Inhibitory effect of recombinant tyrosine­sulfated madanin­1, a thrombin inhibitor, on the behavior of MDA­MB­231 and SKOV3 cells in vitro.

Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. In the present study, the effects of the thrombin­targeting recombinant tyrosine­sulfated madanin­1 on cancer cell behavior and signaling pathways compared with madanin­1 wild­type (WT) were investigated. Recombinant madanin­1 2 sulfation (madanin­1 2S) and madanin­1 WT proteins were generated using Escherichia coli. SKOV3 and MDA­MB­231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by wound healing assay and Transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDA­MB­231 cells, which were significantly suppressed by madanin­1 2S (P<0.05). Madanin­1 2S also significantly suppressed thrombin­induced expression of phosphorylated (p)­Akt and p­extracellular signal­regulated kinase in both cell lines (P<0.05), whereas madanin­1 WT had no effect on the expression levels of these proteins in MDA­MB­231 cells. Furthermore, madanin­1 2S significantly reversed the effects of thrombin on E­cadherin, N­cadherin and vimentin expression in MDA­MB­231 cells (P<0.05), whereas madanin­1 WT did not show any effect. In conclusion, madanin­1 2S suppressed the migration and invasion of cancer cells more effectively than madanin­1 WT. It is hypothesized that inhibiting thrombin via the sulfated form of madanin­1 may be a potential candidate for enhanced cancer therapy; however, further in vivo validation is required.

Sulfated CXCR3 Peptide Trap Use as a Promising Therapeutic Approach for Age-Related Macular Degeneration.

BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.

Recombinant sulfated CCR2 peptide trap reduces retinal degeneration in mice.

Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C-C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.

HIF1α-mediated TRAIL Expression Regulates Lacrimal Gland Inflammation in Dry Eye Disease.

PURPOSE: The purpose of this study was to investigate the expression of death ligands in the lacrimal glands (LGs), identify upstream factors that regulate their expression, and determine the functional roles of these factors in the pathogenesis of dry eye disease (DED). METHODS: For DED experiment, ex vivo coculture system with LG and in vivo murine model using a controlled environment chamber were utilized. C57BL/6 mice and hypoxia-inducible factor (HIF)-1α conditional knockout (CKO) mice were used. Immunohistochemical staining, polymerase chain reaction, and immunoblotting were performed to determine levels of death ligands including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DED-induced LGs. Additionally, acinar cell and CD45+ cell apoptosis was determined with neutralizing TRAIL treatment. RESULTS: Desiccating stress significantly increased HIF-1α expression in LG-acinar cells. Furthermore, HIF-1α deficiency significantly enhanced the infiltration of CD45+ inflammatory cells in LG and induced LG-acinar cell death. Meanwhile, only TRAIL expression was increased in DED-LG, but abrogated in HIF-1α CKO. Interestingly, the main source of TRAIL was the CD45- LG-acinar cells, but not CD45+ immune cells after DED induction. Using ex vivo coculture system, we confirmed LG-induced apoptosis of immune cells via HIF-1α-mediated TRAIL secretion following DED. Consistent with ex vivo, the insufficiency of HIF-1α and TRAIL enhanced recruitment of inflammatory cells to the LG and subsequently exacerbated ocular surface damage in DED mice. CONCLUSIONS: Our findings offer novel insight into the regulatory function of acinar cell-derived TRAIL in limiting inflammatory damage and could be implicated in the development of potential therapeutic strategies for DED.

MicroRNA-27 inhibits adipogenic differentiation in orbital fibroblasts from patients with Graves' orbitopathy.

BACKGROUND: To investigate the role of microRNA (miR)-27a and miR-27b in adipogenesis in an in vitro model of Graves' orbitopathy (GO). METHODS: Orbital fat tissues were harvested from GO and non-GO participants for primary orbital fibroblast cultures. The expression levels of miR-27a and miR-27b between GO and non-GO orbital fat tissues were compared. During adipogenesis of GO orbital fibroblasts, the expression levels of miR-27a and miR-27b were determined, and the effects of mimics of miR-27a and miR-27b transfection on adipogenesis of GO orbital fibroblast were investigated. RESULTS: Real time-polymerase chain reaction showed significantly more decreases in miR-27a and miR-27b levels in orbital fat tissues in GO participants than in non-GO participants (p < 0.05). The expression of both miR-27a and miR-27b was highest in orbital fibroblasts at day 0 and declined gradually after the induction of adipogenic differentiation. The expression levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and C/EBPß were decreased and Oil Red O-stained lipid droplets were lower in GO orbital fibroblasts transfected with miR-27a and miR-27b mimics than in negative controls. CONCLUSIONS: Our results indicated that miR-27a and miR-27b inhibited adipogenesis in orbital fibroblasts from GO patients. Further studies are required to examine the potential of miR-27a and miR-27b as targets for therapeutic strategies.

Transforming Growth Factor-ß1-induced Human Subconjunctival Fibrosis is Mediated by MicroRNA 143/145 Expression.

Purpose: To investigate the roles and pathways of microRNAs 143 and 145 in transforming growth factor (TGF)-ß1-induced human subconjunctival fibrosis. Methods: Human tenon's capsule fibroblasts (HTFs) were obtained from a healthy eye. After treating cultured HTFs with TGF-ß1, the expression of microRNAs 143 and 145 was evaluated using polymerase chain reaction. To identify the pathways of TGF-ß1-induced microRNA 143/145 expression, HTFs were treated with specific inhibitors of p38MAPK, PI3K/Akt, JNK, ERK, and with siRNAs for SMAD2 and SMAD4. Mutagenesis studies were performed to evaluate the role of the CArG box and SMAD-binding element (SBE). To investigate the role of microRNA 143/145 in TGF-ß1-induced myofibroblast transdifferentiation, microRNA 143/145 mimics and microRNA 143/145 inhibitors were applied to the HTFs. Results: Array analysis revealed that TGF-ß1 induced the expression of microRNA 143/145 in a dose- and time-dependent manner. When inhibitors and siRNAs for p38MAPK, PI3K/Akt, ERK, and JNK were applied, the TGF-ß1-induced expression of microRNA 143/145 was inhibited; however, SMAD2 and SMAD4 inhibition did not affect the TGF-ß1-induced expression of these microRNAs. In the mutagenesis studies, both the CArG box and SBE were associated with TGF-ß1-induced expression of microRNA 143/145. Mimics of microRNA 143/145 induced increased myofibroblast formation, whereas their inhibitors had the opposite effect. Conclusions: TGF-ß1-induced human subconjunctival fibrosis was mediated by the expression of microRNA 143/145, mainly via SMAD-independent pathways. Inhibition of TGF-ß1-induced microRNA 143/145 expression in HTFs might represent a novel strategy to prevent subconjunctival fibrosis.

Salmonella­induced miR­155 enhances necroptotic death in macrophage cells via targeting RIP1/3.

Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella), a virulent pathogen, is known to induce host­cell death. Using reverse transcription­quantitative polymerase chain reaction, a 28­fold increase of microRNA (miR)­155 expression in RAW 264.7 macrophages was observed following infection with Salmonella for 24 h. This miR­155 upregulation increased macrophage cell death by up to 40% in 48 h following infection. Western blot analysis revealed that receptor interacting protein 1 (RIP1) and 3 (RIP3) were increased at 18 h following miR­155 transfection to macrophages, similar to Salmonella infection. In addition, inhibition of RIP1 by pre­incubating macrophages with necrostatin­1, a RIP1 specific inhibitor, increased the viability of Salmonella­infected cells and miR­155­transfected cells by up to 20%. The cleavage of poly (adenosine diphosphate­ribose) polymerase­1 (PARP­1) was also enhanced by miR­155 induction upon Salmonella infection. Therefore, it was suggested that RIP1/3­induced necroptosis and PARP­1­mediated necrosis caused by miR­155 induction may represent distinct routes of programmed necrotic cell death of Salmonella­infected macrophages.

Additional role of liver stiffness measurement in stratifying residual hepatocellular carcinoma risk predicted by serum biomarkers in chronic hepatitis B patients under antiviral therapy.

BACKGROUND: The risk of hepatocellular carcinoma (HCC) remains among patients who are treated with antiviral therapy (AVT). The degree of liver fibrosis has been suggested as an important biomarker to stratify the risk of developing HCC. We tested whether liver stiffness (LS) measured using transient elastography is useful over two noninvasive serum biomarkers of fibrosis [the aspartate aminotransferase to platelet ratio index (APRI) and fibrosis-4 (FIB-4)]. PATIENTS AND METHODS: A retrospective cohort of 1014 CHB patients who were under AVT with nucleos(t)ide analogs for at least a year was analyzed. The risk of HCC development according to serum biomarkers (APRI and FIB-4) and LS was compared. RESULTS: The HCC risk was higher for those with a higher degree of liver fibrosis, as estimated by the LS, APRI, and FIB-4. When the two serum biomarkers were used to group the patients, the 3-year HCC incidence rates were 7.3, 3.0, and 1.3% for both high APRI (≥0.5) and FIB-4 (≥1.45) scores, either a high APRI or FIB-4 score, and both low APRI and FIB-4 scores, respectively (P<0.001). Among the 758 patients with discordant or both low APRI and FIB-4 scores, the LS value was high (>6) for a significant proportion of the patients (39.9%). The HCC risk was significantly different according to the LS value (3-year HCC incidence rate of 1.1, 2.0, and 6.8% for LS <6, 6-9, and >9, respectively, P<0.001). CONCLUSION: Among CHB patients under AVT, LS could stratify risk for HCC, including patients with discordant or both low APRI and FIB-4 score. This finding indicates that LS measurement plays an additional role over the serum biomarkers in stratifying the residual risk of HCC.

Unsaturated fatty acids protect trophoblast cells from saturated fatty acid-induced autophagy defects.

Dysregulated serum fatty acids are associated with a lipotoxic placental environment, which contributes to increased pregnancy complications via altered trophoblast invasion. However, the role of saturated and unsaturated fatty acids in trophoblastic autophagy has yet to be explored. Here, we demonstrated that prolonged exposure of saturated fatty acids interferes with the invasiveness of human extravillous trophoblasts. Saturated fatty acids (but not unsaturated fatty acids) inhibited the fusion of autophagosomes and lysosomes, resulting in the formation of intracellular protein aggregates. Furthermore, when the trophoblast cells were exposed to saturated fatty acids, unsaturated fatty acids counteracted the effects of saturated fatty acids by increasing degradation of autophagic vacuoles. Saturated fatty acids reduced the levels of the matrix metalloproteinases (MMP)-2 and MMP-9, while unsaturated fatty acids maintained their levels. In conclusion, saturated fatty acids induced decreased trophoblast invasion, of which autophagy dysfunction plays a major role.

Role of microRNA-146a in regulation of fibrosis in orbital fibroblasts from patients with Graves' orbitopathy.

AIM: To examine the role of microRNA-146a (miR-146a) in the regulation of fibrosis in an in vitro model of Graves' orbitopathy (GO). METHODS: Orbital fat/connective tissues were harvested from patients with GO and non-GO for primary orbital fibroblast cultures. The effects of transforming growth factor-ß (TGF-ß), a potent cytokine that promotes fibrosis, on miR-146a expression were analysed in GO and non-GO orbital fibroblasts using quantitative real-time PCR. The effects of overexpressed miR-146a on TGF-ß-induced fibrotic markers were examined in GO orbital fibroblasts by western blot analysis. Expression ofSma and Mad related family (Smad) 4/tumour necrosis factor receptor-associated factor 6 (TRAF6) after transfection of miR-146a mimics or inhibitors were examined. RESULTS: TGF-ß induced an increase in miR-146a expression in orbital fibroblasts from patients with GO in a time-dependent and concentration-dependent manner. miR-146a mimics further decreased the production of TGF-ß-induced fibronectin, collagen Iα and α-smooth muscle actin protein. The Smad4 and TRAF6 protein levels were significantly decreased by miR-146a mimics, compared with control mimics, and significantly increased on inhibition of miR-146a production compared with a control. CONCLUSIONS: miR-146a plays a role as a negative regulator in the production of TGF-ß-induced fibrotic markers. Thus, miR-146a may be involved in the regulation of fibrosis in orbital fibroblasts from patients with GO.

Non-invasive tests for liver disease severity and the hepatocellular carcinoma risk in chronic hepatitis B patients with low-level viremia.

BACKGROUND & AIMS: We tested whether non-invasive tests for liver disease severity can stratify hepatocellular carcinoma (HCC) risk in chronic hepatitis B virus (HBV)-infected patients showing low-level viremia (LLV, HBV DNA <2000 IU/mL). METHODS: A retrospective cohort of 1006 chronic hepatitis B patients showing persistently LLV, defined by at least two consecutive assessments in the year before enrolment, was assessed for HCC development. Two non-invasive serum biomarkers, the aspartate aminotransferase to platelet ratio index (APRI) and the Fibrosis-4 (FIB-4), were tested. Cirrhosis was defined with ultrasonography. RESULTS: During a median 5.1 years of follow-up, HCC developed in 36 patients. HCC incidence rate at 5 years was significantly higher for cirrhotic patients (19/139, 13.7%), but was not null for non-cirrhotic patients (17/867, 2.0%, P<.001). APRI at a cut-off of 0.5 was more specific but less sensitive for HCC development, and FIB-4 at a cut-off of 1.45 was more sensitive but less specific. When both APRI and FIB-4 were used to group patients, the 5-year cumulative HCC incidence rate was 13.9%, 1.4% and 1.2% for both high, any high, and both low APRI and FIB-4 score among all patients (n=1006, P<.001), respectively, and was 11.4%, 1.5% and 0.4% in the same respective order among non-cirrhotic patients (n=867, P<.001). CONCLUSIONS: The combined use of two non-invasive serum biomarkers (APRI and FIB-4) could stratify HCC risk for chronic HBV-infected patients with LLV.

Sphingosine-1-Phosphate Mediates Fibrosis in Orbital Fibroblasts in Graves' Orbitopathy.

Purpose: To investigate the effect of sphingosine-1-phosphate (S1P) on fibrosis in orbital fibroblasts in Graves' orbitopathy (GO). Methods: Orbital fibroblasts were cultured from orbital adipose/connective tissues of patients with GO and healthy control subjects. Effects of treatment with TGF-ß and cigarette smoke extract (CSE) on S1P receptor (S1PR) messenger RNA (mRNA) and S1P expression were evaluated by real-time polymerase chain reaction and Western blotting. To evaluate the role of S1P in fibrosis, cells were pretreated with W146 (S1PR1 antagonist); JTE013 (S1PR2 antagonist); FTY720 (S1PR1 modulator); or 5C (sphingosine kinase-1 blocker) for 1 hour before stimulation with TGF-ß, CSE, or IL-1ß. Expression of fibrosis-related proteins (collagen Iα, fibronectin, and α-smooth muscle actin [SMA]) and tissue remodeling-related proteins (matrix metalloproteinases [MMPs] and tissue inhibitor of metalloproteinase [TIMP]-1) was then evaluated by Western blotting. Results: Expression levels of S1PR mRNA and S1P in GO orbital fibroblasts increased upon TGF-ß and CSE treatment. Treatment with S1PR blockers and 5C inhibited TGF-ß and CSE-induced expression of collagen Iα, fibronectin, and α-SMA, as well as IL-1ß-induced expression of MMP-1, MMP-2, MMP-9, and TIMP-1. Exogenous S1P treatment without profibrotic stimulants upregulated collagen Iα, fibronectin, α-SMA, MMP-1, MMP-2, MMP-9, and TIMP-1 expression in a dose-dependent manner. Conclusions: Blocking of S1PR activity and inhibition of S1P synthesis led to decreased expression of fibrosis and tissue remodeling-related proteins in primary cultures of orbital fibroblasts derived from patients with GO. Thus, modulation of S1P activity might have therapeutic potential in the suppression of fibrosis in GO.

Sphingosine-1-phosphate is involved in inflammatory reactions in patients with Graves' orbitopathy.

OBJECTIVE: Graves' orbitopathy (GO) is initiated by excessive amount of various inflammatory mediators produced by orbital fibroblasts. This study aimed to assess the crucial role of sphingosine-1-phosphate (S1P) in the inflammatory process of GO. METHODS: Orbital adipose/connective tissue samples were obtained from 10 GO patients and 10 normal control individuals during surgery. Primary orbital fibroblast culture was done. After the expression of S1P receptors and sphingosine kinase (SphK) was assessed with the treatment of interleukin (IL)-1ß, we evaluated the expression of pro-inflammatory factors [intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2) and IL-6] after treating S1P. S1P receptor antagonists and SphK 1 inhibitor were pretreated and the expression of the pro-inflammatory factors was assessed. RESULTS: IL-1ß exacerbated the inflammatory process by enhancing the expression of S1P receptors and SphK in GO orbital fibroblasts. IL-1ß also induced the expressions of ICAM-1, COX-2, and IL-6 in GO orbital fibroblasts, and these expressions were effectively inhibited by S1P receptor antagonists and SphK1 inhibitor. CONCLUSION: S1P has an important role in the pathological inflammatory process of GO, which is mediated through the SphK1-S1P- S1P receptor pathway. SphK1 inhibitors and S1P receptors or antagonists could be potential approaches for controlling the inflammatory process of GO.

Expression of microRNAs in fibroblast of pterygium.

AIM: To screen microRNAs (miRNAs) and set up target miRNAs in pterygium. METHODS: Primary fibroblasts were isolated from pterygium and Tenon's capsule and cultured. Immunocytochemical analysis and Western blotting were performed to confirm the culture of fibroblasts. In all, 1733 miRNAs were screened in the first step by using GeneChip(®) miRNA3.0 Array. Specific miRNAs involved in the pathogenesis of pterygium were subsequently determined using the following criteria: 1) high reproducibility in a repetitive test; 2) base log value of >7.0 for both control and pterygial fibroblasts; and 3) log ratio of >1.0 between pterygial fibroblasts and control fibroblasts. RESULTS: Primary screening showed that 887/1733 miRNAs were up-regulated and 846/1733 miRNAs were down-regulated in pterygial fibroblasts compared with those in control fibroblasts. Of the 1733 miRNAs screened, 4 miRNAs, namely, miRNA-143a-3p, miRNA-181a-2-3p, miRNA-377-5p and miRNA-411a-5p, met the above-mentioned criteria. Primary screening showed that these 4 miRNAs were up-regulated in pterygial fibroblasts compared with control fibroblasts and that miRNA-143a-3p had the highest mean ratio compared with the miRNAs in control fibroblasts. CONCLUSION: miRNA-143a-3p, miRNA-181a-2-3p, miRNA-377-5p and miRNA-411a-5p are up-regulated in pterygial fibroblasts compared with control fibroblasts, suggesting their involvement in the pathogenesis of pterygium.

Role of miR-146a in the Regulation of Inflammation in an In Vitro Model of Graves' Orbitopathy.

PURPOSE: To investigate the role of microRNA 146a (miR-146a) in the regulation of inflammation in an in vitro model of Graves' orbitopathy (GO). METHODS: The level of miR-146a expression in orbital adipose tissue was compared between GO and non-GO by quantitative real-time PCR (qPCR). The effects of interleukin 1ß (IL-1ß) on miR-146a expression were analyzed in orbital fibroblasts by qPCR. To investigate the molecular mechanism underlying IL-1ß-induced miR-146a expression, the effects of inhibitors of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase/extracellular signal-regulated kinases (MEK)-1/2, c-Jun N-terminal kinases (JNK)-1/2, p38 MAP kinase, and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) were analyzed. The effects of miR-146a mimics and inhibitors on IL-1ß-induced IL-6 release were examined by ELISA and Western blotting. RESULTS: The level of miR-146a expression was significantly higher in GO orbital adipose tissue than in non-GO (P = 0.032). Interleukin 1ß induced a time- and concentration-dependent increase in miR-146a expression. Interleukin 1ß (10 ng/mL, 16 hours) induced an approximately 17.5-fold increase in miR-146 expression. The increase in miR-146a expression by IL-1ß was significantly inhibited by NF-κB, JNK-1/2, and PI3K inhibitors (1.94  ±  0.25, 5.28  ±  0.34 and 9.73  ±  2.32-fold, respectively, P < 0.05 compared with IL-1ß-induced miR-146 expression, independent t-test). Interleukin 1ß-induced IL-6 protein production was further decreased by miR-146a mimics, but not by inhibitors of miR-146a. CONCLUSIONS: MicroRNA 146a was upregulated by inflammatory stress in orbital fibroblasts. Our results indicated that miR-146a had a positive effect on the anti-inflammatory process. MicroRNA 146a may play a role in the regulation of inflammation in orbital fibroblasts, and may participate in the pathogenesis of GO.

Effect of Floor Space Allowance on Pig Productivity across Stages of Growth: A Field-scale Analysis.

A total of 152 pig farms were randomly selected from the five provinces in South Korea. During the experiment, the average temperature and relative humidity was 24.7°C and 74% in summer and 2.4°C and 53% in winter, respectively. The correlation between floor space allowance (FSA) and productivity index was analyzed, including non-productive sow days (NPD), number of weaners (NOW), survival rate (SR), appearance rate of A-grade pork (ARA), and days at a slaughter weight of 110 kg (d-SW) at different growth stages. The objectives of the present study were i) to determine the effect of FSA on the pig productivity index and ii) to suggest the minimum FSA for pigs based on scientific baseline data. For the pregnant sow, NPD could be decreased if pregnant sows were raised with a medium level (M) of FSA (3.10 to 3.67 m(2)/head) while also keeping the pig house clean which improves hygiene, and operating the ventilation system properly. For the farrowing sows, the NOW tended to decrease as the FSA increased. Similarly, a high level of FSA (H) is significantly negative with weaner SR of farrowing sows (p-value = 0.017), indicating this FSA tends to depress SR. Therefore, a FSA of 2.30 to 6.40 m(2)/head (very low) could be appropriate for weaners because a limited space can provide a sense of security and protection from external interruptions. The opposite trend was observed that an increase in floor space (>1.12 m(2)/head) leads to increase the SR of growing pigs. For the fattening pigs, H level of FSA was negatively correlated with SR, but M level of FSA was positively correlated with SR, indicating that SR tended to increase with the FSA of 1.10 to 1.27 m(2)/head. In contrast, ARA of male fattening pigs showed opposite results. H level of FSA (1.27 to 1.47 m(2)/head) was suggested to increase productivity because ARA was most affected by H level of space allowance with positive correlation (R(2) = 0.523). The relationship between the FSA and d-SW of fattening pigs was hard to identify because of the low R(2) value. However, the farms that provided a relatively large floor space (1.27 to 1.54 m(2)/head) during the winter period showed d-SW was significantly and negatively affected by FSA.

The Role of Sphingosine-1-Phosphate in Adipogenesis of Graves' Orbitopathy.

PURPOSE: To investigate the action of sphingosine-1-phosphate (S1P) in adipocyte differentiation of orbital fibroblasts and determine its putative role in the pathogenesis of Graves' orbitopathy (GO). METHODS: Primary preadipocyte orbital fibroblast cultures were stimulated for adipogenesis. Real-time PCR was performed to evaluate the expression of S1P receptor mRNA. To evaluate the effect of S1P and S1P receptor blockers (W146 and FTY720) on adipocyte differentiation, cultures were exposed to each receptor blocker for the first 4 days of the differentiation period. Differentiated cells were stained with Oil Red O, and the production of peroxisome proliferator activator gamma (PPARγ) and CCAAT-enhancer-binding proteins (C/EBP) α and ß were determined by Western blot analysis. RESULTS: Sphingosine-1-phosphate receptor 1, 2, and 3 mRNA expression levels were significantly higher in GO tissue samples than non-GO. Sphingosine-1-phosphate receptor 1 through 5 mRNA expression was significantly increased during the 10 days of adipogenesis. Sphingosine-1-phosphate treatment increased the size and number of adipocytes, and increased the expression of adipogenic transcriptional regulators. Treatment with S1P1 receptor inhibitor (W146) for 4 days after induction of adipogenesis attenuated adipocyte differentiation. Sphingosine-1-phosphate receptor blocker also decreased reactive oxygen species (ROS) production in GO orbital fibroblasts and H2O2-stimulated HO-1 production in GO orbital fibroblasts. S1P1 receptor inhibitor reduced the number of adipocytes and suppressed the accumulation of lipid droplets induced by 10 µM H2O2 or 2% cigarette smoke extract (CSE) treatment. CONCLUSIONS: Sphingosine-1-phosphate could play a role in orbital adipocyte differentiation of GO. Modulation of S1P actions may provide a therapeutic target for the treatment of GO.

Homeobox A9 directly targeted by miR-196b regulates aggressiveness through nuclear Factor-kappa B activity in non-small cell lung cancer cells.

MicroRNAs (miRNAs) are recognized as crucial posttranscriptional regulators of gene expression, and play critical roles as oncogenes or tumor suppressors in various cancers. Here, we show that miR-196b is upregulated in mesenchymal-like-state non-small cell lung cancer (NSCLC) cells and lung cancer tissues. Moreover, miR-196b upregulation stimulates cell invasion and a change in cell morphology to a spindle shape via loss of cell-to-cell contacts. We identified homeobox A9 (HOXA9) as a target gene of miR-196b by using public databases such as TargetScan, miRDB, and microRNA.org. HOXA9 expression is inversely correlated with miR-196b levels in clinical NSCLC samples as compared to that in corresponding control samples, and with the migration and invasion of NSCLC cells. Ectopic expression of HOXA9 resulted in a suppression of miR-196b-induced cell invasion, and HOXA9 reexpression increased E-cadherin expression. Furthermore, HOXA9 potently attenuated the expression of snail family zinc finger 2 (SNAI2/SLUG) and matrix metallopeptidase 9 (MMP9) by controlling the binding of nuclear factor-kappa B to the promoter of SLUG and MMP9 genes, respectively. Therefore, we suggest that HOXA9 plays a central role in controlling the aggressive behavior of lung cancer cells and that miR-196b can serve as a potential target for developing anticancer agents. © 2015 Wiley Periodicals, Inc.

Incidental microscopic bile duct tumor thrombi in hepatocellular carcinoma after curative hepatectomy: a matched study.

In patients with hepatocellular carcinoma (HCC), the presence of bile duct tumor thrombi (BDTT) in the major bile ducts indicates poor prognosis compared with that of HCC patients without BDTT. However, the prognostic significance of incidental microscopic BDTT in the peripheral bile ducts after curative liver resection is not known. We compared the outcomes of HCC patients with and without microscopic BDTT in the peripheral bile ducts who underwent hepatectomy.The electronic medical records of 31 patients with microscopic BDTT (BDTT group) were retrospectively reviewed. To compare the surgical outcomes, 62 patients (No BDTT group) were randomly chosen from the remaining HCC patients without BDTT based on age, sex, etiology of HCC, tumor size, tumor number, and modified Union for International Cancer Control T staging.The 1-year, 2-year, and 3-year disease-free survival rates and overall survival rates were 54.8%, 34.0%, 34.0% and 90.1%, 69.2%, 61.0% in the BDTT group and 66.8%, 59.2%, 42.3% and 86.4%, 84.4%, 84.4% in the No BDTT group (P = 0.089 and P = 0.014, respectively). The overall survival curve in the No BDTT group was higher than that in the BDTT group. Multivariate analysis revealed that predisposing factors for tumor recurrence after curative liver resection included increased levels of the protein induced by vitamin K antagonist-II (PIVKA-II), tumor grades 3 and 4, and the presence of BDTT.This study demonstrates that HCC prognosis is worse in patients with incidental microscopic BDTT in the peripheral bile ducts than it is in those without BDTT. The presence of BDTT should therefore be considered when evaluating a patient's HCC prognosis after curative hepatectomy.

Expression of Lymphangiogenic Markers in Rejected Human Corneal Buttons after Penetrating Keratoplasty.

PURPOSE: To investigate the extent and distribution of lymphangiogenesis in the rejected corneal graft, we determined the expression of several lymphangiogenic markers in rejected human corneal buttons. MATERIAL AND METHODS: Thirty-four corneal buttons were obtained from patients who underwent re-keratoplasty for graft rejection after penetrating keratoplasty. All corneas showed signs of rejection, such as, sudden mutton-fat keratic precipitates (KPs) or lines before re-keratoplasty. The corneas were halved, and one half was used for immunostaining and the other half was used for RT-PCR. Expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGFR-2, VEGFR-3, LYVE-1 and podoplanin were measured as lymphangiogenic markers. Four non-operated normal corneas were used as controls. RESULTS: Numerous podoplanin positive cells were found in the anterior and posterior stroma. However, LYVE-1 positive mature lymphatics were found only in herpetic keratitis (HK)-induced graft rejection, and not in pseudophakic bullous keratopathy (PBK). RT-PCR showed that levels of VEGF-A, VEGF-C, VEGFR-2, and VEGFR-3 mRNAs were elevated in rejected corneal buttons versus the non-operated control corneas. Based upon the pre-keratoplasty pathologic conditions, HK cases showed higher levels of VEGF-A and VEGFR-2 than PBK. The mRNA ratios (keratoplastic cornea/normal cornea) for VEGF-A and VEGFR-2 were 8.9 and 5.8, respectively. CONCLUSIONS: The results suggested that the VEGF-A and the VEGFR-2 may be a more important pathway for lymphangiogenesis in rejected corneal grafts than the VEGFR-3. In addition, organized lymphangiogenesis is more prominent in HK than PBK.