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1.  Magnetic Enrichment of Bronchial Epithelial Cells From Sputum for Lung Cancer Diagnosis 
Cancer  2008;114(4):275-283.
Sputum is an easily accessible diagnostic material for lung cancer early detection by cytologic and molecular genetic analysis of exfoliated airway epithelial cells. However, the use of sputum is limited by its cellular heterogeneity, which includes >95% macrophages and neutrophils and only about 1% bronchial epithelial cells. We propose to obtain concentrated and purified bronchial epithelial cells to improve early detection of lung cancer in sputum samples.
Sputum was collected from patients with stage I nonsmall-cell lung cancer, cancer-free smokers, and healthy nonsmokers. Magnetic-assisted cell sorting (MACS) with anti-CD14 and anti-CD16 antibody beads were used to enrich bronchial epithelial cells by depleting macrophages and neutrophils from sputum. Fluorescence in situ hybridization (FISH) analysis for detection of FHIT deletion and cytology were evaluated in the enriched specimens.
The bronchial epithelial cells were concentrated to 40% purity from 1.1% of the starting population, yielding an average of 36-fold enrichment and at least 2.3 × 105 cells per sample. Detecting FHIT deletions for lung cancer diagnosis produced 58% sensitivity in the enriched sputum, whereas there was 42% sensitivity in the unenriched samples (P = .02). Cytologic examination of the enriched sputum resulted in 53% sensitivity, as compared with 39% sensitivity in unenriched sputum (P = .03). Furthermore, only 2 cytocentrifuge slides of the unenriched sputum were needed for the analyses, as compared with up to 10 cytocentrifuge slides required from the unprocessed specimens.
The enrichment of bronchial epithelial cells could improve the diagnostic value of sputum and the efficiency of genetic and cytologic analysis of lung cancer.
PMCID: PMC4255556  PMID: 18484646
magnetic-assisted cell sorting; bronchial epithelial cells; sputum; lung cancer; diagnosis
2.  Sphingosine-1-Phosphate Regulates the Expression of Adherens Junction Protein E-Cadherin and Enhances Intestinal Epithelial Cell Barrier Function 
Digestive diseases and sciences  2010;56(5):1342-1353.
The regulation of intestinal barrier permeability is important in the maintenance of normal intestinal physiology. Sphingosine-1-phosphate (S1P) has been shown to play a pivotal role in enhancing barrier function in several non-intestinal tissues. The current study determined whether S1P regulated function of the intestinal epithelial barrier by altering expression of E-cadherin, an important protein in adherens junctions.
Studies were performed upon cultured differentiated IECs (IEC-Cdx2L1 line) using standard techniques.
S1P treatment significantly increased levels of E-cadherin protein and mRNA in intestinal epithelial cells (IECs) and also led to E-cadherin localizing strongly to the cell–cell border. S1P also improved the barrier function as indicated by a decrease in 14C-mannitol paracellular permeability and an increase in transepithelial electrical resistance (TEER) in vitro.
These results indicate that S1P increases levels of E-cadherin, both in cellular amounts and at the cell–cell junctions, and leads to improved barrier integrity in cultured intestinal epithelial cells.
PMCID: PMC4140085  PMID: 20936358
Sphingosine; Intestinal barrier; Ca2+ signaling; Intestinal epithelium
3.  Global spatiotemporal and genetic footprint of the H5N1 avian influenza virus 
Since 2005, the Qinghai-like lineage of the highly pathogenic avian influenza A virus H5N1 has rapidly spread westward to Europe, the Middle East and Africa, reaching a dominant level at a global scale in 2006.
Based on a combination of genetic sequence data and H5N1 outbreak information from 2005 to 2011, we use an interdisciplinary approach to improve our understanding of the transmission pattern of this particular clade 2.2, and present cartography of global spatiotemporal transmission footprints with genetic characteristics.
Four major viral transmission routes were derived with three sources— Russia, Mongolia, and the Middle East (Kuwait and Saudi Arabia)—in the three consecutive years 2005, 2006 and 2007. With spatiotemporal transmission along each route, genetic distances to isolate A/goose/Guangdong/1996 are becoming significantly larger, leading to a more challenging situation in certain regions like Korea, India, France, Germany, Nigeria and Sudan. Europe and India have had at least two incursions along multiple routes, causing a mixed virus situation. In addition, spatiotemporal distribution along the routes showed that 2007/2008 was a temporal separation point for the infection of different host species; specifically, wild birds were the main host in 2005–2007/2008 and poultry was responsible for the genetic mutation in 2009–2011. “Global-to-local” and “high-to-low latitude” transmission footprints have been observed.
Our results suggest that both wild birds and poultry play important roles in the transmission of the H5N1 virus clade, but with different spatial, temporal, and genetic dominance. These characteristics necessitate that special attention be paid to countries along the transmission routes.
PMCID: PMC4059878  PMID: 24885233
Spatiotemporal; Genetic; Avian influenza virus; H5N1; Transmission footprint; Global
4.  Diagnosis of lung cancer in individuals with solitary pulmonary nodules by plasma microRNA biomarkers 
BMC Cancer  2011;11:374.
Making a definitive preoperative diagnosis of solitary pulmonary nodules (SPNs) found by CT has been a clinical challenge. We previously demonstrated that microRNAs (miRNAs) could be used as biomarkers for lung cancer diagnosis. Here we investigate whether plasma microRNAs are useful in identifying lung cancer among individuals with CT-detected SPNs.
By using quantitative reverse transcriptase PCR analysis, we first determine plasma expressions of five miRNAs in a training set of 32 patients with malignant SPNs, 33 subjects with benign SPNs, and 29 healthy smokers to define a panel of miRNAs that has high diagnostic efficiency for lung cancer. We then validate the miRNA panel in a testing set of 76 patients with malignant SPNs and 80 patients with benign SPNs.
In the training set, miR-21 and miR-210 display higher plasma expression levels, whereas miR-486-5p has lower expression level in patients with malignant SPNs, as compared to subjects with benign SPNs and healthy controls (all P ≤ 0.001). A logistic regression model with the best prediction was built on the basis of miR-21, miR-210, and miR-486-5p. The three miRNAs used in combination produced the area under receiver operating characteristic curve at 0.86 in distinguishing lung tumors from benign SPNs with 75.00% sensitivity and 84.95% specificity. Validation of the miRNA panel in the testing set confirms their diagnostic value that yields significant improvement over any single one.
The plasma miRNAs provide potential circulating biomarkers for noninvasively diagnosing lung cancer among individuals with SPNs, and could be further evaluated in clinical trials.
PMCID: PMC3175224  PMID: 21864403
5.  Small nucleolar RNA signatures as biomarkers for non-small-cell lung cancer 
Molecular Cancer  2010;9:198.
Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC will improve its outcome. The current techniques for NSCLC early detection are either invasive or have low accuracy. Molecular analyses of clinical specimens present promising diagnostic approaches. Non-coding RNAs (ncRNAs) play an important role in tumorigenesis and could be developed as biomarkers for cancer. Here we aimed to develop small nucleolar RNAs (snoRNAs), a common class of ncRNAs, as biomarkers for NSCLC early detection. The study comprised three phases: (1) profiling snoRNA signatures in 22 NSCLC tissues and matched noncancerous lung tissues by GeneChip Array, (2) validating expressions of the signatures by RT-qPCR in the tissues, and (3) evaluating plasma expressions of the snoRNAs in 37 NSCLC patients, 26 patients with chronic obstructive pulmonary disease (COPD), and 22 healthy subjects.
In the surgical tissues, six snoRNAs were identified, which were overexpressed in all tumour tissues compared with their normal counterparts. The overexpressions of the genes in tumors were confirmed by RT-qPCR. The snoRNAs were stably present and reliably detectable in plasma. Of the six genes, three (SNORD33, SNORD66 and SNORD76) displayed higher plasma expressions in NSCLC patients compared with the cancer-free individuals (All < 0.01). The use of the three genes produced 81.1% sensitivity and 95.8% specificity in distinguishing NSCLC patients from both normal and COPD subjects. The plasma snoRNA expressions were not associated with stages and histological types of NSCLC (All > 0.05).
The identified snoRNAs provide potential markers for NSCLC early detection.
PMCID: PMC2919450  PMID: 20663213
6.  Sphingosine-1-Phosphate Protects Intestinal Epithelial Cells from Apoptosis Through the Akt Signaling Pathway 
Digestive diseases and sciences  2008;54(3):499-510.
The regulation of apoptosis of intestinal mucosal cells is important in maintenance of normal intestinal physiology.
Sphingosine-1-phosphate (S1P) has been shown to play a critical role in cellular protection to otherwise lethal stimuli in several nonintestinal tissues.
The current study determines whether S1P protected normal intestinal epithelial cells (IECs) from apoptosis and whether Akt activation was the central pathway for this effect.
S1P demonstrated significantly reduced levels of apoptosis induced by tumor necrosis factor-alpha (TNF-α)/cycloheximide (CHX). S1P induced increased levels of phosphorylated Akt and increased Akt activity, but did not affect total amounts of Akt. This activation of Akt was associated with decreased levels of both caspase-3 protein levels and of caspase-3 activity. Inactivation of Akt by treatment with the PI3K chemical inhibitor LY294002 or by overexpression of the dominant negative mutant of Akt (DNMAkt) prevented the protective effect of S1P on apoptosis. Additionally, silencing of the S1P-1 receptor by specific siRNA demonstrated a lesser decrease in apoptosis to S1P exposure.
These results indicate that S1P protects intestinal epithelial cells from apoptosis via an Akt-dependent pathway.
PMCID: PMC2696985  PMID: 18654850
Sphingosine; Apoptosis; Akt signaling; Intestinal epithelium
7.  Genomic Profiles in Stage I Primary Non Small Cell Lung Cancer Using Comparative Genomic Hybridization Analysis of cDNA Microarrays1 
Neoplasia (New York, N.Y.)  2004;6(5):623-635.
To investigate the genomic aberrations that are involved in lung tumorigenesis and therefore may be developed as biomarkers for lung cancer diagnosis, we characterized the genomic copy number changes associated with individual genes in 14 tumors from patients with primary non small cell lung cancer (NSCLC). Six squamous cell carcinomas (SQCAs) and eight adenocarcinomas (ADCAs) were examined by high-resolution comparative genomic hybridization (CGH) analysis of cDNA microarray. The SQCAs and ADCAs shared common frequency distributions of recurrent genomic gains of 63 genes and losses of 72 genes. Cluster analysis using 57 genes defined the genomic differences between these two major histologic types of NSCLC. Genomic aberrations from a set of 18 genes showed distinct difference of primary ADCAs from their paired normal lung tissues. The genomic copy number of four genes was validated by fluorescence in situ hybridization of 32 primary NSCLC tumors, including those used for cDNA microarray CGH analysis; a strong correlation with cDNA microarray CGH data emerged. The identified genomic aberrations may be involved in the initiation and progression of lung tumorigenesis and, most importantly, may be developed as new biomarkers for the early detection and classification of lung cancer.
PMCID: PMC1531667  PMID: 15548372
Genomic copy number changes; primary non small cell lung cancer; comparative genomic hybridization; cDNA microarray

Results 1-7 (7)