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1.  A comparison of ARMS and direct sequencing for EGFR mutation analysis and Tyrosine Kinase Inhibitors treatment prediction in body fluid samples of Non-Small-Cell Lung Cancer patients 
Epidermal growth factor receptor (EGFR) mutation is strongly associated with the therapeutic effect of tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Nevertheless, tumor tissue that needed for mutation analysis is frequently unavailable. Body fluid was considered to be a feasible substitute for the analysis, but arising problems in clinical practice such as relatively lower mutation rate and poor clinical correlation are not yet fully resolved.
In this study, 50 patients (32 pleural fluids and 18 plasmas) with TKIs therapy experience and with direct sequencing results were selected from 220 patients for further analysis. The EGFR mutation status was re-evaluated by Amplification Refractory Mutation System (ARMS), and the clinical outcomes of TKIs were analyzed retrospectively.
As compared with direct sequencing, 16 positive and 23 negative patients were confirmed by ARMS, and the other 11 former negative patients (6 pleural fluids and 5 plasmas) were redefined as positive, with a fairly well clinical outcome (7 PR, 3 SD, and 1 PD). The objective response rate (ORR) of positive patients was significant, 81.3% (direct sequencing) and 72.7% (ARMS) for pleural fluids, and 80% (ARMS) for plasma. Notably, even reclassified by ARMS, the ORR for negative patients was still relatively high, 60% for pleural fluids and 46.2% for plasma.
When using body fluids for EGFR mutation analysis, positive result is consistently a good indicator for TKIs therapy, and the predictive effect was no less than that of tumor tissue, no matter what method was employed. However, even reclassified by ARMS, the correlation between negative results and clinical outcome of TKIs was still unsatisfied. The results indicated that false negative mutation still existed, which may be settled by using method with sensitivity to single DNA molecule or by optimizing the extraction procedure with RNA or CTC to ensure adequate amount of tumor-derived nucleic acid for the test.
PMCID: PMC3287118  PMID: 22142557
Body Fluids; EGFR Mutation; Direct Sequencing; ARMS; TKIs; NSCLC
2.  Associations of the uric acid related genetic variants in SLC2A9 and ABCG2 loci with coronary heart disease risk 
BMC Genetics  2015;16(1):4.
Multiple studies investigated the associations between serum uric acid and coronary heart disease (CHD) risk. However, further investigations still remain to be carried out to determine whether there exists a causal relationship between them. We aim to explore the associations between genetic variants in uric acid related loci of SLC2A9 and ABCG2 and CHD risk in a Chinese population.
A case–control study including 1,146 CHD cases and 1,146 controls was conducted. Association analysis between two uric acid related variants (SNP rs11722228 in SLC2A9 and rs4148152 in ABCG2) and CHD risk was performed by logistic regression model. Adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Compared with subjects with A allele of rs4148152, those with G allele had a decreased CHD risk and the association remained significant in a multivariate model. However, it altered to null when BMI was added into the model. No significant association was observed between rs11722228 and CHD risk. The distribution of CHD risk factors was not significantly different among different genotypes of both SNPs. Among subjects who did not consume alcohol, the G allele of rs4148152 showed a moderate protective effect. However, no significant interactions were observed between SNP by CHD risk factors on CHD risk.
There might be no association between the two uric acid related SNPs with CHD risk. Further studies were warranted to validate these results.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-015-0162-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4314773  PMID: 25634581
Coronary heart disease; Uric acid; Polymorphism; Gene-environment interaction
3.  Simultaneous hydrogen and ethanol production from cascade utilization of mono-substrate in integrated dark and photo-fermentative reactor 
Integrating hydrogen-producing bacteria with complementary capabilities, dark-fermentative bacteria (DFB) and photo-fermentative bacteria (PFB), is a promising way to completely recover bioenergy from waste biomass. However, the current coupled models always suffer from complicated pretreatment of the effluent from dark-fermentation or imbalance between dark and photo-fermentation, respectively. In this work, an integrated dark and photo-fermentative reactor (IDPFR) was developed to completely convert an organic substrate into bioenergy.
In the IDPFR, Ethanoligenens harbinese B49 and Rhodopseudomonas faecalis RLD-53 were separated by a membrane into dark and photo chambers, while the acetate produced by E. harbinese B49 in the dark chamber could freely pass through the membrane into the photo chamber and serve as a carbon source for R. faecalis RLD-53. The hydrogen yield increased with increasing working volume of the photo chamber, and reached 3.38 mol H2/mol glucose at the dark-to-photo chamber ratio of 1:4. Hydrogen production by the IDPFR was also significantly affected by phosphate buffer concentration, glucose concentration, and ratio of dark-photo bacteria. The maximum hydrogen yield (4.96 mol H2/mol glucose) was obtained at a phosphate buffer concentration of 20 mmol/L, a glucose concentration of 8 g/L, and a ratio of dark to photo bacteria of 1:20. As the glucose and acetate were used up by E. harbinese B49 and R. faecalis RLD-53, ethanol produced by E. harbinese B49 was the sole end-product in the effluent from the IDPFR, and the ethanol concentration was 36.53 mmol/L with an ethanol yield of 0.82 mol ethanol/mol glucose.
The results indicated that the IDPFR not only circumvented complex pretreatments on the effluent in the two-stage process, but also overcame the imbalance of growth and metabolic rate between DFB and PFB in the co-culture process, and effectively enhanced cooperation between E. harbinense B49 and R. faecalis RLD-53. Moreover, simultaneous hydrogen and ethanol production were achieved by coupling E. harbinese B49 and R. faecalis RLD-53 in the IDPFR. According to stoichiometry, the hydrogen and ethanol production efficiencies were 82.67% and 82.19%, respectively. Therefore, IDPFR was an effective strategy for coupling DFB and PFB to fulfill efficient energy recovery from waste biomass.
PMCID: PMC4308915  PMID: 25648915
Hydrogen production; Ethanol production; Dark-fermentation; Photo-fermentation; Integrated dark and photo-fermentative reactor; Kinetics; Membrane
4.  Anti-inflammatory effects of BHBA in both in vivo and in vitro Parkinson’s disease models are mediated by GPR109A-dependent mechanisms 
Accumulating evidence suggests that neuroinflammation plays an important role in the progression of Parkinson’s disease (PD). Excessively activated microglia produce several pro-inflammatory enzymes and pro-inflammatory cytokines, leading to damage to surrounding neurons and eventually inducing neurodegeneration. Therefore, the inhibition of microglial overactivation may be a potential therapeutic strategy to prevent the further progression of PD. β-Hydroxybutyric acid (BHBA) has been shown to suppress lipopolysaccharide (LPS)-induced inflammation in BV-2 cells and to protect dopaminergic neurons in previous studies, but the underlying mechanisms remain unclear. Thus, in this study, we further investigated this mechanism in LPS-induced in vivo and in vitro PD models.
For the in vitro experiments, primary mesencephalic neuron-glia cultures were pretreated with BHBA and stimulated with LPS. [3H]dopamine (DA) uptake, tyrosine hydroxylase-immunoreactive (TH-ir) neurons and morphological analysis were evaluated and analyzed in primary mesencephalic neuron-glia cultures. In vivo, microglial activation and the injury of dopaminergic neurons were induced by LPS intranigral injection, and the effects of BHBA treatment on microglial activation and the survival ratio and function of dopaminergic neurons were investigated. Four our in vitro mechanistic experiment, primary microglial cells were pretreated with BHBA and stimulated with LPS; the cells were then assessed for the responses of pro-inflammatory enzymes and pro-inflammatory cytokines, and the NF-κB signaling pathway was evaluated and analyzed.
We found that BHBA concentration-dependently attenuated the LPS-induced decrease in [3H]DA uptake and loss of TH-ir neurons in the primary mesencephalic neuron/glia mixed culture. BHBA treatment significantly improved the motor dysfunction of the PD model rats induced by intranigral injection of LPS, and this beneficial effect of BHBA was attributed to the inhibition of microglial overactivation and the protection of dopaminergic neurons in the substantia nigra (SN). Our in vitro mechanistic study revealed that the inhibitory effect of BHBA on microglia was mediated by G-protein-coupled receptor 109A (GPR109A) and involved the NF-κB signaling pathway, causing the inhibition of pro-inflammatory enzyme (iNOS and COX-2) and pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) production.
In conclusion, the present study supports the effectiveness of BHBA in protecting dopaminergic neurons against inflammatory challenge.
PMCID: PMC4310035  PMID: 25595674
BHBA; GPR109A; Parkinson’s disease; neuroinflammation; LPS; NF-κB
5.  Life Table Parameters of Three Mirid Bug (Adelphocoris) Species (Hemiptera: Miridae) under Contrasted Relative Humidity Regimes 
PLoS ONE  2014;9(12):e115878.
The genus Adelphocoris (Hemiptera: Miridae) is a group of important insect pests of Bt cotton in China. The three dominant species are A. lineolatus, A. suturalis, and A. fasciaticollis, and these species have different population dynamics. The causal factors for the differences in population dynamics have not been determined; one hypothesis is that humidity may be important for the growth of Adelphocoris populations. In the laboratory, the demographic parameters of the three Adelphocoris species were compared when the mirid bugs were subjected to various levels of relative humidity (40, 50, 60, 70 and 80% RH). Middle to high levels of RH (60, 70 and 80%) were associated with higher egg and nymph survival rates and increased adult longevity and female fecundity. Lower humidity levels (40 and 50% RH) had negative effects on the survival of nymphs, adult longevity and fecundity. The intrinsic rate of increase (rm), the net reproductive rate (R0) and the finite rate of increase (λ) for each Adelphocoris species increased with increasing RH. Significant positive relationships were found between RH and the life table parameters, rm, R0 and λ for the three Adelphocoris species. These results will help to better understand the phenology of the three Adelphocoris species, and the information can be used in population growth models to optimize pest forecasting and management strategies for these key pests.
PMCID: PMC4277414  PMID: 25541705
6.  Inhibition of Survivin Reduces HIF-1α, TGF-β1 and TFE3 in Salivary Adenoid Cystic Carcinoma 
PLoS ONE  2014;9(12):e114051.
In the present study, we explored the expression and correlation of survivin with HIF-1α, TGF-β1 and TFE3 in adenoid cystic carcinoma (AdCC). The expression of survivin, HIF-1α, TGF-β1 and TFE3 was assessed by immunohistochemical staining of a tissue microarray containing tissue samples of normal salivary gland (NSG), pleomorphic adenoma (PA) and AdCC. Correlation analysis of these proteins revealed that increased survivin expression was associated with the overexpression of HIF-1α (P<0.001, r = 0.5599), TGF-β1 (P<0.001, r = 0.6616) and TFE3 (P<0.001, r = 0.7747). The expression of survivin, HIF-1α, TGF-β1 and TFE3 was not correlated with the pathological type of human AdCC (P>0.05). Selective inhibition of survivin by YM155 and siRNA significantly reduced human SACC-83 cell proliferation, with the corresponding decrease in expression of HIF-1α, TGF-β1 and TFE3. The data indicate that the overexpression of survivin in AdCC is related to HIF-1α, TGF-β1 and TFE3. We hypothesize from these findings that the inhibition of survivin may be a novel strategy for neoadjuvant chemotherapeutic and radiosensitive treatment of AdCC.
PMCID: PMC4259474  PMID: 25485635
7.  miR-142-3p regulates the formation and differentiation of hematopoietic stem cells in vertebrates 
Cell Research  2013;23(12):1356-1368.
Previous studies on developmental hematopoiesis have mainly focused on signaling and transcription factors, while the appreciation of epigenetic regulation including that of microRNAs is recent. Here, we show that in zebrafish and mouse, miR-142-3p is specifically expressed in hematopoietic stem cells (HSCs). Knockdown of miR-142a-3p in zebrafish led to a reduced population of HSCs in the aorta-gonad-mesonephros (AGM) region as well as T-cell defects in the thymus. Mechanistically, miR-142a-3p regulates HSC formation and differentiation through the repression of interferon regulatory factor 7 (irf7)-mediated inflammation signaling. Finally, we show that miR-142-3p is also involved in the development of HSCs in mouse AGM, suggesting that it has a highly conserved role in vertebrates. Together, these findings unveil the pivotal roles that miR-142a-3p plays in the formation and differentiation of HSCs by repressing irf7 signaling.
PMCID: PMC3847575  PMID: 24165894
hematopoietic stem cell; miR-142a-3p; irf7; zebrafish; mouse
8.  A CRISPR/Cas9 toolkit for multiplex genome editing in plants 
BMC Plant Biology  2014;14(1):327.
To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.
We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.
We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0327-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4262988  PMID: 25432517
CRISPR/Cas9; Genome editing; Multiple gene mutations; Assembly of multiple gRNAs
9.  Development of antibody functionalized magnetic nanoparticles for the immunoassay of carcinoembryonic antigen: a feasibility study for clinical use 
Magnetic nanoparticles functionalized antibodies are used for in-vitro assays on bio-markers. This work demonstrates the synthesis of high-quality magnetic nanoparticles coated with antibodies against carcinoembryonic antigen (CEA). Various characterizations, such as particle size, particle suspension, bio-activity and the stability of bio-magnetic nanoparticles suspended in liquid, are studied. The properties for the assay of CEA molecules in serum are also studied. The assay method used is so-called immunomagnetic reduction.
The results show that the effects of common materials in serum that interfere with detected signals are not significant. The low-detection limit is 0.21 ng/ml, which is well below the clinical threshold of 2.5 ng/ml.
The dynamic range for the assay of CEA molecules in serum is 500 ng/ml. By assaying serum CEA molecules from 24 normal controls and 30 colorectal-cancer patients, the threshold for the serum-CEA concentration to diagnose colorectal cancer is 4.05 ng/ml, which results in a clinical sensitivity of 0.90 and specificity of 0.87.
PMCID: PMC4254005  PMID: 25424410
Bio-magnetic nanoparticles; Carcinoembryonic antigen; Assay; Colorectal cancer
10.  SOX1 down-regulates β-catenin and reverses malignant phenotype in nasopharyngeal carcinoma 
Molecular Cancer  2014;13(1):257.
Aberrant activation of the Wnt/β-catenin signaling pathway is an important factor in the development of nasopharyngeal carcinoma (NPC). Previous studies have demonstrated that the developmental gene sex-determining region Y (SRY)-box 1 (SOX1) inhibits cervical and liver tumorigenesis by interfering with the Wnt/β-catenin signaling pathway. However, the role of SOX1 in NPC remains unclear. This study investigates the function of SOX1 in NPC pathogenesis.
Down-regulation of SOX1 was detected in NPC cell lines and tissues. Besides, quantitative methylation-specific polymerase chain reaction revealed that SOX1 promoter was hypermethylated in NPC cell lines. Ectopic expression of SOX1 in NPC cells suppressed colony formation, proliferation and migration in vitro and impaired tumor growth in nude mice. Restoration of SOX1 expression significantly reduced epithelial-mesenchymal transition, enhanced cell differentiation and induced cellular senescence. Conversely, transient knockdown of SOX1 by siRNA in these cells partially restored cell proliferation and colony formation. Notably, SOX1 was found to physically interact with β-catenin and reduce its expression independent of proteasomal activity, leading to inhibition of Wnt/β-catenin signaling and decreased expression of downstream target genes.
SOX1 decreases the expression of β-catenin in a proteasome-independent manner and reverses the malignant phenotype in NPC cells.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-257) contains supplementary material, which is available to authorized users.
PMCID: PMC4326525  PMID: 25427424
SOX1; β-catenin; Methylation; Differentiation; NPC
11.  Inhibition of mTOR reduce Stat3 and PAI related angiogenesis in salivary gland adenoid cystic carcinoma 
Angiogenesis is a complex biological process, which is involved in tumorigenesis and progression. However, the molecular mechanism of underlying angiogenesis remains largely unknown. In this study, we accessed the expression of proteins related angiogenesis by immunohistochemical staining of human tissue microarray which contains 72 adenoid cystic carcinoma (AdCC), 12 pleomorphic adenoma (PMA) and 18 normal salivary gland (NSG) using digital pathological scanner and scoring system. We found that the expression of p-S6S235/236 (a downstream molecule of mTOR), p-Stat3T705, PAI, EGFR, and HIF-1α was significantly increased in AdCC as compared with PMA and (or) NSG (p < 0.05). While, the expression of these proteins was not associated with pathological type of human AdCC (p > 0.05). Correlation analysis of these proteins revealed that p-S6S235/236 up-regulates the expression of EGFR/p-Stat3T705 (p < 0.05) and HIF-1α/PAI (p < 0.05). Moreover, the activation of p-S6S235/236, EGFR/p-Stat3T705 and HIF-1α/PAI associated with angiogenesis (CD34) and proliferation (Ki-67). In vitro, Rapamycin suppressed the expression of p-S6S235/236, EGFR, p-Stat3T705, HIF-1α and PAI. Further more, target inhibition of mTOR by rapamycin effectively reduced tumor growth of SACC-83 cells line nude mice xenograft and decreased the expression of p-S6S235/236, EGFR/p-Stat3T705 and HIF-1α/PAI. Taken together, these data revealed that mTOR signaling pathway regulates tumor angiogenesis by EGFR/p-Stat3T705 and HIF-1α/PAI. Inhibition of mTOR by rapamycin could effectively reduced tumor growth. It is likely that mTOR inhibitors may be a potential candidate for treatment of AdCC.
PMCID: PMC4266710  PMID: 25520866
Adenoid cystic carcinoma; angiogenesis; mTOR; Stat3; PAI; rapamycin
12.  Dax1 and Nanog act in parallel to stabilize mouse embryonic stem cells and induced pluripotency 
Nature Communications  2014;5:5042.
Nanog expression is heterogeneous and dynamic in embryonic stem cells (ESCs). However, the mechanism for stabilizing pluripotency during the transitions between Nanoghigh and Nanoglow states is not well understood. Here we report that Dax1 acts in parallel with Nanog to regulate mouse ESC (mESCs) identity. Dax1 stable knockdown mESCs are predisposed towards differentiation but do not lose pluripotency, whereas Dax1 overexpression supports LIF-independent self-renewal. Although partially complementary, Dax1 and Nanog function independently and cannot replace one another. They are both required for full reprogramming to induce pluripotency. Importantly, Dax1 is indispensable for self-renewal of Nanoglow mESCs. Moreover, we report that Dax1 prevents extra-embryonic endoderm (ExEn) commitment by directly repressing Gata6 transcription. Dax1 may also mediate inhibition of trophectoderm differentiation independent or as a downstream effector of Oct4. These findings establish a basal role of Dax1 in maintaining pluripotency during the state transition of mESCs and somatic cell reprogramming.
The transcription factor Dax1 is often used as an embryonic stem cell (ESC) marker. Here the authors show that Dax1 acts in parallel to the pluripotency transcription factor Nanog in the maintenance of mouse ESC pluripotency and is required for full somatic cell reprogramming.
PMCID: PMC4205889  PMID: 25284313
13.  hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling 
Nucleic Acids Research  2014;42(20):12483-12497.
The increased cap-independent translation of anti-apoptotic proteins is involved in the development of drug resistance in lung cancer but signalling events regulating this are poorly understood. Fibroblast growth factor 2 (FGF-2) signalling-induced S6 kinase 2 (S6K2) activation is necessary, but the downstream mediator(s) coupling this kinase to the translational response is unknown. Here, we show that S6K2 binds and phosphorylates hnRNPA1 on novel Ser4/6 sites, increasing its association with BCL-XL and XIAP mRNAs to promote their nuclear export. In the cytoplasm, phosphoS4/6-hnRNPA1 dissociates from these mRNAs de-repressing their IRES-mediated translation. This correlates with the phosphorylation-dependent association of hnRNPA1 with 14-3-3 leading to hnRNPA1 sumoylation on K183 and its re-import into the nucleus. A non-phosphorylatible, S4/6A mutant prevented these processes, hindering the pro-survival activity of FGF-2/S6K2 signalling. Interestingly, immunohistochemical staining of lung and breast cancer tissue samples demonstrated that increased S6K2 expression correlates with decreased cytoplasmic hnRNPA1 and increased BCL-XL expression. In short, phosphorylation on novel N-term sites of hnRNPA1 promotes translation of anti-apoptotic proteins and is indispensable for the pro-survival effects of FGF-2.
PMCID: PMC4227786  PMID: 25324306
14.  TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells 
Oncotarget  2014;5(20):10127-10139.
High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma.
PMCID: PMC4259410  PMID: 25333256
Melanoma; TRIM16; BRAF inhibitor; cell migration; IFNβ1
15.  Crystal structure of 9-(4-bromo­but­yl)-9H-fluorene-9-carb­oxy­lic acid 
The title compound, C18H17BrO2, is a key inter­mediate in the synthesis of lomitapide mesylate, a microsomal triglyceride transfer protein inhibitor. Its asymmetric unit contains two independent mol­ecules with slightly different conformations; the mean planes of the 4-bromo­butyl and carboxyl­ate groups in the two mol­ecules form dihedral angles of 24.54 (12) and 17.10 (18)°. In the crystal, carboxyl­ate groups are involved in O—H⋯O hydrogen bonding, which leads to the formation of two crystallographically independent centrosymmetric dimers. Weak inter­molecular C—H⋯O inter­actions further link these dimers into layers parallel to the bc plane.
PMCID: PMC4257222  PMID: 25484705
crystal structure; lomitapide mesylate; hydrogen bonding
16.  Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli 
PLoS Pathogens  2014;10(9):e1004404.
Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.
Author Summary
Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhea worldwide and is commonly present as an infection in symptomatic travelers returning from developing countries. The attachment of EAEC to the human intestine is mediated protein filaments extending from the bacterial surface known as aggregative adherence fimbria (AAF). Here we use X-ray crystallography and nuclear magnetic resonance (NMR) structures to provide an atomic structure of the protein fibers made by the two major variants, AAF/I and AAF/II. The structures of the major subunit proteins show that the AAFs assemble into flexible, linear polymers that are capped by a single minor protein subunit at the tip. Biochemical assays reveal that the AAFs recognize a common receptor, the extracellular matrix protein fibronectin, via clusters of positively-charged amino acid residues running along the length of the fimbriae. Our structures suggest a unique mechanism based on ionic interactions for AAF-mediated receptor binding and biofilm formation.
PMCID: PMC4169507  PMID: 25232738
18.  Arsenic trioxide-based therapy in relapsed/refractory multiple myeloma patients: a meta-analysis and systematic review 
OncoTargets and therapy  2014;7:1593-1599.
Multiple myeloma (MM) is a clonal malignancy characterized by the proliferation of malignant plasma cells in the bone marrow and the production of monoclonal immunoglobulin. Although some newly approved drugs (thalidomide, lenalidomide, and bortezomib) demonstrate significant benefit for MM patients with improved survival, all MM patients still relapse. Arsenic trioxide (ATO) is the most active single agent in acute promyelocytic leukemia, the antitumor activity of which is partly dependent on the production of reactive oxygen species. Due to its multifaceted effects observed on MM cell lines and primary myeloma cells, Phase I/II trials have been conducted in heavily pretreated patients with relapsed or refractory MM. Therapy regimens varied dramatically as to the dosage of ATO and monotherapy versus combination therapy with other agents available for the treatment of MM. Although ATO-based combination treatment was well tolerated by most patients, most trials found that ATO has limited effects on MM patients. However, since small numbers of patients were randomized to different treatment arms, trials have not been statistically powered to determine the differences in progression-free survival and overall survival among the experimental arms. Therefore, large Phase III studies of ATO-based randomized controlled trials will be needed to establish whether ATO has any potential beneficial effects in the clinical setting.
PMCID: PMC4166211  PMID: 25246802
multiple myeloma; arsenic trioxide; clinical trial; therapy; meta-analysis
19.  Significance of p53 dynamics in regulating apoptosis in response to ionizing radiation, and polypharmacological strategies 
Scientific Reports  2014;4:6245.
Developing pharmacological strategies for controlling ionizing radiation (IR)-induced cell death is important for both mitigating radiation damage and alleviating the side effects of anti-cancer radiotherapy manifested in surrounding tissue morbidity. Exposure to IR often triggers the onset of p53-dependent apoptotic pathways. Here we build a stochastic model of p53 induced apoptosis comprised of coupled modules of nuclear p53 activation, mitochondrial cytochrome c release and cytosolic caspase activation that also takes into account cellular heterogeneity. Our simulations show that the strength of p53 transcriptional activity and its coupling (or timing with respect) to mitochondrial pore opening are major determinants of cell fate: for systems where apoptosis is elicited via a p53-transcription-independent mechanism, direct activation of Bax by p53 becomes critical to IR-induced-damage initiation. We further show that immediate administration of PUMA inhibitors following IR exposure effectively suppresses excessive cell death, provided that there is a strong caspase/Bid feedback loop; however, the efficacy of the treatment diminishes with increasing delay in treatment implementation. In contrast, the combined inhibition of Bid and Bax elicits an anti-apoptotic response that is effective over a range of time delays.
PMCID: PMC4150106  PMID: 25175563
20.  Synthesis of Three Rimantadine Schiff Bases and Their Biological Effects on Serum Albumin 
Three new rimantadine Schiff bases (RSBs) were prepared, and then the interaction of RSBs with bovine serum albumin (BSA) was investigated using fluorescence, synchronous fluorescence, UV-vis absorption spectroscopy under physiological conditions. The results showed that the three RSBs effectively quenched the intrinsic fluorescence of BSA via static quenching. Binding constant (Ka), number of binding sites (n), and the binding distance (r) between three RSBs and BSA were calculated by Stern-Volmer equation and Förster’s theory in this study. According to the results of displacement experiments of site probes, it was considered that the binding sites were located in hydrophobic cavities in sub-domains IIA of BSA. What is more, synchronous fluorescence studies indicated that the hydrophobicity around tryptophan residues was increased with the addition of rimantadine-o-vanillin (ROV) and rimantadine-4-methoxy-salicylaldehyde (RMS), while there was no apparent change with the addition of rimantadine-salicylaldehyde (RS).
PMCID: PMC4232783  PMID: 25587306
Rimantadine schiff base (RSB); Bovine serum albumin (BSA); Interaction; Fluorescence
21.  Perinodular ductular reaction/epithelial cell adhesion molecule loss in small hepatic nodules 
World Journal of Gastroenterology : WJG  2014;20(31):10908-10915.
AIM: To investigate if loss of epithelial cell adhesion molecule (EpCAM) is associated with microinvasion in hepatocellular carcinomas (HCCs) in the presence of chronic hepatitis B.
METHODS: The expression of EpCAM, cytokeratin 7 (CK7) and CK19 in 112 hepatic nodules was studied, including 20 HCCs with nodules ≤ 3 cm, 26 HCCs with nodules > 3 cm, 20 high-grade dysplastic nodules, 26 cirrhotic, large regenerative nodules and 20 cases of cirrhosis.
RESULTS: Membranes of ductular reaction (DR) hepatobiliary cells, interlobular bile duct and some hepatic cells were positive for EpCAM expression. Active expression of DR/EpCAM was observed in the majority of noninvasive nodules (50/66, 75.76%); however, expression was absent in the major area of invasion in HCCs (42/46, 91.30%). DR/EpCAM loss in HCCs ≤ 3 cm was higher than in high-grade dysplastic nodules (HGDNs) (P < 0.05), cirrhotic, large regenerative nodules and cirrhosis (P < 0.01). Furthermore, patients (20 HCCs ≤ 3 cm, 26 HCCs > 3 cm, 20 HGDNs) with DR/EpCAM expression had a higher overall survival rate (P < 0.01) and lower early recurrence rate (P < 0.01). DR/EpCAM expression showed a close relationship with DR/CK7 and DR/CK19 expression (P < 0.01). The area under the receiver operating characteristic (ROC) curve of DR/EpCAM was similar to that of DR/CK7 and DR/CK19 (P > 0.05). The diagnostic specificity and diagnostic accuracy were both increased when DR/EpCAM, DR/CK7 and DR/CK19 were combined (P < 0.01).
CONCLUSION: DR/EpCAM loss may be a useful marker for determining microinvasion in HCCs ≤ 3 cm, but also for predicting prognosis.
PMCID: PMC4138470  PMID: 25152593
Ductular reaction; Epithelial cell adhesion molecule; Hepatocellular carcinomas; Small hepatic nodule; Microinvasion; Differential diagnosis
22.  High glucose induces podocyte apoptosis by stimulating TRPC6 via elevation of reactive oxygen species 
Biochimica et biophysica acta  2013;1833(6):1434-1442.
Podocyte number is significantly reduced in diabetic patients and animal models, but the mechanism remains unclear. In the present study, we found that high glucose induced apoptosis in control podocytes which express transient receptor potential canonical 6 (TRPC6) channels, but not in TRPC6 knockdown podocytes in which TRPC6 was knocked down by TRPC6 silencing short hairpin RNA (shRNA). This effect was reproduced by treatment of podocytes with the reactive oxygen species (ROS), hydrogen peroxide (H2O2). Single-channel data from cell-attached, patch-clamp experiments showed that both high glucose and H2O2 activated the TRPC6 channel in control podocytes, but not in TRPC6 knockdown podocytes. Confocal microscopy showed that high glucose elevated ROS in podocytes and that H2O2 reduced the membrane potential of podocytes and elevated intracellular Ca2+ via activation of TRPC6. Since intracellular Ca2+ overload induces apoptosis, H2O2-induced apoptosis may result from TRPC6-mediated elevation of intracellular Ca2+. These data together suggest that high glucose induces apoptosis in podocytes by stimulating TRPC6 via elevation of ROS.
PMCID: PMC4134943  PMID: 23499875
ROS; Patch-clamp technique; Confocal microscopy; Hydrogen peroxide; Oxidative stress; Intracellular Ca2+
23.  Prognostic and predictive values of SPP1, PAI and caveolin-1 in patients with oral squamous cell carcinoma 
SPP1, PAI and caveolin-1 are known to be closely associated with tumor progression in several kinds of human tumors. This study aimed to investigate the expression of SPP1, PAI and caveolin-1 in oral squamous cell carcinoma (OSCC), and to evaluate their association with the prognosis in oral carcinoma. Immunohistochemical staining was used to examine the expression of SPP1, PAI and caveolin-1 in 17 normal oral mucosa, 6 oral epithelial dysplasia and 43 OSCC specimens by tissue microarrays. High expression of SPP1, PAI and caveolin-1 was found in OSCC patients, and SPP1 and PAI expression were significantly higher in OSCC than in normal oral mucosa. No significant correlations were found between SPP1, PAI and caveolin-1 expression and clinicopathological factors. Expression of SPP1, PAI and caveolin-1 was also not associated with overall survival. Moreover, SPP1 was closely correlated with PAI, caveolin-1 and Keap1, and PAI had significant correlations with caveolin-1, Keap1 and Nrf2, and caveolin-1 was associated with Keap1 by using the Pearson correlation coefficient test. Our findings suggest that overexpressed SPP1, PAI and caveolin-1 were linked to carcinogenesis and progression, and thus they may serve as potential prognostic factors in OSCC.
PMCID: PMC4203219  PMID: 25337248
SPP1; PAI; caveolin-1; oral squamous cell carcinoma; prognosis
24.  The Effects of Sesquiterpenes-Rich Extract of Alpinia oxyphylla Miq. on Amyloid-β-Induced Cognitive Impairment and Neuronal Abnormalities in the Cortex and Hippocampus of Mice 
As a kind of medicine which can also be used as food, Alpinia oxyphylla Miq. has a long clinical history in China. A variety of studies demonstrated the significant neuroprotective activity effects of chloroform (CF) extract from the fruits of Alpinia oxyphylla. In order to further elucidate the possible mechanisms of CF extract which mainly contains sesquiterpenes with neuroprotection on the cognitive ability, mice were injected with Aβ1−42 and later with CF in this study. The results showed that the long-term treatment of CF enhanced the cognitive performances in behavior tests, increased activities of glutathione peroxidase (GSH-px) and decreased the level of malondialdehyde (MDA), acetylcholinesterase (AChE), and amyloid-β (Aβ), and reversed the activation of microglia, degeneration of neuronal acidophilia, and nuclear condensation in the cortex and hippocampus. These results demonstrate that CF ameliorates learning and memory deficits by attenuating oxidative stress and regulating the activation of microglia and degeneration of neuronal acidophilia to reinforce cholinergic functions.
PMCID: PMC4142297  PMID: 25180067
25.  The Impact of MIR137 on Dorsolateral Prefrontal–Hippocampal Functional Connectivity in Healthy Subjects 
Neuropsychopharmacology  2014;39(9):2153-2160.
A recent mega-analysis combining genome-wide association study data revealed that a variant of microRNA 137 (MIR137) exhibits the most significant association with schizophrenia. Other biological evidence also consistently suggests that MIR137 may have a pivotal role in the pathogenesis of schizophrenia. However, the underlying neural mechanism remains unclear. As the disrupted dorsolateral prefrontal cortex (DLPFC) coupling with the hippocampal formation (HF) has been widely observed in schizophrenia patients, DLPFC-HF dysconnectivity can therefore be thought of as a pivotal intermediate phenotype that links genetic variants of psychiatric risk genes to schizophrenia. This study used resting-state functional magnetic resonance imaging to test whether the MIR137 variant (rs1625579) impacts DLPFC-HF functional connectivity and cognitive performance in 290 young, healthy Han Chinese individuals. To identify functional connectivity between DLPFC and HF, a seed-based functional connectivity analysis was used. The association between DLPFC-HF connectivity and working memory performance was further examined in individuals with different MIR137 genotypes. The individuals who are homozygous for the MIR137 risk allele (TT), which confers a high risk for schizophrenia, exhibited significantly different DLPFC-HF functional connectivity compared with TG individuals. Moreover, the DLPFC-HF connectivity could predict the working memory performance in MIR137 TG individuals, but not in TT individuals. The current findings obtained in a large sample of healthy participants identified potential neural mechanisms linking MIR137 with the risk of developing schizophrenia via the intermediate phenotype of DLPFC-HF connectivity.
PMCID: PMC4104332  PMID: 24625753
MIR137; dorsolateral prefrontal cortex; hippocampus; functional connectivity; working memory

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