Development of a fluorescent probe library enabling efficient screening of tumour-imaging probes based on discovery of biomarker enzymatic activities.

Fluorescent probes that can selectively detect tumour lesions have great potential for fluorescence imaging-guided surgery. Here, we established a library-based approach for efficient screening of probes for tumour-selective imaging based on discovery of biomarker enzymes. We constructed a combinatorial fluorescent probe library for aminopeptidases and proteases, which is composed of 380 probes with various substrate moieties. Using this probe library, we performed lysate-based in vitro screening and/or direct imaging-based ex vivo screening of freshly resected clinical specimens from lung or gastric cancer patients, and found promising probes for tumour-selective visualization. Further, we identified two target enzymes as novel biomarker enzymes for discriminating between tumour and non-tumour tissues. This library-based approach is expected to be an efficient tool to develop tumour-imaging probes and to discover new biomarker enzyme activities for various tumours and other diseases.

Circulating osteocalcin levels were not significantly associated with the risk of incident type 2 diabetes mellitus in elderly Japanese men: The Fujiwara-kyo Osteoporosis Risk in Men (FORMEN) Cohort Study.

INTRODUCTION: Cross-sectional studies have shown that patients with type 2 diabetes mellitus (T2DM) have low circulating levels of osteocalcin (OC) and undercarboxylated OC (ucOC). This longitudinal study aimed to examine whether low OC or ucOC levels at baseline are associated with the risk of incident T2DM. METHODS: We examined 1700 community-dwelling Japanese men (≥65 years) after excluding those with history of diseases (other than T2DM) or medications that affect bone and glucose metabolism. T2DM was defined as fasting plasma glucose (FPG) ≥126 mg/dl or glycated hemoglobin A1c (HbA1c) ≥6.5%. Participants without prevalent T2DM at baseline were invited to follow-up surveys 5 and 10 years after baseline. RESULTS: Among the participants, 309 with prevalent T2DM showed significantly lower serum OC and ucOC levels at baseline than those without. After excluding these participants, 46 and 57 participants with incident T2DM were identified in the first and second follow-up surveys, respectively. These participants did not show significantly different OC and ucOC levels at baseline relative to those without T2DM, although their FPG and HbA1c levels at baseline were significantly higher compared to those without incident T2DM. Increase in glycemic indices preceded decrease in OC and ucOC levels. OC and ucOC levels at baseline were not significantly associated with the risk of incident T2DM identified in the follow-up surveys. CONCLUSIONS: OC and ucOC levels at baseline were not significantly associated with the risk of incident T2DM. Our results do not support the findings of animal studies that ucOC is a hormone regulating glucose metabolism.

Fluorescence Probes for Imaging Basic Carboxypeptidase Activity in Living Cells with High Intracellular Retention.

Basic carboxypeptidases (basic CPs) cleave the C-terminal basic amino acid of peptides, and their activity is upregulated in some types of cancers. Therefore, detecting the activity of basic CPs in living cells would be important not only for studying the physiological functions of these enzymes but also for visualization of cancerous tissues. Here, we report two fluorescein diacetate (FDA)-based activatable fluorescence probes, named 5ArgAF-FDA and 5LysAF-FDA, in which the substrate amino acid arginine or lysine is conjugated to the benzene moiety via an azoformyl linker. In live-cell fluorescence imaging of CPM, one of the seven basic CPs, 5ArgAF-FDA showed a larger intracellular fluorescence increase than did 5LysAF-FDA within a few minutes. This increase was inhibited by coincubation with 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), an inhibitor of basic CPs. When 5ArgAF-FDA was applied to a coculture of two breast cancer cell lines with different CPM activities, the fluorescence increase in individual cells was correlated with the expression level of CPM, suggesting that 5ArgAF-FDA has the ability to distinguish cell lines having different levels of CPM activity, owing to its high intracellular retention. We believe these probes will be useful for imaging cancers with upregulated basic CP activity.

Maxillary Swing Approach for Removal of Palatal Carcinoma: A Modified Procedure.

INTRODUCTION: We report a modification of the maxillary swing approach to remove a palatal tumor while preserving the anterior alveolar area. METHODS: Case report using clinical records. RESULTS: The patient was a 54-year-old male. TNM grade was T4bN0M0, and invasion to the base of the pterygoid process was seen. Two courses of induction chemotherapy were administered prior to the operation. Because there was no evidence of anterior maxillary invasion, the maxillary swing approach was chosen. The left anterior maxilla was cut and swung laterally, preserving the blood supply. After removal of the palatal tumor, the maxilla was repositioned and the defect was restored with an anterior lateral thigh flap. Postoperative course was typical, and facial appearance, speech, and masticatory function were satisfactory. CONCLUSIONS: This technique is particularly useful for preserving appearance as well as speech and mastication.