The critical roles of tumor-initiating cells and the lymph node stromal microenvironment in human colorectal cancer extranodal metastasis using a unique humanized orthotopic mouse model.

Colorectal cancer (CRC) is the second-most common cause of cancer-related mortality. The most important prognostic factors are lymph node (LN) involvement and extranodal metastasis. Our objective is to investigate the interactions between CD133(+)CXCR4(+) (CXC receptor 4) colorectal cancer tumor-initiating cells (Co-TICs) and the LN stromal microenvironment in human CRC extranodal metastasis. We established a unique humanized orthotopic xenograft model. Luciferase-tagged CRC cell lines and human cancer cells were injected intrarectally into nonobese diabetic/SCID mice. Mesenteric LN stromal cells, stromal cell line HK, or CXCL12 knockdown HK (HK-KD-A3) cells were coinoculated with CRC cells. Tumor growth and metastasis were monitored by bioluminescent imaging and immunohistochemistry. We found that this model mimics the human CRC metastatic pattern with CRC cell lines or patient specimens. Adding LN stromal cells promotes CRC tumor growth and extranodal metastasis (P < 0.001). Knocking down CXCL12 impaired HK cell support of CRC tumor formation and extranodal metastasis. When HK cells were added, sorted CD133(+)CXCR4(+) Co-TICs showed increased tumor formation and extranodal metastasis capacities compared to unseparated and non-Co-TIC populations. In conclusion, both Co-TIC and LN stromal factors play crucial roles in CRC metastasis through the CXCL12/CXCR4 axis. Blocking Co-TIC/LN-stromal interactions may lead to effective therapy to prevent extranodal metastasis.

A noncommercial polymerase chain reaction-based method to approach one hundred percent recombinant clone selection efficiency.

Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones.

Routine cholecystectomy is not mandatory during morbid obesity surgery.

BACKGROUND: Controversy exists concerning the utility of routine cholecystectomy during bariatric surgery. We report our series of bariatric surgical procedures at our institution without concurrent cholecystectomy. METHODS: From October 2003 to August 2005, 621 morbidly obese patients underwent a weight loss operation. Preoperatively, each patient had undergone abdominal ultrasound (AUS) to evaluate for abnormal gallbladder findings. Patients with previous cholecystectomy were excluded. Symptomatic patients with AUS findings consistent with gallbladder disease underwent concomitant cholecystectomy and bariatric surgery. Asymptomatic patients, despite AUS findings, did not undergo cholecystectomy with their bariatric operation. A comparison between the preoperative AUS-positive and AUS-negative, asymptomatic patients after bariatric surgery was performed. RESULTS: Of the 621 patients who underwent bariatric surgery, 170 (27%) had undergone previous cholecystectomy and were excluded. Of the remaining 451 patients, 17 with positive AUS findings and symptoms underwent cholecystectomy during bariatric surgery. The range of follow-up was 4-25 months. Of the 451 patients, 324 were asymptomatic and had negative AUS findings and 102 were asymptomatic and had positive AUS findings for gallbladder abnormalities. Postoperatively, 29 asymptomatic/AUS-negative patients (9%) developed symptoms and had positive AUS findings. Nine asymptomatic patients with AUS positive findings (9%) developed symptoms. Finally, 38 patients (8.4%) went on to undergo elective cholecystectomy. These 2 groups were not signficantly different statistically. CONCLUSIONS: In this study, the development of symptomatic/AUS-positive gallbladder abnormalities was low after obesity surgery, suggesting that mandatory cholecystectomy is not required at bariatric surgery.

Class I histone deacetylase Thd1p affects nuclear integrity in Tetrahymena thermophila.

Class I histone deacetylases (HDACs) participate in the regulation of DNA-templated processes such as transcription and replication. Members of this class can act locally at specific sites, or they can act more globally, contributing to a baseline acetylation state, both of which actions may be important for genome maintenance and organization. We previously identified a macronuclear-specific class I HDAC in Tetrahymena thermophila called Thd1p, which is expressed early in the development of the macronucleus when it initially becomes transcriptionally active. To test the idea that Thd1p is important for global chromatin integrity in an active macronucleus, Tetrahymena cells reduced in expression of Thd1p were generated. We observed phenotypes that indicated loss of chromatin integrity in the mutant cells, including DNA fragmentation and extrusion of chromatin from the macronucleus, variable macronuclear size and shape, enlarged nucleoli, and reduced phosphorylation of histone H1 from bulk chromatin. Macronuclei in mutant cells also contained more DNA. This observation suggests a role for Thd1p in the control of nuclear DNA content, a previously undescribed role for class I HDACs. Together, these phenotypes implicate Thd1p in the maintenance of macronuclear integrity in multiple ways, probably through site-specific changes in histone acetylation since no change in the acetylation levels of bulk histones was detected in mutant cells.