Persistent Fluorescence Switching of a Probe Using a Photochromic Quencher with High Photostability Assisted by Protein-Surface Modification.

Photoswitchable fluorescent molecules (PSFMs) are widely applicable in the life sciences for super-resolution imaging. Owing to the large and hydrophobic molecular structures of PSFMs that may aggregate in a biological medium, the development of synthetic PSFMs with persistent reversible photoswitching is challenging. Here, we established a protein-surface-assisted photoswitching strategy that allows for persistent reversible fluorescence photoswitching of a PSFM in an aqueous solution. As a first step, we applied the photochromic chromophore furylfulgimide (FF) as a photoswitchable fluorescence quencher and developed a Förster resonance energy transfer-based PSFM, named FF-TMR. Most importantly, the protein-surface modification strategy allows FF-TMR to exhibit persistent reversible photoswitching performance in an aqueous solution. In fixed cells, the fluorescence intensity of FF-TMR bound to antitubulin antibody was repetitively modulated. The protein-surface-assisted photoswitching strategy will be a useful platform to broaden the utility of functionalized synthetic chromophores enabling persistent fluorescence switching that inherits their high resistance to light irradiation.

No-wash fluorogenic labeling of proteins for reversible photoswitching in live cells.

Photoswitchable fluorescent molecules (PSFMs) are positioned as valuable tools for biomolecule localization tracking and super-resolution imaging technologies due to their unique ability to reversibly control fluorescence intensity upon light irradiation. Despite the high demand for PSFMs that are suitable for live-cell imaging, no general method has been reported that enables reversible fluorescence control on proteins of interest in living cells. Herein, we have established a platform to realize reversible fluorescence switching in living cells by adapting a protein labeling system. We have developed a new PSFM, named HTL-Trp-BODIPY-FF, which exhibits strong fluorogenicity upon recognition of Halo-tag protein and reversible fluorescence photoswitching in living cells. This is the first example of a PSFM that can be applicable to a general-purpose Halo-tag protein labeling system for no-wash live-cell imaging.

Clinical role of FDG-PET for HCC: relationship of glucose metabolic indicator to Japan Integrated Staging (JIS) score.

BACKGROUND/AIMS: The purpose of this study was to validate the usefulness of positron emission tomography with fluorine-18-fluorodeoxyglucose (FDG-PET) in predicting degree of malignancy and prognosis, evaluated in terms of Japan Integrated Staging (JIS) score, in cirrhotic patients with hepatocellular carcinoma (HCC). METHODOLOGY: FDG-PET was performed in 50 patients with HCC. The activity within regions of interest placed over tumors was measured, and standardized uptake values (SUVs) were calculated by dividing the tissue activity by injected dose of radioactivity per unit body weight. SUV ratio (SUVR) was expressed as tumor-to-nontumor ratio of SUV. Patients were allocated to 3 groups of similar size: group A, SUVR < or = 1.1; group B, 1.1 < SUVR < 1.6; and group C, 1.6 < or = SUVR. RESULTS: SUVR significantly correlated with tumor node metastasis stage score (p < 0.001) or JIS score (p = 0.022). Survival rate in SUVR group C was significantly lower than that in group A (p < 0.001), and close to being significantly lower than that in group B. On multivariate analysis, JIS scores 2 and > or = 3, SUVR group C were significantly related to survival (All p < 0.05). CONCLUSIONS: SUVR was well associated with a tumor staging which is the factor of JIS score and with survival, and indicated malignancy and prognosis of patients especially with high-grade HCC.

Detection of local residual tumor after laryngeal cancer treatment using FDG-PET.

OBJECTIVE: Fluorine-18-fluorodeoxyglucose positron emission tomography (FDG-PET) is sometimes used as a means of follow-up after diagnosis and treatment of cancers of the head and neck region. The present study was undertaken to evaluate the ability of FDG-PET to detect local residual tumor after treatment of laryngeal cancer. METHODS: Thirty-six patients with laryngeal cancer underwent FDG-PET before and after initial treatment. Of these patients, 20 received FDG-PET before treatment and 28 received it after treatment. The relationship between standardized uptake values (SUV) and the presence or absence of local residual tumor was investigated by setting the cut-off value of the SUV using the receiver operating characteristics (ROC) curve. RESULTS: When the pre-treatment SUV threshold for laryngeal cancer was set at 7.20, the detection of local residual tumor after treatment using FDG-PET had a sensitivity of 77.78%, specificity of 81.82%, false positive rate of 18.18%, false negative rate of 22.22%, accuracy of 80% and a p value of 0.02. When the post-treatment SUV threshold for the larynx was set at 3.35, the test had a sensitivity of 93.75%, specificity of 91.67%, false positive rate of 8.33%, false negative rate of 6.25%, accuracy of 92.86% and a p value of 0.0001. CONCLUSIONS: FDG-PET was found to be useful for determining the presence of local residual tumor after treatment of laryngeal cancer.

A case of hepatic inflammatory pseudotumor identified by FDG-PET.

A 53-year-old man with a history of nausea and elevated liver functions presented to our clinic. A CT scan showed a small tumor in the right lobe of the liver. Fluorine-18-fluorodeoxyglucose (18F-FDG) positron emission tomography confirm abnormal metabolic activity with a high standardized uptake value of 7.3 in the lesion. These findings could indicate a malignancy such as well-differentiated hepatocellular carcinoma or cholangiocarcinoma, or a benign lesion such as hepatic abscess. He was diagnosed by histopathological examination as having an epithelioid granuloma with many inflammatory cells. This is the rare report of hepatic inflammatory pseudotumor featuring markedly increased 18F-FDG uptake.

Distinguishing benign from malignant gallbladder wall thickening using FDG-PET.

OBJECTIVE: Because thickening of the gallbladder wall is observed not only in patients with gallbladder cancer but also in those with benign diseases such as chronic cholecystitis and gallbladder adenomyosis, it is difficult to distinguish between benign and malignant gallbladder wall thickening by conventional techniques of diagnostic imaging such as computed tomography (CT), magnetic resonance imaging (MRI), and abdominal ultrasonography (US). In the present study, we attempted to distinguish between benign and malignant gallbladder wall thickening by means of fluorine-18-fluorodeoxyglucose (FDG)-Positron emission tomography (PET). METHODS: FDG-PET was performed in 12 patients with gallbladder wall thickening detected by CT or US, to determine whether it was benign or malignant. Emission scans were taken, beginning 45 minutes after intravenous administration of FDG, and SUV was calculated as an indicator of glucose metabolism. RESULTS: Of the 12 patients, 4 showed positive uptake of FDG in the gallbladder wall. Of these 4 patients, 3 had gallbladder cancer. The remaining one, who had chronic cholecystitis, had false-positive findings. The other 8 patients had negative uptake of FDG in the gallbladder wall. Two of these 8 underwent surgical resection, which yielded a diagnosis of chronic cholecystitis. The other 6 patients exhibited no sign of gallbladder malignancy and have been followed without active treatment. CONCLUSIONS: FDG-PET appears able to distinguish between benign and malignant gallbladder wall thickening.

3DSRT evaluation of responses of Alzheimer type dementia to donepezil hydrochloride therapy.

BACKGROUND: Dementia of Alzheimer type (DAT) has been diagnosed objectively by using single photon emission tomography (SPECT). Donepezil hydrochloride (donepezil) is available for the symptomatic treatment of DAT. In a quantitative evaluation of therapeutic response in DAT, to compare with regional cerebral blood flows (rCBF) of various lesions before and after treatment, uptake in some sorts of cerebral regions of interests (ROIs) were used to be measured. But ROI analysis has problems such as poor reproducibility and lack of objectivity. The aim of this study was to investigate the evaluation of therapeutic response by three-dimensional stereotaxic ROI template (3DSRT), fully automated ROI analysis software, which can objectively estimate rCBF. METHODS: SPECT studies and Alzheimer's Disease Assessment Scale Japan cognitive Subscale function test ADAS-Jcog, as recognitive function test were performed for 22 patients (16 females, 6 males mean age = 73.6 years) who were diagnosed as DAT. On 3DSRT, we compared ratios of the rCBF values of the parietal lobes, temporo-occipital lobes, hippocampus, corpus callosum and the frontal lobes/cerebellar hemispheres before and after medical treatment. To determine a cut-off number of areas exhibiting improved blood flow optimal as an indicator of improvement in cognitive function in response to treatment, receiver operating characteristics (ROC) analysis of number of areas improved blood flow was performed. RESULTS: The number of cases exhibiting changes in cognitive function was greatest when the cut-off number of areas exhibiting improved blood flow was set at 5. CONCLUSIONS: The possibility of evaluation of therapeutic response to Donepezil in patients with DAT using 3DSRT was thus demonstrated by our study.

Natural history of major complications in hepatitis C virus-related cirrhosis evaluated by per-rectal portal scintigraphy.

AIM: To examine the correlation between the porto-systemic hypertension evaluated by portal shunt index (PSI) and life-threatening complications, including hepatocellular carcinoma (HCC), liver failure (Child-Pugh stage progression), and esophagogastric varices. METHODS: Two hundred and twelve consecutive subjects with HCV-related cirrhosis (LC-C) underwent per-rectal portal scintigraphy. They were allocated into three groups according to their PSI: group I, PSI< or =10%; group II, 10%

Correlation of amino-acid uptake using methionine PET and histological classifications in various gliomas.

OBJECTIVE: The uptake of L-methyl-11C-methionine (MET) by gliomas is greater than that by intact tissue, making methionine very useful for evaluation of tumor extent. If the degree of malignancy of brain tumors can be evaluated by MET-PET, the usefulness of MET-PET as a means of diagnosing brain tumors will increase. METHODS: We performed this study on 67 glioma patients between 3 and 69 years of age (36 males and 31 females). Tumors included diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, ependymoma, oligodendroglioma, medulloblastoma, dysembryoplastic neuroepithelial tumor, choroid plexus papilloma, central neurocytoma, optic glioma, gliomatosis cerebri, pleomorphic xanthoastrocytoma, and ganglioglioma. Tumor activity and degree of malignancy were evaluated using Ki-67LI (LI: labeling index) and Kaplan-Meier survival curves. The correlations between methionine uptake and tumor proliferation (tumor versus contralateral gray matter ratio (T/N) and Ki-67LI) were determined for the group of all subjects. The existence of significant correlations between T/N and Ki-67LI and between SUV and Ki-67LI was determined for astrocytic tumors. Receiver operating characteristics (ROC) analysis of T/N and standardized uptake value (SUV) was performed for the group of astrocytic tumors. We also determined the ROC cut-off levels to ensure high accuracy of the analysis. RESULTS: For the 67 cases of glioma, the degree of accumulation was variable. Ki-67LI differed significantly between the high-grade group and low-grade group at T/N levels between 1.5 and 1.8 on analysis using tumor proliferative potential (p = 0.019-0.031). The prognosis differed significantly between the high-grade and low-grade groups when T/N was in the range of 1.6-1.8 (p = 0.028-0.032). The accuracy thus calculated was highest (85.7%) when T/N was 1.5 as determined by ROC analysis. CONCLUSIONS: When analysis was confined to cases of astrocytic tumor, a correlation was noted between methionine accumulation and Ki-67LI. For the astrocytic tumors, T/N ratio seemed to be more useful as a diagnostic indicator than SUV. The cut-off level of T/N ratio for distinction between high-grade and low-grade astrocytoma appears to lie between 1.5 and 1.6.

A case of diffuse hepatic angiosarcoma diagnosed by FDG-PET.

A 76-year-old woman was admitted to our hospital with a 2-month history of increasing abdominal distension, leg edema, and dyspnea. The serum transaminase level was about twice the upper limit of normal. The CT showed no tumor. Fluorine-18 2-deoxy-2-fluoro-D-glucose (FDG)-positron emission tomography (PET) showed diffuse abnormal accumulation throughout the entire liver. She was diagnosed by histopathological examination as having hepatic angiosarcoma causing veno-occlusive disease (VOD). This is the first report of hepatic angiosarcoma with FDG-PET.

Splenic accumulation of Tc-99m HMDP in a patient with severe alcoholic cirrhosis of the liver.

The authors report unusual splenic accumulation of Tc-99m hydroxymethylene diphosphonate (HMDP) on bone scintigraphy of a patient with alcoholic cirrhosis of the liver. Laboratory studies showed hemolytic anemia and hemochromatosis resulting from alcohol abuse, both of which are thought to be related to the diffuse splenic uptake observed. When diffuse splenic accumulation by Tc-99m HMDP is seen, the existence of alcoholic hepatopathy might be considered.

Natural course of changes in hepatic functional reserve in patients with chronic liver diseases evaluated by scintigraphy with GSA.

Hepatic functional reserve can be evaluated in a noninvasive way by scintigraphy with 99mTc-DTPA-galactosyl human serum albumin (99mTc-GSA). We monitored hepatic functional reserve in patients with chronic hepatitis and cirrhosis using scintigraphy with 99mTc-GSA to determine the natural course of changes in their hepatic functional reserve. Computer acquisition of gamma-camera data was started before the injection of 185 MBq of 99mTc-GSA and was stopped 20 min later. Time-activity curves were generated from ROI for the heart and liver. A receptor index and index of blood clearance were calculated from radioactivity in the heart and liver. Scintigraphy with 99mTc-GSA was performed in 12 healthy subjects, 86 patients with chronic hepatitis, and 226 patients with cirrhosis. Seventy-two patients (23 with chronic hepatitis, 32 with cirrhosis in Child-Pugh stage A, 15 in stage B, and 2 in stage C) were examined at least twice with 12-72 months intervening. The receptor index was lower for more severe disorders, decreasing in the order of chronic hepatitis and cirrhosis in stages A, B, and C. The index of blood clearance was higher for more severe disorders, increasing in the order of chronic hepatitis and cirrhosis in stages A, B, and C. The mean annual change in the receptor index with chronic hepatitis was -0.0007, that with cirrhosis in stage A was -0.0023, and that with cirrhosis in stage B or C was -0.0117. The difference between the median annual change with cirrhosis in stage B or C and that with chronic hepatitis or cirrhosis in stage A was not significant (P=0.064 and 0.251, respectively). The mean annual change in the index of blood clearance with chronic hepatitis was 0.0018, that with cirrhosis in stage A was 0.0060, and that with cirrhosis in stage B or C was 0.0330. The difference between the median annual change in the index of blood clearance with cirrhosis in stage B or C and that with chronic hepatitis or cirrhosis in stage A was significant (P=0.004 and 0.007, respectively). Hepatic receptor imaging with 99mTc-GSA could be used to noninvasively evaluate the hepatic reserve of various liver diseases. Changes in hepatic functional reserve were not steady; it decreased gradually as disease advanced from chronic hepatitis to cirrhosis in Child-Pugh stage A, and decrease rapidly after development of stage B cirrhosis.

Evaluation of ammonia metabolism in the skeletal muscles of patients with cirrhosis using N-13 ammonia PET.

OBJECTIVE: Skeletal muscle is said to compensate for the decreased ammonia metabolism in patients with cirrhosis. Branched-chain amino acids (BCAA) are being used as a treatment for hyperammonemia, and are believed to decrease blood ammonia by consumption of BCAA in skeletal muscles. We examined ammonia metabolism of the skeletal muscles in patients with liver cirrhosis after administration of BCAA using 13N-ammonia positron emission tomography (PET). METHODS: The subjects were patients with compensated or decompensated liver cirrhosis. PET studies were performed before and 2 hours after injection of BCAA. Serial dynamic PET scans (2 min x 10 frames) were started simultaneously with 13N-ammonia injection. The standardized uptake value (SUV) of both thighs was calculated. RESULTS: In the patient with compensated liver cirrhosis, there was little difference in the rate of increase in SUV before to after administration of BCAA. However, in the patient with decompensated liver cirrhosis, the rate of increase in SUV after administration was higher than that before administration of BCAA. CONCLUSION: Ammonia metabolism in the muscle of patients with liver cirrhosis could be examined noninvasively under physiological conditions using 13N-ammonia PET. The muscles were found to metabolize ammonia partially, and the role of this contribution to metabolism of ammonia in patients with decompensated liver cirrhosis is particularly important.

A case of gastric duplication evaluated by gastric emptying scintigraphy.

Gastric duplications are relatively rare, and communication with the gastric lumen is extremely rare. A 67-year-old man was referred to our hospital because of recurrence of epigastric pain and fullness. An upper gastrointestinal contrast study revealed a double compartment stomach, with gastric duplication starting at the esophagogastric junction outside the greater curvature. Computed tomography of the stomach with gastrografin as contrast demonstrated complete communication of the gastric duplication and primary stomach. The patient was diagnosed with complete gastric duplication. Gastric emptying scintigraphy with Tc-99m diethyltriamine pentaacetic acid was performed. Test meal entered the primary stomach and duplication cyst simultaneously, and radioactivity in the primary stomach decreased linearly and gastric emptying was not delayed. In the duplication cyst, about 70% of the food that entered the cyst once was immediately evacuated from it, but the remaining 30% remained in the cyst for a long time. Gastric emptying of the primary stomach was not affected by formation of the duplication cyst.

A case of small cell carcinoma of the esophagus detected incidentally by FDG-PET.

Small cell carcinoma (SmC) of the esophagus is rare, and is sometimes impossible to detect by macroscopic inspection using an endoscope or histological examination of biopsied specimens. A 73-year-old man received F-18 fluorodeoxyglucose positron emission tomography (FDG-PET) to evaluate the response to radiofrequency thermal ablation therapy for lung cancer. FDG-PET showed abnormal accumulation in the posterior mediastinum. Endoscopy disclosed ulcerous lesions with marginal elevation in the middle segment of the esophagus, but the biopsy specimen taken concurrently was not malignant histologically. FDG-PET, performed two months later, revealed abnormal accumulation in the suspect area, and the extent of accumulation was wider than previously. Histological examination of the specimen biopsied during the endoscopy led to a diagnosis of SmC. FDG-PET thus proved useful in the early detection of SmC.

Methionine positron emission tomography for differentiation of recurrent brain tumor and radiation necrosis after stereotactic radiosurgery--in malignant glioma.

OBJECT: Following stereotactic radiosurgery (SRS), we examined how to differentiate radiation necrosis from recurrent malignant glioma using positron emission tomography (PET) with 11C-methionine (Met). METHODS: Met-PET scans were obtained from 11 adult cases of recurrent malignant glioma or radiation injury, suspected on the basis of magnetic resonance images (MRI). Patients had previously been treated with SRS after primary treatment. PET images were obtained as a static scan of 10 minutes performed 20 minutes after injection of Met. We defined two visual grades (e.g., positive or negative Met accumulation). On Met-PET scans, the portion of the tumor with the highest accumulation was selected as the region of interest (ROI), tumor-versus-normal ratio (TN) was defined as the ratio of average radioisotope counts per pixel in the tumor (T), divided by average counts per pixel in normal gray matter (N). The standardized uptake value (SUV) was calculated over the same tumor ROI. Met-PET scan accuracy was evaluated by correlating findings with subsequent histological analysis (8 cases) or, in cases without surgery or biopsy, by the subsequent clinical course and MR findings (3 cases). RESULTS: Histological examinations in 8 cases showed viable glioma cells with necrosis in 6 cases, and necrosis without viable tumor cells in 2 cases. Three other cases were considered to have radiation necrosis because they exhibited stable neurological symptoms with no sign of massive enlargement of the lesion on follow-up MR after 5 months. Mean TN was 1.31 in the radiation necrosis group (5 cases) and 1.87 in the tumor recurrence group (6 cases). Mean SUV was 1.81 in the necrosis group and 2.44 in the recurrence group. There were no statistically significant differences between the recurrence and necrosis groups in TN or SUV. Furthermore, we made a 2 x 2 factorial cross table (accumulation or no accumulation, recurrence or necrosis). From this result, the Met-PET sensitivity, specificity, and accuracy in detecting tumor recurrence were determined to be 100%, 60%, and 82% respectively. In a false positive-case, glial fibrillary acidic protein (GFAP) immunostaining showed a positive finding. CONCLUSION: There were no significant differences between recurrent malignant glioma and radiation necrosis following SRS in Met-PET. However, this study shows Met-PET has a sensitivity and accuracy for differentiating between recurrent glioma and necrosis, and presents important information for developing treatment strategies against post radiation reactions.

Poor labeling of Tc-99m red blood cells in vivo in a radionuclide intestinal bleeding study of a patient who had recently undergone frequent blood transfusions.

The authors report poor labeling of Tc-99m red blood cells (RBCs) in vivo in a radionuclide intestinal bleeding study of a patient who had recently undergone frequent blood transfusions. The existence of RBC antibodies, as a result of the recent blood transfusions in this patient, was one of the causes of the poor labeling. In radionuclide bleeding studies with patients with recent blood transfusion, Tc-99m HSA-D must be chosen instead of Tc-99m RBCs in vivo.