Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1alpha (hepatocyte nuclear factor 1alpha). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1alpha exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1alpha are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1alpha expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1alpha, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.

A multiplex real-time PCR (qPCR) assay was developed for simultaneous detection and differentiation of the three most important Campylobacter species in chickens. Three novel sets of PCR primers and TaqMan probes were designed to amplify the unique DNA sequences within the hipO, cdtA, and pepT genes which are specific to Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, respectively. To avoid competition in the multiple target amplifications, the concentrations of primers and probes were optimized. By using the optimized qPCR conditions together with a minor-groove binding probe of pepT, amplification efficiency greater than 92% and detection sensitivity of 38 genome copies/reaction have been achieved for all three targets. The assay was highly specific for C. jejuni, C. coli, and C. lari with testing of 33 Campylobacter strains and 20 non-Campylobacter strains. In chicken samples spiked with known quantities of Campylobacter cells, the assay was able to detect 1 CFU/ g after a 24-h enrichment. Application of the assay in food was further evaluated using 21 fresh chicken samples obtained from local supermarkets. The results revealed that, after a 24-h or 48-h enrichment, 14 samples (66.7%)were positive for C. jejuni, five samples (23.8%) were positive for C. coli, and none of the samples was contaminated by C. lari. Taken together, the multiplex qPCR assay combined with an enrichment step is a sensitive, species-specific, and non-labor-intensive method suitable for rapid detection of C. jejuni, C. coli, and C. lari in chicken samples.