Detecting Bacterial Biofilms Using Fluorescence Hyperspectral Imaging and Various Discriminant Analyses.

Biofilms formed on the surface of agro-food processing facilities can cause food poisoning by providing an environment in which bacteria can be cultured. Therefore, hygiene management through initial detection is important. This study aimed to assess the feasibility of detecting Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium) on the surface of food processing facilities by using fluorescence hyperspectral imaging. E. coli and S. typhimurium were cultured on high-density polyethylene and stainless steel coupons, which are the main materials used in food processing facilities. We obtained fluorescence hyperspectral images for the range of 420-730 nm by emitting UV light from a 365 nm UV light source. The images were used to perform discriminant analyses (linear discriminant analysis, k-nearest neighbor analysis, and partial-least squares discriminant analysis) to identify and classify coupons on which bacteria could be cultured. The discriminant performances of specificity and sensitivity for E. coli (1-4 log CFU·cm-2) and S. typhimurium (1-6 log CFU·cm-2) were over 90% for most machine learning models used, and the highest performances were generally obtained from the k-nearest neighbor (k-NN) model. The application of the learning model to the hyperspectral image confirmed that the biofilm detection was well performed. This result indicates the possibility of rapidly inspecting biofilms using fluorescence hyperspectral images.

Non-Destructive Detection of Bone Fragments Embedded in Meat Using Hyperspectral Reflectance Imaging Technique.

Meat consumption has shifted from a quantitative to a qualitative growth stage due to improved living standards and economic development. Recently, consumers have paid attention to quality and safety in their decision to purchase meat. However, foreign substances which are not normal food ingredients are unintentionally incorporated into meat. These should be eliminated as a hazard to quality or safety. It is important to find a fast, non-destructive, and accurate detection technique of foreign substance in the meat processing industry. Hyperspectral imaging technology has been regarded as a novel technology capable of providing large-scale imaging and continuous observation information on agricultural products and food. In this study, we considered the feasibility of the short-wave near infrared (SWIR) hyperspectral reflectance imaging technique to detect bone fragments embedded in chicken meat. De-boned chicken breast samples with thicknesses of 3, 6, and 9-mm and 5 bone fragments with lengths of about 20-30-mm are used for this experiment. The reflectance spectra (in the wavelength range from 987 to 1701-nm) of the 5 bone fragments embedded under the chicken breast fillet are collected. Our results suggested that these hyperspectral imaging technique is able to detect bone fragments in chicken breast, particularly with the use of a subtraction image (corresponding to image at 1153.8-nm and 1480.2-nm). Thus, the SWIR hyperspectral reflectance imaging technique can be potentially used to detect foreign substance embedded in meat.

Dietary flavonoids differentially reduce oxidized LDL-induced apoptosis in human endothelial cells: role of MAPK- and JAK/STAT-signaling.

Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized LDL promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. This study investigated multiple mitogen-activated protein kinase (MAPK)-responsive death/survival signaling pathways, through which flavonoids of (-)epigallocatechin gallate (EGCG) and hesperetin exerted antiapoptosis in endothelial cells exposed to oxidized LDL. EGCG and hesperetin substantially diminished the oxidized LDL-induced 2',7'-dichlorofluorecein staining, suggesting that these flavonoids inhibited intracellular accumulation of oxidized LDL-triggered reactive oxygen species and consequent apoptosis. The Western-blot data revealed that oxidized LDL upregulated c-Jun N-terminal kinase (JNK) phosphorylation, which was rapidly reversed by EGCG and hesperetin. They mitigated the consequent activation of the JNK downstream on p53 and c-Jun. Moreover, oxidized LDL increased luciferase activity of p53 in endothelial cells transfected with a p53 promoter construct, the increase of which was strikingly downregulated by EGCG and hesperetin. Surprisingly, hesperetin but not EGCG attenuated phosphorylation of p38MAPK and its downstream c-myc and signal transducers and activators of transcription (STAT)1 evoked by oxidized LDL. This study also attempted to explore a linkage of Janus kinase (JAK)2/STAT3 activation to MAPK signaling in oxidized LDL-induced endothelial apoptosis. Notably, we found that the JAK2 inhibitor substantially blocked the JNK activation. Our findings suggest that EGCG and hesperetin may act as antiatherogenic agents blocking oxidized LDL-induced endothelial apoptosis via differential cellular apoptotic machinery. These data provide evidence that the interplay between p38MAPK and JAK-STAT pathways is involved in dietary flavonoid protection against oxidized LDL through hampering MAPK-dependent pathways involving the activation of JAK2.

Attenuation of monocyte adhesion and oxidised LDL uptake in luteolin-treated human endothelial cells exposed to oxidised LDL.

Oxidative modification of LDL is causally involved in the development of atherosclerosis and occurs in vivo in the blood as well as within the vascular wall. The present study attempted to explore whether polyphenolic flavonoids influence monocyte-endothelium interaction and lectin-like oxidised LDL receptor 1 (LOX-1) expression involved in the early development of atherosclerosis. The flavones luteolin and apigenin inhibited THP-1 cell adhesion onto oxidised LDL-activated human umbilical vein endothelial cells (HUVEC), while the flavanols of (-)epigallocatechin gallate and (+)catechin, the flavonols of quercetin and rutin, and the flavanones of naringin, naringenin, hesperidin and hesperetin did not have such effects. Consistently, Western blot analysis revealed that the flavones at 25 microM dramatically and significantly abolished HUVEC expression of vascular cell adhesion molecule-1 and E-selectin evidently enhanced by oxidised LDL; these inhibitory effects were exerted by drastically down regulating mRNA levels of these cell adhesion molecules. In addition, quercetin and luteolin significantly attenuated expression of LOX-1 protein up regulated in oxidised LDL-activated HUVEC with a fall in transcriptional mRNA levels of LOX-1. In addition, quercetin and luteolin clearly blunted oxidised LDL uptake by HUVEC treated with oxidised LDL. The results demonstrate that the flavones luteolin and apigenin as well as quercetin were effective in the different initial steps of atherosclerosis process by inhibiting oxidised LDL-induced endothelial monocyte adhesion and/or oxidised LDL uptake. Therefore, certain flavonoids qualify as anti-atherogenic agents in LDL systems, which may have implications for strategies attenuating endothelial dysfunction-related atherosclerosis.

Chronic blockade of the angiotensin II receptor has a differential effect on adipose and vascular PAI-1 in OLETF rats.

Angiotensinogen (AGT) and plasminogen activator inhibitor-1 (PAI-1) are expressed in both vascular and adipose tissues. Angiotensin II (AG II) has an adipogenic effect and increases PAI-1 expression. To evaluate the chronic effects of AG II type 1 receptor (AT(1)R) antagonism on adipose mass and PAI-1 expression in vascular and adipose tissues, losartan (30mg/kg/day) was administered to Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes, for 20 weeks. Adipose mass and regional fat distribution in the abdomen did not change after chronic AT(1)R antagonism in OLETF rats. AGT and PAI-1 mRNA expressions in adipose tissue of OLETF rats were significantly increased compared with Long-Evans Tokushima Otsuka (LETO) rats, the normal control. Chronic losartan therapy further increased the level of adipose AGT in OLETF rats, but did not affect the level of adipose PAI-1 mRNA. In contrast, aortic PAI-1 expression in OLETF rats was attenuated by chronic losartan therapy. Our results have two implications. First, adipose tissue may be an important source of AG II in metabolic syndrome even after chronic losartan therapy. Second, chronic AT(1)R antagonism with losartan causes differential effects on vascular and adipose PAI-1 expression.

The effects of thiazolidinedione treatment on the regulations of aquaglyceroporins and glycerol kinase in OLETF rats.

Aquaporins (AQPs) that transport glycerol in addition to water are classified as aquaglyceroporins (AQP3, 7, 9). AQP7 in the adipose tissue and AQP9 in the liver may coordinately contribute to the increase in hepatic gluconeogenesis in states of insulin resistance. Thiazolidinedione (TZD) has been shown to increase adipose AQP7 and induce glycerol kinase (GlyK) which is nearly absent in adipocytes. In the present study, we analyzed both GlyK and AQP gene expression in adipose and hepatic tissues, and AQP3 in kidneys from Long-Evans Tokushima Otsuka (LETO), Otsuka Long-Evans Tokushima Fatty (OLETF), and rosiglitazone (RSG)-treated OLETF (RSG-OLETF) rats. We also evaluated AQP9 protein expression in cultured human hepatoma cells treated with oleic acid, Wy14643, or RSG. A 2-week RSG treatment increased AQP7 mRNA levels in the mesenteric fat, but not in the epididymal fat of OLETF rats. Rosiglitazone treatment markedly increased GlyK expression in both fat depots, with a greater increase in the mesenteric fat. The magnitudes of GlyK induction by RSG were greater than that of AQP7 in both adipose tissues (P < .05, each). AQP9 and GlyK levels in the liver were not affected by RSG treatment in OLETF rats. Oleic acid and Wy14643 upregulated AQP9 protein expression in cultured human hepatoma cells in a dose-dependent manner. AQP3 mRNA levels tended to increase in the outer medulla of the RSG-OLETF rats. These results indicate that in the adipose tissue TZD has an important role in the glycerol metabolic pathway through the regulation of AQP and GlyK, especially by GlyK induction. Free fatty acids may directly enhance glycerol availability in the liver via the upregulation of AQP9 levels. Renal AQP3 may be related to the fluid retention caused by TZD.

Flavones mitigate tumor necrosis factor-alpha-induced adhesion molecule upregulation in cultured human endothelial cells: role of nuclear factor-kappa B.

Flavones have been classified as anti-atherogenic agents that inhibit monocyte adhesion to stimulated endothelium, possibly by blocking induction of cell adhesion molecules (CAM). This anti-atherogenic feature of these flavonoids appears to be related to their chemical structures. Flavones may interfere with key signaling events involved in endothelial cell activation by inflammatory mediators. This study examined the effects of flavones on the induction of CAM and the translocation and DNA binding of nuclear factor-kappa B (NF-kappa B) in TNF-alpha-activated human umbilical vein endothelial cells (HUVEC). The effects of flavones, luteolin and apigenin, on adhesion of THP-1 monocytes to the TNF-alpha-activated HUVEC, protein expression and mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin, and nuclear appearance and DNA binding activity of NF-kappa B were determined. Flavanols, flavonols, and flavanones were used for comparison. TNF-alpha significantly induced HUVEC protein expression of VCAM-1, ICAM-1, and E-selectin with increasing mRNA levels. Luteolin and apigenin inhibited the TNF-alpha-induced upregulation of THP-1 adhesion and VCAM-1 expression; these inhibitory effects were dose-dependent. The flavones at doses of > or =25 micromol/L almost completely abolished the increased CAM protein and mRNA regardless of their anti-oxidative activity. With the exception of the flavonol quercetin, flavonoids had no such effect; quercetin substantially attenuated the CAM induction. The flavones inhibited nuclear translocation and DNA binding activity of the NF-kappa B-containing binding site in the promoter region of the CAM genes in TNF-alpha-activated HUVEC. The inhibition of endothelial CAM induction by flavones is mediated by their interference with the NF-kappa B-dependent transcription pathway. Thus, the flavones may hamper initial atherosclerotic events involving endothelial CAM induction.

Polyphenolic flavonoids differ in their antiapoptotic efficacy in hydrogen peroxide-treated human vascular endothelial cells.

Oxidative injury induces cellular and nuclear damage that leads to apoptotic cell death. Agents or antioxidants that can inhibit production of reactive oxygen species can prevent apoptosis. We tested the hypothesis that flavonoids can inhibit H(2)O(2)-induced apoptosis in human umbilical vein endothelial cells. A 30-min pulse treatment with 0.25 mmol/L H(2)O(2) decreased endothelial cell viability within 24 h by approximately 40% (P < 0.05) with distinct nuclear condensation and DNA fragmentation. In the H(2)O(2) apoptosis model, the addition of 50 micro mol/L of the flavanol (-)epigallocatechin gallate and the flavonol quercetin, which have in vitro radical scavenging activity, partially (P < 0.05) restored cell viability with a reduction in H(2)O(2)-induced apoptotic DNA damage. In contrast, the flavones, luteolin and apigenin, at the nontoxic dose of 50 micro mol/L, intensified cell loss (P < 0.05) after exposure to H(2)O(2) and did not protect cells from oxidant-induced apoptosis. The flavanones, hesperidin and naringin, did not have cytoprotective effects. The antioxidants, (-)epigallocatechin gallate and quercetin, inhibited endothelial apoptosis, enhanced the expression of bcl-2 protein and inhibited the expression of bax protein and the cleavage and activation of caspase-3. Therefore, flavanols and flavonols, in particular (-)epigallocatechin gallate and quercetin, qualify as potent antioxidants and are effective in preventing endothelial apoptosis caused by oxidants, suggesting that flavonoids have differential antiapoptotic efficacies. The antiapoptotic activity of flavonoids appears to be mediated at the mitochondrial bcl-2 and bax gene level.

Involvement of transcription factors in plasma HDL protection against TNF-alpha-induced vascular cell adhesion molecule-1 expression.

Plasma HDL has been reported to protect against the initial development of inflammatory atherosclerosis. Although cellular and molecular mechanisms of this HDL protection are not fully understood, they may involve prevention of the endothelial trafficking of circulating leukocytes via its down-regulation of inflammatory genes. To test this hypothesis, HDL inhibition of monocyte adherence to the endothelium and of the expression of vascular cell adhesion molecule-1 (VCAM-1) and its transcriptional regulatory mechanism were elucidated in human umbilical vein endothelial cells (HUVECs) stimulated by pro-inflammatory TNF-alpha. Plasma HDL (1mg/ml apo A1) markedly prevented the monocyte adhesion to TNF-alpha-activated HUVECs with blunting the increased expression levels of VCAM-1 protein and its mRNA at a maximal TNF-alpha stimulation. Electrophoretic mobility shift assay showed that in the activated HUVECs HDL substantially inhibited DNA binding activities of transcription factors of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) that have binding sites in the promoter region of the VCAM-1 gene. In addition, immunocytochemical staining analysis confirmed that HDL inhibited TNF-alpha-induced VCAM-1 expression via reduced nuclear translocation of NF-kappaB. These results suggest that HDL down-regulates the expression of VCAM-1 gene in TNF-alpha-activated HUVECs at transcriptional levels via blunted translocation and transactivation of NF-kappaB and AP-1 transcription factors. Plasma HDL may block the initial atherosclerotic process via inhibited monocyte adhesion to the endothelium, which is independent of modulating the cholesterol reverse transport of plasma HDL.

Antioxidant alpha-keto-carboxylate pyruvate protects low-density lipoprotein and atherogenic macrophages.

Oxidative modification of low-density lipoprotein (oxLDL) plays a pathogenic role in atherogenesis. Classical antioxidants such as L-ascorbic acid can inhibit formation of oxLDL. Alpha-Keto-carboxylates such as pyruvate and congeners also display antioxidant properties in some cell-free and intact cell systems. We tested the hypothesis that pyruvate or alpha-keto-glutarate may function as antioxidants with respect to LDL incubated with 5 or 10 microM Cu2+ alone or in combination with THP-1-derived macrophages. alpha-Hydroxy-carboxylates (L-lactate), linear aliphatic monocarboxylates (acetate/caprylate) and L-ascorbic acid served as controls. The oxLDL formation was ascertained by electrophoretic mobility and oxLDL cytotoxicity was judged by macrophage viability and thiobarbituric acid reactive substances (TBARS) formation. Cu2+ alone was not cytotoxic but increased electrophoretic mobility of cell-free LDL, stimulating TBARS. Millimolar pyruvate, alpha-ketoglutarate, or micromolar L-ascorbic acid partially inhibited oxLDL formation, while alpha-hydroxy-carboxylate or the aliphatic mono-carboxylates had no measurable antioxidant properties in cell-free LDL. Co-culture of LDL with macrophages and Cu2+ augmented TBARS release and resulted in 95% macrophage death. Pyruvate improved macrophage viability with 5 microM Cu2+ up to 60%. L-Ascorbic acid (> or = 100 microM) protected macrophages up to 80%. When > or = 100 microM L-ascorbic acid was combined with pyruvate, oxLDL formation and macrophage death were fully prevented. Thus, alpha-keto-carboxylates, but not physiological alpha-hydroxy-carboxylates or aliphatic monocarboxylates qualify as antioxidants in LDL systems. Since alpha-keto-carboxylates enhanced the antioxidant power of L-ascorbic acid, our findings may have implications for strategies attenuating atherosclerosis.