Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

Association between body composition and chronic low back pain in Korean adults aged over 50 years: The Korea National Health and Nutrition Examination Survey (KNHANES) 2010-2011.

Background The relationship between overweight or obesity and low back pain (LBP) has previously been investigated. Several recent studies have focused on the relationship between other indicators of obesity, particularly indicators of fat and the risk of LBP. However, the results of body composition and LBP have been inconsistent. Methods All data for the present retrospective, cross-sectional study was extracted from the Korea National Health and Nutrition Examination Survey (KNHANES) versions V-1 and 2 conducted in 2010 and 2011 by the Korean Centers for Disease Control and Prevention. In KNHANES V-1 (2010) and V-2 (2011), those over 50 years of age completed the surveys on LBP, body weight, and body composition assessed using dual-energy X-ray absorptiometry (DXA) were included. The multivariable logistic regression analysis was used to examine the relationship between the presence of chronic LBP and body composition adjusting for confounders. Results We analyzed 3,579 persons who completed the question. In the multivariable analyses adjusting for age and sex, none of the variables, including fat mass and fat-free mass, remained positively or negatively associated with LBP. Additionally, when depression, smoking, alcohol intake, physical activity, diabetes mellitus, and fat or lean tissue mass were included in the multivariable logistic model, no significant associations were found between all measures of fat mass, fat-free mass, and LBP Conclusion This study is contrary to previous studies that concluded that there is a correlation between obesity and fat mass and LBP. LBP is not associated with increased levels of obesity and fat mass.

Cancer-Targeted Azo Dye for Two-Photon Photodynamic Therapy in Human Colon Tissue.

Inappropriate cancer management can be prevented by simultaneous cancer diagnosis, treatment, and real-time assessment of therapeutic processes. Here, we describe the design of a two-photon (TP) photosensitizer (PS), ACC-B, for high temporal and spatioselective near-infrared cancer therapy. ACC-B consisting of a biotin unit significantly enhanced the cancer sensitivity of the PS. Upon TP irradiation, ACC-B generated reactive oxygen species (ROS) through the type I photodynamic therapy (PDT) process and triggered highly selective cancer ablation. In addition, fluorescence microscopy images revealed that ACC-B-loaded live human colon tissues showed a marked difference in ACC-B uptake between normal and cancer tissues, and this property was used for real-time imaging. Upon 770 nm TP treatment, ACC-B generated ROS efficiently in live colon cancer tissues with high spatial selectivity. During PDT, ACC-B can provide in situ spatioselective visualization of cellular behavior and molecular information for therapeutic assessment in specific regions.

Highly Stable Red-Emissive Ratiometric Probe for Monitoring ß-Galactosidase Activity Using Fluorescence Microscopy and Flow Cytometry.

ß-Galactosidase (ß-gal), well known as a useful reporter enzyme, is a potent biomarker for various diseases such as colorectal and ovarian cancers. We have developed a highly stable red-emissive ratiometric fluorescent probe (CCGal1) for quantitatively monitoring the ß-gal enzyme activity in live cells and tissues. This ratiometric probe showed a fast emission color change (620-662 nm) in response to ß-gal selectively, which was accompanied by high enzyme reaction efficacy, cell-staining ability, and outstanding stability with minimized cytotoxicity. Confocal fluorescence microscopy ratiometric images, combined with fluorescence-activated cell sorting flow cytometry, demonstrated that CCGal1 could provide useful information for the diagnosis, prognosis, and treatment of ß-gal enzyme activity-related diseases such as colorectal and ovarian cancers. Further, it may yield meaningful strategies for designing and modifying multifunctional bioprobes with different biomedical applications.

A Diagnostic Method for Gastric Cancer Using Two-Photon Microscopy With Enzyme-Selective Fluorescent Probes: A Pilot Study.

BACKGROUND: Endoscopy is the most important tool for gastric cancer diagnosis. However, it relies on naked-eye evaluation by endoscopists, and the histopathologic confirmation is time-consuming. We aimed to visualize and measure the activity of various enzymes through two-photon microscopy (TPM) using fluorescent probes and assess its diagnostic potential in gastric cancer. METHODS: ß-Galactosidase (ß-gal), carboxylesterase (CES), and human NAD(P)H: quinone oxidoreductase (hNQO1) enzyme activities in the normal mucosa, ulcer, adenoma, and gastric cancer biopsy samples were measured using two-photon enzyme probes. The fluorescence emission ratio at long and short wavelengths (Ch2/Ch1) for each probe was comparatively analyzed. Approximately 8,000 - 9,000 sectional images in each group were obtained by measuring the Ch2/Ch1 ratio according to the tissue depth. Each probe was cross-validated by measuring enzymatic activity from a solution containing lysed tissue. RESULTS: Total of 76 subjects were enrolled in this pilot study (normal 21, ulcer 18, adenoma 17, and cancer 20 patients, respectively). There were significant differences in the mean ratio values of ß-gal (0.656 ± 0.142 vs. 1.127 ± 0.109, P < 0.001) and CES (0.876 ± 0.049 vs. 0.579 ± 0.089, P < 0.001) between the normal and cancer, respectively. The mean ratio value of cancer tissues was different compared to ulcer and adenoma (P < 0.001). The hNQO1 activity showed no significant difference between cancer and other conditions. Normal mucosa and cancer were visually and quantitatively distinguished through ß-gal and CES analyses using TPM images, and enzymatic activity according to depth, was determined using sectional TPM ratiometric images. The results obtained from lysis buffer-treated tissue were consistent with TPM results. CONCLUSIONS: TPM imaging using ratiometric fluorescent probes enabled the discrimination of gastric cancer from normal, ulcer, and adenoma. This novel method can help in a visual differentiation and provide quantitative depth profiling in gastric cancer diagnosis.

Two-Photon Fluorescent Probes for Detecting Enzyme Activities in Live Tissues.

Enzyme regulation is crucial in living organisms to catalyze various biosyntheses to maintain several physiological functions. On the contrary, abnormal enzyme activities can affect bioactivities leading to various serious disorders including cancer, Alzheimer's disease, Parkinson's disease, heart disease, and so on. This biological significance led to the development of various techniques to map specific enzyme activities in living systems to understand their role and distribution. Two-photon microscopy (TPM) in particular has emerged as a promising system for in situ real-time bioimaging owing to its robustness, high sensitivity, and noninvasiveness. It was achieved through the use of a two-photon (TP) light source of an optical window (700-1450 nm) beneficial in deeper light penetration and extraordinary spatial selectivity. Therefore, developing enzyme sensors utilized in TPM has significance in obtaining in vivo enzyme activities with minimal perturbation. The development of an efficient detection tool for enzymes has been continuously reported in the previous literature; here, we meticulously review the TP design strategies that have been attempted by researchers to develop enzyme TP fluorescent sensors that are proving very useful in providing insights for enzyme investigation in the biological system. In this review, the representative TP enzymatic probes that have been made in the past 5 years and their applications in tissue imaging are discussed in brief. In addition, the prospects and challenges of TP enzymatic probe development are also discussed.

Novel Method for S1 Transforaminal Epidural Steroid Injection.

BACKGROUND: S1 transforaminal epidural steroid injection (S1-TFESI) results in positive clinical outcomes for the treatment of pain associated with the S1 nerve root. S1-TFESI via the transforaminal approach is commonly performed under fluoroscopic guidance. Ultrasound guidance is an alternative to mitigate radiation exposure. However, performing spinal procedures under ultrasound guidance has some limitations in confirming the position of the needle tip and vascular uptake. New techniques are therefore needed to make ultrasound and fluoroscopy complementary. Our objective was to describe a novel technique for S1-TFESI and confirm its reproducibility. METHODS: Records of patients with S1 radiculopathy were reviewed retrospectively; those treated using the new S1-TFESI technique were selected. Initially, ultrasound was used to distinguish anatomy of the sacral foramen and guide initial placement of the needle entry point. Fluoroscopy was subsequently used to confirm needle tip position and vascular injection. The number of times the needle required reinsertion was recorded, and ultrasound and C-arm images were stored. RESULTS: Sixty-seven S1-TFESIs were performed in 56 patients. All injections exhibited epidural spread of contrast media, not only to the S1 nerve. The cephalad angle was 16.25 ± 6.75° (range, 5-27°), the oblique angle was 2.48 ± 2.62° (range, 0-7°), and the mean number of attempts was 1.24 ± 1.25. CONCLUSIONS: The new technique, involving the use of ultrasound to guide initial placement of the needle entry point, followed by confirmatory imaging and any needed adjustment with the use of fluoroscopy, can be a technique to complement the shortcomings of using ultrasound or fluoroscopy alone.

Discrimination between Human Colorectal Neoplasms with a Dual-Recognitive Two-Photon Probe.

Colorectal cancer is a major cause of cancer-related deaths worldwide. Histologic diagnosis using biopsy samples of colorectal neoplasms is the most important step in determining the treatment methods, but these methods have limitations in accuracy and effectiveness. Herein, we report a dual-recognition two-photon probe and its application in the discrimination between human colorectal neoplasms. The probe is composed of two monosaccharides, d-glucosamine and ß-d-galactopyranoside, in a fluorophore for the monitoring of both glucose uptake and ß-gal hydrolysis. In vitro/cell imaging studies revealed the excellent selectivity and sensitivity of the probe for glucose transporter-mediated glucose uptake and ß-gal activity. Cancer-specific uptake was monitored by increased fluorescence intensity, and additional screening of cancer cells was achieved by changes in emission ratio owing to the higher activity of ß-gal. Using human colon tissues and two-photon microscopy, we found that the plot of intensity versus ratio can accurately discriminate between colorectal neoplasms in the order of cancer progression (normal, adenoma, and carcinoma).

Near-IR Fluorescent Tracer for Glucose-Uptake Monitoring in Live Cells.

Fluorescent tracers for glucose-uptake monitoring could be used as chemical tools for diagnosis and for discovery of novel therapeutic agents via the development of phenotypic screening systems. Here we present a new near-infrared fluorescent glucose tracer, Glc-SiR-CO2H, for monitoring the cellular glucose uptake. By conjugating glucosamine with two different silicon rhodamine fluorochromes, we found that the net charge of fluorochromes has considerable effects on cellular uptake of the probe. Competition assay with d/l-glucose as well as Western blot analysis implied GLUT-dependent uptake mechanism of this probe. Finally, Glc-SiR-CO2H not only differentiates cancer cells from normal cells, but also allows monitoring anticancer effects in live cells.

The Application of REDOR NMR to Understand the Conformation of Epothilone B.

The structural information of small therapeutic compounds complexed in biological matrices is important for drug developments. However, structural studies on ligands bound to such a large and dynamic system as microtubules are still challenging. This article reports an application of the solid-state NMR technique to investigating the bioactive conformation of epothilone B, a microtubule stabilizing agent, whose analog ixabepilone was approved by the U.S. Food and Drug Administration (FDA) as an anticancer drug. First, an analog of epothilone B was designed and successfully synthesized with deuterium and fluorine labels while keeping the high potency of the drug; Second, a lyophilization protocol was developed to enhance the low sensitivity of solid-state NMR; Third, molecular dynamics information of microtubule-bound epothilone B was revealed by high-resolution NMR spectra in comparison to the non-bound epothilone B; Last, information for the macrolide conformation of microtubule-bound epothilone B was obtained from rotational-echo double-resonance (REDOR) NMR data, suggesting the X-ray crystal structure of the ligand in the P450epoK complex as a possible candidate for the conformation. Our results are important as the first demonstration of using REDOR for studying epothilones.

Development of imidazoline-2-thiones based two-photon fluorescence probes for imaging hypochlorite generation in a co-culture system.

We designed and prepared the imidazoline-2-thione containing OCl(-) probes, PIS and NIS, which operate through specific reactions with OCl(-) that yield corresponding fluorescent imidazolium ions. Importantly, we demonstrated that PIS can be employed to image OCl(-) generation in macrophages in a co-culture system. We have also employed two-photon microscopy and PIS to image OCl(-) in live cells and tissues, indicating that this probe could have wide biological applications.

A cysteamine-selective two-photon fluorescent probe for ratiometric bioimaging.

We report a two-photon fluorescent probe for ratiometric imaging of cysteamine in situ. This probe can detect the levels of endogenous cysteamine with statistical significance in live cells and brain hippocampal tissues, revealing that cysteamine is localized mainly in the perikaria of the pyramidal neurons and the granule cells.

MMHD [(S,E)-2-methyl-1-(2-methylthiazol-4-yl) hexa-1,5-dien-ol], a novel synthetic compound derived from epothilone, suppresses nuclear factor-kappaB-mediated cytokine expression in lipopolysaccharide-stimulated BV-2 microglia.

The effects of MMHD [(S,E)-2-methyl-1-(2-methylthiazol-4-yl) hexa-1,5-dien-ol], a novel synthetic compound derived from epothilone, was investigated for its effects on the expression of proinflammatory mediators in lipopolysaccharide-stimulated BV-2 microglia. MMHD attenuated the expressions of inducible nitric oxide synthase and cyclooxygenase-2 mRNA and protein without affecting cell viability. Moreover, MMHD suppressed nuclear factor-kappaB (NF-kappaB) activation via the translocation of p65 into the nucleus. These results indicate that MMHD exerts anti-inflammatory properties by suppressing the transcription of proinflammatory cytokine genes through the NF-kappaB signaling pathway.

Cancer stem cell markers CD133 and CD24 correlate with invasiveness and differentiation in colorectal adenocarcinoma.

AIM: To verify that CD markers are available for detecting cancer stem cell populations and to evaluate their clinical significance in colon cancer. METHODS: Immunohistochemistry for CD133, CD24 and CD44 was performed on the tissue microarray of 523 colorectal adenocarcinomas. Medical records were reviewed and clinicopathological analysis was performed. RESULTS: In colorectal adenocarcinoma, 128 of 523 cases (24.5%) were positive and 395 cases (75.5%) were negative for CD133 expression. Two hundred and sixty-four of 523 cases (50.5%) were positive and 259 cases (49.5%) were negative for CD24 expression. Five hundred and two of 523 cases (96%) were negative and 21 cases (4%) were positive for CD44 expression. Upon clinicopathological analysis, CD133 expression was present more in male patients (P = 0.002) and in advanced T stage cancer (P = 0.024). Correlation between CD24 expression and clinicopathological factors was seen in the degree of differentiation (P = 0.006). Correlation between CD44 expression and clinicopathological factors was seen in the tumor size (P = 0.001). Survival was not significantly related to CD133, CD24 and CD44 expression. CONCLUSION: CD markers were related to invasiveness and differentiation of colorectal adenocarcinoma. However, CD expression was not closely related to survival.

Distal urethral polypropylene sling surgical management for urodynamic stress incontinence in Korean women.

INTRODUCTION: The purpose of the present study was to assess the objective and subjective efficacy of the distal urethral polypropylene sling (DUPS) for urodynamic stress incontinence (USI) in Korean women. PATIENTS AND METHODS: We performed DUPS in 89 consecutive patients with USI. The Incontinence Impact Questionnaire (IIQ-7) and the Urogenital Distress Inventory (UDI-6) were used to evaluate the surgical outcomes. RESULTS: The mean operative time was 29.4 min (range 25-40). Concomitant procedures were performed including rectocele repair (n = 48), laparoscopically assisted vaginal hysterectomy (n = 12) and laparoscopic myomectomy (n = 1). There were no intraoperative complications or major postoperative complications. The average follow-up was 15 months (range 12-18). Both mean IIQ-7 and UDI-6 scores decreased significantly after DUPS. In addition, 87% of the patients reported no symptoms of USI under any circumstances and 95% of the patients reported never or rarely being bothered by USI. CONCLUSIONS: DUPS is a safe, inexpensive, simple, and effective surgical method for USI. The procedure provides a high cure rate in Korean women.

Synthesis of chroman-2-carboxylic acid N-(substituted)phenylamides and their inhibitory effect on nuclear factor-kappaB (NF-kappaB) activation.

A series of chroman-2-carboxylic acid N-(substituted)phenylamides (2a-s, 3a-j) were synthesized. Their ability to inhibit nuclear factor-kappaB (NF-kappaB) activity was evaluated in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells and their antioxidant activity was examined. NF-kappaB inhibition by chroman compounds was not related to their antioxidant activity. Compounds with -H, -NO2 monosubstituents and -OCH3, -CF3 disubstituents on the phenyl ring were poor inhibitors of NF-kB activity. Compounds with -CH3, -CF3, -CI monosubstituents or -CI, -CH3 disubstituents exhibited moderate to good NF-kappaB activity inhibition (IC50: 18.2-95.8 microM). The most active NF-kappaB inhibitor, 2s, contained a 4-CI (IC50: 18.2 microM) substituent on the phenyl ring and was slightly more potent than the compound KL-1156 (1) (IC50: 43.9 microM).

Epothilones induce human colon cancer SW620 cell apoptosis via the tubulin polymerization independent activation of the nuclear factor-kappaB/IkappaB kinase signal pathway.

Molecular mechanisms underlying epothilone-induced apoptotic cell death were investigated in SW620 human colon cancer cells. Treatment with epothilone B and D at different concentrations (1-100 nmol/L) dose-dependently inhibited cell growth and caused cell cycle arrest at G2-M, which was followed by apoptosis. Consistent with this induction of apoptotic cell death, epothilone B and D enhanced the constitutional activation of nuclear factor-kappaB (NF-kappaB) via IkappaB degradation through IkappaB kinase (IKKalpha and IKKbeta) activation, and this resulted in p50 and p65 translocation to the nucleus. Moreover, cells treated with sodium salicylic acid, an IKK inhibitor, or transiently transfected with mutant IKKalpha and beta did not show epothilone-induced cell growth inhibition or p50 translocation, although p65 was still translocated to the nucleus. Treatment with epothilone B and D also enhanced beta-tubulin polymerization and the formation of p50/beta-tubulin complex. However, beta-tubulin polymerization was not inhibited in the cells treated by sodium salicylic acid or transiently transfected with mutant IKKalpha and beta. Moreover, epothilone B and D increased the expressions of NF-kappaB-dependent apoptotic cell death regulatory genes, i.e., Bax, p53, and the active form of caspase-3, but reduced Bcl-2 expression, and these actions were partially reversed by salicylic acid. In addition, caspase-3 inhibitor reduced epothilone B-induced cell death and NF-kappaB activation. These findings suggest that the activation of NF-kappaB/IKK signals plays an important role in the epothilone-induced apoptotic cell death of SW620 colon cancer cells in a tubulin polymerization-independent manner.