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.

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.

Nosocomial infections caused by vancomycin-resistant Enterococcus in a Japanese general hospital and molecular genetic analysis.

INTRODUCTION: Vancomycin-resistant Enterococcus (VRE) is a rare bacterium in Japan, but an outbreak due to nosocomial transmission in medical facilities has been reported in recent years. Here, we report the outbreak of vanA vancomycin-resistant Enterococcus faecium (VREfm) in multiple wards of Nara Prefectural General Medical Center in 2019 and results of the molecular epidemiology analysis. METHODS: An aggressive screening program was conducted after the first VREfm was detected in a patient in the A ward. During the outbreak, 6000 rectal swab samples were screened for VRE by culture. Isolates from 60 patients with VREfm detected were clustered using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: PFGE revealed a cluster consisting of three major clusters and four multi-strains. The first major cluster consisted of 26 isolates, the second consisted of 10 isolates, the third consisted of 6 isolates, and the remaining 4 clusters consisted of 2 isolates. MLST identified an allele profile (ST80) in most clusters of clone types P01-P06 but an allele profile (ST992) in cluster P07. CONCLUSION: Based on the PFGE pattern, this case was considered to be a nosocomial infection due to multiple clones. Later, in addition to screening, sharing of hospital information, cohorting of patients and staff, and strengthening of environmental cleanup were carried out, and horizontal infection was suppressed.

A case of Bordetella trematum and Kerstersia gyiorum infections in a patient with congestive dermatitis.

Bordetella trematum and Kerstersia gyiorum are rare gram-negative bacilli that are not frequently detected in human infections. In this report, we describe a case of a 48-year-old man who presented to our hospital with an infected wound on his leg. Discharges from the cracks of the granulation were collected and evaluated in our microbiology laboratory. Gram staining of the specimen showed polymorphonuclear leukocytes and abundant gram-negative bacilli. Three types of colonies were isolated on blood agar and were identified as B. trematum and Alcaligenes faecalis using VITEK MS. Moreover, K. gyiorum and B. trematum were identified and confirmed via 16S ribosomal RNA (rRNA) gene sequencing. The patient successfully recovered following application of meropenem antibacterial therapy and surgical debridement. This is the first reported case of complex wound infection caused by both B. trematum and K. gyiorum. Identification of B. trematum has recently been made possible by routine bacterial identification using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, K. gyiorum isolation is still rare, and species identification requires 16S rRNA sequencing. Thus, this case highlighted the importance of using multiple methods, such as MALDI-TOF MS and 16S rRNA gene sequencing, for identification of rarely isolated species from clinical specimens.

Magnesium bis(trifluoromethanesulfonyl)amide complexes with triglyme and asymmetric homologues: phase behavior, coordination structures and melting point reduction.

The phase behavior of binary mixtures of triglyme (G3) and Mg[TFSA]2 (TFSA: bis(trifluoromethanesulfonyl)amide) was investigated, towards the development of a Mg2+-based room-temperature solvate ionic liquid (SIL) electrolyte. In a 1 : 1 molar ratio, G3 and Mg[TFSA]2 form a thermally stable complex (decomposition temperature, Td: 240 °C) with a melting point (Tm) of 70 °C, which is considerably lower than that of the analogous tetraglyme (G4) system (137 °C). X-ray crystallography of a single crystal of [Mg(G3)][TFSA]2 revealed that a single Mg2+ cation is coordinated by a single, distorted, tetradentate G3 molecule from one side, and two monodentate [TFSA]- anions, with transoid conformation, from the reverse side to form an ion pair. Raman spectra of [Mg(G3)][TFSA]2 in the molten state revealed the presence of different coordination structures, as the liquid exhibits changes in the vibrational modes corresponding to G3 and the [TFSA]- anion compared to those observed for the solid. Investigation of the ion pair stabilization energies by DFT calculations suggests that higher stability cation complexes and ion pairs co-exist in the molten state than those observed in the crystalline state. These results imply that the coordination structures of the ion pairs play a key role in providing SILs with low Tm. To decrease the Tm further, several asymmetric homologues of G3, which have higher conformational flexibility than G3, were investigated. Notably, a 1 : 1 mixture of Mg[TFSA]2 with G3Bu (where one of the terminal methyl groups of G3 is substituted for a butyl group) formed a thermally stable complex (Td: 251 °C) without any distinct Tm and showed reasonable ionic conductivity at room-temperature, indicating partial dissociation of ions. In this electrolyte, which showed high oxidative stability, quasi-reversible Mg deposition/dissolution was achieved, indicating that Mg2+-based room-temperature SILs can be utilized as a new class of Mg electrolyte.

Effect of the cation on the stability of cation-glyme complexes and their interactions with the [TFSA]- anion.

The interactions of glymes with alkali or alkaline earth metal cations depend strongly on the metal cations. For example, the stabilization energies (Eform) calculated for the formation of cation-triglyme (G3) complexes with Li+, Na+, K+, Mg2+, and Ca2+ at the MP2/6-311G** level were -95.6, -66.4, -52.5, -255.0, and -185.0 kcal mol-1, respectively, and those for the cation-tetraglyme (G4) complexes were -107.7, -76.3, -60.9, -288.3 and -215.0 kcal mol-1, respectively. The electrostatic and induction interactions are the major source of the attraction in the complexes; the contribution of the induction interactions to the attraction is especially significant in the divalent cation-glyme complexes. The binding energies of the cation-G3 complexes with Li+, Na+, K+, Mg2+, and Ca2+ and the bis(trifluoromethylsulfonyl)amide anion ([TFSA]-) were -83.9, -86.6, -80.0, -196.1, and -189.5 kcal mol-1, respectively, and they are larger than the binding energies of the corresponding cation-G4 complexes (-73.6, -75.0, -77.4, -172.1, and -177.2 kcal mol-1, respectively). The binding energies and conformational flexibility of the cation-glyme complexes also affect the melting points of equimolar mixtures of glyme and TFSA salts. Furthermore, the interactions of the metal cations with the oxygen atoms of glymes significantly decrease the HOMO energy levels of glymes. The HOMO energy levels of glymes in the cation-glyme-TFSA complexes are lower than those of isolated glymes, although they are higher than those of the cation-glyme complexes.

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.

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)[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.

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.

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.

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.

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.

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.

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.

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.

Comparison of the Drug-Induced Efficacies between Omidenepag Isopropyl, an EP2 Agonist and PGF2α toward TGF-ß2-Modulated Human Trabecular Meshwork (HTM) Cells.

To compare the drug-induced efficacies between omidenepag (OMD), an EP2 agonist, and prostaglandin F2α (PGF2α) on glaucomatous trabecular meshwork (TM) cells, two- and three-dimensional (2D and 3D) cultures of TGF-ß2-modulated human trabecular meshwork (HTM) cells were used. The following analyses were performed: (1) transendothelial electrical resistance (TEER) and FITC-dextran permeability measurements (2D), (2) the size and stiffness of the 3D spheroids, and (3) the expression (both 2D and 3D) by several extracellular matrix (ECM) molecules including collagen (COL) 1, 4 and 6, and fibronectin (FN), and α smooth muscle actin (αSMA), tight junction (TJ)-related molecules, claudin11 (Cldn11) and ZO1, the tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9 and 14, connective tissue growth factor (CTGF), and several endoplasmic reticulum (ER) stress-related factors. TGF-ß2 significantly increased the TEER values and decreased FITC-dextran permeability, respectively, in the 2D HTM monolayers, and induced the formation of downsized and stiffer 3D HTM spheroids. TGF-ß2-induced changes in TEER levels and FITC-dextran permeability were remarkably inhibited by PGF2α. PGF2α induced increases in the sizes and stiffness of the TGF-ß2-treated 3D spheroids, but OMD enhanced only spheroid size. Upon exposure to TGF-ß2, the expression of most of the molecules that were evaluated were significantly up-regulated, except some of ER stress-related factors were down-regulated. TJ-related molecules or ER stress-related factors were significantly up-regulated (2D) or down-regulated (3D), and down-regulated (2D) by PGF2α and OMD, while both drugs altered the expression of some of the other genes in the 3D spheroids in a different manner. The findings presented herein suggest that PGF2α and OMD differently modulate the permeability of the TGFß2-modulated 2D monolayers and the physical properties of the 3D HTM spheroids.

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.

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.