TGF-ß Isoforms and Local Environments Greatly Modulate Biological Nature of Human Retinal Pigment Epithelium Cells.

To characterize transforming growth factor-ß (TGF-ß) isoform (TGF-ß1~3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including collagen 1 (COL1), COL4, and COL6; α-smooth muscle actin (αSMA); hypoxia-inducible factor 1α (HIF1α); and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α), a master regulator for mitochondrial respiration;, tight junction-related molecules, Zonula occludens-1 (ZO1) and E-cadherin; and vascular endothelial growth factor (VEGF); (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-ß isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-ß1 and TGF-ß3 caused a marked increase in TEER values, and TGF-ß2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-ß isoforms, among which TGF-ß1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-ß2 and TGF-ß3 or by TGF-ß1 and TGF-ß3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-ß isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-ß-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-ß isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.

Crystal structure and Hirshfeld surface analysis of (1H-imidazole-κN3)[4-methyl-2-({[2-oxido-5-(2-phenyl-diazen-1-yl)phen-yl]methyl-idene}amino)penta-noate-κ3O,N,O']copper(II).

The title copper(II) complex, [Cu(C18H19N3O3)(C3H4N2)], consists of a tridentate ligand synthesized from l-leucine and azo-benzene-salicyl-aldehyde. One imidazole mol-ecule is additionally coordinated to the copper(II) ion in the equatorial plane. The crystal structure features N-H⋯O hydrogen bonds. A Hirshfeld surface analysis indicates that the most important contributions to the packing are from H⋯H (52.0%) and C⋯H/H⋯C (17.9%) contacts.

Impact of Multiplex Polymerase Chain Reaction Test in Patients With Meningitis or Encephalitis.

Background: The objective of this study was to evaluate the impact of the FilmArray meningitis/encephalitis panel (FAME) on length of stay (LOS) and duration of antimicrobial treatment in children and adults in a Japanese community hospital. Methods: This retrospective cohort study was conducted in Japan between January 2016 and December 2022. We included hospitalized patients with cerebrospinal fluid (CSF) samples and those aged <2 months or who had 5 or more white blood cells/µL in the CSF. To compare the days of therapy (DOT) and LOS between the pre-FAME and FAME periods, multivariate Poisson regression analyses were conducted without an offset term. Results: The number of cases undergoing pathogen-specific polymerase chain reaction increased from 3.7% in the pre-FAME period to 57.5% in the FAME period (P < .001). The pathogen identification rate also increased during the FAME period, from 0.4% to 18.7% (P < .001). While the antibacterial DOT was not statistically different between the 2 periods (adjusted rate ratio [aRR], 1.06 [95% confidence interval {CI}, 1.00-1.13]; P = .063]), the antiviral DOT was significantly shorter in the FAME period (aRR, 0.80 [95% CI, .71-.89]; P < .001). Conclusions: This study revealed a significant reduction in antiviral use during the FAME period, whereas LOS and antibacterial use did not decrease. Given the possibility of factors (eg, the COVID-19 pandemic) affecting the epidemiology of meningitis and encephalitis, the indications and impact of the FAME test should be evaluated with continuous monitoring of the epidemiology of meningitis and encephalitis and its clinical impact.

Crystal structure and Hirshfeld surface analysis of (1H-imidazole-κN3)[N-(2-oxido-benzyl-idene)tyrosinato-κ3O,N,O']copper(II).

The title copper(II) complex, [Cu(C16H13NO4)(C3H4N2)], consists of a tridentate ligand synthesized from l-tyrosine and salicyl-aldehyde. One imidazole mol-ecule is additionally coordinating to the copper(II) ion. The crystal structure features N-H⋯O, O-H⋯O and C-H⋯O hydrogen bonds. The Hirshfeld surface analysis indicates that the most important contributions to the packing are from H⋯H (37.9%), C⋯H (28.2%) and O⋯H/H⋯O (21.2%) contacts.

Pasteurella bettyae infection requiring finger amputation due to rapid deterioration and tissue damage.

We report a case of infection of the middle finger of a 69-year-old man who visited our hospital. Pus was collected from the erythematous and swollen area of the nail cage of the left-hand middle finger and evaluated in our microbiology laboratory. Gram staining of the specimen revealed multinucleated leukocytes and abundant gram-negative bacilli. Isolated colonies were identified as Pasteurella bettyae using VITEK MS and 16 S ribosomal RNA (rRNA) gene sequencing. The patient's blood test results improved after treatment with penicillin, but the local factors affecting the finger did not improve, and amputation of the middle finger had to be performed. This case represents a report of a very rare hand infection caused by P. bettyae. Polymorphic identification methods, such as MALDI-TOF MS and 16 S rRNA gene sequencing, are needed for members of the genus Pasteurella isolated from severe infections and abnormal sites, and further studies are warranted.

Crystal structure and Hirshfeld surface analysis of mono/bis-(aqua-κO)[N-(2-oxido-benzyl-idene)valinato-κ3 O,N,O']copper(II): dimeric Schiff base copper(II) complexes having different numbers of coordinated water mol-ecules.

The mol-ecular structure of the title compound, [Cu(C12H13N2O3)(H2O)2]·[Cu(C12H13N2O3)(H2O)], consists of two different mol-ecules in the asymmetric unit. Both of the structures consist of a tridentate ligand synthesized from l-valine and salicyl-aldehyde, and one water mol-ecule or two water mol-ecules coordinating to CuII. They have a square-planar (mol-ecule 1) or a square-pyramidal (mol-ecule 2) coordination geometry. In the crystal, the mol-ecules form intra- and inter-molecular O-H⋯O hydrogen bonds involving the coordinated water mol-ecules and other sites. A Hirshfeld surface analysis indicated that the most important contributions to the packing are from H⋯H [52.9% (mol-ecule 1) and 51.1% (mol-ecule 2)] and H⋯O/ O⋯H [21.2% (mol-ecule 1) and 25.8% (mol-ecule 2)] contacts. In addition, an electrostatic potential map was also obtained from DFT calculations to support the discussion of the inter-molecular inter-actions.

Epidemiology of respiratory tract infections using multiplex PCR in a Japanese acute care hospital during the COVID19 pandemic.

Introduction: We aimed to investigate the epidemiology of respiratory infections by season and age during the COVID-19 pandemic in a Japanese acute care hospital using multiplex PCR testing. Methods: We detected 21 pathogens in specimens from outpatients with respiratory symptoms at the Nara Prefecture General Medical Center using the multiplex PCR-based FilmArray Respiratory Panel 2.1 (bioMérieux). Results: Of the 3177 cases, 1215 (38.2%) were infected with at least one causative virus, and 1641 viruses were detected. The most common viruses detected were human rhinovirus/enterovirus (n = 655) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (n = 264). Additionally, 321 (10.1%) of these cases were infected with two or more overlapping viruses. There were 23 cases of co-infection with SARS-CoV-2 and other viruses. In the winter months from December 2020 to March 2021, the number of detected viruses was relatively low, followed by the surge of human rhinovirus/enterovirus, respiratory syncytial virus (RSV), and parainfluenza type 3 in the spring and summer of 2021. While the number of human rhinovirus/entero-virus remained relatively high after the 2021 summer, the number of other viruses detected since September 2021 was low. After December 2021, the number of SARS-CoV-2 increased rapidly. Conclusions: Continuous monitoring of the epidemiology of respiratory infection is important to understand the prolonged impact of the COVID-19 pandemic.

Impact of the COVID-19 pandemic and multiplex polymerase chain reaction test on outpatient antibiotic prescriptions for pediatric respiratory infection.

This study aimed to evaluate the impact of the prolonged COVID-19 pandemic on outpatient antibiotic prescriptions for pediatric respiratory infections at an acute care hospital in Japan in order to direct future pediatric outpatient antibiotic stewardship. The impact of the COVID-19 pandemic and the FilmArray Respiratory Panel (RP) on outpatient antibiotic prescriptions was assessed from January 2019 to December 2021 using an interrupted time series analysis of children <20 years. The overall antimicrobial prescription rate decreased from 38.7% to 22.4% from the pre-pandemic period to the pandemic. The pandemic (relative risk [RR] level, 0.97 [0.58-1.61]; P = 0.90; RR slope, 1.05 [0.95-1.17] per month; P = 0.310) and FilmArray RP (RR level, 0.90 [0.46-1.75]; P = 0.75; RR slope, 0.95 [0.85-1.06] per month; P = 0.330) had no significant effect on the monthly antibiotic prescription rates. The COVID-19 pandemic was not significantly related to the antibiotic prescription rate, suggesting that it did not impact physicians' behavior toward antibiotic prescriptions. Replacing rapid antigen tests with the FilmArray RP introduced on December 1, 2020, did not affect the magnitude of the reduction in antibiotic prescription rate for pediatric respiratory infections.

Improvement of the SOD activity of the Cu2+ complexes by hybridization with lysozyme and its hydrogen bond effect on the activity enhancement.

We prepared L-amino acids (L-valine and L-serine, respectively) based on the Schiff base Cu2+ complexes CuSV and CuSS in the absence/presence of hydroxyl groups and their imidazole-bound compounds CuSV-Imi and CuSS-Imi to reveal the effects of hydroxyl groups on SOD activity. The structural and spectroscopic features of the Cu2+ complexes were evaluated using X-ray crystallography, UV-vis spectroscopy, and EPR spectroscopy. The spectroscopic behavior upon addition of lysozyme indicated that both CuSV and CuSS were coordinated by the imidazole group of His15 in lysozyme at their equatorial position, leading to the formation of hybrid proteins with lysozyme. CuSS-Imi showed a higher SOD activity than CuSV-Imi, indicating that the hydroxyl group of CuSS-Imi played an important role in the disproportionation of O2 - ion. Hybridization of the Cu2+ complexes CuSV and CuSS with lysozyme resulted in higher SOD activity than that of CuSV-Imi and CuSS-Imi. The improvements in SOD activity suggest that there are cooperative effects between Cu2+ complexes and lysozyme.

Comparison of MALDI-TOF mass spectrometry and rpoB gene sequencing for the identification of clinical isolates of Aeromonas spp.

Aeromonas spp., widely present in rivers and soil, cause mild gastroenteritis, severe septicemia, and soft tissue infections in humans. Treatment of these infections require accurate identification of pathogenic Aeromonas spp. However, identification at the species level using conventional methods is highly challenging. In this study, we aimed to compare the accuracy of two different approaches developed for bacterial identification: (i) housekeeping gene sequencing (rpoB) in conjunction with phylogenetic analysis and (ii) matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS) (MALDI Biotyper and VITEK MS), for differentiating Aeromonas spp. We analyzed 58 Aeromonas isolates recovered from patients at different medical institutions in Japan using both identification methods. The rpoB sequencing method was the most accurate, identifying all Aeromonas isolates at the species level. Meanwhile, the MALDI Biotyper system correctly identified 53 (91.4%) isolates at the genus level and an additional 30 (51.7%) at the species level. The VITEK MS system correctly identified 58 (100%) isolates at the genus level and an additional 34 (58.6%) at the species level. Thus, MALDI Biotyper and VITEK MS accurately identified isolates at the genus level, but differences were found in the accuracy of identification of species. However, the low cost and ease of analysis make MALDI-TOF MS-based methods strong candidates for use in clinical laboratories that require easy-to-use identification methods.

FGF-2 enhances fibrogenetic changes in TGF-ß2 treated human conjunctival fibroblasts.

The objective of the current study was to examine the effects of fibroblast growth factor-2 (FGF-2) on conjunctival fibrogenesis that was induced by the presence of transforming growth factor-ß2 (TGF-ß2). Two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) were used for this purpose. The 2D and 3D cultured HconF were characterized by transendothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D), real-time metabolic analyses (2D), size and stiffness measurements (3D), and the mRNA expression of extracellular matrix molecules, their modulators, Tissue inhibitor of metalloproteinases and matrix metalloproteinases and ER-stress related genes (2D and 3D). FGF-2 significantly increased planar proliferation, as evidenced by TEER values and FITC dextran permeability, and shifted glucose metabolism to the energetic phenotype of 2D HconF cells, and the stiffness of the 3D spheroids, and these effects were further enhanced in the presence of TGF-ß2. Analyses of the expression of possible candidate molecules involved in cell architecture and stress indicated that some additive effects caused by both factors were also recognized in some of these molecules. The findings reported herein indicate that the FGF-2, either along or additively with TGF- ß2 increased the fibrogenetic changes on the plane as well as in the spatial space of HconF cells.

All-trans Retinoic Acids Synergistically and Beneficially Affect In Vitro Glaucomatous Trabecular Meshwork (TM) Models Using 2D and 3D Cell Cultures of Human TM Cells.

We report herein on the effects of all-trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor ß2 (TGF-ß2). In the presence of 5 ng/mL TGF-ß2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans-endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-ß2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-ß2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-ß2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-ß2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-ß2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

All Trans-Retinoic Acids Facilitate the Remodeling of 2D and 3D Cultured Human Conjunctival Fibroblasts.

Vitamin A derivative, all-trans-retinoic acid (ATRA), is known to be a potent regulator of the growth and differentiation of various types of cells. In the present study, the unidentified effects of ATRA on superficial and vertical spreading conjunctival scarring were examined. The study involved the use of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells in the presence or absence of TGF-ß2. The effects of ATRA (1 µM) on superficial or vertical spreading conjunctival scarring were evaluated by the barrier function by trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements and real-time metabolic analysis, as well as the physical properties, namely, the size and stiffness, of 3D spheroids, respectively. In addition, the expressions of several related molecules, including extracellular matrix (ECM) molecules, ECM modulators including a tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), and ER stress-related factors, were examined. ATRA significantly induced (1) an increase in TEER values and a decrease in FITC dextran permeability, respectively, in the 2D monolayers, and (2) relatively and substantially increased the size and stiffness, respectively, of the 3D spheroids. These ATRA-induced effects were further enhanced in the TGF-ß2-treated cells, whereas the TGF-ß2-induced enhancement in glycolytic capacity was canceled by the presence of ATRA. Consistent with these physical and morphological effects, the mRNA expressions of several molecules were significantly but differently induced between 2D and 3D cultures by ATRA, although the presence of TGF-ß2 did not substantially affect these gene expression levels. The findings reported in this study indicate that ATRA may exacerbate both superficial and vertical conjunctival fibrosis spreading independently of TGF-ß2-induced changes.

An α2-Adrenergic Agonist, Brimonidine, Beneficially Affects the TGF-ß2-Treated Cellular Properties in an In Vitro Culture Model.

We report herein on the effects of brimonidine (BRI), an α2-adrenergic agonist, on two-dimensional (2D) and three-dimensional (3D) cell-cultured TGF-ß2-untreated and -treated human trabecular meshwork (HTM) cells. In the presence of TGF-ß2 (5 ng/mL), (1) the effects of BRI on (1) the 2D HTM monolayers' barrier function were investigated as estimated using trans-endothelial electrical resistance (TEER) measurement and FITC dextran permeability; (2) real-time analyses of cellular metabolism using a Seahorse Bioanalyzer; (3) the largeness and hardness of 3D spheroids; and (4) the expression of genes that encode extracellular matrix (ECM) proteins, including collagens (COL) 1, 4, and 6; fibronectin (FN) and α-smooth muscle actin (α-SMA); ECM modulators, including a tissue inhibitor of matrix proteinase (TIMP) 1-4; matrix metalloproteinase (MMP) 2, 9, and 14; and several endoplasmic reticulum (ER) stress-related genes, including the X-box-binding protein 1 (XBP1), the spliced XBP1 (sXBP1), glucose-regulated protein (GRP)78, GRP94, and CCAAT-enhancer-binding protein homologous protein (CHOP). BRI markedly inhibited the TGF-ß2-induced increase in the values of TEER of the 2D cell monolayer and the hardness of the 3D spheroids, although it had no effect on their sizes. BRI also cancelled the TGF-ß2-induced reduction in mitochondrial maximal respiration but had no effect on the glycolytic capacity. In addition, the gene expression of these molecules was quite different between the 2D and 3D cultures of HTM cells. The present observations found in this study indicate that BRI may beneficially affect TGF-ß2-induced changes in both cultures, 2D and 3D, of HTM cells, although their structural and functional properties that were altered varied significantly between both cultures of HTM cells.

Fatty acid metabolism is involved in both retinal physiology and the pathology of retinal vascular diseases.

To study the pathophysiological roles of the fatty acid-binding proteins (FABPs) within the retina, we performed; (1) immunolabeling of human retinas, wild type (WT) rat and mouse retinas, rat models for diabetic retinopathy (DR) and retinitis pigmentosa (RP) with anti-FABP3, FABP4, FABP5, FABP7, FABP8 and FABP12, (2) electroretinogram (ERG) measurements of WT and FABP4-deficient (Fabp4-/-) mice, (3) ELISA or gas chromatography measurements of plasma (P-) and vitreous (V-) levels of FABP4 and vascular endothelial growth factor A (VEGFA), and fatty acids (FAs) from patients with retinal vascular disease (RVD) including proliferative DR (PDR, n = 30) and retinal vein occlusion (RVO, n = 18) and non-RVD (n = 18). Within the human retina, diverse expressions of FABP3, FABP4, FABP7 and FABP8 were identified. In contrast, positive immunoreactivities toward only FABP4 and FABP12 were detected in the cases of rat and mouse retinas, and interestingly, the FABP4 labeling patterns for the WT, DR and RP rat retinas were different. The ERG amplitudes of Fabp4-/- mice were enhanced compared with those of WT mice. The concentrations of V-FABP4, V-VEGFA and total FAs were significantly higher in RVD patients than in non-PDR patients (P < 0.05). The V-FAs levels of each were significantly and positively correlated with V-FABP4 and V-VEGFA, although no significant correlation between vitreous (V-) and plasma (P-) FABP4, VEGFA and FAs were detected. The current study reveals that V-FAs appear to have significant roles in both retinal physiology as well as the pathogenesis of RVD with FABP4, which is commonly expressed within the retina in most species.

Human Trabecular Meshwork (HTM) Cells Treated with TGF-ß2 or Dexamethasone Respond to Compression Stress in Different Manners.

To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-ß2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-ß2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0-10 min) and subsequent downregulation (10-30 min) followed by another upregulation (30-60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-ß2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-ß2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures.

Hypoxia Differently Affects TGF-ß2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells.

The hypoxia associated with the transforming growth factor-ß2 (TGF-ß2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O2 gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-ß2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-ß2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-ß2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-ß2 increased proton leakage. The findings reported herein indicate that the TGF-ß2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.

ROCK 1 and 2 affect the spatial architecture of 3D spheroids derived from human corneal stromal fibroblasts in different manners.

The objective of the current study was to examine the roles of ROCK1 and 2 on the spatial architecture of human corneal stroma. We examined the effects of a pan-ROCK inhibitor (pan-ROCK-i), ripasudil, and a ROCK2 inhibitor (ROCK2-i), KD025 on the expression of genes that encode for ECM proteins including collagen (COL) 1, 4, 6, and fibronectin (FN), their regulators, a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9 and 14, and ER stress-related factors of two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs), and the physical properties of 3D HCSF spheroids. A gene expression analysis using ROCK-is indicated that KD025 (ROCK2 selective ROCK inhibitor) induced more significant changes than Rip (ripasudil, pan-ROCK inhibitor), suggesting that ROCK2 might be more extensively involved in the metabolism of ECM proteins and cell architectures of the 2D cultured HCSFs than ROCK1. In terms of the physical properties, size and stiffness of the 3D HCSFs spheroids, Rip caused a significant enlargement and this enhancement was concentration-dependent while KD025 also exerted a similar but less pronounced effect. In contrast, Rip and KD025 modulated physical stiffness differently, in that Rip caused a substantial decrease and KD025 caused an increase. Such diverse effects between Rip and KD025 were also observed for the gene expressions of ECM proteins, their regulators, and ER-stress related factors. The findings presented herein suggest that the ROCK1 and 2 influence the spatial architecture of 3D HCFS spheroids in different manners.

Fatty Acid-Binding Proteins 4 and 5 Are Involved in the Pathogenesis of Retinal Vascular Diseases in Different Manners.

This study reports on the pathological significance of the vitreous fatty acid-binding protein (Vt-FABP) 4 and 5, and vascular endothelial growth factor A (Vt-VEGFA) in patients with retinal vascular diseases (RVDs) including proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO). Subjects with PDR (n = 20), RVO (n = 10), and controls (epiretinal membrane, n = 18) who had undergone vitrectomies were enrolled in this study. The levels of Vt-FABP4, Vt-FABP5, and Vt-VEGFA were evaluated by enzyme-linked immunosorbent assays (ELISA). Retinal circulation levels were measured by a laser-speckle flow analyzer (LSFA) and other relevant data were collected. The Vt-FABP5 levels were significantly (p < 0.05) elevated in patients with RVDs compared to control patients. This elevation was more evident in patients with RVO than with PDR. Log Vt-FABP5 was significantly correlated negatively or positively with all the LSFA retinal circulation indexes or Log triglycerides (r = 0.31, p = 0.031), respectively. However, the elevations in the Vt-FABP4 and Vt-VEGFA levels were more evident in the PDR group (p < 0.05) and these factors were correlated positively with Log fasting glucose and negatively with some of the LSFA retinal circulation indexes. Multivariable regression analyses indicated that the LSFA blood flows of the optic disc at baseline was an independent effector with Log Vt-FABP5 other than several possible factors including age, gender, Log triglycerides, Log Vt-FABP4 and Log Vt-VEGFA. These current findings suggest that Vt-FABP5 is involved in the pathogenesis of RVD in a manner that is different from that for Vt-FABP4 and Vt-VEGFA, presumably by regulating retinal circulation.

Identification and Functional Analysis of GTP Cyclohydrolase II in Candida glabrata in Response to Nitrosative Stress.

Reactive nitrogen species (RNS) are signal molecules involved in various biological events; however, excess levels of RNS cause nitrosative stress, leading to cell death and/or cellular dysfunction. During the process of infection, pathogens are exposed to nitrosative stress induced by host-derived RNS. Therefore, the nitrosative stress resistance mechanisms of pathogenic microorganisms are important for their infection and pathogenicity, and could be promising targets for antibiotics. Previously, we demonstrated that the RIB1 gene encoding GTP cyclohydrolase II (GCH2), which catalyzes the first step of the riboflavin biosynthesis pathway, is important for nitrosative stress resistance in the yeast Saccharomyces cerevisiae. Here, we identified and characterized the RIB1 gene in the opportunistic pathogenic yeast Candida glabrata. Our genetic and biochemical analyses indicated that the open reading frame of CAGL0F04279g functions as RIB1 in C. glabrata (CgRIB1). Subsequently, we analyzed the effect of CgRIB1 on nitrosative stress resistance by a growth test in the presence of RNS. Overexpression or deletion of CgRIB1 increased or decreased the nitrosative stress resistance of C. glabrata, respectively, indicating that GCH2 confers nitrosative stress resistance on yeast cells. Moreover, we showed that the proliferation of C. glabrata in cultures of macrophage-like cells required the GCH2-dependent nitrosative stress detoxifying mechanism. Additionally, an infection assay using silkworms as model host organisms indicated that CgRIB1 is indispensable for the virulence of C. glabrata. Our findings suggest that the GCH2-dependent nitrosative stress detoxifying mechanism is a promising target for the development of novel antibiotics.