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1.  Rare cell isolation and analysis in microfluidics 
Lab on a chip  2014;14(4):626-645.
Rare cells are low-abundance cells in a much larger population of background cells. Conventional benchtop techniques have limited capabilities to isolate and analyze rare cells because of their generally low selectivity and significant sample loss. Recent rapid advances in microfluidics have been providing robust solutions to the challenges in the isolation and analysis of rare cells. In addition to the apparent performance enhancements resulting in higher efficiencies and sensitivity levels, microfluidics provides other advanced features such as simpler handling of small sample volumes and multiplexing capabilities for high-throughput processing. All of these advantages make microfluidics an excellent platform to deal with the transport, isolation, and analysis of rare cells. Various cellular biomarkers, including physical properties, dielectric properties, as well as immunoaffinities, have been explored for isolating rare cells. In this Focus article, we discuss the design considerations of representative microfluidic devices for rare cell isolation and analysis. Examples from recently published works are discussed to highlight the advantages and limitations of the different techniques. Various applications of these techniques are then introduced. Finally, a perspective on the development trends and promising research directions in this field are proposed.
PMCID: PMC3991782  PMID: 24406985
2.  Biogeographic Patterns of Structural Traits and C:N:P Stoichiometry of Tree Twigs in China’s Forests 
PLoS ONE  2015;10(2):e0116391.
There have been a number of studies on biogeographic patterns of plant leaf functional traits; however, the variations in traits of other plant organs such as twigs are rarely investigated. In this study, we sampled current-year twigs of 335 tree species from 12 forest sites across a latitudinal span of 32 degrees in China, and measured twig specific density (TSD), twig dry matter content (TDMC), and carbon (C), nitrogen (N) and phosphorous (P) contents, to explore the latitudinal and environmental patterns of these twig traits. The overall mean of TSD and TDMC was 0.37 g cm−3 and 41%, respectively; mean twig C, N and P was 472 mg g−1, 9.8 mg g−1 and 1.15 mg g−1, respectively, and mean N:P mass ratio was 10.6. TSD was positively correlated with TDMC which was positively associated with twig C but negatively with twig N and P. There were no significant differences in TSD between conifer, deciduous-broadleaf and evergreen-broadleaf plants, but evergreen-broadleaf plants had the lowest and conifers the highest TDMC. Conifer twigs were lowest in C, N, P and N:P, whereas deciduous-plant twigs were highest in N and P and evergreen-plant twigs were highest in C and N:P. As latitude increased or temperature/precipitation dropped, TDMC and P increased, but N:P ratio decreased. Our results also showed that the patterns of twig P and N:P stoichiometry were consistent with those reported for leaves, but no significant trends in twig N were observed along the gradient of latitude, climate and soils. This study provides the first large-scale patterns of the twig traits and will improve our understanding of the biogeochemistry of carbon and other key nutrients in forest ecosystems.
PMCID: PMC4321987  PMID: 25664764
3.  Proteomic Analysis of Seedling Roots of Two Maize Inbred Lines That Differ Significantly in the Salt Stress Response 
PLoS ONE  2015;10(2):e0116697.
Salinity is a major abiotic stress that limits plant productivity and quality throughout the world. Roots are the sites of salt uptake. To better understand salt stress responses in maize, we performed a comparative proteomic analysis of seedling roots from the salt-tolerant genotype F63 and the salt-sensitive genotype F35 under 160 mM NaCl treatment for 2 days. Under salinity conditions, the shoot fresh weight and relative water content were significantly higher in F63 than in F35, while the osmotic potential was significantly lower and the reduction of the K+/Na+ ratio was significantly less pronounced in F63 than in F35. Using an iTRAQ approach, twenty-eight proteins showed more than 2.0- fold changes in abundance and were regarded as salt-responsive proteins. Among them, twenty-two were specifically regulated in F63 but remained constant in F35. These proteins were mainly involved in signal processing, water conservation, protein synthesis and biotic cross-tolerance, and could be the major contributors to the tolerant genotype of F63. Functional analysis of a salt-responsive protein was performed in yeast as a case study to confirm the salt-related functions of detected proteins. Taken together, the results of this study may be helpful for further elucidating salt tolerance mechanisms in maize.
PMCID: PMC4320067  PMID: 25659111
4.  Novel Agent Nitidine Chloride Induces Erythroid Differentiation and Apoptosis in CML Cells through c-Myc-miRNAs Axis 
PLoS ONE  2015;10(2):e0116880.
The proto-oncogene c-Myc plays critical roles in human malignancies including chronic myeloid leukemia (CML), suggesting that the discovery of specific agents targeting c-Myc would be extremely valuable for CML treatment. Nitidine Chloride (NC), a natural bioactive alkaloid, is suggested to possess anti-tumor effects. However, the function of NC in leukemia and the underlying molecular mechanisms have not been established. In this study, we found that NC induced erythroid differentiation, accompanied by increased expression of erythroid differentiation markers, e. g. α-, ε-, γ-globin, CD235a, CD71 and α-hemoglobin stabilizing protein (AHSP) in CML cells. We also observed that NC induced apoptosis and upregulated cleaved caspase-3 and Parp-1 in K562 cells. These effects were associated with concomitant attenuation of c-Myc. Our study showed that NC treatment in CML cells enhanced phosphorylation of Thr58 residue and subsequently accelerated degradation of c-Myc. A specific group of miRNAs, which had been reported to be activated by c-Myc, mediated biological functions of c-Myc. We found that most of these miRNAs, especially miR-17 and miR-20a showed strong decrement after NC treatment or c-Myc interference. Furthermore, overexpression of c-Myc or miR-17/20a alleviated NC induced differentiation and apoptosis in K562 cells. More importantly, NC enhanced the effects of imatinib in K562 and primary CML cells. We further found that even imatinib resistant CML cell line (K562/G01) and CML primary cells exhibited high sensitivity to NC, which showed potential possibility to overcome imatinib resistance. Taken together, our results clearly suggested that NC promoted erythroid differentiation and apoptosis through c-Myc-miRNAs regulatory axis, providing potential possibility to overcome imatinib resistance.
PMCID: PMC4315404  PMID: 25647305
5.  New Clustered Regularly Interspaced Short Palindromic Repeat Locus Spacer Pair Typing Method Based on the Newly Incorporated Spacer for Salmonella enterica 
Journal of Clinical Microbiology  2014;52(8):2955-2962.
A clustered regularly interspaced short palindromic repeat (CRISPR) typing method has recently been developed and used for typing and subtyping of Salmonella spp., but it is complicated and labor intensive because it has to analyze all spacers in two CRISPR loci. Here, we developed a more convenient and efficient method, namely, CRISPR locus spacer pair typing (CLSPT), which only needs to analyze the two newly incorporated spacers adjoining the leader array in the two CRISPR loci. We analyzed a CRISPR array of 82 strains belonging to 21 Salmonella serovars isolated from humans in different areas of China by using this new method. We also retrieved the newly incorporated spacers in each CRISPR locus of 537 Salmonella isolates which have definite serotypes in the Pasteur Institute's CRISPR Database to evaluate this method. Our findings showed that this new CLSPT method presents a high level of consistency (kappa = 0.9872, Matthew's correlation coefficient = 0.9712) with the results of traditional serotyping, and thus, it can also be used to predict serotypes of Salmonella spp. Moreover, this new method has a considerable discriminatory power (discriminatory index [DI] = 0.8145), comparable to those of multilocus sequence typing (DI = 0.8088) and conventional CRISPR typing (DI = 0.8684). Because CLSPT only costs about $5 to $10 per isolate, it is a much cheaper and more attractive method for subtyping of Salmonella isolates. In conclusion, this new method will provide considerable advantages over other molecular subtyping methods, and it may become a valuable epidemiologic tool for the surveillance of Salmonella infections.
PMCID: PMC4136180  PMID: 24899040
6.  Anterior segment optical coherence tomography evaluation of ocular graft-versus-host disease: a case study 
To explore ocular graft-versus-host disease (GVHD), anterior segment optical coherence tomography (AS-OCT) imaging of eyelids, tear meniscus, cornea and conjunctiva is performed in subsequent sessions on a patient who has ocular GVHD after allogeneic related donor stem cell transplant. The OCT results are presented together with those from a normal subject. OCT imaging is promising in visualizing several ocular GVHD manifestations, such as abnormal meibomian gland orifice (MGO), conjunctival keratinization, conjunctival hyperemia and chemosis, corneal epithelium opacification, thinning and sloughing. This case study demonstrates the capability of AS-OCT in the imaging and monitoring of ocular GVHD, which may be useful in the development of current ocular GVHD staging system and the clinical management for GVHD treatment.
PMCID: PMC4312287
Medical and biological imaging; optical coherence tomography (OCT); ophthalmology; ocular graft-versus-host disease (GVHD)
7.  Pulsatile motion of trabecular meshwork in a patient with iris cyst by phase-sensitive optical coherence tomography: a case report 
A 45-year-old man was diagnosed with a 3 mm × 3 mm iris cyst located at 9 o’clock behind iris and protruding into temporal angle by slit lamp examination, gonioscopy, and ultrasound biomicroscopy (UBM). Phase-sensitive optical coherence tomography (PhS-OCT) was applied on this case for the quantitative measurements of trabecular meshwork (TM) motion. The frequency of TM motion was with the same rhythm of the patient’s peripheral pulse. Its amplitude on the closed angle region showed significant smaller than the open angle region. PhS-OCT can be a useful tool for the diagnosis and follow-up in ocular diseases surrounding iridocorneal angle.
PMCID: PMC4312295
Trabecular meshwork (TM); iris cyst; phase-sensitive optical coherence tomography (PhS-OCT)
8.  The world's second largest population of humpback dolphins in the waters of Zhanjiang deserves the highest conservation priority 
Scientific Reports  2015;5:8147.
Chinese white dolphins (Sousa chinensis) inhabiting shallow coastal waters are vulnerable to impacts from human activities in the near shore waters. This study examined the population of Chinese white dolphins occurring off the coast of Zhanjiang in the northern South China Sea. A total of 492 Chinese white dolphins were identified, 176 of which were photographed on more than one occasion. The Zhanjiang Chinese white dolphin population is isolated from populations of conspecifics along the Guangdong coast. It is composed of approximately 1485 individuals (95% CI = 1371–1629; SE = 63.8), with estimates of mean representative range and core area of 168.51 and 44.26 km2, respectively. The high site fidelity and long-term residence of Chinese white dolphins in the study area are well established. A review of all available data indicates that based on what is currently known, the Zhanjiang Chinese white dolphin population is the second largest of the species and genus in the world. However, the recent industrial boom along the Zhanjiang coast has increased concerns regarding the conservation of the Zhanjiang Chinese white dolphin population. We recommend the designation of a national nature reserve as a most urgent measure for protecting Chinese white dolphins in Zhanjiang waters.
PMCID: PMC4311443  PMID: 25634769
9.  Sub-micrometer-precision, three-dimensional (3D) hydrodynamic focusing via “microfluidic drifting” 
Lab on a chip  2013;14(2):415-423.
In this article, we demonstrate single-layered, “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing devices with particle/cell focal positioning approaching submicron precision along both lateral and vertical directions. By systematically optimizing channel geometries and sample/sheath flow rates, a series of “microfluidic drifting” based 3D hydrodynamic focusing devices with different curvature angles are designed and fabricated. Their performances are then evaluated by confocal microscopy, fast camera imaging, and side-view imaging techniques. Using a device with a curvature angle of 180°, we have achieved a standard deviation of ±0.45 µm in particle focal position and a coefficient of variation (CV) of 2.37% in flow cytometric measurements. To the best of our knowledge, this is the best CV that has been achieved by a microfluidic flow cytometry device. Moreover, the device showed the capability to distinguish 8 peaks when subjected to a stringent 8-peak rainbow calibration test, signifying the ability to perform sensitive, accurate tests similar to commercial flow cytometers. We have further tested and validated our device by detection of HEK-293 cells. With its advantages in simple fabrication (i.e., single-layered device), precise 3D hydrodynamic focusing (i.e., submicrometer precision along both lateral and vertical directions), and high detection resolution (i.e., low CV), our method could serve as an important basis for high-performance, mass-producible microfluidic flow cytometry.
PMCID: PMC3989543  PMID: 24287742
10.  Interleukin-1β regulates the expression of glucocorticoid receptor isoforms in nasal polyps in vitro via p38 MAPK and JNK signal transduction pathways 
To explore the upstream signal transduction mechanisms responsible for the imbalanced expression of glucocorticoid receptor (GR) isoforms in chronic rhinosinusitis (CRS) mucosa.
An in vitro model of Glucocorticoid resistance was established by inducing nasal polyp tissue with IL-1β. Changes in the protein and mRNA expression of GRα, GRβ and the key enzymes in the p38 MAPK and JNK signal pathways were measured, respectively, before and after being induced with different doses of IL-1β and specific inhibitors of p38 MAPK, JNK, ERK, PI3K and PKC. The Glucocorticoid sensitivity was measured using in vitro Glucocorticoid binding assay. Analysis of variance (ANOVA) was used to analyze the data.
The mRNA and protein expression levels of GRα, GRβ and key enzymes of the p38 MAPK and JNK pathways increased both in time- and concentration-dependent manners in IL-1β-induced nasal polyp tissue. The expression of GRβ increased more significantly than that of GRα, and the GRα/GRβ ratio decreased in time- and concentration-dependent manners. Statistically significant differences were found in the GRα/GRβ ratio and the mRNA expression of phospho-p38 MAPK and phospho-JNK between the IL-1β-induced groups and the control groups (P < 0.05). Either a specific inhibitor of the p38 MAPK pathway or a specific inhibitor of the JNK pathway increased the GRα/GRβ ratio and the Glucocorticoid affinity. None of the specific inhibitors of ERK, PI3K or PKC had any influence on the expression of GR isoforms.
Our results demonstrated that the imbalanced expression of GR isoforms in nasal polyp tissue induced by IL-1β in vitro is mediated through the p38 MAPK and JNK signal pathways.
PMCID: PMC4308899  PMID: 25632272
Chronic rhinosinusitis (CRS); Nasal polyp (NP); Signal transduction; Mitogen-activated protein kinase (MAPK)
11.  Emergence and Prevalence of Non-H2S-Producing Salmonella enterica Serovar Senftenberg Isolates Belonging to Novel Sequence Type 1751 in China 
Journal of Clinical Microbiology  2014;52(7):2557-2565.
Salmonella enterica serovar Senftenberg is a common nontyphoidal Salmonella serotype which causes human Salmonella infections worldwide. In this study, 182 S. Senftenberg isolates, including 17 atypical non-hydrogen sulfide (H2S)-producing isolates, were detected in China from 2005 to 2011. The microbiological and genetic characteristics of the non-H2S-producing and selected H2S-producing isolates were determined by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis. The phs operons were amplified and sequenced. The 17 non-H2S-producing and 36 H2S-producing isolates belonged to 7 sequence types (STs), including 3 new STs, ST1751, ST1757, and ST1758. Fourteen of the 17 non-H2S-producing isolates belonged to ST1751 and had very similar PFGE patterns. All 17 non-H2S-producing isolates had a nonsense mutation at position 1621 of phsA. H2S-producing and non-H2S-producing S. Senftenberg isolates were isolated from the same stool sample from three patients; isolates from the same patients displayed the same antimicrobial susceptibility, ST, and PFGE pattern but could be discriminated based on CRISPR spacers. Non-H2S-producing S. Senftenberg isolates belonging to ST1751 have been prevalent in Shanghai, China. It is possible that these emerging organisms will disseminate further, because they are difficult to detect. Thus, we should strengthen the surveillance for the spread of this atypical S. Senftenberg variant.
PMCID: PMC4097678  PMID: 24829240
12.  Peripheral Blood Mononuclear Cell Traffic Plays a Crucial Role in Mother-to-Infant Transmission of Hepatitis B Virus 
The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.
PMCID: PMC4323366
Hepatitis B virus; mother-to-infant transmission; peripheral blood mononuclear cell; fetomaternal cellular traffic.
13.  Twist1 correlates with poor differentiation and progression in gastric adenocarcinoma via elevation of FGFR2 expression 
World Journal of Gastroenterology : WJG  2014;20(48):18306-18315.
AIM: To explore the correlation between Twist-related protein (Twist)1, fibroblast growth factor receptor (FGFR)2 and gastric adenocarcinoma differentiation and progression.
METHODS: We evaluated Twist1 and FGFR2 in 52 gastric adenocarcinoma samples by immunohistochemistry and quantitative real time polymerase chain reaction, and analyzed the correlation between Twist1, FGFR2 and cancer differentiation. We also detected Twist1 and FGFR2 expression in gastric adenocarcinoma cell lines, and evaluated Twist1 influence on FGFR2 expression. In addition, we studied the role of FGFR2 in Twist1-promoted cancer progression, including proliferation, invasion and epithelial-mesenchymal transition (EMT).
RESULTS: Twist1 and FGFR2 were detected in almost all the gastric adenocarcinoma samples. Twist1 (P = 0.0213) and FGFR2 (P = 0.0310) mRNA levels had a significant association with gastric adenocarcinoma differentiation. Moreover, Twist1 and FGFR2 expression in poorly differentiated cells (SNU-1 and SNU-16) was notably higher than in well-differentiated cells (MKN-7 and MKN-28). In poorly differentiated gastric adenocarcinomas, FGFR2 mRNA level was significantly positively correlated with Twist1 mRNA level (P = 0.004). Twist1 was proved to promote FGFR2 by regulating Twist1 expression by knockdown and overexpression. Additionally, Twist1 could induce proliferation, invasion and EMT in gastric cancer; of these, FGFR2 was required for invasion and EMT, rather than proliferation.
CONCLUSION: Twist1 and FGFR2 are highly associated with differentiation of gastric adenocarcinoma; Twist1 can facilitate invasion and EMT in gastric adenocarcinoma via promotion of FGFR2 expression.
PMCID: PMC4277967  PMID: 25561797
Twist-related protein 1; Fibroblast growth factor receptor 2; Gastric adenocarcinoma; Cancer differentiation; Cancer progression
The annals of applied statistics  2014;8(2):974-998.
Large case/control genome-wide association studies (GWAS) often include groups of related individuals with known relationships. When testing for associations at a given locus, current methods incorporate only the familial relationships between individuals. Here, we introduce the chromosome-based Quasi Likelihood Score (cQLS) statistic that incorporates local Identity-By-Descent (IBD) to increase the power to detect associations. In studies robust to population stratification, such as those with case/control sibling pairs, simulations show that the study power can be increased by over 50%. In our example, a GWAS examining late-onset Alzheimers disease, the p-values among the most strongly associated SNPs in the APOE gene tend to decrease, with the smallest p-value decreasing from 1.23 × 10−8 to 7.70 × 10−9. Furthermore, as a part of our simulations, we reevaluate our expectations about the use of families in GWAS. We show that, although adding only half as many unique chromosomes, genotyping affected siblings is more efficient than genotyping randomly ascertained cases. We also show that genotyping cases with a family history of disease will be less beneficial when searching for SNPs with smaller effect sizes.
PMCID: PMC4275846  PMID: 25544865
cQLS; GWAS; related individuals; case-control
15.  Insights from systems pharmacology into cardiovascular drug discovery and therapy 
BMC Systems Biology  2014;8(1):141.
Given the complex nature of cardiovascular disease (CVD), information derived from a systems-level will allow us to fully interrogate features of CVD to better understand disease pathogenesis and to identify new drug targets.
Here, we describe a systematic assessment of the multi-layer interactions underlying cardiovascular drugs, targets, genes and disorders to reveal comprehensive insights into cardiovascular systems biology and pharmacology. We have identified 206 effect-mediating drug targets, which are modulated by 254 unique drugs, of which, 43% display activities across different protein families (sequence similarity < 30%), highlighting the fact that multitarget therapy is suitable for CVD. Although there is little overlap between cardiovascular protein targets and disease genes, the two groups have similar pleiotropy and intimate relationships in the human disease gene-gene and cellular networks, supporting their similar characteristics in disease development and response to therapy. We also characterize the relationships between different cardiovascular disorders, which reveal that they share more etiological commonalities with each other rooted in the global disease-disease networks. Furthermore, the disease modular analysis demonstrates apparent molecular connection between 227 cardiovascular disease pairs.
All these provide important consensus as to the cause, prevention, and treatment of various CVD disorders from systems-level perspective.
Electronic supplementary material
The online version of this article (doi:10.1186/s12918-014-0141-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4297424  PMID: 25539592
Cardiovascular disease; Network pharmacology; Network analysis; Drug discovery; Drug-target network; Gene-disease network
16.  Transplantation of human amniotic epithelial cells repairs brachial plexus injury: pathological and biomechanical analyses 
Neural Regeneration Research  2014;9(24):2159-2163.
A brachial plexus injury model was established in rabbits by stretching the C6 nerve root. Immediately after the stretching, a suspension of human amniotic epithelial cells was injected into the injured brachial plexus. The results of tensile mechanical testing of the brachial plexus showed that the tensile elastic limit strain, elastic limit stress, maximum stress, and maximum strain of the injured brachial plexuses were significantly increased at 24 weeks after the injection. The treatment clearly improved the pathological morphology of the injured brachial plexus nerve, as seen by hematoxylin eosin staining, and the functions of the rabbit forepaw were restored. These data indicate that the injection of human amniotic epithelial cells contributed to the repair of brachial plexus injury, and that this technique may transform into current clinical treatment strategies.
PMCID: PMC4316449  PMID: 25657737
nerve regeneration; peripheral nerve injury; brachial plexus injury; animal model; human amniotic epithelial cells; forepaw function; morphology; tensile mechanics; neural regeneration
17.  Resistant mutants induced by adefovir dipivoxil in hepatitis B virus isolates 
World Journal of Gastroenterology : WJG  2014;20(45):17100-17106.
AIM: To investigate the loci of adefovir dipivoxil (ADV)-induced resistance in hepatitis B virus (HBV) isolates and optimize the management of ADV-treated patients.
METHODS: Between June 2008 and August 2010, a cross-sectional control study was conducted comprising 79 patients with chronic HBV infection-related liver disease who had been administered ADV monotherapy. Patients underwent liver imaging. Serum DNA extracts were analyzed for HBV DNA levels, genotypes, and serology markers, and deep sequencing of the HBV P gene was performed.
RESULTS: ADV-resistant patients were found either with a single mutated locus, or with coexisting mutated loci. The most prevalent mutations were rtA181T, rtV214A, and rtN236T. Twenty-six patients had more than two mutated loci. The mutants were distributed among the patients without any significant affinity for gender, age, end-stage of liver disease, complications of non-alcoholic fatty liver disease, or HBV DNA levels. Patients with the rtA181T mutant were primarily infected with genotype C and e-antigen negative HBV, while patients with the rtN236T mutant were primarily infected by genotype B HBV (χ2 = 6.004, 7.159; P = 0.023, 0.007). The duration of treatment with ADV was shorter in the single mutant group compared with the multi-mutant group (t = 2.426, P = 0.018).
CONCLUSION: Drug-resistant HBV mutants are complex and diverse. Patients should receive the standard and first-line antiviral treatment, strictly comply with medication dosage, and avoid short-term withdrawal.
PMCID: PMC4258578  PMID: 25493022
Hepatitis B virus; Adefovir dipivoxil; Drug-resistant mutant; Gene sequencing
18.  A Microfluidic Localized, Multiple Cell Culture Array using Vacuum Actuated Cell Seeding: Integrated Anticancer Drug Testing 
Biomedical microdevices  2013;15(6):10.1007/s10544-013-9779-3.
In this study, we introduced a novel and convenient approach to culture multiple cells in localized arrays of microfluidic chambers using one-step vacuum actuation. In one device, we integrated 8 individually addressable regions of culture chambers, each only requiring one simple vacuum operation to seed cells lines. Four cell lines were seeded in designated regions in one device via sequential injection with high purity (99.9%-100%) and cultured for long-term. The on-chip simultaneous culture of HuT 78, Ramos, PC-3 and C166-GFP cells for 48 h was demonstrated with viabilities of 92%+/−2%, 94%+/−4%, 96%+/−2% and 97%+/−2%, respectively. The longest culture period for C166-GFP cells in this study was 168 h with a viability of 96%+/−10%. Cell proliferation in each individual side channel can be tracked. Mass transport between the main channel and side channels was achieved through diffusion and studied using fluorescein solution. The main advantage of this device is the capability to perform multiple cell-based assays on the same device for better comparative studies. After treating cells with staurosporine or anti-human CD95 for 16 h, the apoptotic cell percentage of HuT 78, CCRF-CEM, PC-3 and Ramos cells were 36%+/−3%, 24%+/−4%, 12%+/−2%, 18%+/−4% for staurosporine, and 63%+/−2%, 45%+/−1%, 3%+/−3%, 27%+/−12% for anti-human CD95, respectively. With the advantages of enhanced integration, ease of use and fabrication, and flexibility, this device will be suitable for long-term multiple cell monitoring and cell based assays.
PMCID: PMC3830671  PMID: 23813077
microfluidics; multiple cell seeding; vacuum actuation; on-chip drug test
19.  BCDP: Budget Constrained and Delay-Bounded Placement for Hybrid Roadside Units in Vehicular Ad Hoc Networks 
Sensors (Basel, Switzerland)  2014;14(12):22564-22594.
In vehicular ad hoc networks, roadside units (RSUs) placement has been proposed to improve the the overall network performance in many ITS applications. This paper addresses the budget constrained and delay-bounded placement problem (BCDP) for roadside units in vehicular ad hoc networks. There are two types of RSUs: cable connected RSU (c-RSU) and wireless RSU (w-RSU). c-RSUs are interconnected through wired lines, and they form the backbone of VANETs, while w-RSUs connect to other RSUs through wireless communication and serve as an economical extension of the coverage of c-RSUs. The delay-bounded coverage range and deployment cost of these two cases are totally different. We are given a budget constraint and a delay bound, the problem is how to find the optimal candidate sites with the maximal delay-bounded coverage to place RSUs such that a message from any c-RSU in the region can be disseminated to the more vehicles within the given budget constraint and delay bound. We first prove that the BCDP problem is NP-hard. Then we propose several algorithms to solve the BCDP problem. Simulation results show the heuristic algorithms can significantly improve the coverage range and reduce the total deployment cost, compared with other heuristic methods.
PMCID: PMC4299028  PMID: 25436656
roadside unit; facility placement; delay bound; vehicular sensor networks
20.  Glytan decreases portal pressure via mesentery vasoconstriction in portal hypertensive rats 
World Journal of Gastroenterology : WJG  2014;20(44):16674-16682.
AIM: To investigate the effects of Glytan on splanchnic hemodynamics and its reduction of portal pressure in portal hypertensive rats.
METHODS: Glytan (Ganluotong in Chinese), is composed of salvianolic acid B and diammonium glycyrrhizinate. Portal hypertension (PHT) was induced in the rats by common bile duct ligation (BDL). Hemodynamic studies were performed using the colored microsphere method. Radioimmunoassay (RIA) was used to determine endothelin (ET)-1 levels in the mesenteric circulation. Western blotting methods were used to investigate the effect of Glytan on ET A receptor (ETAR), ET B receptor (ETBR), endothelial NO synthase (eNOS), G-protein-coupled receptor kinase (GRK)2, and β-arrestin 2 expression in the mesentery. The mRNA of ETAR and ETBR was determined using real-time polymerase chain reaction.
RESULTS: Treatment with Glytan reduced portal pressure (PP) and portal territory blood flow (PTBF) and increased both mean arterial pressure (MAP) and splanchnic vascular resistance (SVR). Especially at 4 wk, PP decreased by about 40%, while MAP increased by 13%, SVR increased by 12%, and PTBF decreased by about 21%. The effect of blood flow reduction was greatest in the mesentery (about 33%) at 4 wk. The mesenteric circulation ET-1 levels of BDL rats were lower and negatively correlated with PP at 4 wk. Glytan can increase mesenteric ET-1 content and inhibit ETBR, eNOS, GRK2, and β-arrestin 2 expression in the mesentery. Moreover, Glytan showed no effect on the expression of ETAR protein and mRNA.
CONCLUSION: The decreased PP and PTBF observed after Glytan treatment were related to increased mesenteric vasoconstriction and increased receptor sensitivity to vasoconstrictor.
PMCID: PMC4248211  PMID: 25469036
Glytan; Portal hypertension; Hemodynamics; Mesentery; Endothelin-1; Receptor; Sensitivity
21.  Urine-derived stem cells: A novel and versatile progenitor source for cell-based therapy and regenerative medicine 
Genes & diseases  2014;1(1):8-17.
Engineered functional organs or tissues, created with autologous somatic cells and seeded on biodegradable or hydrogel scaffolds, have been developed for use in individuals with tissue damage suffered from congenital disorders, infection, irradiation, or cancer. However, in those patients, abnormal cells obtained by biopsy from the compromised tissue could potentially contaminate the engineered tissues. Thus, an alternative cell source for construction of the neo-organ or functional recovery of the injured or diseased tissues would be useful. Recently, we have found stem cells existing in the urine. These cells are highly expandable, and have self-renewal capacity, paracrine properties, and multi-differentiation potential. As a novel cell source, urine-derived stem cells (USCs) provide advantages for cell therapy and tissue engineering applications in regeneration of various tissues, particularly in the genitourinary tract, because they originate from the urinary tract system. Importantly, USCs can be obtained via a non-invasive, simple, and low-cost approach and induced with high efficiency to differentiate into three dermal cell lineages.
PMCID: PMC4234168  PMID: 25411659
Cell therapy; Genitourinary tract; Stem cells; Tissue regeneration; Urine
22.  Intestinal GUCY2C Prevents TGF-β Secretion Coordinating Desmoplasia and Hyperproliferation in Colorectal Cancer 
Cancer research  2013;73(22):10.1158/0008-5472.CAN-13-0887.
Tumorigenesis is a multi-step process that reflects intimate reciprocal interactions between epithelia and underlying stroma. However, tumor-initiating mechanisms coordinating transformation of both epithelial and stromal components are not defined. In humans and mice, initiation of colorectal cancer is universally associated with loss of guanylin and uroguanylin, the endogenous ligands for the tumor suppressor guanylyl cyclase C (GUCY2C), disrupting a network of homeostatic mechanisms along the crypt-surface axis. Here, we reveal that silencing GUCY2C in human colon cancer cells increases Akt-dependent TGF-β secretion, activating fibroblasts through TGF-β type I receptors and Smad3 phosphorylation. In turn, activating TGF-β signaling induces fibroblasts to secrete hepatocyte growth factor (HGF), reciprocally driving colon cancer cell proliferation through cMET-dependent signaling. Elimination of GUCY2C signaling in mice (Gucy2c-/-) produces intestinal desmoplasia, with increased reactive myofibroblasts, which is suppressed by anti-TGF-β antibodies or genetic silencing of Akt. Thus, GUCY2C coordinates intestinal epithelial-mesenchymal homeostasis through reciprocal paracrine circuits mediated by TGF-β and HGF. In that context, GUCY2C signaling constitutes a direct link between the initiation of colorectal cancer and the induction of its associated desmoplastic stromal niche. The recent regulatory approval of oral GUCY2C ligands to treat chronic gastrointestinal disorders underscores the potential therapeutic opportunity for oral GUCY2C hormone replacement to prevent remodeling of the microenvironment essential for colorectal tumorigenesis.
PMCID: PMC3838632  PMID: 24085786
GUCY2C; TGF-β; desmoplasia; hepatocyte growth factor; colorectal cancer
24.  Cidea Control of Lipid Storage and Secretion in Mouse and Human Sebaceous Glands 
Molecular and Cellular Biology  2014;34(10):1827-1838.
Sebaceous glands are skin appendages that secrete sebum onto hair follicles to lubricate the hair and maintain skin homeostasis. In this study, we demonstrated that Cidea is expressed at high levels in lipid-laden mature sebocytes and that Cidea deficiency led to dry hair and hair loss in aged mice. In addition, Cidea-deficient mice had markedly reduced levels of skin surface lipids, including triacylglycerides (TAGs) and wax diesters (WDEs), and these mice were defective in water repulsion and thermoregulation. Furthermore, we observed that Cidea-deficient sebocytes accumulated a large number of smaller-sized lipid droplets (LDs), whereas overexpression of Cidea in human SZ95 sebocytes resulted in increased lipid storage and the accumulation of large LDs. Importantly, Cidea was highly expressed in human sebaceous glands, and its expression levels were positively correlated with human sebum secretion. Our data revealed that Cidea is a crucial regulator of sebaceous gland lipid storage and sebum lipid secretion in mammals and humans.
PMCID: PMC4019032  PMID: 24636991
25.  Genetic Association Analysis of 30 Genes Related to Obesity in a European American Population 
Obesity, which is frequently associated with diabetes, hypertension, and cardiovascular diseases, is primarily the result of a net excess of caloric intake over energy expenditure. Human obesity is highly heritable, but the specific genes mediating susceptibility in non-syndromic obesity remain unclear. We tested candidate genes in pathways related to food intake and energy expenditure for association with body mass index (BMI).
We re-analyzed 355 common genetic variants of 30 candidate genes in 7 molecular pathways related to obesity in 1,982 unrelated European Americans from the New York Health Project. Data were analyzed by using a Bayesian hierarchical generalized linear model. The BMIs were log-transformed and then adjusted for covariates including age, age2, gender, and diabetes status. The single nucleotide polymorphisms (SNPs) were modeled as additive effects.
With the stipulated adjustments, nine SNPs in eight genes were significantly associated with BMI: GHRL (rs35683), AGRP (rs5030980), CPE (rs1946816 and rs4481204), GLP1R (rs2268641), HTR2A (rs912127), NPY5R (Y5R1c52), SOCS3 (rs4969170), and STAT3 (rs4796793). We also found a gender-by-SNP interaction (rs1745837 in HTR2A), which indicated that variants in the gene HTR2A had a stronger association with BMI in males. In addition, NPY1R was detected as having a significant gene effect even though none of the SNPs in this gene was significant.
Variations in genes AGRP, CPE, GHRL, GLP1R, HTR2A, NPY1R, NPY5R, SOCS3, and STAT3 showed modest associations with BMI in European Americans. The pathways in which these genes participate regulate energy intake and thus these associations are mechanistically plausible in this context.
PMCID: PMC3909018  PMID: 23900445
Obesity; genetic association; single nucleotide polymorphism (SNP); Bayesian hierarchical generalized linear model (BhGLM)

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