPORT: A 16-month-old boy was referred for treatment of a persistent corneal epithelial defect on his left eye. Magnetic resonance imaging revealed the apparent absence of the trigeminal nerve on the left side, and the patient was therefore diagnosed with neurotrophic keratopathy. Treatment with eye drops containing the tetrapeptides FGLM-NH2 and SSSR derived from the neuropeptide substance P and the growth factor IGF-1, respectively, resulted in resurfacing of the corneal epithelial defect. DISCUSSION: The trigeminal nerve anomaly of the patient likely gave rise to neurotrophic keratopathy as a result of a deficiency of neural factors, emphasizing the importance of neural regulation in corneal epithelial homeostasis.

Spontaneous healing of corneal perforation after temporary discontinuation of erlotinib treatment.

PURPOSE: To report a case of corneal perforation associated with oral administration of erlotinib and its spontaneous healing after temporary discontinuation of drug treatment. CASE REPORT: A 65-year-old man with metastatic lung cancer was treated with erlotinib (150 mg/day), a specific tyrosine kinase inhibitor of the epidermal growth factor receptor. He was referred to our corneal service for the treatment of bilateral corneal disorders, diagnosed with mild aqueous-deficient dry eye, and treated by insertion of punctal plugs. His corneal epithelial disorders initially improved, but subsequently worsened, as manifested by the development of bilateral corneal ulceration with corneal perforation in the right eye. The oral administration of erlotinib was interrupted in preparation for tectonic keratoplasty, but 2 days later the corneal perforation of the right eye and the bilateral epithelial defects had healed spontaneously. Treatment with erlotinib was resumed at half the initial dose, and the cornea of both eyes has remained apparently healthy. DISCUSSION: Erlotinib may be secreted into tear fluid and thereby adversely affect the corneal epithelium. The development of corneal epithelial disorders in patients receiving this drug may be reversed by reducing its dose.

Cys34-cysteinylated human serum albumin is a sensitive plasma marker in oxidative stress-related chronic diseases.

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.