A Case of Primary Paraganglioma that Arose in the Pancreas: the Color Doppler Ultrasonography and Dynamic CT Features

Paragangliomas rarely originate from the pancreas and they are characterized on imaging studies as well-marginated, hypervascular masses with cystic areas. We herein report on a case report of pancreatic paraganglioma in a 57-year-old woman, which was confirmed on pathology. Color Doppler ultrasonography and dynamic CT demonstrated a well-demarcated, extremely hypervascular mass with prominent intratumoral vessels and early contrast filling of the draining veins from the mass. Endoscopic retrograde cholangiopancreatography showed that the main pancreatic duct was displaced and mildly dilated

Strategy for <i>in</i><i>vivo</i> cloning by the recruitment of the translation initiation sequence for antibiotic marker expression.

<p>(A) The receiver plasmid, pRMT, contains a silent chloramphenicol resistance gene (Cmr) that is activated by homologous recombination with the target DNA in <i>E. coli</i> cells. The DNA insert is comprised of 5'-homologous sequences (H1), target gene sequences, the RBS plus ATG for activation of the silent selection marker, and 3'-homologous sequences (H2). F and R indicate primers with specific sequences for the amplification of the target DNA. P_T7 and T_T7 represent the T7 promoter and T7 terminator, respectively. (B) Shown are the sequences of the homologous region used to prepare the inserts with the target gene and the region for homologous recombination with the silent chloramphenicol resistance gene in receiver plasmid, pRMT and pRMT-sacB.</p

A Cell–Cell Communication-Based Screening System for Novel Microbes with Target Enzyme Activities

The development of synthetic biological devices has increased rapidly in recent years and the practical benefits of such biological devices are becoming increasingly clear. Here, we further improved the design of a previously reported high-throughput genetic enzyme screening system by investigating device-compatible biological components and phenol-mediated cell–cell communication, both of which increased the efficiency and practicality of the screening device without requiring the use of flow cytometry analysis. A sensor cell was designed to detect novel microbes with target enzyme activities on solid media by forming clear, circular colonies with fluorescence around the unknown microbes producing target enzymes. This mechanism of detection was enabled by the combination of pre-effector phenolic substrate treatment in the presence of target enzyme-producing microbes and control of the growth and fluorescence of remote sensor cells <i>via</i> phenol-mediated cell–cell communication. The sensor cells were applied to screen soil bacteria with phosphatase activity using phenyl phosphate as phenolic substrates. The sensor cells facilitated successful visualization of phosphatase activity in unknown microbes, which were identified by 16S rRNA analysis. Enzyme activity assays confirmed that the proposed screening technique was able to find 23 positive clones out of 33 selected colonies. Since many natural enzymatic reactions produce phenolic compounds from phenol-derived substrates, we anticipate that the proposed technique may have broad applications in the assessment and screening of novel microbes with target enzymes of interest. This method also can provide insights into the identification of novel enzymes for which screening assays are not yet available

The homogeneity problem and the solution using a counter selection marker, <i>sacB</i>.

<p>(A) Shown are the genetic structure and scheme of the pRMT and pRMT-sacB plasmids when the GFPuv gene was recombined into both plasmids. (B) Shown is the restriction analysis of the target plasmids from selected hits. upper and lower panels represent the electrophoresis results from the pRMT and pRMT-sacB plasmids, respectively, digested with <i>Bgl</i>II and <i>Sca</i>I. The C- and C+ represent the indicator bands of one without and one with the insert, respectively while the GFPuv gene was used as a target gene.</p

Generating <i>In Vivo</i> Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

<div><p>A robust method for the <i>in vivo</i> cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into <i>Escherichia coli</i> cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the <i>sacB</i> gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the <i>E. coli</i> cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into <i>E. coli</i> cells.</p> </div

Restored antibiotic resistance and GFPuv expression by homologous recombination.

<p>(A) <i>E. coli</i> JM109 cells containing the pRMT plasmid were sensitive to 25 μg/l of chloramphenicol in LB broth because the chloramphenicol resistance gene is silent in the receiver plasmid. (B) GFPuv expression is observed in the positive colonies. (C) The effect of the amount of DNA insert on <i>in </i><i>vivo</i> cloning. The DNA inserts were transformed into the cells containing the pRMT vector by electroporation. The colony number increases with higher concentrations of the insert used for electroporation. (D) The effect of the homologous sequence length on the pRMT system. </p

Size dependency and the cloning of the carotenoid synthetic gene clusters.

<p>(A) Shown are the genetic structures of the various carotenoid synthetic gene clusters used in this study. P_T7 in synthetic gene cluster represent the T7 promoter. P1, P2, and P3 refer to additional promoters. (B) Shown are <i>E. coli</i> colonies expressing the various target genes: the 2-kb insert exhibited green fluorescence, while the 4-kb insert produced yellow-pigmented colonies. The 6-kb and 10-kb inserts showed mixed colonies of yellow and red, the colors of β-carotenoid and astaxanthin, respectively. (C) Analysis of the effect of insert size on the cloning efficiency.</p