Multiphoton imaging of the monosachharide induced formation of fluorescent advanced glycation end products in tissues.

We studied the in vitro rate of fluorescent advanced glycation end products (fAGEs) formation with multiphoton microscopy in different porcine tissues (aorta, cornea, kidney, dermis, and tendon). These tissues were treated with d-glucose, d-galactose, and d-fructose, three primary monosaccharides found in human diets. We found that the use of d-fructose resulted in the highest glycation rate, followed by d-galactose and then d-glucose. Moreover, compared to non-collagen tissue constituents such as elastic fibers and cells, the rate of tissue glycation was consistently higher in collagen, suggesting that collagen is a more sensitive target for fAGE formation. However, we also found that collagen in different tissues exhibits different rates of fAGE formation, with slower rates observed in tightly packed tissues such as cornea and tendon. Our study suggests that for fAGE to be developed into a long-term glycemic biomarker, loosely organized collagen tissues located in the proximity of vasculature may be the best targets.

Hemoglobin autofluorescence as potential long-term glycemic marker in the rat animal model.

Diabetes is a serious disease whose patients often require long-term care. Blood glucose and intermediate glycation product of glycated hemoglobin (HbA1c) are, at best, surrogate biomarkers of disease progression. There is indication that advanced glycation end products (AGEs) better reflect diabetic risks. In this study, we explored the use of red blood cells (RBCs) and lysed hemoglobin (Hb) autofluorescence (AF) as potential biomarkers of diabetic complication. AF spectra measured under 370 nm excitation reveals that both RBC and Hb fluorescence in the 420 to 600 nm region. At early time points following diabetic induction in rats, AF increase in lysed Hb is more dramatic compared to that of RBCs. Moreover, we found significance variance of Hb autofluorescence despite relatively constant HbA1c levels. Furthermore, we found that although a correlation exists between AGE autofluorescence and HbA1c levels, the lack of complete correspondence suggests that the rate of AGE production differs significantly among different rats. Our results suggest that with additional development, both RBC and Hb autofluorescence from lysed RBCs may be used act long-term glycemic markers for diabetic complications in patients.

Multiphoton imaging of the effect of monosaccharide diffusion and formation of fluorescent advanced end products in porcine aorta.

Prolonged exposure of tissues to elevated blood sugar levels lead to the formation of advanced glycation end products (AGEs), thus contributing to diabetic complications. Since the vascular system is in immediate contact with blood, diabetic effects on aorta is a major health concern. However, the relative effect of the diffusion of sugar molecular through the vascular wall and the rate of AGE formation is not known. In this study, we aim to address this issue by incubating excised porcine aorta in D-glucose, D-galactose, and D-fructose solutions for different periods. The tissue specimens were then excised for multiphoton imaging of autofluorescence intensity profiles across the aorta wall. We found that for Days 4 to 48 incubation, autofluorescence is constant along the radial direction of the aorta sections, suggesting that monosaccharide diffusion is rapid in comparison to the rate of formation of fluorescent AGEs (fAGEs). Moreover, we found that in porcine aorta, the rate of fAGE formation of D-fructose and D-glucose are factors 2.08 and 1.14 that of D-galactose. Our results suggest that for prolonged exposure of the cardiovascular system to elevated monosaccharides 4 days or longer, damage to the aorta is uniform throughout the tissues.

Dynamic visualization of the recovery of mouse hepatobiliary metabolism to acetaminophen-overdose damage.

Acetaminophen (APAP) overdose is one of the world's leading causes of drug-induced hepatotoxicity. Although traditional methods such as histological imaging and biochemical assays have been successfully applied to evaluate the extent of APAP-induced liver damage, detailed effect of how APAP overdose affect the recovery of hepatobiliary metabolism and is not completely understood. In this work, we used intravital multiphoton microscopy to image and quantify hepatobiliary metabolism of the probe 6-carboxyfluorescein diacetate in APAP-overdose mice. We analyzed hepatobiliary metabolism for up to 7 days following the overdose and found that the excretion of the probe molecule was the most rapid on Day 1 following APAP overdose and slowed down on Days 2 and 3. On Day 7, probe excretion capability has exceeded that of the normal mice, suggesting that newly regenerated hepatocytes have higher metabolic capabilities. Our approach may be further developed applied to studying drug-induced hepatotoxicity in vivo.

Nb2O5 and Ti-Doped Nb2O5 Charge Trapping Nano-Layers Applied in Flash Memory.

High-k material charge trapping nano-layers in flash memory applications have faster program/erase speeds and better data retention because of larger conduction band offsets and higher dielectric constants. In addition, Ti-doped high-k materials can improve memory device performance, such as leakage current reduction, k-value enhancement, and breakdown voltage increase. In this study, the structural and electrical properties of different annealing temperatures on the Nb2O5 and Ti-doped Nb2O5(TiNb2O7) materials used as charge-trapping nano-layers in metal-oxide-high k-oxide-semiconductor (MOHOS)-type memory were investigated using X-ray diffraction (XRD) and atomic force microscopy (AFM). Analysis of the C-V hysteresis curve shows that the flat-band shift (∆VFB) window of the TiNb2O7 charge-trapping nano-layer in a memory device can reach as high as 6.06 V. The larger memory window of the TiNb2O7 nano-layer is because of a better electrical and structural performance, compared to the Nb2O5 nano-layer.

In vivo multiphoton kinetic imaging of the toxic effect of carbon tetrachloride on hepatobiliary metabolism.

We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mice. The acquired images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. We found that with progression of hepatotoxicity, the spatial gradient of hepatic function disappeared. A CCl4-induced damage mechanism involves the compromise of membrane functions, resulting in accumulation of processed 6-carboxyfluorescein molecules. At day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.

Multiphoton dynamic imaging of the effect of chronic hepatic diseases on hepatobiliary metabolism in vivo.

In this study, intravital multiphoton microscopy was used to quantitatively investigate hepatobiliary metabolism in chronic pathologies of the liver. Specifically, through the use of the probe molecule 6-carboxyfluorescein diacetate, the effects of liver fibrosis, fatty liver, and hepatocellular carcinoma on the metabolic capabilities of mouse liver were investigated. After the acquisition of time-lapse images, a first order kinetic model was used to calculate rate constant resolved images of various pathologies. It was found that the ability of the liver to metabolically process the probe molecules varies among different pathologies, with liver fibrosis and fatty liver disease negatively impacted the uptake, processing, and excretion of molecules. The approach demonstrated in this work allows the study of the response of hepatic functions to different pathologies in real time and is useful for studying processes such as pharmacokinetics through direct optical imaging.

Direct visualization of functional heterogeneity in hepatobiliary metabolism using 6-CFDA as model compound.

Hepatobiliary metabolism is one of the major functions of the liver. However, little is known of the relationship between the physiological location of the hepatocytes and their metabolic potential. By the combination of time-lapse multiphoton microscopy and first order kinetic constant image analysis, the hepatocellular metabolic rate of the model compound 6-carboxyfluorescein diacetate (6-CFDA) is quantified at the single cell level. We found that the mouse liver can be divided into three zones, each with distinct metabolic rate constants. The sinusoidal uptake coefficients k1 of Zones 1, 2, and 3 are respectively 0.239 ± 0.077, 0.295 ± 0.087, and 0.338 ± 0.133 min-1, the apical excreting coefficients k2 of Zones 1, 2, and 3 are 0.0117 ± 0.0052, 0.0175 ± 0.0052, and 0.0332 ± 0.0195 min-1, respectively. Our results show not only the existence of heterogeneities in hepatobiliary metabolism, but they also show that Zone 3 is the main area of metabolism.

Visualizing and quantifying difference in cytoplasmic and nuclear metabolism in the hepatobiliary system in vivo.

The liver is a major organ responsible for performing xenobiotic metabolism. In this process, xenobiotic is uptaken and processed in hepatocytes and subsequently excreted into the bile canaliculi. However, the intracellular heterogeneity in such metabolic processes is not known. We use the molecular probe 6-carboxyfluorescein diacetate (6-CFDA) to investigate xenobiotic metabolism in hepatocytes with intravital multiphoton fluorescence microscopy. 6-CFDA is processed by intracellular esterase to fluorescent 6-CF, which can be imaged and quantified. We found that compared to the nucleus, cytoplasmic 6-CF fluorescence intensity reached a maximum earlier (cytoplasm: 11.3 ± 4.4 min; nucleus: 14.7 ± 4.9 min) following 6-CFDA injection. We also found a slight difference in the rate of 6-CFDA metabolism as the rates of 6-CF decay at rates of 1.43 ± 0.75 and 1.27 ± 0.72 photons/min for the cytoplasm and nucleus, respectively. These results indicate that molecular transport to the nucleus is additionally hindered and can affect drug transport there

Luteolin attenuates TGF-ß1-induced epithelial-mesenchymal transition of lung cancer cells by interfering in the PI3K/Akt-NF-κB-Snail pathway.

AIMS: Luteolin is a natural flavonoid that possesses a variety of pharmacological activities, such as anti-inflammatory and anti-cancer abilities. Whether luteolin regulates the transformation ability of lung cancer cells remains unclear. The current study aims to uncover the effects and underlying mechanisms of luteolin in regulation of and epithelial-mesenchymal transition of lung cancer cells. MAIN METHODS: The lung adenocarcinoma A549 cells were used in this experiment; the cells were pretreated with luteolin followed by administration with TGF-ß1. The expression levels of various cadherin and related upstream regulatory modules were examined. KEY FINDINGS: Pretreatment of luteolin prevented the morphological change and downregulation of E-cadherin of A549 cells induced by TGF-ß1. In addition, the activation of PI3K-Akt-IκBa-NF-κB-Snail pathway which leads to the decline of E-cadherin induced by TGF-ß1 was also attenuated under the pretreatment of luteolin. SIGNIFICANCE: We provide the mechanisms about how luteolin attenuated the epithelial-mesenchymal transition of A549 lung cancer cells induced by TGF-ß1. This finding will strengthen the anti-cancer effects of flavonoid compounds via the regulation of migration/invasion and EMT ability of various cancer cells.