High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood.

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%-90.0%) and excellent linear performance (R2 = 0.8189-0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

The apicoplast housekeeping machinery, specifically apicoplast DNA replication, transcription, and translation, was targeted by ciprofloxacin, thiostrepton, and rifampin, respectively, in the in vitro cultures of four Babesia species. Furthermore, the in vivo effect of thiostrepton on the growth cycle of Babesia microti in BALB/c mice was evaluated. The drugs caused significant inhibition of growth from an initial parasitemia of 1% for Babesia bovis, with 50% inhibitory concentrations (IC(50)s) of 8.3, 11.5, 12, and 126.6 µM for ciprofloxacin, thiostrepton, rifampin, and clindamycin, respectively. The IC(50)s for the inhibition of Babesia bigemina growth were 15.8 µM for ciprofloxacin, 8.2 µM for thiostrepton, 8.3 µM for rifampin, and 206 µM for clindamycin. The IC(50)s for Babesia caballi were 2.7 µM for ciprofloxacin, 2.7 µM for thiostrepton, 4.7 µM for rifampin, and 4.7 µM for clindamycin. The IC(50)s for the inhibition of Babesia equi growth were 2.5 µM for ciprofloxacin, 6.4 µM for thiostrepton, 4.1 µM for rifampin, and 27.2 µM for clindamycin. Furthermore, an inhibitory effect was revealed for cultures with an initial parasitemia of either 10 or 7% for Babesia bovis or Babesia bigemina, respectively. The three inhibitors caused immediate death of Babesia bovis and Babesia equi. The inhibitory effects of ciprofloxacin, thiostrepton, and rifampin were confirmed by reverse transcription-PCR. Thiostrepton at a dose of 500 mg/kg of body weight resulted in 77.5% inhibition of Babesia microti growth in BALB/c mice. These results implicate the apicoplast as a potential chemotherapeutic target for babesiosis.

Primary hepatic carcinoid tumor (a case report).

A primary hepatic carcinoid tumor arising in a 77-year-old woman is reported. The patient was admitted with a huge tumor in the right lobe of the liver and treated by an extended right lobectomy of the liver. Light microscopic findings showed that the tumor cells had small oval-shaped nuclei and eosinophilic cytoplasm with small granules forming trabecular, glandular, and rosette patterns. Immunohistochemically, tumor cells were stained positive with neuron-specific enolase and synaptophysin and were stained slightly positive with chromogranin and carcinoembryonic antigen. Careful examination before and after the operation revealed no other origin of the tumor. Based on the findings, the liver tumor was diagnosed as a primary carcinoid. The clinical features and diagnosis of this rare tumor are discussed in this report.

Indication of hepatectomy for cirrhotic patients with hepatocellular carcinoma classified as Child-Pugh class B.

We clarified the indication of partial hepatectomy in hepatocellular carcinoma (HCC) patients with liver cirrhosis classified as Child-Pugh class B. Univariate analysis revealed that adverse prognostic factors were (1) the presence of ascites, (2) elevated total bilirubin (1.5 mg/dl or higher), (3) reduced choline esterase (160 IU/ or lower), (4) elevated alpha-fetoprotein (AFP) (400 ng/ml or higher), (5) microscopic vascular invasion, and (6) non-curative hepatectomy. Microvascular invasion was excluded in the multivariate analysis because this factor could not be predicted before hepatectomy. Multivariate analysis revealed that independent adverse prognostic factors were (1) elevated total bilirubin (1.5 mg/dl or higher), (2) presence of ascites, (3) elevated AFP (400 ng/ml or higher), and (4) non-curative hepatectomy. The overall 5-year survival rate of patients with none of or only one of the four adverse prognostic factors was 45.8%. The overall 5-year survival rate of patients with two or more adverse prognostic factors was only 7.0%. Partial hepatectomy is the first choice of treatment for patients with none of or only one of the four adverse prognostic factors, whereas orthotopic liver transplantation or other conservative treatment should be considered for patients with two or more adverse prognostic factors.

Enzymatic degradation of p-chlorophenol in a two-phase flow microchannel system.

Enzymatic degradation of p-chlorophenol was carried out in a two-phase flow in a microchannel (100 microm width, 25 microm depth) fabricated on a glass plate (70 mm x 38 mm). This is the first report on the enzymatic reaction in a two-phase flow on a microfluidic device. The surface of the microchannel was partially modified with octadecylsilane groups to be hydrophobic, thus allowing clear phase separation at the end-junction of the microchannel. The enzyme (laccase), which is surface active, was solubilized in a succinic aqueous buffer and the substrate (p-chlorophenol) was in isooctane. The degradation of p-chlorophenol occurred mainly at the aqueous-organic interface in the microchannel. We investigated the effects of flow velocity and microchannel shape on the enzymatic degradation of p-chlorophenol. Assuming that diffusion of the substrate (p-chlorophenol) is the rate-limiting step in the enzymatic degradation of p-chlorophenol in the microchannel, we proposed a simple theoretical model for the degradation in the microchannel. The calculated degradation values agreed well with the experimental data.

Gasless laparoscopic hepatic resection for cirrhotic patients with solid liver tumors.

Gasless laparoscopic hepatic resection with a 5-cm minilaparotomy was performed in 10 cirrhotic patients with small liver tumors. To maintain good visualization and working space during hepatic resections, we developed a simple retraction system. Mean operative time and blood loss were 291 minutes and 249 mL, respectively. No blood transfusion was required during the operations. No serious complications occurred such as gas embolism. Our laparoscopic procedures had various advantages. Blood, smoke, and water vapor could be aspirated by suction without disturbance of the visual field. There was no risk of gas embolism. It was possible to use conventional instruments through the ports or the wound made by a minilaparotomy. Hemostasis therefore could be performed easily. The procedure could be applicable to cirrhotic patients with some complications. This laparoscopic procedure is recommended for patients with small HCCs associated with liver cirrhosis who are not candidates for major hepatectomy.

Significance of the molar ratio of branched-chain amino acids to tyrosine before hepatectomy.

BACKGROUND/AIMS: To predict the degree of liver fibrosis and select a safe operative procedure, the correlation between liver fibrotic ratio and preoperative liver function variables were analyzed, and the significance of the molar ratio of branched-chain amino acids to tyrosine determined preoperatively was investigated. METHODOLOGY: Forty-four patients with hepatectomy were enrolled in this study. Liver tissue specimens excised from the patients were stained by Azan-Mallory's method. The liver fibrotic ratio was measured using an Image Cytometer. The correlation between liver fibrotic ratio and preoperative liver function values, including the serum level of branched-chain amino acids to tyrosine, were determined. RESULTS: The degrees of liver fibrosis (normal group, 1.4 +/- 0.7%; chronic hepatitis group, 4.5 +/- 2.8%; cirrhosis group, 8.4 +/- 2.4%) correlated significantly with preoperative liver function values such as branched-chain amino acids to tyrosine, hepaplastin test, type IV collagen 7s domain, total bilirubin, indocyanine green clearance retention rate at fifteen minutes, and platelet count. The serum level of branched-chain amino acids to tyrosine showed the most-significant correlation with the degree of liver fibrosis. CONCLUSIONS: The serum level of branched-chain amino acids to tyrosine correlates well with the degree of liver fibrosis, and this value is a useful preoperative parameter for predicting the degree of liver fibrosis and for selecting a safe operative procedure.