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author:("Lu, liaoning")
1.  EMMPRIN Regulates Cytoskeleton Reorganization and Cell Adhesion in Prostate Cancer 
The Prostate  2011;72(1):72-81.
Background
Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment.
Methods
In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) vs. malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization.
Results
EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins.
Conclusions
Our results suggest that EMMPRIN regulates cell adhesion, invasion and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis.
doi:10.1002/pros.21408
PMCID: PMC3158271  PMID: 21563192
Prostate Cancer; EMMPRIN; Cytoskeleton; shRNA; Filopodia
2.  Genome-wide association study for serum urate concentrations and gout among African Americans identifies genomic risk loci and a novel URAT1 loss-of-function allele 
Human Molecular Genetics  2011;20(20):4056-4068.
Serum urate concentrations are highly heritable and elevated serum urate is a key risk factor for gout. Genome-wide association studies (GWAS) of serum urate in African American (AA) populations are lacking. We conducted a meta-analysis of GWAS of serum urate levels and gout among 5820 AA and a large candidate gene study among 6890 AA and 21 708 participants of European ancestry (EA) within the Candidate Gene Association Resource Consortium. Findings were tested for replication among 1996 independent AA individuals, and evaluated for their association among 28 283 EA participants of the CHARGE Consortium. Functional studies were conducted using 14C-urate transport assays in mammalian Chinese hamster ovary cells. In the discovery GWAS of serum urate, three loci achieved genome-wide significance (P< 5.0 × 10−8): a novel locus near SGK1/SLC2A12 on chromosome 6 (rs9321453, P= 1.0 × 10−9), and two loci previously identified in EA participants, SLC2A9 (P= 3.8 × 10−32) and SLC22A12 (P= 2.1 × 10−10). A novel rare non-synonymous variant of large effect size in SLC22A12, rs12800450 (minor allele frequency 0.01, G65W), was identified and replicated (beta −1.19 mg/dl, P= 2.7 × 10−16). 14C-urate transport assays showed reduced urate transport for the G65W URAT1 mutant. Finally, in analyses of 11 loci previously associated with serum urate in EA individuals, 10 of 11 lead single-nucleotide polymorphisms showed direction-consistent association with urate among AA. In summary, we identified and replicated one novel locus in association with serum urate levels and experimentally characterize the novel G65W variant in URAT1 as a functional allele. Our data support the importance of multi-ethnic GWAS in the identification of novel risk loci as well as functional variants.
doi:10.1093/hmg/ddr307
PMCID: PMC3177647  PMID: 21768215
3.  Genetic Association for Renal Traits among Participants of African Ancestry Reveals New Loci for Renal Function 
PLoS Genetics  2011;7(9):e1002264.
Chronic kidney disease (CKD) is an increasing global public health concern, particularly among populations of African ancestry. We performed an interrogation of known renal loci, genome-wide association (GWA), and IBC candidate-gene SNP association analyses in African Americans from the CARe Renal Consortium. In up to 8,110 participants, we performed meta-analyses of GWA and IBC array data for estimated glomerular filtration rate (eGFR), CKD (eGFR <60 mL/min/1.73 m2), urinary albumin-to-creatinine ratio (UACR), and microalbuminuria (UACR >30 mg/g) and interrogated the 250 kb flanking region around 24 SNPs previously identified in European Ancestry renal GWAS analyses. Findings were replicated in up to 4,358 African Americans. To assess function, individually identified genes were knocked down in zebrafish embryos by morpholino antisense oligonucleotides. Expression of kidney-specific genes was assessed by in situ hybridization, and glomerular filtration was evaluated by dextran clearance. Overall, 23 of 24 previously identified SNPs had direction-consistent associations with eGFR in African Americans, 2 of which achieved nominal significance (UMOD, PIP5K1B). Interrogation of the flanking regions uncovered 24 new index SNPs in African Americans, 12 of which were replicated (UMOD, ANXA9, GCKR, TFDP2, DAB2, VEGFA, ATXN2, GATM, SLC22A2, TMEM60, SLC6A13, and BCAS3). In addition, we identified 3 suggestive loci at DOK6 (p-value = 5.3×10−7) and FNDC1 (p-value = 3.0×10−7) for UACR, and KCNQ1 with eGFR (p = 3.6×10−6). Morpholino knockdown of kcnq1 in the zebrafish resulted in abnormal kidney development and filtration capacity. We identified several SNPs in association with eGFR in African Ancestry individuals, as well as 3 suggestive loci for UACR and eGFR. Functional genetic studies support a role for kcnq1 in glomerular development in zebrafish.
Author Summary
Chronic kidney disease (CKD) is an increasing global public health problem and disproportionately affects populations of African ancestry. Many studies have shown that genetic variants are associated with the development of CKD; however, similar studies are lacking in African ancestry populations. The CARe consortium consists of more than 8,000 individuals of African ancestry; genome-wide association analysis for renal-related phenotypes was conducted. In cross-ethnicity analyses, we found that 23 of 24 previously identified SNPs in European ancestry populations have the same effect direction in our samples of African ancestry. We also identified 3 suggestive genetic variants associated with measurement of kidney function. We then tested these genes in zebrafish knockdown models and demonstrated that kcnq1 is involved in kidney development in zebrafish. These results highlight the similarity of genetic variants across ethnicities and show that cross-species modeling in zebrafish is feasible for genes associated with chronic human disease.
doi:10.1371/journal.pgen.1002264
PMCID: PMC3169523  PMID: 21931561
4.  Using Digital Crumbs from an Electronic Health Record to Identify, Study and Improve Health Care Teams 
We have developed a novel approach, the Digital Crumb Investigator, for using data collected as a byproduct of Electonic Health Record (EHR) use to help define care teams and care processes. We are developing tools and methods to utilize these routinely collected data to visualize and quantify care networks across acute care and ambulatory settings We have chosen a clinical care domain where clinicians use EHRs in their offices, on the maternity wards and in the neonatal intensive care units as a test paradigm for this technology. The tools and methods we deliver should readily translate to other health care settings that collect behind-the-scenes electronic metadata such as audit trails. We believe that by applying the methods of social networking to define clinical relationships around a patient’s care we will enable new areas of research into the usage of EHRs to promote patient safety and other improvements in care.
PMCID: PMC3243159  PMID: 22195103
5.  Mechanistic Studies on the Triggered Release of Liposomal Contents by Matrix Metalloproteinase-9 
Journal of the American Chemical Society  2008;130(32):10633-10642.
Matrix metalloproteinases (MMPs) are a class of extracellular matrix degrading enzymes over-expressed in many cancers and contribute to the metastatic ability of the cancer cells. We have recently demonstrated that liposomal contents can be released when triggered by the enzyme MMP-9. Herein, we report our results on the mechanistic studies of the MMP-9 triggered release of the liposomal contents. We synthesized peptides containing the cleavage site for MMP-9 and conjugated them with fatty acids to prepare the corresponding lipopeptides. By employing Circular Dichroism spectroscopy, we demonstrate that the lipopeptides, when incorporated in liposomes, are de-mixed in the lipid bilayers and generate triple helical structures. MMP-9 cleaves the triple helical peptides, leading to the release of the liposomal contents. Other MMPs, which cannot hydrolyze triple helical peptides, failed to release the contents from the liposomes. We also observed that the rate and the extent of release of the liposomal contents depend on the mismatch between acyl chains of the synthesized lipopeptide and phospholipid components of the liposomes. Circular Dichroism spectroscopic studies imply that the observed differences in the release reflect the ability of the liposomal membrane to anneal the defects following the enzymatic cleavage of the liposome-incorporated lipopeptides.
doi:10.1021/ja801548g
PMCID: PMC2644422  PMID: 18642903
Triggered release; liposomes; matrix metalloproteinases; phase separation; triple helical lipopeptides
6.  Tube-Gel Digestion 
This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl β-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.
doi:10.1074/mcp.M500138-MCP200
PMCID: PMC1360194  PMID: 16150870

Results 1-6 (6)