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1.  Deletion of C7L and K1L Genes Leads to Significantly Decreased Virulence of Recombinant Vaccinia Virus TianTan 
PLoS ONE  2013;8(7):e68115.
The vaccinia virus TianTan (VTT) has been modified as an HIV vaccine vector in China and has shown excellent performance in immunogenicity and safety. However, its adverse effects in immunosuppressed individuals warrant the search for a safer vector in the following clinic trails. In this study, we deleted the C7L and K1L genes of VTT and constructed six recombinant vaccinia strains VTT△C7L, VTT△K1L, VTT△C7LK1L, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. The pathogenicity and immunogenicity of these recombinants were evaluated in mouse and rabbit models. Comparing to parental VTT, VTT△C7L and VTT△K1L showed significantly decreased replication capability in CEF, Vero, BHK-21 and HeLa cell lines. In particular, replication of VTT△C7LK1L decreased more than 10-fold in all four cell lines. The virulence of all these mutants were decreased in BALB/c mouse and rabbit models; VTT△C7LK1L once again showed the greatest attenuation, having resulted in no evident damage in mice and erythema of only 0.4 cm diameter in rabbits, compared to 1.48 cm for VTT. VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag elicited as strong cellular and humoral responses against HIV genes as did VTKgpe, while humoral immune response against the vaccinia itself was reduced by 4-8-fold. These data show that deletion of C7L and K1L genes leads to significantly decreased virulence without compromising animal host immunogenicity, and may thus be key to creating a more safe and effective HIV vaccine vector.
doi:10.1371/journal.pone.0068115
PMCID: PMC3698190  PMID: 23840887
2.  Synergistic antitumor effect of AAV-mediated TRAIL expression combined with cisplatin on head and neck squamous cell carcinoma 
BMC Cancer  2011;11:54.
Background
Adeno-associated virus-2 (AAV-2)-mediated gene therapy is quite suitable for local or regional application in head and neck cancer squamous cell carcinoma (HNSCC). However, its low transduction efficiency has limited its further development as a therapeutic agent. DNA damaging agents have been shown to enhance AAV-mediated transgene expression. Cisplatin, one of the most effective chemotherapeutic agents, has been recognized to cause cancer cell death by apoptosis with a severe toxicity. This study aims to evaluate the role of cisplatin in AAV-mediated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and the effect on HNSCC both in vitro and in vivo.
Methods
Five human HNSCC cell lines were treated with recombinant soluble TRAIL (rsTRAIL) and infected with AAV/TRAIL to estimate the sensitivity of the cancer cells to TRAIL-induced cytotoxicity. KB cells were infected with AAV/EGFP with or without cisplatin pretreatment to evaluate the effect of cisplatin on AAV-mediated gene expression. TRAIL expression was detected by ELISA and Western blot. Cytotoxicity was measured by MTT assay and Western blot analysis for caspase-3 and -8 activations. Following the in vitro experiments, TRAIL expression and its tumoricidal activity were analyzed in nude mice with subcutaneous xenografts of HNSCC.
Results
HNSCC cell lines showed different sensitivities to rsTRAIL, and KB cells possessed both highest transduction efficacy of AAV and sensitivity to TRAIL among five cell lines. Preincubation of KB cells with subtherapeutic dosage of cisplatin significantly augmented AAV-mediated transgene expression in a heparin sulfate proteoglycan (HSPG)-dependent manner. Furthermore, cisplatin enhanced the killing efficacy of AAV/TRAIL by 3-fold on KB cell line. The AAV mediated TRAIL expression was observed in the xenografted tumors and significantly enhanced by cisplatin. AAV/TRAIL suppressed the tumors growth and cisplatin augmented the tumoricidal activity by two-fold. Furthermore, Combination treatment reduced cisplatin-caused body weight loss in nude mice.
Conclusion
The combination of AAV-mediated TRAIL gene expression and cisplatin had synergistic therapeutic effects on head and neck cancers and reduced the potential toxicity of cisplatin. These findings suggest that the combination of AAV/TRAIL and cisplatin may be a promising strategy for HNSCC therapy.
doi:10.1186/1471-2407-11-54
PMCID: PMC3044652  PMID: 21291526
3.  Crystallization and preliminary crystallographic analysis of Gibberella zeae extracellular lipase 
G. zeae extracellular lipase has been overexpressed, purified and crystallized. Diffraction data were collected to 2.8 Å resolution.
Fusarium head blight, one of the most destructive crop diseases, is mainly caused by Fusarium graminearum (known in its sexual stage as Gibberella zeae). F. graminearum secretes various extracellular enzymes that have been hypothesized to be involved in host infection. One of the extracellular enzymes secreted by this organism is the G. zeae extracellular lipase (GZEL), which is encoded by the FGL1 gene. In order to solve the crystal structure of GZEL and to gain a better understanding of the biological functions of the protein and of possible inhibitory mechanisms of lipase inhibitors, recombinant GZEL was crystallized at 291 K using PEG 3350 as a precipitant. A data set was collected to 2.8 Å resolution from a single flash-cooled crystal (100 K). The crystal belonged to space group P212121, with unit-cell parameters a = 78.4, b = 91.0, c = 195.8 Å, α = β = γ = 90°. The presence of four molecules was assumed per asymmetric unit, which gave a Matthews coefficient of 2.6 Å3 Da−1.
doi:10.1107/S1744309108019283
PMCID: PMC2531274  PMID: 18765911
extracellular lipases; Fusarium graminearum; Gibberella zeae; fusarium head blight
4.  Research on focal nodular hyperplasia with MSCT and postprocessing 
AIM: To investigate and evaluate the pathological features and diagnostic value of focal nodular hyperplasia (FNH) with multi-section spiral computed tomography (MSCT) and postprocessing.
METHODS: A total of 25 patients with FNH who had undergone MSCT and postprocessing were included in the investigation. All patients had been pathologically or clinically confirmed with FNH. A number of 75 cases of hepatic carcinomas, hemangiomas and adenomas were randomly selected at a same period for a comparative study.
RESULTS: There was a single focus in 22 cases and multiple foci in 3 cases. On the plain scan, 17 lesions showed hypodensity, 7 isodensity and 4 hyperdensity (the case with fatty liver). With contrast, 28 lesions were enhanced evenly or in the nodules in the arterial phase; 13 lesions still showed hyperdensity, 11 lesions isodensity and 4 lesions hypodensity in the parenchymatous phase; in the delayed phase only 5 lesions showed hyperdensity but 9 lesions showed isodensity or slight hypodensity and 14 lesions showed hypodensity. Twelve lesions of 28 had central asteroid scars. Thickened feeding arteries in postprocessing were seen in 24 lesions, and were integrated into the parenchymatous lesions with a gradual and smooth course. On the contrary, there were no artery penetrated into the lesion found in any of comparative hepatic tumors.
CONCLUSION: Doctors could make a correct diagnosis and differentiation of FNH on evaluation of the characteristic appearance on MSCT with postprocessing.
doi:10.3748/wjg.15.4838
PMCID: PMC2761565  PMID: 19824121
Angiography; Computer-assisted image processing; Focal nodular hyperplasia; Liver diseases; X-ray; Computed tomography
5.  Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein PevD1 from Verticillium dahliae  
The overexpression, purification, crystallization and preliminary X-ray diffraction analysis of protein elicitor PevD1 from Verticillium dahliae are reported.
The effector protein PevD1 from the pathogenic fungus Verticillium dahliae was purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 M sodium formate. A native data set was collected at 1.9 Å resolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 M potassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.
doi:10.1107/S1744309112020556
PMCID: PMC3388926  PMID: 22750869
PevD1; effector proteins; Verticillium dahliae
6.  Deformed grids for single-particle cryo-electron microscopy of specimens exhibiting a preferred orientation 
Journal of structural biology  2013;182(3):255-258.
For biological samples showing a preferred orientation on the carbon support film of an electron microscope (EM) grid, accurate three-dimensional (3D) reconstructions by single-particle cryo-EM require data collection in which the specimen grids are tilted in the microscope, to obtain adequate numbers of particles that cover the high-degree angular distribution. However, image drift caused by the electron beam interacting with the cryo specimen becomes severe when grids are tilted to high angles (> 30°). We produced deformed grids by applying a deliberate mechanical deformation to EM grids containing a thin carbon film supported by a thick holey carbon film. We applied cryo-EM using deformed grids to the isolated cardiac ryanodine receptor, an ion channel complex known to assume a preferred orientation on the carbon support film. These grids contained more particles having high Euler angle orientations without the need to tilt the specimen grids. Meanwhile, the drifting that was apparent in the images was reduced from that typical of images from tilted regular EM grids. This was achieved by imaging particles in holes close to the deformed areas, where carbon films were locally bent, offering planes of inclination with various angles. The deformed grids improve the efficiency and quality of data collection for single-pahrticle cryo-EM of samples showing a limited range of orientations.
doi:10.1016/j.jsb.2013.03.005
PMCID: PMC3665629  PMID: 23537848
single-particle cryo-EM; deformed EM grids; preferred orientation; ryanodine receptor
7.  Chlorella zofingiensis as an Alternative Microalgal Producer of Astaxanthin: Biology and Industrial Potential 
Marine Drugs  2014;12(6):3487-3515.
Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione), a high-value ketocarotenoid with a broad range of applications in food, feed, nutraceutical, and pharmaceutical industries, has been gaining great attention from science and the public in recent years. The green microalgae Haematococcus pluvialis and Chlorella zofingiensis represent the most promising producers of natural astaxanthin. Although H. pluvialis possesses the highest intracellular astaxanthin content and is now believed to be a good producer of astaxanthin, it has intrinsic shortcomings such as slow growth rate, low biomass yield, and a high light requirement. In contrast, C. zofingiensis grows fast phototrophically, heterotrophically and mixtrophically, is easy to be cultured and scaled up both indoors and outdoors, and can achieve ultrahigh cell densities. These robust biotechnological traits provide C. zofingiensis with high potential to be a better organism than H. pluvialis for mass astaxanthin production. This review aims to provide an overview of the biology and industrial potential of C. zofingiensis as an alternative astaxanthin producer. The path forward for further expansion of the astaxanthin production from C. zofingiensis with respect to both challenges and opportunities is also discussed.
doi:10.3390/md12063487
PMCID: PMC4071588  PMID: 24918452
astaxanthin; Chlorella zofingiensis; fed-batch; genetic engineering; mass cultivation; microalgae; stress
8.  Hemostatic Effects of Microbubble-Enhanced Low-Intensity Ultrasound in a Liver Avulsion Injury Model 
PLoS ONE  2014;9(5):e95589.
Objectives
Microbubble-enhanced therapeutic ultrasound (MEUS) can block the blood flow in the organs. The aim of this study was to evaluate the hemostatic effect of microbubble-enhanced pulsed, low-intensity ultrasound in a New Zealand White rabbit model of avulsion trauma of the liver. The therapeutic ultrasound (TUS) transducer was operated with the frequency of 1.2 MHz and an acoustic pressure of 3.4 MPa. Microbubble-(MB) enhanced ultrasound (MEUS) (n = 6) was delivered to the distal part of the liver where the avulsion was created. Livers were treated by TUS only (n = 4) or MB only (n = 4) which served as controls. Bleeding rates were measured and contrast enhanced ultrasound (CEUS) was performed to assess the hemostatic effect, and liver hemoperfusion before and after treatment. Generally, bleeding rates decreased more than 10-fold after the treatment with MEUS compared with those of the control group (P<0.05). CEUS showed significant declines in perfusion. The peak intensity value and the area under the curve also decreased after insonation compared with those of the control group (P<0.05). Histological examination showed cloudy and swollen hepatocytes, dilated hepatic sinusoids, perisinusoidal spaces with erythrocyte accumulation in small blood vessels, obvious hemorrhage around portal areas and scattered necrosis in liver tissues within the insonation area of MEUS Group. In addition, necrosis was found in liver tissue 48 h after insonation. We conclude that MEUS might provide an effective hemostatic therapy for serious organ trauma such as liver avulsion injury.
doi:10.1371/journal.pone.0095589
PMCID: PMC4006836  PMID: 24788757
9.  Wnt modulates MCL1 to control cell survival in triple negative breast cancer 
BMC Cancer  2014;14:124.
Background
Triple negative breast cancer (TNBC) has higher rates of recurrence and distant metastasis, and poorer outcome as compared to non-TNBC. Aberrant activation of WNT signaling has been detected in TNBC, which might be important for triggering oncogenic conversion of breast epithelial cell. Therefore, we directed our focus on identifying the WNT ligand and its underlying mechanism in TNBC cells.
Methods
We performed large-scale analysis of public microarray data to screen the WNT ligands and the clinical significance of the responsible ligand in TNBC. WNT5B was identified and its overexpression in TNBC was confirmed by immunohistochemistry staining, Western blot and ELISA. ShRNA was used to knockdown WNT5B expression (shWNT5B). Cellular functional alteration with shWNT5B treatment was determined by using wound healing assay, mammosphere assay; while cell cycle and apoptosis were examined by flowcytometry. Mitochondrial morphology was photographed by electron microscope. Biological change of mitochondria was detected by RT-PCR and oxygen consumption assay. Activation of WNT pathway and its downstream targets were evaluated by liciferase assay, immunohistochemistry staining and immunoblot analysis. Statistical methods used in the experiments besides microarray analysis was two-tailed t-test.
Results
WNT5B was elevated both in the tumor and the patients’ serum. Suppression of WNT5B remarkably impaired cell growth, migration and mammosphere formation. Additionally, G0/G1 cell cycle arrest and caspase-independent apoptosis was observed. Study of the possible mechanism indicated that these effects occurred through suppression of mitochondrial biogenesis, as evidenced by reduced mitochondrial DNA (MtDNA) and compromised oxidative phosphorylation (OXPHOS). In Vivo and in vitro data uncovered that WNT5B modulated mitochondrial physiology was mediated by MCL1, which was regulated by WNT/β-catenin responsive gene, Myc. Clinic data analysis revealed that both WNT5B and MCL1 are associated with enhanced metastasis and decreased disease-free survival.
Conclusions
All our findings suggested that WNT5B/MCL1 cascade is critical for TNBC and understanding its regulatory apparatus provided valuable insight into the pathogenesis of the tumor development and the guidance for targeting therapeutics.
doi:10.1186/1471-2407-14-124
PMCID: PMC3936852  PMID: 24564888
WNT5B; MCL1; WNT/β-catenin pathway; Triple negative breast cancer (TNBC)
10.  Clara cell 10-kDa protein inhibits TH17 responses through modulating dendritic cells in the setting of allergic rhinitis 
Background
TH17 responses have recently been implicated to play a role in allergic airway diseases, but their local expression in the setting of allergic rhinitis (AR) and their regulation in allergic airway diseases remain unclear.
Objective
We sought to investigate the regulatory role of Clara cell 10-kDa protein (CC10), an endogenous regulator of airway inflammation, on TH17 responses in the setting of AR.
Methods
Wild-type and homozygous CC10-null mice were used to establish an ovalbumin (OVA)–induced AR model. Human recombinant CC10 was given during sensitization or challenge. TH17 responses in human subjects and mice were examined by using flow cytometry, quantitative RT-PCR assay, immunohistochemistry, and ELISA. The direct effect of CC10 on TH17 cells and CD11c+ dendritic cells (DCs) was studied by means of cell culture. Adoptive transfer was used to examine the influence of CC10-conditioned DCs on airway inflammation. The regulatory effect of CC10 on the expression of the CCL20 gene was tested by using the BEAS-2B cell line.
Results
Compared with those of control subjects, TH17 responses were enhanced in the nasal mucosa of patients with AR. CC10-null mice with AR showed enhanced TH17 responses, and CC10 treatment significantly decreased TH17 responses. CC10 had no direct effect on in vitro TH17 cell differentiation. CC10 could significantly decrease the expression of OX40 ligand, IL-23, and IL-6 but enhance CD86 and TGF-β expression in DCs. Importantly, CC10 was able to inhibit TH17 cell polarization in the presence of OVA-pulsed DCs. CC10 pretreatment inhibited TH17 responses elicited by adoptive transfer of OVA-pulsed DCs. Furthermore, CC10 decreased the expression of CCL20 in BEAS-2B cells induced by inflammatory cytokines.
Conclusion
TH17 responses are enhanced in patients with AR, and CC10 inhibits TH17 responses through modulation of the function of DCs.
doi:10.1016/j.jaci.2012.11.027
PMCID: PMC3633083  PMID: 23273949
Allergic rhinitis; Clara cell 10-kDa protein; dendritic cell; inhibition; TH17 response
11.  2-[(1H-Benzimidazol-2-yl)imino­meth­yl]-4,6-dibromo­phenol ethanol hemisolvate 
The title compound, C14H9Br2N3O·0.5C2H5OH, crystallizes with two 2-[(1H-benzimidazol-2-yl)imino­meth­yl]-4,6-dibromo­phenol mol­ecules and one ethanol solvent mol­ecule in the asymmetric unit. The benzene and benzimidazole rings subtend dihedral angles of 4.5 (3) and 5.2 (2)° in the two mol­ecules. In the crystal, one mol­ecule presents π–π stacking with the equivalent mol­ecule related by inversion, at a distance of 3.30 Å (separation between mol­ecular mean planes). A three-dimensional network is formed through N—H⋯N, N—H⋯O and O—H⋯N hydrogen bonds.
doi:10.1107/S1600536813002031
PMCID: PMC3588477  PMID: 23476552
12.  Differential Expression Profiling and Functional Analysis of microRNAs through Stage I-III Papillary Thyroid Carcinoma 
Objective: To elucidate the mechanisms undergoing the pathogenesis of PTC, this study try to find stage specific microRNAs (miRNAs) using microarray chip in stage I, II and III papillary thyroid carcinoma (PTC) tissues as well predict miRNAs binding target genes and their molecular functions.
Methods: PTC specimens of stage I, II, and III and their paired adjacent non-tumor tissue (one patient for each stage) were collected. The expressions of miRNAs were examined using miRNA microarray chip. The most significant changed miRNAs from microarray were verified by using quantitative RT-PCR. The Potential miRNAs regulating target genes and their preliminary biological functions were forecasted with variety function prediction software.
Results: Ten miRNAs exhibited sequential up regulation expression profiles and five miRNAs performed sequential down regulation throughout stage I to III (p<0.05). After normalization, Fifteen miRNAs showed significant different compared to adjacent non-tumor tissues (p<0.05). Among of them, the most significant up regulation and down regulation miRNAs were miR-146b-5p and miR-335, respectively. Both of them were verified with qRT-PCR. 34 target genes for miR-146-5p and 36 target genes for miR-335 was predicted.
Conclusion: MicroRNA profile assay successfully detected a branch of differential expression miRNAs between PTC and normal tissue. Some of them also showed stage specific. Biological function analysis showed that target genes were involved in five aspects including cell proliferation, differentiation, apoptosis, cycle, and signaling transduction pathway, suggesting the regulatory role of abnormal expression of critical miRNAs in the pathogenesis of PTC.
doi:10.7150/ijms.5794
PMCID: PMC3607244  PMID: 23533107
papillary thyroid carcinoma; microRNA; microarray; qRT-PCR; bioinformatics.
13.  The Expression Levels of Plasma micoRNAs in Atrial Fibrillation Patients 
PLoS ONE  2012;7(9):e44906.
Background
MicroRNA (miRNA) has been found in human blood. It has been increasingly suggested that miRNAs may serve as biomarkers for diseases. We examined the potential of circulating miRNA to serve as predictors of atrial fibrillation (AF).
Methodology/Principal Findings
During the discovery stage of this project, we used massively parallel signature sequencing (MPSS) to carry out an in-depth analysis of the miRNA expression profile (miRNome) in 5 healthy controls, 5 patients with paroxysmal atrial fibrillation (PAF) alone, and 5 patients with persistent atrial fibrillation (PersAF) alone. Twenty-two specific miRNAs were found to be dysregulated in each PAF group, PersAF group, or control group. Four candidate microRNAs (miRNA-146a, miRNA-150, miRNA-19a, and miRNA-375) met our selection criteria and were evaluated in an independent cohort of 90 plasma samples using TaqMan miRNA quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). We found miRNA-150 levels to be reduced by a factor of approximately 17 in PAF relative to controls and a factor of approximately 20 in PersAF relative to controls (P<.0001). Logistic regression analyses were carried out to evaluate the reduced miRNA-150 expression levels (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.5 to 3.57, P<0.001), age (OR 1.1, 95% CI 1.36 to 2.73, P<0.001), and Left atrial diameter (LAD) (OR 1.5, 95% CI 1.36 to 1.8, P<0.001). Each was independently associated with AF. Much of the identified target genes related to AF were part of the inflammatory response system. We found that plasma levels of CRP were negatively correlated with the plasma levels of miRNA-150.
Conclusions/Significance
In summary, we firstly found that plasma miRNA-150 levels in from AF patients were substantially lower than that from healthy people. Circulating reduced miRNA-150 was significantly associated with AF.
doi:10.1371/journal.pone.0044906
PMCID: PMC3445571  PMID: 23028671
14.  Population Dynamics and Genetic Diversity of C4 Strains of Human Enterovirus 71 in Mainland China, 1998–2010 
PLoS ONE  2012;7(9):e44386.
Background
Since 1997, several countries within the Asian Pacific region have been affected by one or more massive outbreaks of Hand Foot and Mouth Disease (HFMD). Virus typing experiments revealed that these outbreaks were caused by strains of human enterovirus 71 (EV71) belonging to several different, recently emerged subgenogroups. In mainland China, a different situation was observed. The first outbreak, localized in Shangdong Province, was reported in 2007, and was followed by a wide-spread outbreak in mainland China in 2008. Since then, numbers of reported HFMD cases have been persistently high.
Methodology/Principal Findings
To gain insight in the epidemiological behavior of EV71 in China, we studied genetic diversity and EV71 population dynamics to address whether the increase in number of reported EV71 infections reflects a real increase in viral spread or is just the result of increased awareness and surveillance. We used systematically collected VP1 gene sequences of 257 EV71 strains collected in Guangdong province from 2008 to 2010 as part of HFMD surveillance activities, and supplemented them with 305 GenBank EV71 reference stains collected in China from 1998 to 2010. All isolates from Guangdong Province belonged to subgenogroup C4. Viral population dynamics indicated that the increased reporting of HFMD in China since 2007 reflects a real increase in viral spread and continued replacement of viral lineages through time. Amino acid sequence comparisons revealed substitution of amino acid in residues 22, 145 and 289 through time regularly with the VP1 gene of EV71 strains isolated in mainland China from 1998 to 2010.
Conclusions
EV71 strains isolated in mainland China mainly belonged to subgenogroup C4. There was exponential growth of the EV71 virus population in 2007 and 2008. There was amino acid substitution through time regularly with the VP1 gene which possibly increased viral spread and/or ability of the virus to circulate persistently among the Chinese population.
doi:10.1371/journal.pone.0044386
PMCID: PMC3440427  PMID: 22984501
15.  Transmission Electron Microscopy Studies of Cellular Responses to Entry of Virions: One Kind of Natural Nanobiomaterial 
Virions are one kind of nanoscale pathogen and are able to infect living cells of animals, plants, and bacteria. The infection is an intrinsic property of the virions, and the biological process provides a good model for studying how these nanoparticles enter into cells. During the infection, the viruses employ different strategies to which the cells have developed respective responses. For this paper, we chose Bombyx mori cypovirus 1 (BmCPV-1) interactions with midgut cells from silkworm, and severe acute respiratory syndrome (SARS) associated coronavirus interactions with Vero E6 cells, as examples to demonstrate the response of eukaryotic cells to two different types of virus from our previous studies. The bacteriophage-bacteria interactions are also introduced to elucidate how the bacteriophage conquers the barrier of cell walls in the prokaryotic cells to transport genome into the host.
doi:10.1155/2012/596589
PMCID: PMC3332201  PMID: 22567012
16.  The expression of osteopontin and its association with Clara cell 10-kDa protein in allergic rhinitis 
SUMMARY
Background
Osteopontin (OPN) is a multifunctional protein which has recently been linked to allergic diseases. Clara cell 10-kDa protein (CC10) is another protein linked to allergy, and has been suggested to have an inhibitory role in inflammatory airway diseases. At this time, it is not known whether OPN is involved in allergic rhinitis (AR) or if there is any association between CC10 and OPN in AR.
Objective
To study the expression of OPN and its potential association with CC10 in AR.
Methods
The expression of CC10 and OPN in nasal mucosa of AR patients was investigated. AR animal models were established by employing wild-type and CC10-knockout mice. In some experiments, human recombinant CC10 protein was given to AR mice during either sensitization or challenge. The phenotypic changes were examined by histology and real-time RT-PCR. The direct effect of CC10 on OPN expression in spleen mononuclear cells and on OPN-induced inflammatory cytokine expression in BEAS-2B cells was measured through in vitro cell culture.
Results
OPN expression was up-regulated, with a concomitant down-regulation of CC10, in AR patients, showing a significant negative correlation between their expression. Compared with control mice sensitized with PBS, OPN expression was significantly increased in AR mice; such increase was more prominent in CC10-knockout mice, compared to wild-type. Administration of CC10 during both sensitization and challenge could markedly ameliorate Th2-skewed inflammation and OPN expression in nasal mucosa. CC10 administration at the sensitization phase could also reduce spleen OPN expression. The in vitro study showed that CC10 directly down-regulated OPN expression in spleen mononuclear cells stimulated with OVA and suppressed OPN-induced expression of Th2 cytokines and proinflammatory cytokines in BEAS-2B cells.
Conclusion
In the context of allergic airway responses, CC10 can inhibit OPN expression and suppress the Th2 promoting function of OPN, resulting in CC10’s inhibitory biological effects.
doi:10.1111/j.1365-2222.2010.03549.x
PMCID: PMC2948078  PMID: 20553297
allergic rhinitis; Clara cell 10-kDa protein; osteopontin; regulation
17.  Clara Cell 10-kD Protein Suppresses Chitinase 3-Like 1 Expression Associated with Eosinophilic Chronic Rhinosinusitis 
Rationale: Clara cell 10-kD (CC10) protein, an antiinflammatory molecule, is involved in inflammatory upper airway diseases, but its regulatory role is unclear, particularly in the process of chronic rhinosinusitis (CRS).
Objectives: To investigate the regulatory mechanisms of CC10 in eosinophilic CRS (ECRS) using an allergic mouse model.
Methods: Homozygous CC10-knockout mice were used to establish an allergic ECRS model. Phenotypic changes were examined by histology, cytokine ELISA, and gene microarray analysis. Differential expression of chitinase 3-like 1 (CHI3L1) was verified by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. The functional role of CHI3L1 in vivo was assessed by the use of anti-CHI3L1 antibody in ECRS mice. CHI3L1 gene expression regulated by inflammatory cytokines and CC10 protein was performed using BEAS-2B cell line.
Measurements and Main Results: Compared with wild-type mice, a significantly greater extent of inflammatory cell infiltration and tissue remodeling was found in CC10-knockout ECRS mice, which was associated with significantly higher levels of various cytokines and eotaxin-1. CHI3L1 was up-regulated in ECRS mice with a significant further increase in CC10-knockout mice. Anti-CHI3L1 treatment markedly ameliorated eosinophilic inflammation. Furthermore, nasal mucosal CC10 gene transfer in CC10-knockout mice attenuated eosinophilic inflammation and suppressed the levels of CHI3L1. Moreover, significantly up-regulated expression of CHI3L1 was noted in human ECRS. IL-1β, tumor necrosis factor–α, and IL-13 were found to up-regulate CHI3L1 expression in BEAS-2B cells, whereas CC10 inhibited such up-regulation.
Conclusions: These results suggest that CHI3L1 is a novel molecule involved in ECRS and that CC10 plays a regulatory role in ECRS, presumably by attenuating CHI3L1 expression.
doi:10.1164/rccm.200904-0597OC
PMCID: PMC2862304  PMID: 20093645
inflammation; tissue remodeling; mouse
18.  Modeling of Diffusion Transport through Oral Biofilms with the Inverse Problem Method 
Aim
The purpose of this study was to develop a mathematical model to quantitatively describe the passive transport of macromolecules within dental biofilms.
Methodology
Fluorescently labeled dextrans with different molecular mass (3 kD, 10 kD, 40 kD, 70 kD, 2 000 kD) were used as a series of diffusion probes. Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum were used as inocula for biofilm formation. The diffusion processes of different probes through the in vitro biofilm were recorded with a confocal laser microscope.
Results
Mathematical function of biofilm penetration was constructed on the basis of the inverse problem method. Based on this function, not only the relationship between average concentration of steady-state and molecule weights can be analyzed, but also that between penetrative time and molecule weights.
Conclusion
This can be used to predict the effective concentration and the penetrative time of anti-biofilm medicines that can diffuse through oral biofilm. Furthermore, an improved model for large molecule is proposed by considering the exchange time at the upper boundary of the dental biofilm.
doi:10.4248/IJOS10075
PMCID: PMC3499001  PMID: 21404968
oral biofilm; diffusion model; boundary condition; inverse problem method
19.  2-(2-{[2-(4-Pyridylcarbon­yl)hydrazinyl­idene]meth­yl}phen­oxy)acetic acid 
In the title compound, C15H13N3O4, the pyridine and benzene rings are nearly perpendicular [dihedral angle = 84.24 (5)°]. In the crystal structure, classical O—H⋯N hydrogen bonding between the OH group of the carboxyl unit and a neighbouring pyridine ring N atom and N—H⋯O hydrogen bonding between the imine NH group and a neighbouring O atom of an acyl unit, together with complementary non-classical C—H⋯O hydrogen bonds between carboxyl O atoms and neighbouring CH groups, link the mol­ecules into a three-dimensional system.
doi:10.1107/S1600536810017083
PMCID: PMC2979532  PMID: 21579430
20.  Organochlorine Pesticides in Consumer Fish and Mollusks of Liaoning Province, China: Distribution and Human Exposure Implications 
Fish and mollusk samples were collected from markets located in 12 cities in Liaoning province, China, during August and September 2007, and 22 organochlorine pesticides (OCPs) were detected. DDT, HCH, endosulfan, chlordane, and HCB were the dominating OCPs, with mean concentrations and ranges of, respectively, 15.41 and 0.57 to 177.56 ng/g, 0.84 and below detection limit (BDL) to 22.99 ng/g, 1.31 and BDL to 13.1 ng/g, 1.05 and BDL to 15.68 ng/g, and 0.63 and BDL to 9.21 ng/g in all fish and mollusk samples. The concentrations of other OCPs generally were low and were detectable in a minority of samples, reflecting the low levels of these OCPs in the study region. In general, OCP concentrations were obviously higher in fish than in mollusks, and higher in freshwater fish than in marine fish, which indicated, first, that freshwater fish are more easily influenced than seawater fish and mollusks by OCP residues in agricultural areas and, second, that there are different biota accumulation factors for OCPs between fish and mollusk. To learn the consumption of fish and mollusk, 256 questionnaires were sent to families in 12 cities of Liaoning province. Using the contamination data, average estimated daily intakes of OCPs via fish and mollusk consumption were calculated, which were used for exposure assessment. The public health risks caused by exposure to OCPs in the course of fish and mollusk consumption were compared to noncancer benchmarks and cancer benchmarks.
doi:10.1007/s00244-010-9504-7
PMCID: PMC2928924  PMID: 20352204
21.  2-(2-{[2-(2-Pyridylcarbon­yl)hydrazono]meth­yl}phen­oxy)acetic acid 
In the title compound, C15H13N3O4, the pyridine and benzene rings are nearly coplanar [dihedral angle = 4.92 (12)°]. The maximum deviation from the best least-squares plane calculated for the main mol­ecular skeleton is 0.1722 (1) Å for the carbonyl O atom. In the crystal, inter­molecular O—H⋯O hydrogen bonds connect the mol­ecules into a chain, while π–π stacking inter­actions between the pyridine and benzene rings [centroid–centroid distance = 3.9162 (8) Å and offset angle = 27.20°] complete a two-dimensional network.
doi:10.1107/S160053680905082X
PMCID: PMC2980243  PMID: 21580133
22.  Dichlorido[N′-(3,5-dichloro-2-hydroxy­benzyl­idene)pyridine-4-carbohydrazide-κN](1,10-phenanthroline-κ2 N,N′)cobalt(II) methanol monosolvate 
In the title compound, [CoCl2(C13H9Cl2N3O2)2(C12H8N2)]·CH3OH, the CoII atom is octahedrally coordinated by two N atoms from the pyridyl rings of the tridentate N′-(3,5-dichloro-2-hydroxy­benzyl­idene)pyridine-4-carbohydrazide (H2 L) ligand, two N atoms from the 1,10-phenanthroline ligand and two chloride ions. The acyl­hydrazone groups are not involved into the coordination of the metal ion. In the crystal packing an extended three-dimensional network formed by N—H⋯Cl, N—H⋯O, O—H⋯N, O—H⋯N and O—H⋯Cl hydrogen bonds is observed.
doi:10.1107/S1600536809047680
PMCID: PMC2972077  PMID: 21578623
23.  ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance 
PLoS Genetics  2014;10(6):e1004414.
Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD+) levels and blocked acquired resistance by inhibiting the activity of the NAD+-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.
Author Summary
Acquired resistance through genetic mutations is a major mechanism for cancer drug resistance and accounts for the short life of targeted therapy in several types of human cancer. Mechanistically, however, very little is understood about how resistant mutations are actually acquired during cancer therapy. In this manuscript, we used chronic myelogenous leukemia (CML) as a disease model and showed that mutation acquisition process is accompanied by global genome transcriptional reprogramming and reduction of cellular differentiation status. Forced cell differentiation by all-trans retinoic acid (ATRA) potently blocks acquisition of genetic mutations and CML acquired resistance. ATRA effect is mediated, in part, through stimulating CD38 gene expression, which reduces cellular cofactor nicotinamide adenine dinucleotide (NAD+) content and thus the activity of NAD+-dependent protein deacetylase SIRT1 that promotes error-prone DNA damage repair and mutagenesis. Our findings provide novel insight of mutation acquisition process during targeted therapy for CML. This study has translational implication in clinical treatment of CML, and perhaps other malignancies, by combining a differentiation agent to overcome mutation-mediated drug resistance if possible.
doi:10.1371/journal.pgen.1004414
PMCID: PMC4072521  PMID: 24967705
24.  Preparation of Anti-Tumor Nanoparticle and Its Inhibition to Peritoneal Dissemination of Colon Cancer 
PLoS ONE  2014;9(6):e98455.
Background
5-Fluorouracil (5-FU) is one of the most classic chemotherapy drugs. Nanoparticle drug delivery vehicles offer superiority over target effect enhancement and abatement of side effects. Little is known however as to the specific effect of nanoparticle on peritoneal dissemination of colon cancer. The aim of this study is to prepare one NPs (nanoparticles) loaded with 5-FU and investigate the characteristic of NPs and the role of it in peritoneal metastasis nodules formation of human colon cancer.
Methodology/Principal Findings
Prepared the NPs (nanoparticles) loaded with 5-FU (5-Fluorouracil) by PEG-PLGA with the method of double emulsion. Then evaluate the characteristics of the NPs by scanning electron microscopy, analyzing the particle diameter distribution and determining the loading efficiency. Detect the release features of NPs in vitro and in vivo. Nude mice with peritoneal metastases were treated with 5-FU solution or 5-FU-NPs through peritoneal cavity. Count the nodules on peritoneum and mesenterium and survey the size of them. We got NPs with average-diameter of 310 nm. In vitro release test shows NPs can release equably for 5 days with release rate of 99.2%. In vivo, NPs group can keep higher plasma concentration of 5-FU longer than it in solution group. The number of peritoneal dissemination nodule below 1 mm in 5-FU-sol group(17.3±3.5) and 5-FU-NP group(15.2±3.2) is less than control group(27.2±4.7)(P<0.05). The total number of nodules in 5-FU-NP group(28.7±4.2) is significantly smaller than in 5-FU-sol group(37.7±6.3) (P<0.05).
Conclusions/Significance
The novel anti-tumor nanoparticles loaded with 5-FU by PEG-PLGA can release maintain 5 days and have inhibitory action to peritoneal dissemination of colon cancer in mice.
doi:10.1371/journal.pone.0098455
PMCID: PMC4045714  PMID: 24896096
25.  Synthesis of (S)-FTY720 vinylphosphonate analogues and evaluation of their potential as sphingosine kinase 1 inhibitors and activators 
Bioorganic & medicinal chemistry  2013;21(9):2503-2510.
Sphingosine kinase 1 (SK1) is over-expressed in many cancers where it provides a selective growth and survival advantage to these cells. SK1 is thus a target for anti-cancer agents that can promote apoptosis of cancer cells. In previous work, we synthesized a novel allosteric SK1 inhibitor, (S)-FTY720 vinylphosphonate. We now report a more expeditious route to this inhibitor which features B-alkyl Suzuki coupling as a key step and show that replacement of the amino group in (S)-FTY720 vinylphosphonate with an azido group converts the vinylphosphonate from an allosteric inhibitor to an activator of SK1 at low micromolar concentrations. Our results demonstrate the feasibility of using the (S)-FTY720 vinylphosphonate scaffold to define structure-activity relationships in the allosteric site of SK1.
doi:10.1016/j.bmc.2013.02.042
PMCID: PMC3627827  PMID: 23541833
Sphingosine 1-phosphate; B-alkyl Suzuki cross-coupling; FTY720; Epoxide opening; Allosterism

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