A Novel Fundus Image Reading Tool for Efficient Generation of a Multi-dimensional Categorical Image Database for Machine Learning Algorithm Training.

BACKGROUND: We described a novel multi-step retinal fundus image reading system for providing high-quality large data for machine learning algorithms, and assessed the grader variability in the large-scale dataset generated with this system. METHODS: A 5-step retinal fundus image reading tool was developed that rates image quality, presence of abnormality, findings with location information, diagnoses, and clinical significance. Each image was evaluated by 3 different graders. Agreements among graders for each decision were evaluated. RESULTS: The 234,242 readings of 79,458 images were collected from 55 licensed ophthalmologists during 6 months. The 34,364 images were graded as abnormal by at-least one rater. Of these, all three raters agreed in 46.6% in abnormality, while 69.9% of the images were rated as abnormal by two or more raters. Agreement rate of at-least two raters on a certain finding was 26.7%-65.2%, and complete agreement rate of all-three raters was 5.7%-43.3%. As for diagnoses, agreement of at-least two raters was 35.6%-65.6%, and complete agreement rate was 11.0%-40.0%. Agreement of findings and diagnoses were higher when restricted to images with prior complete agreement on abnormality. Retinal/glaucoma specialists showed higher agreements on findings and diagnoses of their corresponding subspecialties. CONCLUSION: This novel reading tool for retinal fundus images generated a large-scale dataset with high level of information, which can be utilized in future development of machine learning-based algorithms for automated identification of abnormal conditions and clinical decision supporting system. These results emphasize the importance of addressing grader variability in algorithm developments.

Effect of a Caregiver's Education Program on Stroke Rehabilitation.

OBJECTIVE: To evaluate effects of caregiver's education program on their satisfaction, as well as patient functional recovery, performed in addition to daily conventional rehabilitation treatment. METHODS: Three hundred eleven subjects diagnosed with first-onset stroke and transferred to the Department of Physical Medicine and Rehabilitation of Inha University Hospital were surveyed. In 2015, caregivers attended an education program for acute and subacute stroke patients. Patients who received an additional rehabilitation therapy were assigned to the experimental group (n=81), whereas the control group (n=100) consisted of transfer cases in 2014 with only conventional treatment. The experimental group was classified by severity using the Korean version of the National Institutes of Health Stroke Scale (K-NIHSS), which was administered to all 181 subjects, in addition to, the Korean version of the Mini Mental Status Examination (K-MMSE), a Modified Barthel Index (K-MBI), and the Berg Balance Scale (K-BBS). Caregiver satisfaction and burden before and after education programs were assessed using the Canadian Occupational Performance Measure (COPM), as well as family burden and caregiver burnout scales. RESULTS: No significant intergroup difference was observed between initial K-NIHSS, K-MMSE, K-BBS, K-MBI scores, and times from admission to transfer. Those with moderate or severe strokes under the experimental condition showed a more significant improvement than the control group as determined by the K-NIHSS and K-BBS, as well as tendential K-MMSE and K-MBI score increases. Satisfaction was significantly greater for family members and formal caregivers of patients with strokes of moderate severity in the experimental group. CONCLUSION: The caregiver's education program for stroke subjects had a positive outcome on patients' functional improvement and caregiver satisfaction. The authors believe that the additional rehabilitation therapy with the education program aids patients to achieve functional improvements for an optimal return to social life.

Mechanism of anti-platelet activity of Oligoporus tephroleucus oligoporin A: involvement of extracellular signal-regulated kinase phosphorylation and cyclic nucleotide elevation.

This study investigated the inhibitory effects of oligoporin A on platelet aggregation and the mechanism of its action on downstream signaling molecules. Oligoporin A was isolated from the fruiting bodies of Oligoporus tephroleucus (Polyporaceae). The anti-platelet activities of oligoporin A were studied using rat platelets. The effects of oligoporin A on intracellular Ca(2+) mobilization, ATP release, production of the cyclic nucleotides cAMP and cGMP, extracellular signal-regulated kinase (ERK) 2 phosphorylation, and fibrinogen binding to active integrin α(II)(b)ß(3) were assessed. Oligoporin A, but not oligoporins B and C, inhibited collagen-induced platelet aggregation in a concentration-dependent manner. Interestingly, oligoporin A did not affect ADP- and thrombin-induced platelet aggregations, which act on different types of membrane receptors. Granule secretion analysis demonstrated that oligoporin A significantly and dose-dependently reduced collagen-induced ATP release and intracellular Ca(2+) mobilization. Additionally, oligoporin A induced the dynamic increase in cAMP and cGMP. Increased cGMP production was further confirmed by the simultaneous production of nitric oxide. Pretreatment with oligoporin A significantly blocked collagen-induced ERK2 phosphorylation. Finally, oligoporin A vaguely diminished the binding of fibrinogen to its cognate receptor, integrin α(II)(b)ß(3). The results indicate that oligoporin A inhibits only collagen-induced platelet aggregation mediated through the modulation of downstream signaling molecules. Oligoporin A may be beneficial against cardiovascular disease provoked by aberrant platelet activation.

Phellinus baumii ethyl acetate extract inhibits lipopolysaccharide-induced iNOS, COX-2, and proinflammatory cytokine expression in RAW264.7 cells.

Mushrooms are valuable sources of biologically active compounds possessing anticancer, antiplatelet, and anti-inflammatory properties. Phellinus baumii is a mushroom used in folk medicine for a variety of human diseases. However, its potential anti-inflammatory effect has remained unclear. Therefore, we studied the effect of P. baumii ethyl acetate extract (PBEAE) on inflammatory mediator and proinflammatory cytokine protein and/or mRNA expression levels using the nitric oxide (NO) assay, enzyme immunoassay (EIA), western blot, and reverse transcription polymerase chain reaction (RT-PCR) in lipopolysaccharide (LPS)-stimulated macrophage like RAW264.7 cells. PBEAE markedly inhibited NO generation and prostaglandin E(2) (PGE(2)) synthesis in a concentration-dependent pattern without any cytotoxic effect at the concentration range used. PBEAE also suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. In addition, LPS-induced iNOS and COX-2 mRNA expression levels were dose-dependently inhibited by PBEAE pretreatment. Furthermore, PBEAE attenuated the mRNA expression levels of proinflammatory cytokines, specifically interleukin (IL)-1ß, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF), in a concentration-dependent fashion. Our study suggests that P. baumii might exhibit anti-inflammatory properties by downregulating proinflammatory mediators. Thus, further study on compounds isolated from PBEAE is warranted to investigate the associated molecular mechanisms and identify the potential therapeutic targets.

Korean Red Ginseng Saponin Fraction Downregulates Proinflammatory Mediators in LPS Stimulated RAW264.7 Cells and Protects Mice against Endotoxic Shock.

Korean red ginseng has shown therapeutic effects for a number of disease conditions. However, little is known about the antiinflammatory effect of Korean red ginseng saponin fraction (RGSF) in vitro and in vivo. Therefore, in this study, we showed that RGSF containing 20(S)-protopanaxadiol type saponins inhibited nitric oxide production and attenuated the release of tumor necrotic factor (TNF)-α, interleukin (IL)-6, granulocyte monocyte colony stimulating factor (GMCSF), and macrophage chemo-attractant protein-1 in lipopolysaccharide (LPS) stimulated murine macrophage RAW264.7 cells. Moreover, RGSF down-regulated the mRNA expressions of inducible nitric oxide synthase, cyclooxyginase-2, IL-1ß, TNF-α, GMCSF, and IL-6. Furthermore, RGSF reduced the level of TNF-α in the serum and protected mice against LPS mediated endotoxic shock. In conclusion, these results indicated that ginsenosides from RGSF and their metabolites could be potential sources of therapeutic agents against inflammation.

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase.

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

Microglial peroxiredoxin V acts as an inducible anti-inflammatory antioxidant through cooperation with redox signaling cascades.

Reactive oxygen species (ROS) actively participate in microglia-mediated pathogenesis as pro-inflammatory molecules. However, little is known about the involvement of specific antioxidants in maintaining the microglial oxidative balance. We demonstrate that microglial peroxiredoxin (Prx) 5 expression is up-regulated by lipopolysaccharide (LPS) through activation of the ROS-sensitive signaling pathway and is involved in attenuation of both microglial activation and nitric oxide (NO) generation. Unlike in stimulation of oxidative insults with paraquat and hydrogen peroxide, Prx V expression is highly sensitive to LPS-stimulation in microglia. Reduction of ROS level by treatment with either NADPH oxidase inhibitor or antioxidant ablates LPS-mediated Prx V up-regulation in BV-2 microglial cells and is closely associated with the activation of the c-jun N-terminal kinase (JNK) signaling pathway. This suggests the involvement of ROS/JNK signaling in LPS-mediated Prx V induction. Furthermore, NO induces Prx V up-regulation that is ablated by the addition of inducible nitric oxide synthase inhibitor or deleted mutation of inducible nitric oxide synthase in LPS-stimulated microglia. Therefore, these results suggest that Prx V is induced by cooperative action among the ROS, RNS, and JNK signaling cascades. Interestingly, knockdown of Prx V expression causes the acceleration of microglia activation, including augmented ROS generation and JNK-dependent NO production. In summary, we demonstrate that Prx V plays a key role in the microglial activation process through modulation of the balance between ROS/NO generation and the corresponding JNK cascade activation.

Automated control of blood glucose in the OR and surgical ICU.

A device which integrates existing intravenous continuous glucose monitors and infusion pumps into a central hub for automated intravenous intensive insulin therapy, targeting non-diabetic critically-ill patients is presented. Additionally, a fuzzy logic based controller that is capable of automatically making closed-loop decisions to achieve tight glycemic control between a euglycemic range of 90 to 120 mg/dl is presented. Initial bench top testing shows a significant improvement in glycemic control with fuzzy logic control when compared to manual infusion protocols currently used in hospitals; future animal testing will be performed to verify these results in vivo.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of phosphoinositide 3-kinase and p38 mitogen-activated protein kinase signal pathways is required for lipopolysaccharide-induced microglial phagocytosis.

The importance of microglial reactive oxygen species (ROS) signaling in neuroinflammatory processes has been well demonstrated; however, relatively little is known regarding the related mechanisms underlying these processes. Here, we show that ROS-dependent signal pathways that govern microglial phagocytosis are highly dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activation. Specifically, phagocytosis was greatly reduced by both antioxidant and Nox inhibitor treatments in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Additionally, there was a marked reduction in intracellular ROS content. These results suggest that Nox is the main ROS source for LPS-induced microglial phagocytosis. More decisive evidence for the involvement of ROS in phagocytosis was obtained from an examination of phosphatidyl inositol 3-kinase (PI3-K) and p38 mitogen-activated protein kinase (MAPK) signal pathway activation under reduced ROS levels. These two kinases were activated by LPS treatment and inhibited by ROS neutralization and Nox inhibition. We conclude that microglial phagocytosis requires ROS-dependent PI3-K and p38 MAPK activation and that Nox-derived ROS functions as an upstream regulator of both PI3-K and p38 MAPK. These findings will provide a fundamental basis for a therapeutic modality in inflammation-mediated neurodiseases.

Peroxiredoxin I is an indicator of microglia activation and protects against hydrogen peroxide-mediated microglial death.

Imbalance between oxidative stress and antioxidative defence system is generally known as one of mechanisms causing an oxidative stress-medieated neuropathogenesis. Peroxiredoxins (Prxs), a family of antioxidative enzymes neutralizing cellular hydroperoxides, was characterized recently, but their distributions and roles have not been resolved clearly or controversial in the central nervous system, Therefore, the present study was carried out to determine the specific cell types that express Prx I in the mouse brain and primary neural cells, and to examine its antioxidative role in the preferential cell types. Immunohistochemical reactivity for Prx I was detected dominantly in oligodendrocytes and rarely in microglia, whereas strong and specific immunoreactivity for Prx I was observed exclusively in microglia of primary neural cell culture. Further evidences for Prx I specificity were its relatively high expression in BV-2 microglial cells and its upregulated expression in microglia after lipopolysaccharide (LPS) stimulation. These results imply that Prx I can be used as an indicator of microglial activation. Inhibition of p38 MAPK ablated LPS-mediated Prx I upregulation and sensitized the microglia to H(2)O(2)-mediated cell death. These findings indicate that Prx I function as a scavenger for H(2)O(2) generated during microglial activation. The results of this study will help in unraveling the neuropathologic roles of the six Prx isoforms in neural function.

Radical scavenging and anti-inflammatory activity of extracts from Opuntia humifusa Raf.

Opuntia humifusa Raf. (O. humifusa Raf.) is a member of the Cactaceae family. To determine the antioxidative and anti-inflammatory effects of this herb, various solvent fractions (methanol, hexane, chloroform, ethyl acetate, butanol, and water) prepared from the leaves of cacti were tested using DPPH (2,2-diphenyl-l-picrylhydrazyl radical) and xanthine oxidase assays, and nitric oxide (NO)-producing macrophage cells. We found that O. humifusa Raf. displayed potent antioxidative and anti-inflammatory activity. Thus, all solvent fractions, except for the water layer, showed potent scavenging effects. The scavenging effect of the ethyl acetate fraction was higher than that of the other fractions, with IC50 values of 3.6 and 48.2 microg mL(-1). According to activity-guided fractionation, one of the active radical scavenging principles in the ethyl acetate fraction was found to be quercetin. In contrast, only two fractions (chloroform and ethyl acetate) significantly suppressed nitric oxide production from the lipopolysaccharide (LPS)-activated RAW264.7 cells. In addition, chloroform and ethyl acetate fractions significantly blocked the expression of inducible nitric oxide synthetase (iNOS) and interleukin-6 (IL-6) from the RAW264.7 cells stimulated by LPS. Moreover, ethyl acetate fractions significantly blocked the expression of IL-1beta from the RAW264.7 cells stimulated by LPS. Therefore, the results suggested that O. humifusa Raf. may modulate radical-induced toxicity via both direct scavenging activity and the inhibition of reactive species generation, and the modulation of the expression of inflammatory cytokines. Finally, O. humifusa Raf. may be useful as a functional food or drug against reactive species-mediated disease.

Histone deacetylase 1 contributes to cell cycle and apoptosis.

Histone deacetylases (HDACs) are generally thought to play important roles in human disease. However, little information is available concerning the specific functions of individual HDACs. We previously reported on transgenic mice that expressed human HDAC1 and experienced steatosis and nuclear pleomorphism in their hepatic tissues. To find out if the over-expression of HDAC1 contributes to the expression of genes related to the cell cycle, apoptosis, and lipid metabolism that eventually contribute to the pathological changes in the livers of the transgenic mice, the expression profiles of the related genes in liver tissues were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The activated human HDAC1 significantly induced the expression levels of mRNA for p53, PPAR-gamma and Bak and reduced the p21 expression level compared with the levels in control littermates. However, the protein levels of p53 and PPAR-gamma were significantly decreased. In conclusion, our results indicate that HDAC1 can regulate gene expression at the mRNA and protein levels independently and that this may be a potential cytopathic factor for hepatic tissue in transgenic mice that over-express HDAC1.

Gender-dependent hepatic alterations in H-ras12V transgenic mice.

BACKGROUND/AIMS: Although it has been proposed that Ras and related signal pathways play important roles in hepatocarcinogenesis, appropriate in vivo models are lacking. METHODS: Two hepatocellular carcinoma lines were established using pronuclear microinjection techniques to create an insertion of the H-ras12V transgene under the control of the albumin enhancer/promoter. The resulting phenotypes and related molecular events were then examined. RESULTS: Male (but not female) transgenic mice older than 2 months showed hepatic alterations with a high degree of reproducibility, as compared to the wild-type mice. The liver/body-weight ratios were lower for the females than for the males. The transgene-carrying line 28 was investigated extensively with respect to molecular differences between the genders. Male hepatocytes showed higher Ras activity and higher reactive oxygen species (ROS) levels than female hepatocytes. The female hepatocytes showed higher expression levels of p53 and p21Waf1/Cip1, enhanced cytochrome c release, which correlated with cell cycle arrest, and higher levels of hypodiploid cell formation, as compared to the male hepatocytes. CONCLUSIONS: The gender-related differences in molecular responses to activated Ras may have implications for the prevalence of hepatic alterations in males. Our transgenic mice represent a potentially valuable animal model for future investigations.

Inhibitory role of peroxiredoxin II (Prx II) on cellular senescence.

Reactive oxygen species (ROS) were generated in all oxygen-utilizing organisms. Peroxiredoxin II (Prx II) as one of antioxidant enzymes may play a protective role against the oxidative damage caused by ROS. In order to define the role of Prx II in organismal aging, we evaluated cellular senescence in Prx II(-/-) mouse embryonic fibroblast (MEF). As compared to wild type MEF, cellular senescence was accelerated in Prx II(-/-) MEF. Senescence-associated (SA)-beta-galactosidase (Gal)-positive cell formation was about 30% higher in Prx II(-/-) MEF. N-Acetyl-l-cysteine (NAC) treatment attenuated SA-beta-Gal-positive cell formation. Prx II(-/-) MEF exhibited the higher G2/M (41%) and lower S (1.6%) phase cells as compared to 24% and 7.3% [corrected] in wild type MEF, respectively. A high increase in the p16 and a slight increase in the p21 and p53 levels were detected in PrxII(-/-) MEF cells. The cellular senescence of Prx II(-/-) MEF was correlated with the organismal aging of Prx II(-/-) mouse skin. While extracellular signal-regulated kinase (ERK) and p38 activation was detected in Prx II(-/-) MEF, ERK and c-Jun N-terminal kinase (JNK) activation was detected in Prx II(-/-) skin. These results suggest that Prx II may function as an enzymatic antioxidant to prevent cellular senescence and skin aging.

Transgenic expression of Korean type hepatitis C virus core protein and related mutants in mice.

Hepatitis C virus (HCV) is a major causative agent in liver disease. In order to investigate if Korean type HCV core protein and its related mutants, S99Q and S1161, are cytopathic to liver, three types of transgenic mice were established. The expression of transgenes was confirmed by HCV specific RT-PCR and Western immunoblotting. The livers of all wild type core and S1161 transgenic lineages remained largely histologically normal. However, the livers of the S99Q transgenic mice showed significant high level of cell dysplasia associated with the transgene expression in hepatocytes largely located around the central veins by in situ hybridization analysis. In conclusion, the mutant HCV core protein at S99Q may contribute to the progress of HCV induced liver disease.

Studies on the effects of biomedicinal agents on serum concentration of Ca2+, P and ALP activity in osteoporosis-induced rats.

The present study was carried out to investigate the effects of biomedicinal agents on Ca2+, P and alkaline phosphatase (ALP) levels in ovariectomized rats. Rats were ovariectomized bilaterally and were fed up with Ca2+ and P-free diet during 8(9,10) weeks to induce osteoporosis. Osteoporosis was determined by the extent of bone density and by lowering the concentrations of serum Ca2+, P and ALP activity every week. Rats in antler, safflower, ipriflavon, or co-administrated with estrogen groups were administrated with feed supplement for 5 weeks to elucidate the protective and therapeutic effects against osteoporosis. The bone tissue was examined with electron microscope to determine the effects of each treatment on osteoporosis. 1. The levels of serum Ca2+ and P in osteoporosis-induced rats, administrated with antler, ipriflavon and estrogen groups, were little higher than those of control rats. However, the levels of serum Ca and P in ovariectomized rats were significantly higher than those of control group (p<0.05). 2. The activities of serum ALP in osteoporosis-induced rats, administrated with antler extract, safflower, ipriflavon, or co-administrated with estrogen, were little increased in comparing with those of control group, but were significantly decreased in with combination of estrogen for 5 weeks. However, The connections were interrupted and the bone matrix was destroyed in the osteoporosis-induced rats. 3. The inter-trabecular connections were examined under electron microscope. The connections were well maintained and bone loss was without in the administration with antler, safflower, and ipriflavon with combination of estrogen for 5 weeks. However, The connections were interrupted and the bone matrix was destroyed in the osteoporosis-induced rats.

SR144528 as inverse agonist of CB2 cannabinoid receptor.

It is now well established that several G protein- coupled receptors can signal without agonist stimulation (constitutive receptors). Inverse agonists have been shown to inhibit the activity of such constitutive G protein-coupled receptor signaling. Agonist activation of the G(i/o)-coupled peripheral cannabinoid receptor CB2 normally inhibits adenylyl cyclase type V and stimulates adenylyl cyclase type II. Using transfected COS cells, we show here that application of SR144528, an inverse agonist of CB2, leads to a reverse action (stimulation of adenylyl cyclase V and inhibition of adenylyl cyclase II). This inverse agonism of SR144528 is dependent on the temperature, as well as on the concentration of the cDNA of CB2 transfected. Pertussis toxin blocked the regulation of adenylyl cyclase activity by SR 144528.