Non-lethal exposure to short-wavelength light-emitting diodes modulates tight-junction structure in human corneal epithelial cells via cAMP-dependent signaling.

Light-emitting diodes (LED)-derived lights have been widely used as a medical treatment in photobiomodulation (PBM). However, the PBM effects in ophthalmology are less well investigated. Herein, we explored the effect of LED-generated light on the tight-junction (TJ) formation in human corneal epithelial cells (HCEs). The HCEs were separately exposed to monochromatic LEDs at wavelengths of 365 nm (UVA), 420 nm (violet), 470 nm (blue), 530 nm (green), 590 nm (amber), 660 nm (deep red), and 740 nm (far red) at 10 J/cm2/day for 1 and 2 days. Long-term cultivation of HCEs without LED exposure for up to 14 days was established as a control. The effects of both LED wavelength and culture duration on cell morphology, cAMP-regulated proteins, TJ-associated proteins, and cell growth-associated proteins were also analyzed. Together with the increase in cell number during prolonged cultivation, cAMP, ZO-1, ZO-2, CLDN1, and CLDN4 all increased significantly during long-term cultivation without LED exposure. There was no difference in HCE viability after exposure to all monochromatic LEDs at an accumulated dose of 20 J/cm2. As determined by immunoblotting, UVA, violet, and blue light increased intracellular cAMP, ZO-1, ZO-2, CLDN1, and CLDN4 expression, respectively. UVA and violet, but not blue, light increased PKAreg-pS77 expression. However, none of the other treatments changed the expression of PKAcat-pT197, VASP-pS157, Bax, Bcl-2, or Bcl-xL. Immunofluorescence staining confirmed the formation of TJ structures. The expressions of ZO-1, ZO-2, CLDN1, and CLDN4 as well as TJ structures 2 days following UVA, violet, and blue exposure were similar to those of control cells after 9 days of cultivation. We conclude that short-wavelength LEDs at non-lethal exposure intensities accelerated the formation of TJ structure in HCEs via a cAMP-dependent regulatory cascade.

Comparing the sensitivity of fatigue and sleep disturbance assessment tools in women with advanced cancer undergoing chemotherapy.

Objective: This study aims to investigate the variations in fatigue and sleep disturbances among female patients with advanced lung cancer (ALC) and advanced breast cancer (ABC) during chemotherapy. Methods: A total of 36 female patients with ALC and 36 with ABC, all of whom had completed their first cycle of chemotherapy, were included. Fatigue was assessed using the General Fatigue Scale (GFS), and sleep disturbances were evaluated using the Pittsburgh Sleep Quality Index (PSQI) at designated time points throughout the chemotherapy process. Results: Linear regression analysis indicated that variables such as age, education level, employment status, cancer type, clinical stage, and symptom distress had no significant correlation with either fatigue or sleep disturbances. The GFS significantly discriminated fatigue among the ALC, ABC, and combined groups, while the PSQI demonstrated a significant distinction in sleep disturbance only within the ALC and combined groups. Conclusions: In summary, when considering the findings of both assessments in this study, the GFS score exhibited greater sensitivity in detecting fatigue than the PSQI score did for identifying sleep disturbances in advanced cancer patients undergoing chemotherapy.

Identification and Isolation of an Intermediate Metabolite with Dual Antioxidant and Anti-Proliferative Activity Present in the Fungus Antrodia cinnamomea Cultured on an Alternative Medium with Cinnamomum kanehirai Leaf Extract.

The fungus Antrodia cinnamomea has been used as a folk medicine for various diseases, especially cancer. When A. cinnamomea is cultured on the original host, an endangered woody plant Cinnamomum kanehirai Hayata, the fungus produces more active ingredients, but its growth is slow. Here, C. kanehirai leaf ethanol extract (KLEE) was used as a substitute for C. kanehirai wood to culture A. cinnamomea on solid medium to shorten the culture period and produce active metabolites en masse. The antioxidant activities of methanol extracts from A. cinnamomea cultured on KLEE (MEAC-KLEE) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, reducing power, and ferrous ion-chelating effect, and the effective concentration (EC50) values were 0.27, 0.74, and 0.37 mg mL-1, respectively. MEAC-KLEE exhibited specific anti-proliferative activity against a non-small-cell lung cancer cell line (A549) by Annexin V assay. A secondary metabolite (2,4-dimethoxy-6-methylbenzene-1,3-diol, DMMB) present in the extract (MEAC-KLEE) was purified by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) spectra. DMMB exhibited moderate antioxidant activity against DPPH radicals and reducing power, with EC50 values of 12.97 and 25.59 µg mL-1, respectively, and also induced apoptosis in A549 cells. Our results provide valuable insight into the development of DMMB for nutraceutical biotechnology.

Mitomycin C modulates intracellular matrix metalloproteinase-9 expression and affects corneal fibroblast migration.

Mitomycin C (MMC) is often used to prevent postoperative corneal haze and subconjunctival fibrosis in ocular surgery. It also affects the motility and viability of the residual ocular cells, including corneal stromal cells. Extracellular matrix metalloproteinase-9 (MMP-9) contributes to the promotion of cell movement in macrophage and cancer cells, but the intracellular role of MMP-9 remained unclear. Herein, we illustrated the novel role of intracellular MMP-9 in MMC-suppressed cell migration using isolated human corneal fibroblasts (HCFs). In HCFs, MMC enhanced intracellular MMP-9 at transcriptional and protein levels. Using co-immunoprecipitation analysis, we confirmed that MMC enhanced the association between intracellular MMP-9 and inactive FAK/paxillin (PXN) complexes, i.e. PXN without phospho-tyrosine 118 (pY118) and FAK without phospho-tyrosine 397 (pY397). To verify the role of intracellular MMP-9 in migration, its gene was directly isolated from HCFs and highly expressed in HCFs by a lentivirus-based pseudovirus system with encephalomyocarditis virus (EMCV)-driven enhanced green fluorescent protein (GFP) as the MMP-9-IG-versus IG-expressing cells. Compared with the IG-expressing cells, higher intracellular MMP-9 expression in the MMP-9-IG-expressing HCFs proliferated and migrated more slowly. Phosphorylation of FAK at Y397 and PXN at both Y31 and Y118 were significantly less in the MMP-9-IG-expressing HCFs. These suggested that MMC-upregulated intracellular MMP-9 clutched inactive FAK/PXN complexes at focal adhesion sites to form a new "inactive" trimer, prohibited FAK/PXN complexes phosphorylation and retarded corneal fibroblast migration.

Chemotherapy agents induce tartrate-resistant acid phosphatase 5a contributing to the symptom distress in lung cancer patients.

Tartrate-resistant acid phosphatase 5a (TRACP5a) is mainly secreted by activated macrophages in chronic inflammation. Serum TRACP5a is associated with symptom distress in lung cancer patients during chemotherapy. Therefore, this study aimed to investigate whether chemotherapy drugs modulate TRACP5a as an inducible marker for symptom distress in lung cancer patients during chemotherapy. In clinical analysis, lung cancer participants completely received the six-cycle chemotherapy process (n = 42). Clinical determinations for TRACP5a, C-reactive protein (CRP), interleukin-6 (IL-6), white blood cells, monocytes, and hemoglobin were analyzed at six time points: BL, C1d8, C2d1, C4d1, C4d8, and Ed28. Meanwhile, five questionnaires for fatigue, sleep disturbance, pain, depression, and confusion were finished before drug treatment. For monocyte-to-macrophage differentiation, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA). TRACP5a secretion in THP-1 cells was determined at the following days up to 6 days after 1-day incubation of chemotherapy drugs by dot blotting. Clinical analysis revealed that TRACP5a significantly increased at C1d8 and C4d8, but dropped at C2d1 and Ed28. CRP and IL-6 displayed a broad-range variation, resulting in no significant difference among the assessment time points. In contrast, monocytes decreased at C1d8 and C4d8, but rose again at C2d1 and Ed28. In symptom distress, the changes only in fatigue and sleep disturbance were positively associated with the trend in TRACP5a. In PMA-treated THP-1 cells, TRACP5a significantly increased after stimulation with gemcitabine and paclitaxel. Taken together, induction of TRACP5a by chemotherapy drugs might be generated from monocyte-differentiated macrophages, further causing clinical symptom distress in lung cancer patients.

Evaluation of Patent Blue as the Vital Dye in an Animal Model of Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To evaluate the safety and feasibility of patent blue (PB) as the vital dye in Descemet membrane endothelial keratoplasty (DMEK). METHODS: Bovine corneal endothelial cells were incubated with different concentrations (0.02%-2.5%) of PB. The cell viability, which was assessed by Cell Counting Kit-8 assay, was compared with that of untreated control and 0.06% to 0.4% trypan blue. The dyes were also used for graft preparation and implantation in the porcine eye model to evaluate stain quality, dye retention, and the feasibility of using PB in DMEK surgery. RESULTS: No obvious increase in cytotoxicity was detected for 0.06% to 0.4% trypan blue and PB at concentrations up to 1.0%, but the cell viability after incubating with 1.5% to 2.5% PB was significantly reduced. PB at 0.5% to 1.0% generated good staining quality that can be used to facilitate graft implantation. Although the staining quality of 0.5% to 1.0% PB faded to an intermediate level after a 30-minute wash in phosphate-buffered saline, dye retention persisted for up to 24 hours. CONCLUSIONS: PB at 0.5% to 1.0% is biocompatible and can stain the graft sufficiently, making it an alternative for DMEK surgery.

The Relationship Between Inflammatory Biomarkers and Symptom Distress in Lung Cancer Patients Undergoing Chemotherapy.

BACKGROUND: Symptom distress often occurs in lung cancer patients undergoing chemotherapy. However, a biomarker has not been identified to reflect the severity of their symptom distress. OBJECTIVE: The aim of this study was to investigate the relationship between symptom distress and serum inflammatory biomarkers in lung cancer patients undergoing chemotherapy. METHODS: A longitudinal, repeated-measures design was used to assess subjective symptoms (fatigue, sleep disturbance, pain, depression, and confusion), serum biomarkers (tartrate-resistant acid phosphatase 5a [TRACP5a], interleukin 6 [IL-6], IL-8, and C-reactive protein), and white blood cells in 62 lung cancer patients recruited from a single medical center at 3 time points: T1 was the baseline, T2 was the eighth day after the first chemotherapy cycle, and T3 was prior to the second cycle. Symptom distress was measured individually by 5 questionnaires (General Fatigue Scale, Pittsburgh Sleep Quality Index, Brief Pain Inventory, Profile of Mood States-Depressive, and Confusion). RESULTS: The trend of TRACP5a was positively correlated to the trend of the patients' symptom distress. However, the trends of IL-6 and IL-8 did not correlate. CONCLUSIONS: Serum TRACP5a was associated with symptom distress in lung cancer patients. Therefore, TRACP5a might be a potential biomarker to assess symptom distress of lung cancer patients undergoing chemotherapy. IMPLICATIONS FOR PRACTICE: Oncology nurses may be able to apply TRACP5a expression to predict or monitor multiple distress symptoms in lung cancer patients undergoing chemotherapy. Furthermore, nurses can use these study findings to better understand the patients who need more attention to improve their quality of life.

Molecular mechanism of fluoroquinolones modulation on corneal fibroblast motility.

Topical fluoroquinolones are widely used to prevent ocular infections after ophthalmic surgery. However, they have been shown to affect the corneal cell motility, whose mechanism remains indefinite. The purpose of this study was to investigate how fluoroquinolones affect corneal stromal cell motility. Human corneal fibroblasts (HCFs) were incubated in ciprofloxacin (CIP), levofloxacin (LEV), or moxifloxacin (MOX) at 0, 10, 50, and 100 µg/ml for up to 3 days. Effect of CIP, LEV, or MOX on HCF migration was monitored using migration assay. HCF viability was determined by WST-1 assay. Expression of focal adhesion kinase (FAK), paxillin (PXN), and their phosphorylated forms were analyzed by immunoblotting. Binding affinity between FAK and PXN was determined by co-immunoprecipitation. Our results revealed that CIP and MOX, but not LEV, noticeably retarded HCF migration. HCF proliferation was significantly reduced by CIP (38.2%), LEV (29.5%), and MOX (21.3%), respectively (p = 0.002). CIP and MOX suppressed the phosphorylation of PXN at tyrosines (10.2 ± 4.3%, p < 0.001; 11.7 ± 2.4%, p < 0.001, respectively), including tyrosine 118 (33.3 ± 5.2%, p < 0.001; 34.0 ± 4.4%, p < 0.001, respectively). CIP and MOX diminished the binding affinity between FAK and PXN (8.2 ± 1.8%, p < 0.001; 9.0 ± 4.5%, p < 0.001, respectively). Nevertheless, tyrosine dephosphorylation and FAK dissociation of PXN were not found in LEV-treated HCFs. None of these fluoroquinolones affect phosphorylation of FAK-Y397. We conclude that CIP and MOX, but not LEV, might delay corneal fibroblast migration via interfering with recruitment of PXN to focal adhesions and dephosphorylation of PXN at the tyrosines.

Dexamethasone modifies mitomycin C-triggered interleukin-8 secretion in isolated human Tenon's capsule fibroblasts.

Intraoperative mitomycin C (MMC) is widely used to prevent pterygium recurrence and glaucoma filtering bleb failure, but it has been shown to induce corneal inflammation and cell death. Postoperative dexamethasone (DEX) is advocated to reduce MMC-related inflammation and cell death in corneal fibroblasts. Nevertheless, its long-term regulation mechanism in Tenon's capsule remains to be explored. The purpose of this study was to investigate how DEX modifies MMC's effects in human Tenon's capsule fibroblasts (HTFs). HTFs isolated from the pterygium surgical patients (n = 6) were treated with MMC at 0, 0.1, 0.2, and 0.4 mg/ml for 5 min and incubated in DEX at 10 µM for 0, 1, 2, and 3 days. Recombinant interleukin-8 (IL-8) was used to verify the effect of MMC-related IL-8 secretion. Cell proliferation of all the treated cells was analyzed by WST-1 assay. The amount of IL-8 secretion in HTFs was determined by enzyme-linked immunosorbent assay. Immunoblotting assay was used to analyze the expression of peroxisome-proliferator-activated receptor gamma (PPARγ) and B-cell lymphoma-extra large (Bcl-xL). Our results revealed that MMC significantly reduced the HTF cell proliferation rate. Additionally, MMC significantly upregulated IL-8 secretion in HTFs concentration-dependently. At 3 days post treatment (dpt), 5-min exposures to 0.1, 0.2, and 0.4 mg/ml MMC resulted in 1.4-fold (p = 0.012), 1.6-fold (p = 0.012), and 2.5-fold (p = 0.001) increases of IL-8 secretion. In contrast, DEX reversed the MMC-retarded cell proliferation rate (p = 0.036) and repressed MMC-related IL-8 secretion by 33.5% at 3 dpt (p = 0.003). Addition of recombinant IL-8 noticeably suppressed HTF cell proliferation in a concentration-dependent manner. DEX stimulated upregulation of both PPARγ and Bcl-xL at 1 dpt in normal HTFs and at 2 dpt in MMC-treated HTFs. PPARγ silencing reduced expression of PPARγ and Bcl-xL, but enhanced IL-8 secretion (p < 0.001). On the other hand, Bcl-xL silencing enhanced IL-8 secretion (p < 0.001), but did not affect PPARγ expression. These revealed that IL-8 secretion in HTFs is modulated by PPARγ-dependent Bcl-xL signaling. We conclude that DEX reversed the MMC-inhibited HTF cell proliferation via diminishing the MMC-induced IL-8 secretion, which resulted from a late-phase upregulation of the PPARγ and Bcl-xL. These long-term effects suggest a beneficial postoperative DEX treatment following intraoperative MMC application.

White spot syndrome virus induces metabolic changes resembling the warburg effect in shrimp hemocytes in the early stage of infection.

The Warburg effect is an abnormal glycolysis response that is associated with cancer cells. Here we present evidence that metabolic changes resembling the Warburg effect are induced by a nonmammalian virus. When shrimp were infected with white spot syndrome virus (WSSV), changes were induced in several metabolic pathways related to the mitochondria. At the viral genome replication stage (12 h postinfection [hpi]), glucose consumption and plasma lactate concentration were both increased in WSSV-infected shrimp, and the key enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PDH), showed increased activity. We also found that at 12 hpi there was no alteration in the ADP/ATP ratio and that oxidative stress was lower than that in uninfected controls. All of these results are characteristic of the Warburg effect as it is present in mammals. There was also a significant decrease in triglyceride concentration starting at 12 hpi. At the late stage of the infection cycle (24 hpi), hemocytes of WSSV-infected shrimp showed several changes associated with cell death. These included the induction of mitochondrial membrane permeabilization (MMP), increased oxidative stress, decreased glucose consumption, and disrupted energy production. A previous study showed that WSSV infection led to upregulation of the voltage-dependent anion channel (VDAC), which is known to be involved in both the Warburg effect and MMP. Here we show that double-stranded RNA (dsRNA) silencing of the VDAC reduces WSSV-induced mortality and virion copy number. For these results, we hypothesize a model depicting the metabolic changes in host cells at the early and late stages of WSSV infection.

Effect of mitomycin C on IL-1R expression, IL-1-related hepatocyte growth factor secretion and corneal epithelial cell migration.

PURPOSE: To investigate how mitomycin C (MMC) modulates hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) secretions in human corneal fibroblasts and regulates human corneal epithelial (HCE) cell migration. METHODS: Primary human corneal fibroblasts were treated with MMC (0.05, 0.1, or 0.2 mg/mL for 5 minutes) and were cultivated with or without interleukin (IL)-1beta. Transcript and secretion of HGF and KGF were determined by quantitative real-time RT-PCR and Western blot analysis, respectively. The effect of MMC-treated fibroblasts on HCE cell migration was evaluated using a transwell migration assay. The influence of MMC on HGF expression/secretion and HCE cell migration was further confirmed by RNA interference. The number of IL-1 receptors (IL-1R) on the fibroblast surface was analyzed by flow cytometry. RESULTS: MMC alone did not affect endogenous HGF expression, whereas IL-1beta alone significantly upregulated HGF transcripts and secretion. By modifying IL-1R numbers, MMC further upregulated IL-1beta-related HGF expression at a concentration of 0.05 mg/mL but to a lesser extent at 0.1 and 0.2 mg/mL. KGF transcripts and intracellular expression were suppressed by MMC dose dependently in the presence or absence of IL-1beta, whereas KGF secretion was not affected. Conditioned medium from MMC-treated fibroblasts exerted a similar concentration-dependent effect on HCE cell migration, enhancing migration most significantly at 0.05 mg/mL MMC in the presence of IL-1beta. The MMC dose-dependent modulation of HCE cell migration was abolished in HGF-silenced fibroblasts. CONCLUSIONS: MMC differentially modulated IL-1R expression at various concentrations and regulated HGF and KGF differently. MMC alone did not alter HGF expression. In the presence of IL-1beta, MMC-treated corneal fibroblasts modified HCE cell migration through IL-1beta-induced HGF secretion.

Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling.

The molecular mechanism behind what causes an infection of Enterovirus 71 (EV71) in young children to result in severe neurological diseases is unclear. Herein, we show that Cdk5, a critical signalling effector of various neurotoxic insults in the brain, is activated by EV71 infection of neuronal cells. EV71-induced neuronal apoptosis could be effectively repressed by blocking either Cdk5 kinase activity or its protein expression. Moreover, EV71-induced Cdk5 activation was modulated by c-Abl. The suppression of c-Abl kinase activity by STI571 notably repressed both the Cdk5 activation and neuronal apoptosis in cells infected with EV71. Although EV71 also induces apoptosis in non-neuronal cells, it did not affect Abl and Cdk5 activities in several non-neuronal cell lines. Intriguingly, coxsackievirus A16 (CA16), a genetically closely related serotype to EV71 that usually does not induce severe neurological disorders, could only weakly stimulate Abl, but not Cdk5 kinase activity. Taken together, our data suggest a serotype- and cell type-specific mechanism, by which EV71 induces Abl kinase activity, which in turn triggers Cdk5-signalling for neuronal apoptosis.