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1.  RhI-Catalyzed Decarbonylative Spirocyclization via C–C Cleavage of Benzocyclobutenones: An Efficient Approach to Access Functionalized Spirocycles 
We describe a rhodium-catalyzed all-carbon spirocenter formation through a decarbonylative coupling of trisubstituted cyclic olefins and benzocyclobutenones via C–C activation. A [Rh(CO)2Cl]2/P(C6F5)3 metal-ligand combination was found to catalyze this transformation most efficiently. A range of diverse spirocyclic rings were synthesized in good to excellent yields and many sensitive functional groups were tolerated. Mechanistic study supports the hydrogen-transfer process that occurs via a β-H elimination/decarbonylation pathway.
PMCID: PMC3970409  PMID: 24446067
C–C activation; homogeneous catalysis; rhodium; spirocycle; decarbonylation
2.  Angiogenin Promotes U87MG Cell Proliferation by Activating NF-κB Signaling Pathway and Downregulating Its Binding Partner FHL3 
PLoS ONE  2015;10(2):e0116983.
Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression.
PMCID: PMC4320115  PMID: 25659096
3.  Molecular markers to assess short-term disease local recurrence in nasopharyngeal carcinoma 
Oncology Reports  2015;33(3):1418-1426.
An important challenge in nasopharyngeal carcinoma (NPC) research is to develop effective predictors of tumor recurrence following treatment to determine whether immediate adjuvant therapy is necessary. We retrospectively analyzed archived specimens collected from 45 patients with paired samples of primary NPC (pNPC) and recurrent NPC (rNPC). Clinical samples were collected from the Cancer Center Databases of the First People’s Hospital of Foshan and Shantou Central Hospital (affiliates of Sun Yat-Sen University) between 2001 and 2012. Expression levels of phosphor-Stat3 (p-Stat3), signalosome complex subunit 5 (Jab1/Csn5), Akt1, C/EBP homologous protein (CHOP), Ki-67, and apoptosis were determined by immunohistochemistry in pNPC and rNPC samples from the same patients. Differences in these markers between the short-term interval to recurrence (ITR) group (ITR <18 months) and long-term ITR group (ITR ≥18 months) were further analyzed. In Cox’s regression analysis, the ITR was significantly associated as an independent-negative prognostic factor for overall survival (hazard ratio, 0.211; 95% confidence interval, 0.053–0.841; P=0.027). p-Stat3 was increased in the short-term ITR group (ITR <18 months) and tended to be lower in the long-term ITR group (ITR ≥18 months). In the short-term ITR group, nuclear Akt expression was significantly increased in paired rNPC (P=0.028). In the long-term ITR group, the expression of nuclear Jab1/Csn5 (P=0.047) and assessment of apoptosis measured with TdT-mediated dUTP nick end-labeling (TUNEL) (P=0.003) was significantly increased in paired rNPC. The results suggest that differences between short- and long-term ITR may predict outcome in rNPC. Furthermore, the overexpression of Jab1/Csn5 and Akt may contribute to the carcinogenesis of rNPC, and Akt seems to promote the progression of short-term ITR. Intra-individual changes of Jab1/Csn5, Akt, and TUNEL may help to identify short-term ITR.
PMCID: PMC4324483  PMID: 25607111
nasopharyngeal carcinoma; recurrence; disease-free interval to recurrence
4.  Rhipicephalus microplus serine protease inhibitor family: annotation, expression and functional characterisation assessment 
Rhipicephalus (Boophilus) microplus evades the host’s haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes.
The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays.
A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15).
This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.
PMCID: PMC4322644  PMID: 25564202
Genome; Protease inhibitor; Rhipicephalus microplus; serpin; Cattle tick
5.  High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7 
The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic E. coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR+ open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10–100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.
PMCID: PMC3913999  PMID: 24361397
Escherichia coli O157; multiple antibiotic resistance; mutation; genes; repressor protein; DNA
6.  Antibacterial and anti-biofilm activities of thiazolidione derivatives against clinical staphylococcus strains 
Both Staphylococcus aureus and Staphylococcus epidermidis can form biofilms on natural surfaces or abiotic surfaces, such as medical implants, resulting in biofilm-associated diseases that are refractory to antibiotic treatment. We previously reported a promising antibacterial compound (Compound 2) and its derivatives with bactericidal and anti-biofilm activities against both S. epidermidis and S. aureus. We have further evaluated the antibacterial activities of four Compound 2 derivatives (H2-38, H2-39, H2-74 and H2-81) against 163 clinical strains of S. epidermidis and S. aureus, including methicillin-susceptible and methicillin-resistant strains, as well as biofilm-forming and non-biofilm-forming strains. The four derivatives inhibited the planktonic growth of all of the clinical staphylococcal isolates, including methicillin-resistant S. aureus and methicillin-resistant S. epidermidis and displayed bactericidal activities against both immature (6 h) and mature (24 h) biofilms formed by the strong biofilm-forming strains. The derivatives, which all target YycG, will help us to develop new antimicrobial agents against multidrug-resistant staphylococci infections and biofilm-associated diseases.
PMCID: PMC4317670
antibacterial; anti-biofilm activity; methicillin-resistant Staphylococcus aureus; minimal inhibitory concentration; MIC50; Staphylococcus epidermidis
7.  Removal of the Tag from His-tagged ILYd4, a Human CD59 Inhibitor, Significantly Improves its Physical Properties and its Activity 
Current pharmaceutical design  2012;18(27):4187-4196.
Complement dependent cytotoxicity (CDC) significantly contributes to Rituximab (RTX) and Ofatumumab (OFA) efficacies in the treatment of B-cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex and thereby inhibits CDC. hCD59 is an important determinant of the sensitivity of NHL and CLL to RTX and OFA treatment. Recently, we developed a specific and potent hCD59 inhibitor, His-tagged ILYd4, which consists of 30 amino acid sequences extending from the N-terminus of ILYd4. Our previously published results indicate that His-tagged ILYd4 can be used as a lead candidate to further develop a potential therapeutic adjuvant for RTX and OFA treatment of RTX-resistant NHL and CLL. However, these studies were conducted using ILYd4 tagged on the N-terminus with 30 additional amino acids (AA) containing 6 X His used for immobilized metal affinity chromatograph. As a further step towards the development of ILYd4-based therapeutics, we investigated the impact of the removal of this extraneous sequence on the anti-hCD59 activity. In this paper, we report the generation and characterization of tag-free ILYd4. We demonstrate that tag-free ILYd4 has over three-fold higher anti-hCD59 activities than the His-tagged ILYd4. The enhanced RTX-mediated CDC effect on B-cell malignant cells comes from tag-free ILYd4’s improved functionality and physical properties including better solubility, reduced tendency to aggregation, and greater thermal stability. Therefore, tag-free ILYd4 is a better candidate for the further development for the clinical application.
PMCID: PMC4279449  PMID: 22642361
Rituximab; complement; CD59; intermedilysin; his-tag
8.  Protein-Losing Enteropathy in Systemic Lupus Erythematosus: 12 Years Experience from a Chinese Academic Center 
PLoS ONE  2014;9(12):e114684.
Protein-losing enteropathy (PLE) is a complication in some systemic lupus erythematosus (SLE) patients that is often misdiagnosed. With this study, we provide insight into clinical characteristics, laboratory characteristics, diagnostic tests, risk factors, treatment, and prognosis of the disease.
A retrospective, case-control study was performed in 44 patients with SLE-related PLE (PLE group) and 88 patients with active SLE (control group) admitted to our care from January 2000−January 2012. Risk factors for SLE-related PLE were examined, and we analyzed the accuracy of single and combined laboratory characteristics in discriminating SLE-related PLE from active SLE. Serum albumin and C3 levels were measured as outcome during and after treatment with corticosteroids and immunosuppressive agents.
The PLE group had lower mean serum albumin and 24-hour urine protein levels, higher mean total plasma cholesterol levels, and greater frequencies of anti-SSA and SSB seropositivity compared with the control group. Anti-SSA seropositivity, hypoalbuminemia, and hypercholesterolemia were independent risk factors for SLE-related PLE. The simultaneous presence of serum albumin (<22 g/l) and 24-hour urine protein (<0.8 g/24 h) had high specificity, positive predictive value, negative predictive value, and positive likelihood ratio, a low negative likelihood ratio and no significant reduction in sensitivity. High dosage of glucocorticosteroid combined with cyclophosphomide were mostly prescribed for SLE-related PLE.
SLE-related PLE should be considered when an SLE patient presents with generalized edema, anti-SSA antibody seropositivity, hypercholesterolemia, severe hypoalbuminemia, and low 24-hour urine protein levels. Aggressive treatment for lupus might improve prognosis.
PMCID: PMC4260872  PMID: 25490025
9.  Staphylococcus epidermidis SrrAB Regulates Bacterial Growth and Biofilm Formation Differently under Oxic and Microaerobic Conditions 
Journal of Bacteriology  2014;197(3):459-476.
SrrAB expression in Staphylococcus epidermidis strain 1457 (SE1457) was upregulated during a shift from oxic to microaerobic conditions. An srrA deletion (ΔsrrA) mutant was constructed for studying the regulatory function of SrrAB. The deletion resulted in retarded growth and abolished biofilm formation both in vitro and in vivo and under both oxic and microaerobic conditions. Associated with the reduced biofilm formation, the ΔsrrA mutant produced much less polysaccharide intercellular adhesion (PIA) and showed decreased initial adherence capacity. Microarray analysis showed that the srrA mutation affected transcription of 230 genes under microaerobic conditions, and 51 genes under oxic conditions. Quantitative real-time PCR confirmed this observation and showed downregulation of genes involved in maintaining the electron transport chain by supporting cytochrome and quinol-oxidase assembly (e.g., qoxB and ctaA) and in anaerobic metabolism (e.g., pflBA and nrdD). In the ΔsrrA mutant, the expression of the biofilm formation-related gene icaR was upregulated under oxic conditions and downregulated under microaerobic conditions, whereas icaA was downregulated under both conditions. An electrophoretic mobility shift assay further revealed that phosphorylated SrrA bound to the promoter regions of icaR, icaA, qoxB, and pflBA, as well as its own promoter region. These findings demonstrate that in S. epidermidis SrrAB is an autoregulator and regulates biofilm formation in an ica-dependent manner. Under oxic conditions, SrrAB modulates electron transport chain activity by positively regulating qoxBACD transcription. Under microaerobic conditions, it regulates fermentation processes and DNA synthesis by modulating the expression of both the pfl operon and nrdDG.
PMCID: PMC4285975  PMID: 25404696
10.  Evaluating the Role of PTH in Promotion of Chondrosarcoma Cell Proliferation and Invasion by Inhibiting Primary Cilia Expression 
Chondrosarcoma is characterized by secretion of a cartilage-like matrix, with high proliferation ability and metastatic potential. Previous studies have shown that parathyroid hormone-related protein (PTHrP) has a close relationship with various tumor types. The objectives of this study were to research the function played by PTHrP in human chondrosarcoma, especially targeting cell proliferation and invasion, and to search for the potential interaction between PTHrP and primary cilia in tumorigenesis. Surgical resection tissues and the human chondrosarcoma cell line SW1353 were used in the scientific research. Cells were stimulated with an optimum concentration of recombinant PTH (1-84), and siRNA was used to interfere with internal PTHrP. Cell proliferation and invasion assays were applied, including MTS-8 cell proliferation assay, Western blot, RT-PCR, Transwell invasion assay, and immunohistochemistry and immunofluorescence assays. A high level of PTHrP expression was found in human chondrosarcoma tissues, and recombinant PTH exhibited positive promotion in tumor cell proliferation and invasion. In the meantime, PTHrP could inhibit the assembly of primary cilia and regulate downstream gene expression. These findings indicate that PTHrP can regulate tumor cell proliferation and invasion ability, possibly through suppression of primary cilia assembly. Thus, restricting PTHrP over-expression is a feasible potential therapeutic method for chondrosarcoma.
PMCID: PMC4264140  PMID: 25365173
chondrosarcoma; primary cilia expression; parathyroid hormone-related protein; PTHrP
11.  Apolipoprotein E Gene Polymorphisms Are Associated with Primary Hyperuricemia in a Chinese Population 
PLoS ONE  2014;9(10):e110864.
Primary hyperuricemia, an excess of uric acid in the blood, is a major public health problem. In addition to the morbidity that is attributable to gout, hyperuricemia is also associated with metabolic syndrome, hypertension, and cardiovascular disease. This study aims to assess the genetic associations between Apolipoprotein E (APOE) polymorphisms and hyperuricemia in a Chinese population.
A total of 770 subjects (356 hyperuricemic cases and 414 normouricemic controls) were recruited from the Ningxia Hui Autonomous Region, China. A physical examination was performed and fasting blood was collected for biochemical tests, including determination of the levels of serum lipid, creatinine, and uric acid. Multi-ARMS PCR was applied to determine the APOE genotypes, followed by an investigation of the distribution of APOE genotypes and alleles frequencies in the controls and cases.
The frequencies of the APOE-ε2ε3 genotype (17.70% vs. 10.39%, P = 0.003) and the APOE-ε2 allele (10.53% vs. 5.80%, P = 0.001) were significantly higher in the hyperuricemic group than in the normouricemic group. Furthermore, male cases were more likely to have the APOE-ε2ε3 genotype and APOE-ε2 allele, compared with male controls. In both Han and Hui subjects, cases were more likely to have the APOE-ε2ε3 genotype and the APOE-ε2 allele compared with controls. Furthermore, multivariate logistic regression showed that carriers of the APOE-ε2ε3 genotype (P = 0.001, OR = 2.194) and the ε2 allele (P = 0.001, OR = 2.099) were significantly more likely to experience hyperuricemia than carriers of the ε3/ε3 genotype and the ε3 allele after adjustment for sex, body mass index (BMI), diastolic blood pressure (DBP), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), creatinine (Cr) and fasting blood glucose(FBG).
The APOE-ε2ε3 genotype and the APOE-ε2 allele are associated with serum uric acid levels in Chinese subjects, indicating that individuals carrying the APOE-ε2 allele have a higher risk of hyperuricemia than non-carriers.
PMCID: PMC4214707  PMID: 25356596
12.  Novel MiRNA and PhasiRNA Biogenesis Networks in Soybean Roots from Two Sister Lines That Are Resistant and Susceptible to SCN Race 4 
PLoS ONE  2014;9(10):e110051.
The soybean cyst nematode (SCN), Heterodera glycines, is the most devastating pathogen of soybean worldwide. SiRNAs (small interfere RNAs) have been proven to induce the silencing of cyst nematode genes. However, whether small RNAs from soybean root have evolved a similar mechanism against SCN is unknown. Two genetically related soybean sister lines (ZP03-5373 and ZP03-5413), which are resistant and susceptible, respectively, to SCN race 4 infection were selected for small RNA deep sequencing to identify small RNAs targeted to SCN. We identified 71 less-conserved miRNAs-miRNAs* counterparts belonging to 32 families derived from 91 loci, and 88 novel soybean-specific miRNAs with distinct expression patterns. The identified miRNAs targeted 42 genes representing a wide range of enzymatic and regulatory activities. Roots of soybean conserved one TAS (Trans-acting siRNA) gene family with a similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profile. In addition, we found that six miRNAs (gma-miR393, 1507, 1510, 1515, 171, 2118) guide targets to produce secondary phasiRNAs (phased, secondary, small interfering RNAs) in soybean root. Multiple targets of these phasiRNAs were predicted and detected. Importantly, we also found that the expression of 34 miRNAs differed significantly between the two lines. Seven ZP03-5373-specific miRNAs were differentially expressed after SCN infection. Forty-four transcripts from SCN were predicted to be potential targets of ZP03-5373-specific differential miRNAs. These findings suggest that miRNAs play an important role in the soybean response to SCN.
PMCID: PMC4214822  PMID: 25356812
13.  Rare incidence of primary adrenocortical carcinosarcoma: A case report and literature review 
Oncology Letters  2014;9(1):153-158.
Adrenocortical carcinoma (ACC) is a rare, but highly aggressive type of tumor with an incidence of one to two per million annually. Adrenocortical carcinosarcoma is an exceptional variant of ACC, which is characterized by the presence of histological regions of carcinoma and sarcoma. To date, to the best of our knowledge, there have only been 12 reported cases of adrenocortical carcinosarcoma. In the present study, a case of primary, non-functional adrenocortical carcinosarcoma is described, as well as a review of the literature to raise awareness of this particularly rare type of malignant neoplasm that is associated with a worse diagnosis and prognosis than adrenocortical carcinoma. In the present study, the patient underwent a laparoscopic left adrenalectomy and the tumor was dissected without complication from the left kidney. Microscopic observations showed the tumor comprised of epithelial and spindle cell components. The patient did not exhibit signs of tumor recurrence at the one-month follow-up. The potential diagnosis of adrenocortical carcinosarcoma must be considered when diagnosing adrenal malignancies in adults. In addition, comphrensive imunohistochemical staining may be required to identify possible sarcomatous patterns. To the best of our knowledge, the present case is the first to report an incidence of adrenocortical carcinosarcoma in China. Details of the patient are presented and the pathology of adrenocortical carcinosarcoma is discussed.
PMCID: PMC4247116  PMID: 25435950
adrenocortical carcinosarcoma; adrenocortical carcinoma; pathology
14.  Upregulation of CD147 promotes cell invasion, epithelial-to-mesenchymal transition and activates MAPK/ERK signaling pathway in colorectal cancer 
Colorectal cancer (CRC) is one of the most common cancers in the world. CD147, a transmembrane protein, has been reported to be correlated with various cancers. In this study, we aimed to investigate the mechanism of CD147 in regulating drug resistance, cell invasion and epithelial-to-mesenchymal transition (EMT) in CRC cells. qRT-PCR and western blotting were used to evaluated the expression of CD147 in 40 CRC cases and 4 cell lines. Increased expression of CD147 at both mRNA and protein levels was found in CRC samples, and the level of CD147 was correlated with lymph node metastasis. CD147 overexpression increased the 5-Fluorouracil (5-FU) resistance, enhanced the invasion and EMT of CRC cells by regulating EMT markers and MMPs. Adverse results were obtained in CD147 knockdown CRC cell line. Further investigation revealed that CD147 activated MAPK/ERK pathway, ERK inhibitor U0126 suppressed the CD147-induced cell invasion, migration and MMP-2, MMP-9 expression. Taken together, our study indicates that CD147 promotes the 5-FU resistance, and MAPK/ERK signaling pathway is involved in CD147-promoted invasion and EMT of CRC cells.
PMCID: PMC4270601  PMID: 25550778
CD147; 5-FU resistance; invasion; EMT; MAPK/ERK pathway; colorectal cancer
15.  Integration of Cardiac Proteome Biology and Medicine by a Specialized Knowledgebase 
Circulation research  2013;113(9):1043-1053.
Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts.
The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and web portals, thereby serving as a new resource to advance cardiovascular biology and medicine.
Methods and Results
We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB), a centralized platform of high quality cardiac proteomic data, bioinformatics tools and relevant cardiovascular phenotypes. Currently, COPaKB features eight organellar modules, comprising 4,203 LC-MS/MS experiments from human, mouse, drosophila and C. elegans as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest.
COPaKB ( provides an innovative and interactive resource, which connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified web server, non-proteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.
PMCID: PMC4076475  PMID: 23965338
Cardiovascular Proteomics; COPaKB; Spectral Library; Omics Science; knowledge translation; bioinformatics; organelle; proteomics; mitochondria
16.  In Situ Atom Scale Visualization of Domain Wall Dynamics in VO2 Insulator-Metal Phase Transition 
Scientific Reports  2014;4:6544.
A domain wall, as a device, can bring about a revolution in developing manipulation of semiconductor heterostructures devices at the atom scale. However, it is a challenge for these new devices to control domain wall motion through insulator-metal transition of correlated-electron materials. To fully understand and harness this motion, it requires visualization of domain wall dynamics in real space. Here, domain wall dynamics in VO2 insulator-metal phase transition was observed directly by in situ TEM at atom scale. Experimental results depict atom scale evolution of domain morphologies and domain wall exact positions in (202) and (040) planes referring to rutile structure at 50°C. In addition, microscopic mechanism of domain wall dynamics and accurate lattice basis vector relationship of two domains were investigated with the assistance of X-ray diffraction, ab initio calculations and image simulations. This work offers a route to atom scale tunable heterostructure device application.
PMCID: PMC4189024  PMID: 25292447
17.  Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth 
Cell Research  2014;24(10):1164-1180.
An increased population of CD4+CD25highFoxp3+ regulatory T cells (Tregs) in the tumor-associated microenvironment plays an important role in cancer immune evasion. However, the underlying mechanism remains unclear. Here we observed an increased secretion of miR-214 in various types of human cancers and mouse tumor models. Tumor-secreted miR-214 was sufficiently delivered into recipient T cells by microvesicles (MVs). In targeted mouse peripheral CD4+ T cells, tumor-derived miR-214 efficiently downregulated phosphatase and tensin homolog (PTEN) and promoted Treg expansion. The miR-214-induced Tregs secreted higher levels of IL-10 and promoted tumor growth in nude mice. Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-214 resulted in enhanced immune suppression and tumor implantation/growth in mice. The MV delivery of anti-miR-214 antisense oligonucleotides (ASOs) into mice implanted with tumors blocked Treg expansion and tumor growth. Our study reveals a novel mechanism through which cancer cell actively manipulates immune response via promoting Treg expansion.
PMCID: PMC4185347  PMID: 25223704
secreted microRNA; regulatory T cell; PTEN; microvesicle; immune evasion; tumor
18.  Uncovering Community Structures with Initialized Bayesian Nonnegative Matrix Factorization 
PLoS ONE  2014;9(9):e107884.
Uncovering community structures is important for understanding networks. Currently, several nonnegative matrix factorization algorithms have been proposed for discovering community structure in complex networks. However, these algorithms exhibit some drawbacks, such as unstable results and inefficient running times. In view of the problems, a novel approach that utilizes an initialized Bayesian nonnegative matrix factorization model for determining community membership is proposed. First, based on singular value decomposition, we obtain simple initialized matrix factorizations from approximate decompositions of the complex network’s adjacency matrix. Then, within a few iterations, the final matrix factorizations are achieved by the Bayesian nonnegative matrix factorization method with the initialized matrix factorizations. Thus, the network’s community structure can be determined by judging the classification of nodes with a final matrix factor. Experimental results show that the proposed method is highly accurate and offers competitive performance to that of the state-of-the-art methods even though it is not designed for the purpose of modularity maximization.
PMCID: PMC4182427  PMID: 25268494
19.  Cas9-Based Tools for Targeted Genome Editing and Transcriptional Control 
Development of tools for targeted genome editing and regulation of gene expression has significantly expanded our ability to elucidate the mechanisms of interesting biological phenomena and to engineer desirable biological systems. Recent rapid progress in the study of a clustered, regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein system in bacteria has facilitated the development of newly facile and programmable platforms for genome editing and transcriptional control in a sequence-specific manner. The core RNA-guided Cas9 endonuclease in the type II CRISPR system has been harnessed to realize gene mutation and DNA deletion and insertion, as well as transcriptional activation and repression, with multiplex targeting ability, just by customizing 20-nucleotide RNA components. Here we describe the molecular basis of the type II CRISPR/Cas system and summarize applications and factors affecting its utilization in model organisms. We also discuss the advantages and disadvantages of Cas9-based tools in comparison with widely used customizable tools, such as Zinc finger nucleases and transcription activator-like effector nucleases.
PMCID: PMC3957621  PMID: 24389925
20.  Proteomic Investigation into Betulinic Acid-Induced Apoptosis of Human Cervical Cancer HeLa Cells 
PLoS ONE  2014;9(8):e105768.
Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.
PMCID: PMC4141803  PMID: 25148076
21.  Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility 
Malaria Journal  2014;13(1):315.
Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown.
Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163.
615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway.
This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.
Electronic supplementary material
The online version of this article (doi:10.1186/1475-2875-13-315) contains supplementary material, which is available to authorized users.
PMCID: PMC4150949  PMID: 25124718
Gamete; Plasmodium; Glycolysis; Flagellum; Energy metabolism
24.  Fabrication and characterization of bio-engineered cardiac pseudo tissues 
Biofabrication  2009;1(3):035001.
We report to fabricate functional three-dimensional (3D) tissue constructs by using an inkjet based bio-prototyping method. With the use of the modified inkjet printers, contractile cardiac hybrids that exhibit the forms of the 3D rectangular sheet and even the “half heart” (with two connected ventricles) have been fabricated by arranging alternate layers of biocompatible alginate hydrogels and mammalian cardiac cells according to pre-designed 3D patterns. In this study, primary feline adult and H1 cardiomyocytes were used as model cardiac cells. Alginate hydrogels with controlled micro-shell structures were built by spraying cross-linkers in micro drops onto un-gelled alginic acid. The cells remained viable in constructs as thick as 1 cm due to the programmed porosity. Microscopic and macroscopic contractile functions of these cardiomyocytes constructs were observed in vitro. These results suggest that the inkjet bio-prototyping method could be used for hierarchical design of functional cardiac pseudo tissues, balanced with porosity for mass transport and structural support.
PMCID: PMC4120951  PMID: 20811105
25.  Human Keratinocyte Cultures in the Investigation of Early Steps of Human Papillomavirus Infection 
Human papillomaviruses (HPVs) are non-enveloped DNA viruses that are highly tropic for mucosal and cutaneous epithelia. The HPV life cycle is tightly linked to epithelial cell differentiation, where HPVs only infect the basal proliferating keratinocytes, and progeny virus assembly and release only occurs in differentiated upper-layer keratinocytes. Therefore, human keratinocyte monolayer cultures provide a useful model to study the early stages of HPV infection. However, previous reports have shown some conflicting results of virus-host interactions during HPV entry, which may be partly attributable to the different cell culture models used to examine these steps of HPV infection. Thus, there is a need to have a standardized in vitro model system to study virus-host interactions during HPV entry. Here, we describe the 3 most widely accepted keratinocyte models for studying HPV infection: primary human foreskin keratinocytes (HFK), normal immortalized keratinocytes (NIKS), and transformed HaCaT keratinocytes. We also describe methods to genetically manipulate these cells, enabling the study of candidate host genes that may be important during HPV infection. Lastly, we will outline simple and robust methods to assay HPV infectivity, which can be used to determine whether knockdown or overexpression of a particular gene affects HPV entry.
PMCID: PMC4076347  PMID: 24281871
Keratinocyte; HFK; NIKS; HaCaT; Lentivirus; Transduction; Transfection; Puromycin; Papillomavirus; HPV

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