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1.  CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes 
Scientific Reports  2015;5:8366.
The era of genomics has demanded the development of more efficient and timesaving approaches to validate gene function in disease. Here, we utilized the CRISPR-Cas9 system to generate Kcnj13 mutant mice by zygote injection to verify the pathogenic role of human KCNJ13, mutations of which are thought to cause Leber congenital amaurosis (LCA), an early-onset form of blindness. We found that complete loss of Kcnj13 is likely postnatal lethal. Among surviving F0-generation mice examined, 80% show mosaic KCNJ13 expression in the retinal pigment epithelium (RPE). Mosaic expression correlates with decreased response to light and photoreceptor degeneration, indicating that Kcnj13 mutant mice mimic human KCNJ13-related LCA disease. Importantly, mosaic animals enable us to directly compare Kcnj13 mutant and wild-type RPE cells in the same eye. We found that RPE cells lacking KCNJ13 protein still survive but overlying photoreceptors exhibit cell degeneration. At the same time, wild-type RPE cells can rescue neighboring photoreceptor cells that overlie mutant RPE cells. These results suggest that KCNJ13 expression is required for RPE cells to maintain photoreceptor survival. Moreover, we show that CRISPR-Cas9 engineered mosaicism can be used to rapidly test candidate gene function in vivo.
PMCID: PMC4322368  PMID: 25666713
2.  Local Metric Learning in 2D/3D Deformable Registration With Application in the Abdomen 
IEEE transactions on medical imaging  2014;33(8):1592-1600.
In image-guided radiotherapy (IGRT) of disease sites subject to respiratory motion, soft tissue deformations can affect localization accuracy. We describe the application of a method of 2D/3D deformable registration to soft tissue localization in abdomen. The method, called registration efficiency and accuracy through learning a metric on shape (REALMS), is designed to support real-time IGRT. In a previously developed version of REALMS, the method interpolated 3D deformation parameters for any credible deformation in a deformation space using a single globally-trained Riemannian metric for each parameter. We propose a refinement of the method in which the metric is trained over a particular region of the deformation space, such that interpolation accuracy within that region is improved. We report on the application of the proposed algorithm to IGRT in abdominal disease sites, which is more challenging than in lung because of low intensity contrast and nonrespiratory deformation. We introduce a rigid translation vector to compensate for nonrespiratory deformation, and design a special region-of-interest around fiducial markers implanted near the tumor to produce a more reliable registration. Both synthetic data and actual data tests on abdominal datasets show that the localized approach achieves more accurate 2D/3D deformable registration than the global approach.
PMCID: PMC4321725  PMID: 24771575
Abdomen; image-guided radiotherapy (IGRT); radiation oncology; 2D/3D registration
3.  MicroRNA-153 acts as a prognostic marker in gastric cancer and its role in cell migration and invasion 
OncoTargets and therapy  2015;8:357-364.
MicroRNA (miRNA)-153 (miR-153) has been considered as a novel tumor-related miRNA and is found to be significantly deregulated in human cancers. In this study, we found that the expression levels of miR-153 were obviously lower in gastric cancer tissues than those in matched adjacent nontumor tissues. Otherwise, miR-153 was expressed at significantly lower levels in aggressive tumor tissues. Clinical association analysis indicated that low expression of miR-153 was prominently correlated with poor prognostic features in gastric cancer. Furthermore, we demonstrated that the low expression of miR-153 was correlated with short 5-year survival of gastric cancer patients. Multivariate Cox regression analysis indicated that miR-153 was an independent prognostic marker in gastric cancer. Our in vitro studies showed that upregulation of miR-153 reduced cell migration and invasion in MKN-45 cells. Meanwhile, downregulation of miR-153 promoted SGC-7901 cell migration and invasion. An inverse correlation between miR-153 and SNAI1 expression was observed in gastric cancer tissues. In addition, upregulation of miR-153 reduced SNAI1 expression and subsequently suppressed epithelial–mesenchymal transition (EMT) with elevated expression of E-cadherin and reduced expression of vimentin in MKN-45 cells. Furthermore, downregulation of miR-153 increased SNAI1 expression and promoted EMT in SGC-7901 cells. In conclusion, miR-153 is an independent prognostic marker for predicting survival of gastric cancer patients and may promote gastric cancer cell migration and invasion, by inhibiting SNAI1-induced EMT.
PMCID: PMC4322869
miR-153; prognosis; SNAI1; EMT; tumor metastasis
4.  The general amino acid control pathway regulates mTOR and autophagy during serum/glutamine starvation 
The Journal of Cell Biology  2014;206(2):173-182.
To meet their metabolic needs, starved cells first activate autophagy, but activation in parallel of the general amino acid control pathway increases amino acid uptake, leading to reactivation of mTOR and down-regulation of autophagy.
Organisms have evolved elaborate mechanisms to adjust intracellular nutrient levels in response to fluctuating availability of exogenous nutrients. During starvation, cells can enhance amino acid uptake and synthesis through the general amino acid control (GAAC) pathway, whereas nonessential cellular contents are recycled by autophagy. How these two pathways are coordinated in response to starvation is currently unknown. Here we show that the GAAC pathway couples exogenous amino acid availability with autophagy. Starvation caused deactivation of mTOR, which then activated autophagy. In parallel, serum/glutamine starvation activated the GAAC pathway, which up-regulated amino acid transporters, leading to increased amino acid uptake. This elevated the intracellular amino acid level, which in turn reactivated mTOR and suppressed autophagy. Knockdown of activating transcription factor 4, the major transcription factor in the GAAC pathway, or of SLC7A5, a leucine transporter, caused impaired mTOR reactivation and much higher levels of autophagy. Thus, the GAAC pathway modulates autophagy by regulating amino acid uptake and mTOR reactivation during serum/glutamine starvation.
PMCID: PMC4107793  PMID: 25049270
5.  An ignored variable: solution preparation temperature in protein crystallization 
Scientific Reports  2015;5:7797.
Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.
PMCID: PMC4297974  PMID: 25597864
6.  Versatile On-Resin Synthesis of High Mannose Glycosylated Asparagine with Functional Handles 
Carbohydrate research  2013;383:69-75.
Here we present a synthetic route for solid phase synthesis of N-linked glycoconjugates containing high mannose oligosaccharides which allows the incorporation of useful functional handles on the N-terminus of asparagine. In this strategy, the C-terminus of an Fmoc protected aspartic acid residue is first attached to a solid phase support. The side chain of aspartic acid is protected by a 2-phenylisopropyl protecting group, which allows selective deprotection for the introduction of glycosylation. By using a convergent on-resin glycosylamine coupling strategy, an N-glycosidic linkage is successfully formed on the free side chain of the resin bound aspartic acid with a large high mannose oligosaccharide, Man8GlcNAc2, to yield N-linked high mannose glycosylated asparagine. The use of on-resin glycosylamine coupling provides excellent glycosylation yield, can be applied to couple other types of oligosaccharides, and also makes it possible to recover excess oligosaccharides conveniently after the on-resin coupling reaction. Useful functional handles including an alkene (p-vinylbenzoic acid), an alkyne (4-pentynoic acid), biotin, and 5-carboxyfluorescein are then conjugated onto the N-terminal amine of asparagine on-resin after the removal of the Fmoc protecting group. In this way, useful functional handles are introduced onto the glycosylated asparagine while maintaining the structural integrity of the reducing end of the oligosaccharide. The asparagine side chain also serves as a linker between the glycan and the functional group and preserves the native presentation of N-linked glycan which may aid in biochemical and structural studies. As an example of a biochemical study using functionalized high mannose glycosylated asparagine, a fluorescence polarization assay has been utilized to study the binding of the lectin Concanavalin A (ConA) using 5-carboxyfluorescein labeled high mannose glycosylated asparagine.
PMCID: PMC3974579  PMID: 24326091
Solid Phase Synthesis; Glycosylated Asparagine; High Mannose; Glycosylamine; On-resin Coupling; Functionalized Oligosaccharide
7.  Distinct Splice Variants and Pathway Enrichment in the Cell Line Models of Aggressive Human Breast Cancer Subtypes 
Journal of proteome research  2013;13(1):212-227.
This study was conducted as a part of the Chromosome-Centric Human Proteome Project (C-HPP) of the Human Proteome Organization. The United States team of C-HPP is focused on characterizing the protein-coding genes in chromosome 17. Despite its small size, chromosome 17 is rich in protein-coding genes, it contains many cancer-associated genes, including BRCA1, ERBB2 (Her2/neu), and TP53. The goal of this study was to examine the splice variants expressed in three ERBB2 expressed breast cancer cell line models of hormone receptor negative breast cancers by integrating RNA-Seq and proteomic mass spectrometry data. The cell-lines represent distinct phenotypic variations subtype: SKBR3 (ERBB2+ (over-expression)/ ER−/PR−; adenocarcinoma), SUM190 (ERBB2+ (over-expression)/ER−/PR−; inflammatory breast cancer) and SUM149 (ERBB2 (low expression) ER−/PR −; inflammatory breast cancer). We identified more than one splice variant for 1167 genes expressed in at least one of the three cancer cell lines. We found multiple variants of genes that are in the signaling pathways downstream of ERBB2 along with variants specific to one cancer cell line compared to the other two cancer cell lines and to normal mammary cells. The overall transcript profiles based on read counts indicated more similarities between SKBR3 and SUM190. The top-ranking Gene Ontology and BioCarta pathways for the cell-line specific variants pointed to distinct key mechanisms including: amino sugar metabolism, caspase activity, and endocytosis in SKBR3; different aspects of metabolism, especially of lipids in SUM190; cell- to-cell adhesion, integrin and ERK1/ERK2 signaling, and translational control in SUM149. The analyses indicated an enrichment in the electron transport chain processes in the ERBB2 over-expressed cell line models; and an association of nucleotide binding, RNA splicing and translation processes with the IBC models, SUM190 and SUM149. Detailed experimental studies on the distinct variants identified from each of these three breast cancer cell line models may open opportunities for drug target discovery and help unveil their specific roles in cancer progression and metastasis.
PMCID: PMC3943430  PMID: 24111759
Splice variants (SpV); splice variant protein (SpP); splice variant transcript (SpT); ERBB2+ (Her2/neu); EGFR; proteotypic peptide; I-TASSER; breast cancer subtypes
8.  Toward More Transparent and Reproducible Omics Studies Through a Common Metadata Checklist and Data Publications 
Biological processes are fundamentally driven by complex interactions between biomolecules. Integrated high-throughput omics studies enable multifaceted views of cells, organisms, or their communities. With the advent of new post-genomics technologies, omics studies are becoming increasingly prevalent; yet the full impact of these studies can only be realized through data harmonization, sharing, meta-analysis, and integrated research. These essential steps require consistent generation, capture, and distribution of metadata. To ensure transparency, facilitate data harmonization, and maximize reproducibility and usability of life sciences studies, we propose a simple common omics metadata checklist. The proposed checklist is built on the rich ontologies and standards already in use by the life sciences community. The checklist will serve as a common denominator to guide experimental design, capture important parameters, and be used as a standard format for stand-alone data publications. The omics metadata checklist and data publications will create efficient linkages between omics data and knowledge-based life sciences innovation and, importantly, allow for appropriate attribution to data generators and infrastructure science builders in the post-genomics era. We ask that the life sciences community test the proposed omics metadata checklist and data publications and provide feedback for their use and improvement.
PMCID: PMC3903324  PMID: 24456465
9.  miR-33a promotes glioma-initiating cell self-renewal via PKA and NOTCH pathways 
The Journal of Clinical Investigation  2014;124(10):4489-4502.
Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Glioma-initiating cells (GICs) are stem-like cells that have been implicated in glioblastoma progression and recurrence; however, the distinct properties of GICs and non-GICs within GBM tumors are largely uncharacterized. Here, we evaluated stem cell–associated microRNA (miR) expression in GICs from GBM patients and GICs derived from xenografted human glioma cell lines and determined that miR-33a promotes GIC growth and self-renewal. Moreover, evaluation of a GBM tissue array revealed that higher miR-33a expression was associated with poor prognosis of GBM patients. Antagonizing miR-33a function in GICs reduced self-renewal and tumor progression in immune-compromised mice, whereas overexpression of miR-33a in non-GICs promoted the display of features associated with GICs. We identified the mRNAs encoding phosphodiesterase 8A (PDE8A) and UV radiation resistance–associated gene (UVRAG) as direct miR-33a targets. PDE8A and UVRAG negatively regulated the cAMP/PKA and NOTCH pathways, respectively; therefore, miR-33a–dependent reduction of these proteins promoted growth and self-renewal of GICs by enhancing PKA and NOTCH activity. Furthermore, in GBM specimens, there was an inverse correlation between the expression levels of miR-33a and PDE8A and UVRAG expression. These findings reveal a miR-33a–centered signaling network that promotes GIC maintenance and has potential as a therapeutic target for GBM treatment.
PMCID: PMC4191031  PMID: 25202981
10.  Health Workforce Equity in Urban Community Health Service of China 
PLoS ONE  2014;9(12):e115988.
To reveal the equity of health workforce distribution in urban community health service (CHS), and to provide evidence for further development of community health service in China.
A community-based, cross-sectional study was conducted in China from September to December 2011. In the study, 190 CHS centers were selected from 10 provinces of China via stratified multistage cluster sampling. Human resources profiles and basic characteristics of each CHS centers were collected. Lorenz curves and Gini Coefficient were used to measure the inequality in the distribution of health workforce in community health service centers by population size and geographical area. Wilcoxon rank test for paired samples was used to analyze the differences in equity between different health indicators.
On average, there were 7.37 health workers, including 3.25 doctors and 2.32 nurses per 10,000 population ratio. Significant differences were found in all indicators across the samples, while Beijing, Shandong and Zhejiang ranked the highest among these provinces. The Gini coefficients for health workers, doctors and nurses per 10,000 population ratio were 0.39, 0.44, and 0.48, respectively. The equity of doctors per 10,000 population ratio (G = 0.39) was better than that of doctors per square kilometer (G = 0.44) (P = 0.005). Among the total 6,573 health workers, 1,755(26.7%) had undergraduate degree or above, 2,722(41.4%)had junior college degree and 215(3.3%) had high school education. Significant inequity was found in the distribution of workers with undergraduate degree or above (G = 0.52), which was worse than that of health works per 10000 population (P<0.001).
Health workforce inequity was found in this study, especially in quality and geographic distribution. These findings suggest a need for more innovative policies to improve health equity in Chinese urban CHS centers.
PMCID: PMC4281229  PMID: 25551449
11.  Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy 
PLoS ONE  2014;9(12):e116165.
SIRT1 is central to the lifespan and vascular health, but undergoes degradation that contributes to several medical conditions, including diabetes. How SIRT1 turnover is regulated remains unclear. However, emerging evidence suggests that endothelial nitric oxide synthase (eNOS) positively regulates SIRT1 protein expression. We recently identified NO as an endogenous inhibitor of 26S proteasome functionality with a cellular reporter system. Here we extended this finding to a novel pathway that regulates SIRT1 protein breakdown. In cycloheximide (CHX)-treated endothelial cells, NONOate, an NO donor, and A23187, an eNOS activator, significantly stabilized SIRT1 protein. Similarly, NO enhanced SIRT1 protein, but not mRNA expression, in CHX-free cells. NO also stabilized an autophagy-related protein unc-51 like kinase (ULK1), but did not restore SIRT1 protein levels in ULK1-siRNA-treated cells or in mouse embryonic fibroblasts (MEF) from Ulk1−/− mice. This suggests that ULK1 mediated the NO regulation of SIRT1. Furthermore, adenoviral overexpression of ULK1 increased SIRT1 protein expression, while ULK1 siRNA treatment decreased it. Rapamycin-induced autophagy did not mimic these effects, suggesting that the effects of ULK1 were autophagy-independent. Treatment with MG132, a proteasome inhibitor, or siRNA of β-TrCP1, an E3 ligase, prevented SIRT1 reduction induced by ULK1-siRNA. Mechanistically, ULK1 negatively regulated 26S proteasome functionality, which was at least partly mediated by O-linked-GlcNAc transferase (OGT), probably by increased O-GlcNAc modification of proteasomal subunit Rpt2. The NO-ULK1-SIRT1 axis was likely operative in the whole animal: both ULK1 and SIRT1 protein levels were significantly reduced in tissue homogenates in eNOS-knockout mice (lung) and in db/db mice where eNOS is downregulated (lung and heart). Taken together, the results show that NO stabilizes SIRT1 by regulating 26S proteasome functionality through ULK1 and OGT, but not autophagy, in endothelial cells.
PMCID: PMC4277463  PMID: 25541949
12.  Quantification and Dynamic Monitoring of EGFR T790M in Plasma Cell-Free DNA by Digital PCR for Prognosis of EGFR-TKI Treatment in Advanced NSCLC 
PLoS ONE  2014;9(11):e110780.
Among advanced non-small cell lung cancer (NSCLC) patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA) of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy.
Materials and Methods
We retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS) after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and digital-PCR (D-PCR). Real-time PCR was performed to measure c-MET amplification.
Detection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5%) and post-TKI (43.0% vs. 25.2%) plasma samples. Patients with pre-TKI T790M showed inferior PFS (8.9 vs. 12.1 months, p = 0.007) and overall survival (OS, 19.3 vs. 31.9 months, p = 0.001) compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001) on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes (p = 0.044 and p = 0.015, respectively).
Qualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis.
PMCID: PMC4236040  PMID: 25405807
13.  Spinal coronal profiles and proximal femur bone mineral density in adolescent idiopathic scoliosis 
European Spine Journal  2013;22(11):2433-2437.
Although the occurrence and progression of AIS has been linked to low bone mineral density (BMD), the relationships between spinal curvature and bilateral differences in proximal femur BMD are controversial. Few correlation studies have stratified patients by curve type. The purpose of this study was to evaluate the relationships between spinal coronal profile and bilateral differences in proximal femur BMD in patients with adolescent idiopathic scoliosis (AIS).
This study included 67 patients with AIS who underwent posterior correction and fusion surgery between January 2009 and October 2011. The mean age at the time of surgery was 17.4 ± 4.1 years. Bilateral proximal femur BMD was measured before surgery by dual-energy X-ray absorptiometry. We compared the proximal femur BMDs by determining the bilateral BMD ratio (left proximal femur BMD divided by that of the right). We evaluated correlations between coronal parameters, obtained from preoperative radiographs, and the BMD ratio using Pearson’s correlation analysis.
Patients with Lenke type 1 curve (48; all with a right convex curve) had a mean bilateral proximal femur BMD ratio of 1.00 ± 0.04. Patients with Lenke type 5 curve (19; all with a left convex curve) had a mean bilateral proximal femur BMD ratio of 0.94 ± 0.04, indicating that the BMD in the proximal femur on the right side (concave) was greater than that in the left (convex). Coronal balance was significantly correlated with the BMD ratio in both the Lenke type 1 and type 5 groups, with a correlation coefficient of 0.46 and 0.50, respectively.
The bilateral proximal femur BMD ratio was significantly correlated with the coronal balance in AIS patients. When the C7 plumb line was shifted toward one side, the BMD was greater in the contralateral proximal femur.
PMCID: PMC3886500  PMID: 23764767
Adolescent idiopathic scoliosis; Bone mineral density; Proximal femur
14.  Association between point-of-care CRP testing and antibiotic prescribing in respiratory tract infections: a systematic review and meta-analysis of primary care studies 
The British Journal of General Practice  2013;63(616):e787-e794.
Most patients with respiratory tract infections (RTIs) are prescribed antibiotics in general practice. However, there is little evidence that antibiotics bring any value to the treatment of most RTIs. Point-of-care C-reactive protein testing may reduce antibiotic prescribing.
To systematically review studies that have examined the association between point-of-care (POC) C-reactive protein testing and antibiotic prescribing for RTIs in general practice.
Design and setting
Systematic review and meta-analysis of randomised controlled trials and observational studies.
MEDLINE® and Embase were systematically searched to identify relevant publications. All studies that examined the association between POC C-reactive protein testing and antibiotic prescribing for patients with RTIs were included. Two authors independently screened the search results and extracted data from eligible studies. Dichotomous measures of outcomes were combined using risk ratios (RRs) with 95% confidence intervals (CIs) either by fixed or random-effect models.
Thirteen studies containing 10 005 patients met the inclusion criteria. POC C-reactive protein testing was associated with a significant reduction in antibiotic prescribing at the index consultation (RR 0.75, 95% CI = 0.67 to 0.83), but was not associated with antibiotic prescribing at any time during the 28-day follow-up period (RR 0.85, 95% CI = 0.70 to 1.01) or with patient satisfaction (RR 1.07, 95% CI = 0.98 to 1.17).
POC C-reactive protein testing significantly reduced antibiotic prescribing at the index consultation for patients with RTIs. Further studies are needed to analyse the confounders that lead to the heterogeneity.
PMCID: PMC3809432  PMID: 24267862
antibiotic prescribing; meta-analysis; point-of-care C-reactive protein testing; primary care; respiratory tract infections
15.  Identifying Newly Approved Medications in Medicare Claims Data: A Case Study Using Tocilizumab 
Pharmacoepidemiology and drug safety  2013;22(11):10.1002/pds.3475.
After U.S. licensure, parenterally administered medications are identified using non-specific drug codes. Accurately identifying these medications is critical to safety and effectiveness research. Methods to identify medications prior to assignment of specific drug codes have not been well described.
To describe a generalized approach using non-specific drug codes to identify parenteral therapies in Medicare claims and to assess the ability of that approach to identify tocilizumab (TCZ), a new biologic agent approved in 2010.
We used 2008–2010 Medicare data for a cohort of rheumatoid arthritis patients for algorithm development. Our algorithm classified non-specific drug codes based upon: (1) ICD9 codes; (2) unit values (i.e. dose); (3) codes for infusion/injection procedures; (4) expected versus observed total reimbursement amount and reimbursement per unit. We assessed algorithm performance by linking to an arthritis registry to examine external validity.
Of 472,803 claims with non-specific drug codes, 9,762 claims satisfied the TCZ algorithm. 74.3% of 9,762 claims were classified as TCZ by exact unit price or allowed amount, 4.4% by unique doses, 21.3% by diagnosis code and small deviation from unit price or allowed amount. The algorithm demonstrated good performance characteristics: sensitivity 94% (95% CI 80–99), specificity 100% (99–100) and PPV 97% (84–100).
Claims-based algorithms in Medicare or similar data systems can accurately identify newly approved biologics administered parenterally prior to the assignment of specific drug codes.
PMCID: PMC3856178  PMID: 24038595
rheumatoid arthritis; Medicare; Part D; biologics; tocilizumab; denosumab; certolizumab; linkage; registry
16.  The Association between Bile Salt Export Pump Single-Nucleotide Polymorphisms and Primary Biliary Cirrhosis Susceptibility and Ursodeoxycholic Acid Response 
Disease Markers  2014;2014:350690.
Background. Primary biliary cirrhosis (PBC) is a chronic and progressive cholestasis liver disease. Bile salt export pump (BSEP) is the predominant bile salt efflux system of hepatocytes. BSEP gene has been attached great importance in the susceptibility of PBC and the response rate of ursodeoxycholic acid (UDCA) treatment of PBC patients. Methods. In this study, TaqMan assay was used to genotype four variants of BSEP, and the Barcelona criteria were used for evaluating the response rate of UDCA treatment. Results. Variant A allele of BSEP rs473351 (dominant model, OR = 2.063; 95% CI, 1.254–3.393; P = 0.004) was highly associated with PBC susceptibility. On the contrary, variant A allele of BSEP rs2287618 (dominant model, OR = 0.617; 95% CI, 0.411–0.928; P = 0.020) provided a protective role and Barcelona evaluation criterion indicated that the frequency of variant allele at BSEP rs2287618 was significantly decreased in UDCA-responsive PBC patients (P = 0.021). Conclusion. These results suggested that BSEP rs473351 was closely associated with the susceptibility of PBC and if people with BSEP rs2287618 were diagnosed as PBC, the UDCA treatment was not satisfactory. Larger studies with mixed ethnicity subjects and stratified by clinical and subclinical characteristics are needed to validate our findings.
PMCID: PMC4216684  PMID: 25392597
17.  Computational Advances in Cancer Informatics (A) 
Cancer Informatics  2014;13(Suppl 1):45-48.
PMCID: PMC4216040  PMID: 25484572
18.  GATA3 controls the specification of prosensory domain and neuronal survival in the mouse cochlea 
Human Molecular Genetics  2013;22(18):3609-3623.
HDR syndrome (also known as Barakat syndrome) is a developmental disorder characterized by hypoparathyroidism, sensorineural deafness and renal disease. Although genetic mapping and subsequent functional studies indicate that GATA3 haplo-insufficiency causes human HDR syndrome, the role of Gata3 in sensorineural deafness and auditory system development is largely unknown. In this study, we show that Gata3 is continuously expressed in the developing mouse inner ear. Conditional knockout of Gata3 in the developing inner ear disrupts the morphogenesis of mouse inner ear, resulting in a disorganized and shortened cochlear duct with significant fewer hair cells and supporting cells. Loss of Gata3 function leads to the failure in the specification of prosensory domain and subsequently, to increased cell death in the cochlear duct. Moreover, though the initial generation of cochleovestibular ganglion (CVG) cells is not affected in Gata3-null mice, spiral ganglion neurons (SGNs) are nearly depleted due to apoptosis. Our results demonstrate the essential role of Gata3 in specifying the prosensory domain in the cochlea and in regulating the survival of SGNs, thus identifying a molecular mechanism underlying human HDR syndrome.
PMCID: PMC3749857  PMID: 23666531
19.  DSTYK kinase domain ablation impaired the mice capabilities of learning and memory in water maze test 
DSTYK (Dual serine/threonine and tyrosine protein kinase) is a putative dual Ser/Thr and Tyr protein kinase with unique structural features. It is proposed that DSTYK may play important roles in brain because of its high expression in most brain areas. In the present study, a DSTYK knockout (KO) mouse line with the ablation of C-terminal of DSTYK including the kinase domain was generated to study the physiological function of DSTYK. The DSTYK KO mice are fertile and have no significant morphological defects revealed by Nissl staining compared with wildtype mice. Open field test and rotarod test showed there is no obvious difference in basic motor and balance capacity between the DSTYK homozygous KO mice and DSTYK heterozygous KO mice. In water maze test, however, the DSTYK homozygous KO mice show impaired capabilities of learning and memory compared with the DSTYK heterozygous KO mice.
PMCID: PMC4230056  PMID: 25400726
DSTYK; knockout mice; brain development; learning and memory
20.  The evanescent wavefield part of a cylindrical vector beam 
Optics Express  2013;21(19):22246-22254.
The evanescent wave of the cylindrical vector field is analyzed using the vector angular spectrum of the electromagnetic beam. Comparison between the contributions of the TE and TM terms of both the propagating and the evanescent waves associated with the cylindrical vector field in free space is demonstrated. The physical pictures of the evanescent wave and the propagating wave are well illustrated from the vectorial structure, which provides a new approach to manipulating laser beams by choosing the states of polarization in the cross-section of the field.
PMCID: PMC3796686  PMID: 24104116
(260.5430) Polarization; (260.2110) Electromagnetic optics; (260.0260) Physical optics; (070.7345) Wave propagation; (070.0070) Fourier optics and signal processing; (050.1940) Diffraction
21.  Levels of CD105+ cells increase and cell proliferation decreases during S-phase arrest of amniotic fluid cells in long-term culture 
The present study aimed to improve the characterization of amniotic fluid cells (AFCs) in order to optimize their use in chromosomal prenatal diagnosis and as seed or stem cells for tissue engineering. The AFCs used in the current study were obtained from three females in their second trimester of pregnancy. The cells were cultured independently and characterized by cell morphology, cell markers, cell cycle distribution and chromosome Giemsa banding in an early- and late-passage. The AFCs remained homogeneous in culture and expressed mesenchymal markers, but not endothelial markers along the culture process. In addition, compared with the early-passage cells, the late-passage cells exhibit an increase in CD105 expression, a decrease in cell division and a delay in the cell cycle, and a number of cells underwent cell cycle arrest. However, the cells retained a normal karyotype. Therefore, the current study characterized AFCs in a clinical culture and confirmed that AFCs are mesenchymal precursors. The results obtained may be useful for the application of AFCs in prenatal diagnosis.
PMCID: PMC4186349  PMID: 25289067
amniotic fluid cells; CD105; cell proliferation; long-term culture; S-phase arrest
22.  Promise of Personalized Omics to Precision Medicine 
The rapid development of high-throughput technologies and computational frameworks enables the examination of biological systems in unprecedented detail. The ability to study biological phenomena at omics levels in turn is expected to lead to significant advances in personalized and precision medicine. Patients can be treated according to their own molecular characteristics. Individual omes as well as the integrated profiles of multiple omes, such as the genome, the epigenome, the transcriptome, the proteome, the metabolome, the antibodyome, and other omics information are expected to be valuable for health monitoring, preventative measures, and precision medicine. Moreover, omics technologies have the potential to transform medicine from traditional symptom-oriented diagnosis and treatment of diseases towards disease prevention and early diagnostics. We discuss here the advances and challenges in systems biology-powered personalized medicine at its current stage, as well as a prospective view of future personalized health care at the end of this review.
PMCID: PMC4154620  PMID: 23184638
23.  Whole Exome Sequencing Identifies TTC7A Mutations for Combined Immunodeficiency with Intestinal Atresias 
Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare hereditary disease characterized by intestinal obstructions and profound immune defects.
We sought to determine the underlying genetic causes of CID-MIA by analyzing the exomic sequence of 5 patients and their healthy direct relatives from 5 unrelated families.
We performed whole exome sequencing on 5 CID-MIA patients and 10 healthy direct family members belonging to 5 unrelated families with CID-MIA. We also performed targeted Sanger sequencing for the candidate gene TTC7A on 3 additional CID-MIA patients.
Through analysis and comparison of the exomic sequence of the individuals from these 5 families, we identified biallelic damaging mutations in the TTC7A gene, for a total of 7 distinct mutations. Targeted TTC7A gene sequencing in 3 additional unrelated patients with CID-MIA revealed biallelic deleterious mutations in two of them, as well as an aberrant splice product in the third patient. Staining of normal thymus showed that the TTC7A protein is expressed in thymic epithelial cells as well as in thymocytes. Moreover, severe lymphoid depletion was observed in the thymus and peripheral lymphoid tissues from two patients with CID-MIA.
We identified deleterious mutations of the TTC7A gene in 8 unrelated patients with CID-MIA and demonstrated that the TTC7A protein is expressed in the thymus. Our results strongly suggest that TTC7A gene defects cause CID-MIA.
Clinical Implications
Damaging mutations in the gene TTC7A should be scrutinized in patients with CID-MIA. Characterization of the role of this protein in the immune system and intestinal development, as well as in thymic epithelial cells may have important therapeutic implications.
PMCID: PMC3759618  PMID: 23830146
Combined Immunodeficiency with Multiple Intestinal Atresias; Tetracopeptide Repeat Domain 7A; Whole Exome Sequencing; Thymus
24.  2D/3D Image Registration using Regression Learning 
In computer vision and image analysis, image registration between 2D projections and a 3D image that achieves high accuracy and near real-time computation is challenging. In this paper, we propose a novel method that can rapidly detect an object’s 3D rigid motion or deformation from a 2D projection image or a small set thereof. The method is called CLARET (Correction via Limited-Angle Residues in External Beam Therapy) and consists of two stages: registration preceded by shape space and regression learning. In the registration stage, linear operators are used to iteratively estimate the motion/deformation parameters based on the current intensity residue between the target projec-tion(s) and the digitally reconstructed radiograph(s) (DRRs) of the estimated 3D image. The method determines the linear operators via a two-step learning process. First, it builds a low-order parametric model of the image region’s motion/deformation shape space from its prior 3D images. Second, using learning-time samples produced from the 3D images, it formulates the relationships between the model parameters and the co-varying 2D projection intensity residues by multi-scale linear regressions. The calculated multi-scale regression matrices yield the coarse-to-fine linear operators used in estimating the model parameters from the 2D projection intensity residues in the registration. The method’s application to Image-guided Radiation Therapy (IGRT) requires only a few seconds and yields good results in localizing a tumor under rigid motion in the head and neck and under respiratory deformation in the lung, using one treatment-time imaging 2D projection or a small set thereof.
PMCID: PMC3775380  PMID: 24058278
2D/3D Registration; Regression; Machine Learning; IGRT; Radiation Therapy
25.  Whole-genome haplotyping using long reads and statistical methods 
Nature biotechnology  2014;32(3):261-266.
Rapid growth of sequencing technologies has greatly contributed to increasing our understanding of human genetics. Yet, in spite of this growth, mainstream technologies have been largely unsuccessful in resolving the diploid nature of the human genome. Here we describe statistically aided long read haplotyping (SLRH), a rapid, accurate method based on a simple experimental protocol that requires potentially as little as 30 Gbp of sequencing in addition to a standard (50x coverage) whole-genome analysis for human samples. Using this technology, we phase 99% of single-nucleotide variants in three human genomes into long haplotype blocks of 200 kbp to 1 Mbp in length. As a demonstration of the potential applications of our method, we determine allele-specific methylation patterns in a human genome and identify hundreds of differentially methylated regions that were previously unknown. Such information may offer insight into the mechanisms behind differential gene expression.
PMCID: PMC4073643  PMID: 24561555

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