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1.  BET-bromodomain inhibition of MYC-amplified medulloblastoma 
Purpose
MYC-amplified medulloblastomas are highly lethal tumors. BET bromodomain inhibition has recently been shown to suppress MYC-associated transcriptional activity in other cancers. The compound JQ1 inhibits BET bromodomain-containing proteins, including BRD4. Here we investigate BET bromodomain targeting for the treatment of MYC-amplified medulloblastoma.
Experimental Design
We evaluated the effects of genetic and pharmacological inhibition of BET bromodomains on proliferation, cell cycle, and apoptosis in established and newly generated patient- and GEMM-derived medulloblastoma cell lines and xenografts that harbored amplifications of MYC or MYCN. We also assessed the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity. We assessed in vivo efficacy of JQ1 in orthotopic xenografts established in immunocompromised mice.
Results
Treatment of MYC-amplified medulloblastoma cells with JQ1 decreased cell viability associated with arrest at G1 and apoptosis. We observed down-regulation of MYC expression and confirmed inhibition of MYC-associated transcriptional targets. Exogenous expression of MYC from a retroviral promoter reduced the effect of JQ1 on cell viability, suggesting that attenuated levels of MYC contribute to the functional effects of JQ1. JQ1 significantly prolonged survival of orthotopic xenograft models of MYC-amplified medulloblastoma (p<0.001). Xenografts harvested from mice after five doses of JQ1 had reduced expression of MYC mRNA and a reduced proliferative index.
Conclusion
JQ1 suppresses MYC expression and MYC-associated transcriptional activity in medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-amplified medulloblastoma.
doi:10.1158/1078-0432.CCR-13-2281
PMCID: PMC4198154  PMID: 24297863
BET-bromodomain; JQ1; MYC; MYCN; MYCL1; medulloblastoma
2.  The Association between Hantavirus Infection and Selenium Deficiency in Mainland China 
Viruses  2015;7(1):333-351.
Hemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses and transmitted by rodents is a significant public health problem in China, and occurs more frequently in selenium-deficient regions. To study the role of selenium concentration in HFRS incidence we used a multidisciplinary approach combining ecological analysis with preliminary experimental data. The incidence of HFRS in humans was about six times higher in severe selenium-deficient and double in moderate deficient areas compared to non-deficient areas. This association became statistically stronger after correction for other significant environment-related factors (low elevation, few grasslands, or an abundance of forests) and was independent of geographical scale by separate analyses for different climate regions. A case-control study of HFRS patients admitted to the hospital revealed increased activity and plasma levels of selenium binding proteins while selenium supplementation in vitro decreased viral replication in an endothelial cell model after infection with a low multiplicity of infection (MOI). Viral replication with a higher MOI was not affected by selenium supplementation. Our findings indicate that selenium deficiency may contribute to an increased prevalence of hantavirus infections in both humans and rodents. Future studies are needed to further examine the exact mechanism behind this observation before selenium supplementation in deficient areas could be implemented for HFRS prevention.
doi:10.3390/v7010333
PMCID: PMC4306842  PMID: 25609306
hemorrhagic fever with renal syndrome; selenium; hantavirus; rodents; environmental factors; China
3.  Genome-Scale Transcriptome Analysis in Response to Nitric Oxide in Birch Cells: Implications of the Triterpene Biosynthetic Pathway 
PLoS ONE  2014;9(12):e116157.
Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10−5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis.
doi:10.1371/journal.pone.0116157
PMCID: PMC4281108  PMID: 25551661
4.  Acupuncture at heterotopic acupoints enhances jejunal motility in constipated and diarrheic rats 
World Journal of Gastroenterology : WJG  2014;20(48):18271-18283.
AIM: To investigate the effect and mechanism of acupuncture at heterotopic acupoints on jejunal motility, particularly in pathological conditions.
METHODS: Jejunal motility was assessed using a manometric balloon placed in the jejunum approximately 18-20 cm downstream from the pylorus and filled with approximately 0.1 mL warm water in anesthetized normal rats or rats with diarrhea or constipation. The heterotopic acupoints including LI11 (Quchi), ST37 (Shangjuxu), BL25 (Dachangshu), and the homotopic acupoint ST25 (Tianshu), and were stimulated for 60 s by rotating acupuncture needles right and left at a frequency of 2 Hz. To determine the type of afferent fibers mediating the regulation of jejunal motility by manual acupuncture, the ipsilateral sciatic A or C fibers of ST37 were inactivated by local application of the A-fiber selective demyelination agent cobra venom or the C fiber blocker capsaicin. Methoctramine, a selective M2 receptor antagonist, was injected intravenously to identify a specific role for M2 receptors in mediating the effect of acupuncture on jejunal motility.
RESULTS: Acupuncture at heterotopic acupoints, such as LI11 and ST37, increased jejunal motility not only in normal rats, but also in rats with constipation or diarrhea. In normal rats, manual acupuncture at LI11 or ST37 enhanced jejunal pressure from 7.34 ± 0.19 cmH2O to 7.93 ± 0.20 cmH2O, an increase of 9.05% ± 0.82% (P < 0.05), and from 6.95 ± 0.14 cmH2O to 8.97 ± 0.22 cmH2O, a significant increase of 27.44% ± 1.96% (P < 0.01), respectively. In constipated rats, manual acupuncture at LI11 or ST37 increased intrajejunal pressure from 8.17 ± 0.31 cmH2O to 9.86 ± 0.36 cmH2O, an increase of 20.69% ± 2.10% (P < 0.05), and from 8.82 ± 0.28 cmH2O to 10.83 ± 0.28 cmH2O, an increase of 22.81% ± 1.46% (P < 0.05), respectively. In rats with diarrhea, MA at LI11 or ST37 increased intrajejunal pressure from 11.95 ± 0.35 cmH2O to 13.96 ± 0.39 cmH2O, an increase of 16.82% ± 2.35% (P < 0.05), and tended to increase intrajejunal pressure (from 12.42 ± 0.38 cmH2O to 13.05 ± 0.38 cmH2O, an increase of 5.07% ± 1.08%, P > 0.05), respectively. In contrast, acupuncture ST25, a homotopic acupoint, decreased not only intrajejunal pressure, but also significantly decreased frequency in normal rats and rats with constipation or diarrhea. Following demyelination of Aδ fibers, acupuncture at ST37 again augmented intrajejunal pressure to 121.48% ± 3.06% of baseline. Following capsaicin application for 24 h, acupuncture at ipsilateral ST37 increased intrajejunal pressure significantly to 106.63% ± 1.26% of basal levels when compared to measurements prior to capsaicin treatment (P < 0.05). Acupuncture at LI11, ST37, or BL25 significantly rescued methoctramine-mediated inhibition of jejunal motility amplitude from 42.83% ± 1.65% to 53.43% ± 1.95% of baseline (P < 0.05), from 45.15% ± 2.22% to 70.51% ± 2.34% of baseline (P < 0.01), and from 38.03% ± 2.34% to 70.12% ± 2.22% of baseline (P < 0.01), respectively.
CONCLUSION: Acupuncture at heterotopic acupoints increases the amplitude of jejunal motility in rats. C fibers and M2 receptors predominantly and partially mediate the regulation of jejunal motility by acupuncture, respectively.
doi:10.3748/wjg.v20.i48.18271
PMCID: PMC4277964  PMID: 25561794
Acupuncture; Heterotopic acupoint; LI11; ST37; BL25; Jejunal motility; Constipation; Diarrhea; C fibers; Muscarinic receptors
5.  Effects of a Randomized Intervention to Improve Workplace Social Capital in Community Health Centers in China 
PLoS ONE  2014;9(12):e114924.
Objective
To examine whether workplace social capital improved after implementing a workplace social capital intervention in community health centers in China.
Methods
This study was conducted in 20 community health centers of similar size in Jinan of China during 2012–2013. Using the stratified site randomization, 10 centers were randomized into the intervention group; one center was excluded due to leadership change in final analyses. The baseline survey including 447 staff (response rate: 93.1%) was conducted in 2012, and followed by a six-month workplace social capital intervention, including team building courses for directors of community health centers, voluntarily public services, group psychological consultation, and outdoor training. The follow-up survey in July 2013 was responded to by 390 staff members (response rate: 86.9%). Workplace social capital was assessed with the translated and culturally adapted scale, divided into vertical and horizontal dimensions. The facility-level intervention effects were based on all baseline (n = 427) and follow-up (n = 377) respondents, except for Weibei respondents. We conducted a bivariate Difference-in-Difference analysis to estimate the facility-level intervention effects.
Results
No statistically significant intervention effects were observed at the center level; the intervention increased the facility-level workplace social capital, and its horizontal and vertical dimensions by 1.0 (p = 0.24), 0.4 (p = 0.46) and 0.8 (p = 0.16), respectively.
Conclusions
The comprehensive intervention seemed to slightly improve workplace social capital in community health centers of urban China at the center level. High attrition rate limits any causal interpretation of the results. Further studies are warranted to test these findings.
doi:10.1371/journal.pone.0114924
PMCID: PMC4263705  PMID: 25503627
7.  Investigation of Variants in UCP2 in Chinese Type 2 Diabetes and Diabetic Retinopathy 
PLoS ONE  2014;9(11):e112670.
Purpose
The aim of this study was to investigate variants in UCP2 genes in type 2 diabetes mellitus (DM) and diabetic retinopathy (DR) in Chinese population.
Materials and Methods
We conducted a single nucleotide polymorphism-based and haplotype-based case-control study between the variants of UCP2 and DM and between the variants of UCP2 and DR in 479 Chinese patients with type 2 DM and 479 control subjects without DM. Two SNPs (rs660339 and rs659366) were selected as genetic markers.
Results
The risk allele C at UCP2 rs660339 was closely associated with DM in Chinese population. There was significant difference in rs660339 between DM and controls (P = 0.0016; OR [95%CI]  = 1.37 (1.14–1.65)). Subjects who were homozygous of the C allele were more likely to develop DM. The frequency of C allele was higher in DM (58%) than in control (51%). But this locus didn't have a definite effect on the onset of non-proliferative diabetic retinopathy (NPDR) (P = 0.44; OR [95%CI]  = 0.80 (0.56–1.14)) and proliferative diabetic retinopathy (PDR) (P = 1.00; OR [95%CI]  = 0.99 (0.74–1.34)) comparing to subjects with DM without retinopathy (DWR), respectively. Moreover, the UCP2 rs659366 polymorphism showed no significant difference between DM and control (P = 0.66; OR [95%CI]  = 1.10 (0.91–1.32)). However, there was a significant difference between PDR and DWR (P = 0.016; OR [95%CI]  = 0.66 (0.49–0.90)), but there was no difference between NPDR and DWR (P = 1.00; OR [95%CI]  = 0.96 (0.67–1.37)). Participants who carried the G allele at rs659366 were more likely to develop PDR. For the haplotype, C-A was present more frequently in DM than in control (16% vs 7%), indicating that it was risky, and T-A was present less in DM than in control (29% vs 35%). Haplotype frequencies in DR and DWR showed no significant difference (P = 0.068).
Conclusion
It was indicated that UCP2 may be implicated in the pathogenesis of type 2 DM and DR in Chinese population.
doi:10.1371/journal.pone.0112670
PMCID: PMC4232517  PMID: 25396419
8.  Vertebrate kidney tubules elongate using a planar cell polarity-dependent, rosette-based mechanism of convergent extension 
Nature genetics  2012;44(12):1382-1387.
Cystic kidney diseases are a global public health burden, affecting over 12 million people1. Although much is known about the genetics of kidney development and disease, the cellular mechanisms driving normal kidney tubule elongation remain unclear 2,3. Here, we used in vivo imaging to demonstrate for the first time that mediolaterally-oriented cell intercalation is fundamental to vertebrate kidney morphogenesis. Surprisingly, kidney tubule elongation is driven in large part by a myosin-dependent, multi-cellular rosette-based mechanism, previously only described in Drosophila. In contrast to Drosophila, however, non-canonical Wnt/PCP signaling is required to control rosette topology and orientation during vertebrate kidney tubule elongation. These data resolve longstanding questions concerning the role of PCP signaling in the developing kidney and moreover establish rosette-based intercalation as a deeply conserved cellular engine for epithelial morphogenesis.
doi:10.1038/ng.2452
PMCID: PMC4167614  PMID: 23143599
9.  Relationship between lipids levels and right ventricular volume overload in congestive heart failure 
Background
The relationship between lipids and coronary artery disease has been well established. However, this is not the case between lipids and heart failure. Ironically, high lipid levels are associated with better outcomes in heart failure, but the mechanisms underlying the phenomenon are not fully understood. This study was performed to test the hypothesis that reduced intestinal lipid absorption due to venous congestion may lead to low lipid levels.
Methods
We collected data of clinical characteristics, echocardiograph, and lipid profile in 442 unselected patients with congestive heart failure. Correlations between lipid levels [including total cholesterol (TCL), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG)] and right ventricle end diastolic diameter (RVEDD), left ventricle end diastolic diameter (LVEDD), right atrium diameter (RA), left atrium diameter (LA), or left ventricle ejection fraction (LVEF) were analyzed using Pearson correlation and partial correlation. RVEDD, LVEDD, RA, and LA were indexed to the body surface area.
Results
There was a significantly inverse correlation between TCL levels and RVEDD (r = −0.34, P < 0.001) and RA (r = −0.36, P < 0.001). Other lipids such as LDL-C, HDL-C, and TG had a similar inverse correlation with RVEDD and RA. All these correlations remained unchanged after adjusting for age, gender, smoking status, physical activity levels, comorbidities, and medication use.
Conclusions
Lipid levels were inversely correlated to RVEDD in patients with congestive heart failure; however, because this was an observational study, further investigation is needed to verify our results as well as identify a causal relationship, if any.
doi:10.11909/j.issn.1671-5411.2014.03.011
PMCID: PMC4178509  PMID: 25278966
Lipid levels; Heart failure; Right ventricle; Volume overload; Correlation analysis
10.  Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives 
Oncotarget  2014;5(18):8528-8543.
The molecularly targeted agents, including anti-VEGF or anti-EGFR monoclonal antibody and some inhibitors of EGFR tyrosine kinase, are effective in the treatment of non-small-cell lung cancer (NSCLC) to a certain extent, but the benefit for a proportion of patients is still limited. Hence, it is necessary and urgent to develop more selective and effective molecular targeted agents against lung cancer. Here, we have synthesized novel theanine derivatives, methyl coumarin-3-carboxylyl L-theanine (TMC), ethyl coumarin-3-carboxylyl L-theanine (TEC), ethyl 6-fluorocoumarin- 3-carboxylyl L-theanine (TFC), and ethyl 6-nitrocoumarin-3-carboxylyl L-theanine (TNC), which are fluorescent small molecules, based on their parental compound theanine and studied their anticancer activities in vitro, ex vivo and in vivo models of human and mouse cancers. Our results show that the four theanine derivatives significantly inhibit the lung cancer cell migration and the growth of lung cancer and leukemia cell lines. TFC and TNC display enhanced effects with anticancer drugs cytarabine, vincristine, and methotrexate on inhibition of lung cancer cell growth and no toxicity to the normal human embryonic lung fibroblast and peripheral blood lymphocytes. TFC and TNC exhibit strong suppression of the highly metastatic Lewis lung cancer (LLC) and A549 tumor growth in tumor-bearing mice without toxicity to mice. TFC and TNC can effectively suppress the growth of lung cancer cells in vitro, ex vivo and in vivo by targeting EGFR/VEGFR-Akt/NF-κB pathways. Our study has suggested that TFC and TNC may have the therapeutic and/or adjuvant therapeutic applications in the treatment of lung cancers and other cancer.
PMCID: PMC4226702  PMID: 25138052
Theanine derivatives; lung cancer; growth and migration; xenograft mouse models; inhibition; EGFR/VEGFR-Akt-NF-κB pathways
11.  Malignancy-associated metabolic profiling of human glioma cell lines using 1H NMR spectroscopy 
Molecular Cancer  2014;13(1):197.
Background
Ambiguity in malignant transformation of glioma has made prognostic diagnosis very challenging. Tumor malignant transformation is closely correlated with specific alterations of the metabolic profile. Exploration of the underlying metabolic alterations in glioma cells of different malignant degree is therefore vital to develop metabolic biomarkers for prognosis monitoring.
Methods
We conducted 1H nuclear magnetic resonance (NMR)-based metabolic analysis on cell lines (CHG5, SHG44, U87, U118, U251) developed from gliomas of different malignant grades (WHO II and WHO IV). Several methods were applied to analyze the 1H-NMR spectral data of polar extracts of cell lines and to identify characteristic metabolites, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), fuzzy c-means clustering (FCM) analysis and orthogonal projection to latent structure with discriminant analysis (OPLS-DA). The expression analyses of glial fibrillary acidic protein (GFAP) and matrix metal proteinases (MMP-9) were used to assess malignant behaviors of cell lines. GeneGo pathway analysis was used to associate characteristic metabolites with malignant behavior protein markers GFAP and MMP-9.
Results
Stable and distinct metabolic profiles of the five cell lines were obtained. The metabolic profiles of the low malignancy grade group (CHG5, SHG44) were clearly distinguished from those of the high malignancy grade group (U87, U118, U251). Seventeen characteristic metabolites were identified that could distinguish the metabolic profiles of the two groups, nine of which were mapped to processes related to GFAP and MMP-9. Furthermore, the results from both quantitative comparison and metabolic correlation analysis indicated that the significantly altered metabolites were primarily involved in perturbation of metabolic pathways of tricarboxylic acid (TCA) cycle anaplerotic flux, amino acid metabolism, anti-oxidant mechanism and choline metabolism, which could be correlated with the changes in the glioma cells’ malignant behaviors.
Conclusions
Our results reveal the metabolic heterogeneity of glioma cell lines with different degrees of malignancy. The obtained metabolic profiles and characteristic metabolites are closely associated with the malignant features of glioma cells, which may lay the basis for both determining the molecular mechanisms underlying glioma malignant transformation and exploiting non-invasive biomarkers for prognosis monitoring.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-197) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-197
PMCID: PMC4158044  PMID: 25163530
Glioma cell line; Malignancy; Metabolic profiling; 1H-NMR; Spectroscopy
12.  Solvent induced rapid modulation of micro/nano structures of metal carboxylates coordination polymers: mechanism and morphology dependent magnetism 
Scientific Reports  2014;4:6023.
Rational modulation of morphology is very important for functional coordination polymers (CPs) micro/nanostructures, and new strategies are still desired to achieve this challenging target. Herein, organic solvents have been established as the capping agents for rapid modulating the growth of metal-carboxylates CPs in organic solvent/water mixtures at ambient conditions. Co-3,5-pyridinedicarboxylate (pydc) CPs was studied here as the example. During the reaction, the organic solvents exhibited three types of modulation effect: anisotropic growth, anisotropic growth/formation of new crystalline phase and the formation of new crystalline phase solely, which was due to the variation of their binding ability with metal cations. The following study revealed that the binding ability was critically affected by their functional groups and molecular size. Moreover, their modulation effect could be finely tuned by changing volume ratios of solvent mixtures. Furthermore, they could be applied for modulating other metal-carboxylates CPs: Co-1,3,5-benzenetricarboxylic (BTC), Zn-pydc and Eu-pydc etc. Additionally, the as-prepared Co-pydc CPs showed a fascinating morphology-dependent antiferromagnetic behavior.
doi:10.1038/srep06023
PMCID: PMC4129412  PMID: 25113225
13.  Surgical Technique: Hemilaminectomy and Unilateral Lateral Mass Fixation for Cervical Ossification of the Posterior Longitudinal Ligament 
Background
Surgical approaches for cervical ossification of the posterior longitudinal ligament (OPLL) include anterior, posterior, or combined decompression with or without fusion. The goal of surgery is to decompress the spinal cord while maintaining the stability and sagittal alignment of the cervical spine. C5 palsy has been reported as a postoperative complication of cervical laminectomy or laminoplasty characterized as motor weakness of the muscles supplied with C5 nerve roots. Several studies have shown this phenomenon was partially attributable to posterior shift of spinal cord.
Description of Technique
The rationale for choosing hemilaminectomy is to control postoperative posterior shift of the spinal cord and afford more stability by preserving ligamentous attachments and posterior bony elements as much as possible. After a fixation system of lateral mass screws and rods is installed unilaterally, laminae are removed from the underlying dura using a high-speed burr and Kerrison laminectomy rongeur on the other side. The spinous processes are preserved.
Patients and Methods
Patients with multilevel continuous/mixed cervical OPLL are good candidates for this technique. We retrospectively reviewed 146 patients who had multilevel continuous/mixed cervical OPLL and underwent surgery from January 2006 to January 2010. Neurologic condition was evaluated using the improvement ratio (IR) of the Japanese Orthopaedic Association (JOA) score for cervical myelopathy.
Results
The mean JOA score increased from 10 points before surgery to 14 points at last followup. The mean IR of neurologic function (JOA score) was 59%. C5 palsy was not observed in any patient after decompression, and cervical lordosis changed from 8.7° preoperatively to 9.1° at last followup.
Conclusions
For patients with multilevel continuous/mixed cervical OPLL without fixed kyphosis, multilevel hemilaminectomy with unilateral lateral mass fixation is an effective alternative technique.
Level of Evidence
Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
doi:10.1007/s11999-013-2889-7
PMCID: PMC3676587  PMID: 23467986
14.  AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings 
Plant Methods  2014;10:19.
Background
Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging.
Results
We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts.
Conclusions
AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.
doi:10.1186/1746-4811-10-19
PMCID: PMC4076510  PMID: 24987449
Agrobacterium; Arabidopsis; Transient transformation; Gene expression; Innate immunity; Gain-of-function
15.  Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study 
Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future.
PMCID: PMC4128998  PMID: 25120763
Unclassified renal cell carcinoma; comparative genomic hybridization; exon GeneChip; chromosome imbalance; gene mutation
16.  Regeneration of Solanum nigrum by Somatic Embryogenesis, Involving Frog Egg-Like Body, a Novel Structure 
PLoS ONE  2014;9(6):e98672.
A new protocol was established for the regeneration of Solanum nigrum by frog egg-like bodies (FELBs), which are novel somatic embryogenesis (SE) structures induced from the root, stem, and leaf explants. The root, stem, and leaf explants (93.33%, 85.10%, and 100.00%, respectively) were induced to form special embryonic calli on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid, under dark condition. Further, special embryonic calli from the root, stem, and leaf explants (86.97%, 83.30%, and 99.47%, respectively) were developed into FELBs. Plantlets of FELBs from the three explants were induced in vitro on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine and 0.1 mg/L gibberellic acid, and 100.00% plantlet induction rates were noted. However, plantlet induction in vivo on MS medium supplemented with 20 mg/L thidiazuron showed rates of 38.63%, 15.63%, and 61.30% for the root, stem, and leaf explants, respectively, which were lower than those of the in vitro culture. Morphological and histological analyses of FELBs at different development stages revealed that they are a novel type of SE structure that developed from the mesophyll (leaf) or cortex (stem and root) cells of S. nigrum.
doi:10.1371/journal.pone.0098672
PMCID: PMC4045584  PMID: 24896090
17.  Epidemiologic Features and Environmental Risk Factors of Severe Fever with Thrombocytopenia Syndrome, Xinyang, China 
Background
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease discovered in rural areas of Central China in 2009, caused by a novel bunyavirus, SFTS virus (SFTSV). The disease usually presents as fever, thrombocytopenia, and leukocytopenia, with case-fatality rates ranging from 2.5% to 30%. Haemaphysalis longicornis was suspected to be the most likely vector of SFTSV. By the end of 2012, the disease had expanded to 13 provinces of China. SFTS patients have been reported in Japan and South Korea, and a disease similar to SFTS has been reported in the United States.
Methodology/Principal Findings
We characterized the epidemiologic features of 504 confirmed SFTS cases in Xinyang Region, the most severely SFTS-afflicted region in China from 2011 to 2012, and assessed the environmental risk factors. All cases occurred during March to November, with the epidemic peaking from May to July. The patients' ages ranged from 7 to 87 years (median 61 years), and the annual incidence increased with age (χ2 test for trend, P<0.001). The female-to-male ratio of cases was 1.58, and 97.0% of the cases were farmers who resided in the southern and western parts of the region. The Poisson regression analysis revealed that the spatial variations of SFTS incidence were significantly associated with the shrub, forest, and rain-fed cropland areas.
Conclusions
The distribution of SFTS showed highly significant temporal and spatial heterogeneity in Xinyang Region, with the majority of SFTS cases being elderly farmers who resided in the southern and western parts of the region, mostly acquiring infection between May and July when H. longicornis is highly active. The shrub, rain-fed, and rain-fed cropland areas were associated with high risk for this disease.
Author Summary
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease discovered in rural areas of Central China in 2009, caused by a novel bunyavirus, SFTS virus (SFTSV). The disease usually presents as fever, thrombocytopenia, and leukocytopenia, with case-fatality rates ranging from 2.5% to 30%. By the end of 2012, the disease had expanded to 13 provinces of China. SFTS patients have been reported in Japan and South Korea, and a disease similar to SFTS has been reported in the United States. Here we characterized the epidemiologic features of 504 confirmed SFTS cases in Xinyang, the most severely SFTS-affected region in China from 2011 to 2012, and identified the environmental risk factors. We found the distribution of SFTS cases showed highly significant temporal and spatial heterogeneity, with the majority of SFTS cases being elderly farmers who resided in the southern and western parts of the region, mostly acquiring infection between May and July when H. longicornis is highly active. The shrub, forest, and rain-fed cropland areas were strongly associated with high risk for SFTS.
doi:10.1371/journal.pntd.0002820
PMCID: PMC4014392  PMID: 24810269
18.  Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice 
Cell & Bioscience  2014;4:21.
Background
Steroid receptor coactivator 3 (SRC-3) is a multifunctional protein that plays an important role in malignancy of several cancers and in regulation of bacterial LPS-induced inflammation. However, the involvement of SRC-3 in allergic response remains unclear. Herein we used passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models to assess the role of SRC-3 in allergic response.
Results
SRC-3-deficient mice exhibited more severe allergic response as demonstrated by a significant drop in body temperature and a delayed recovery period compared to wild-type mice in PSA mouse model, whereas no significant difference was observed between two kinds of mice in PCA mouse models. Mast cells play a pivotal role in IgE-mediated allergic response. Antigen-induced aggregation of IgE receptor (FcϵRI) on the surface of mast cell activates a cascade of signaling events leading to the degranulation and cytokine production in mast cells. SRC-3-deficient bone marrow derived mast cells (BMMCs) developed normally but secreted more proinflammatory cytokines such as TNF-α and IL-6 than wild-type cells after antigen stimulation, whereas there was no significant difference in degranulation between two kinds of mast cells. Further studies showed that SRC-3 inhibited the activation of nuclear factor NF-κB pathway and MAPKs including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in antigen-stimulated mast cells.
Conclusions
Our data demonstrate that SRC-3 suppresses cytokine production in antigen-stimulated mast cells as well as PSA in mice at least in part through inhibiting NF-κB and MAPK signaling pathways. Therefore, SRC-3 plays a protective role in PSA and it may become a drug target for anaphylactic diseases.
doi:10.1186/2045-3701-4-21
PMCID: PMC4021842  PMID: 24834318
SRC-3; Mast cell; Passive systemic anaphylaxis; Passive cutaneous anaphylaxis
19.  Novel Antibody against a Glutamic Acid-Rich Human Fibrinogen-Like Protein 2-Derived Peptide near Ser91 Inhibits hfgl2 Prothrombinase Activity 
PLoS ONE  2014;9(4):e94551.
Fibrinogen-like protein 2 (fgl2) is highly expressed in microvascular endothelial cells in diseases associated with microcirculatory disturbances and plays a crucial role in microthrombosis. Previous studies have demonstrated that the Ser89 residue is a critical site for mouse fgl2 prothrombinase activity. The aim of this study was to investigate the prothrombinase inhibitory ability of antibodies against an hfgl2-derived peptide. The peptide was termed NPG-12 because it is located at the N-terminus of membrane-bound hfgl2, contains 12 amino acid residues (corresponding to residues 76 to 87), and is rich in Glu. This peptide was selected as an antigenic determinant to produce antibodies in immunized rabbits using the DNAStar and HomoloGene software program. Abundant hfgl2 expression was induced in human umbilical vein endothelial cells through treatment with TNF-α. The generated anti-NPG-12 antibodies specifically recognize fgl2, as determined by ELISA, Western Blot and immunostaining. Moreover, one-stage clotting and thrombin generation tests provide evidence that the antibodies can reduce the hfgl2 prothrombinase activity without affecting the platelet-poor plasma prothrombin time (PT) or the activated partial thromboplastin time (APTT). In addition, the antibodies exerted undetectable influence on the proliferation or activation of bulk T cell populations. In conclusion, the selected peptide sequence NPG-12 may be a critical domain for hfgl2 prothrombinase activity, and the development of inhibitors against this sequence may be promising for research or management of hfgl2-associated microcirculatory disturbances.
doi:10.1371/journal.pone.0094551
PMCID: PMC3984148  PMID: 24728278
20.  VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation 
PLoS ONE  2014;9(4):e93547.
Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway.
doi:10.1371/journal.pone.0093547
PMCID: PMC3972149  PMID: 24691407
21.  Perfluorocarbon Liquid: Its Application in Vitreoretinal Surgery and Related Ocular Inflammation 
BioMed Research International  2014;2014:250323.
The application of perfluorocarbon liquids has been well acclaimed in vitreoretinal surgery. Its unique physical properties make it an ideal intraoperative tool to improve the efficiency and safety of surgical procedures in complicated cases. The main functions of perfluorocarbon liquids in vitreoretinal surgery include relocating and fixing the detached retina, displacing the subretinal and subchoroidal to fluid anteriorly, revealing proliferative vitreous retinopathy (PVR) for further maneuvers, protecting the macula from exposure to chemicals with potential toxicity, and assisting the removal of foreign body. The related clinical applications include retinal detachment with severe proliferative vitreoretinopathy, giant tear, diabetic retinopathy (DR), retinopathy of prematurity (ROP), and posterior dislocated crystalline and intraocular lenses. The application of perfluorocarbon liquids has been expended over the past fewer years. Several PFCLs related ocular inflammations have been observed in in vitro studies, animal studies, and clinical follow-up. The complete removal of PFCLs is recommended at the end of the surgery in most cases.
doi:10.1155/2014/250323
PMCID: PMC3985162  PMID: 24800216
22.  Influence of Copper Nanoparticles on the Physical-Chemical Properties of Activated Sludge 
PLoS ONE  2014;9(3):e92871.
The physical-chemical properties of activated sludge, such as flocculating ability, hydrophobicity, surface charge, settleability, dewaterability and bacteria extracellular polymer substances (EPS), play vital roles in the normal operation of wastewater treatment plants (WWTPs). The nanoparticles released from commercial products will enter WWTPs and can induce potential adverse effects on activated sludge. This paper focused on the effects of copper nanoparticles (CuNPs) on these specific physical-chemical properties of activated sludge. It was found that most of these properties were unaffected by the exposure to lower CuNPs concentration (5 ppm), but different observation were made at higher CuNPs concentrations (30 and 50 ppm). At the higher CuNPs concentrations, the sludge surface charge increased and the hydrophobicity decreased, which were attributed to more Cu2+ ions released from the CuNPs. The carbohydrate content of EPS was enhanced to defense the toxicity of CuNPs. The flocculating ability was found to be deteriorated due to the increased cell surface charge, the decreased hydrophobicity, and the damaged cell membrane. The worsened flocculating ability made the sludge flocs more dispersed, which further increased the toxicity of the CuNPs by increasing the availability of the CuNPs to the bacteria present in the sludge. Further investigation indicated that the phosphorus removal efficiency decreased at higher CuNPs concentrations, which was consistent with the deteriorated physical-chemical properties of activated sludge. It seems that the physical-chemical properties can be used as an indicator for determining CuNPs toxicity to the bacteria in activated sludge. This work is important because bacteria toxicity effects to the activated sludge caused by nanoparticles may lead to the deteriorated treatment efficiency of wastewater treatment, and it is therefore necessary to find an easy way to indicate this toxicity.
doi:10.1371/journal.pone.0092871
PMCID: PMC3963960  PMID: 24663333
23.  A first generation BAC-based physical map of the half-smooth tongue sole (Cynoglossus semilaevis) genome 
BMC Genomics  2014;15:215.
Background
Half-smooth tongue sole (Cynoglossus semilaevis Günther) has been exploited as a commercially important cultured marine flatfish, and female grows 2–3 times faster than male. Genetic studies, especially on the chromosomal sex-determining system of this species, have been carried out in the last decade. Although the genome of half-smooth tongue sole was relatively small (626.9 Mb), there are still some difficulties in the high-quality assembly of the next generation genome sequencing reads without the assistance of a physical map, especially for the W chromosome of this fish due to abundance of repetitive sequences. The objective of this study is to construct a bacterial artificial chromosome (BAC)-based physical map for half-smooth tongue sole with the method of high information content fingerprinting (HICF).
Results
A physical map of half-smooth tongue sole was constructed with 30, 294 valid fingerprints (7.5 × genome coverage) with a tolerance of 4 and an initial cutoff of 1e-60. A total of 29,709 clones were assembled into 1,485 contigs with an average length of 539 kb and a N50 length of 664 kb. There were 394 contigs longer than the N50 length, and these contigs will be a useful resource for future integration with linkage map and whole genome sequence assembly. The estimated physical length of the assembled contigs was 797 Mb, representing approximately 1.27 coverage of the half-smooth tongue sole genome. The largest contig contained 410 BAC clones with a physical length of 3.48 Mb. Almost all of the 676 BAC clones (99.9%) in the 21 randomly selected contigs were positively validated by PCR assays, thereby confirming the reliability of the assembly.
Conclusions
A first generation BAC-based physical map of half-smooth tongue sole was constructed with high reliability. The map will promote genetic improvement programs of this fish, especially integration of physical and genetic maps, fine-mappings of important gene and/or QTL, comparative and evolutionary genomics studies, as well as whole genome sequence assembly.
doi:10.1186/1471-2164-15-215
PMCID: PMC3998196  PMID: 24650389
Half-smooth tongue sole; Cynoglossus semilaevis; BAC library; Physical map; Genome
24.  Cucurbitacin IIb Exhibits Anti-Inflammatory Activity through Modulating Multiple Cellular Behaviors of Mouse Lymphocytes 
PLoS ONE  2014;9(2):e89751.
Cucurbitacin IIb (CuIIb) is one of the major active compounds in Hemsleyadine tablets which have been used for clinical treatment of bacillary dysentery, enteritis and acute tonsilitis. However, its action mechanism has not been completely understood. This study aimed to explore the anti-inflammatory activity of CuIIb and its underlying mechanism in mitogen-activated lymphocytes isolated from mouse mesenteric lymph nodes. The results showed that CuIIb inhibited the proliferation of concanavalin A (Con A)-activated lymphocytes in a time- and dose-dependent manner. CuIIb treatment arrested their cell cycle in S and G2/M phases probably due to the disruption of the actin cytoskeleton and the modulation of p27Kip1 and cyclin levels. Moreover, the surface expression of activation markers CD69 and CD25 on Con A-activated CD3+ T lymphocytes was suppressed by CuIIb treatment. Both Con A- and phorbol ester plus ionomycin-induced expression of TNF-α, IFN-γ and IL-6 proteins was attenuated upon exposure to CuIIb. Mechanistically, CuIIb treatment suppressed the phosphorylation of JNK and Erk1/2 but not p38 in Con A-activated lymphocytes. Although CuIIb unexpectedly enhanced the phosphorylation of IκB and NF-κB (p65), it blocked the nuclear translocation of NF-κB (p65). In support of this, CuIIb significantly decreased the mRNA levels of IκBα and TNF-α, two target genes of NF-κB, in Con A-activated lymphocytes. In addition, CuIIb downregulated Con A-induced STAT3 phosphorylation and increased cell apoptosis. Collectively, these results suggest that CuIIb exhibits its anti-inflammatory activity through modulating multiple cellular behaviors and signaling pathways, leading to the suppression of the adaptive immune response.
doi:10.1371/journal.pone.0089751
PMCID: PMC3934946  PMID: 24587010
25.  A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis 
PLoS ONE  2014;9(1):e83556.
Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.
doi:10.1371/journal.pone.0083556
PMCID: PMC3885512  PMID: 24416168

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