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1.  Complete Genome Sequence of Porcine Parvovirus N Strain Isolated from Guangxi, China 
Genome Announcements  2015;3(1):e01359-14.
We report here the complete genomic sequence of the porcine parvovirus (PPV) N strain, isolated in 1989 from the viscera of a stillborn fetus farrowed by a gilt in Guangxi, southern China. Phylogenetic analyses suggest that the PPV-N strain is closely related to attenuated PPV NADL-2 strains. The PPV-N strain has good immunogenicity, genetic stability, and safety.
PMCID: PMC4290985  PMID: 25573932
2.  A Complex Recombination Pattern in the Genome of Allotetraploid Brassica napus as Revealed by a High-Density Genetic Map 
PLoS ONE  2014;9(10):e109910.
Polyploidy plays a crucial role in plant evolution. Brassica napus (2n = 38, AACC), the most important oil crop in the Brassica genus, is an allotetraploid that originated through natural doubling of chromosomes after the hybridization of its progenitor species, B. rapa (2n = 20, AA) and B. oleracea (2n = 18, CC). A better understanding of the evolutionary relationship between B. napus and B. rapa, B. oleracea, as well as Arabidopsis, which has a common ancestor with these three species, will provide valuable information about the generation and evolution of allopolyploidy. Based on a high-density genetic map with single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers, we performed a comparative genomic analysis of B. napus with Arabidopsis and its progenitor species B. rapa and B. oleracea. Based on the collinear relationship of B. rapa and B. oleracea in the B. napus genetic map, the B. napus genome was found to consist of 70.1% of the skeleton components of the chromosomes of B. rapa and B. oleracea, with 17.7% of sequences derived from reciprocal translocation between homoeologous chromosomes between the A- and C-genome and 3.6% of sequences derived from reciprocal translocation between non-homologous chromosomes at both intra- and inter-genomic levels. The current study thus provides insights into the formation and evolution of the allotetraploid B. napus genome, which will allow for more accurate transfer of genomic information from B. rapa, B. oleracea and Arabidopsis to B. napus.
PMCID: PMC4214627  PMID: 25356735
3.  Association between Baseline Body Mass Index (BMI) and Overall Survival among Patients over Age 60 with Acute Myeloid Leukemia (AML) 
American journal of hematology  2013;88(8):642-646.
Acute Myeloid Leukemia (AML) is more common and more lethal among patients over the age of 60. Increased body mass index (BMI) has been associated with a higher incidence of various malignancies, including AML. We sought to determine whether patient BMI at the time of AML diagnosis is related to overall survival among elderly patients. We identified 97 patients with AML diagnosed after the age of 60 and treated with cytarabine-based induction chemotherapy. The median age was 68 years (range 60-87); 52% of patients were male, and our study population was predominantly white (89% of patients). The median overall survival (OS) for all patients was 316 days (95% CI 246-459). The hazard ratio for mortality was increased among patients with a BMI < 25 compared to BMI ≥ 30 (HR 2.14, P=0.009, 95% CI 1.21-3.77), as well as with older age (HR 1.76, P=0.015, 95% CI 1.12-2.79) and with secondary versus de novo disease (HR 1.95, P=0.006, 95% CI 1.21-3.14). After multivariable analysis, we did not find a significant association between OS and other potential confounders such as coronary artery disease or diabetes among these patients. We conclude that increased BMI was independently associated with improved OS among older AML patients at our institution.
PMCID: PMC4214755  PMID: 23619915
Acute myeloid leukemia; Body mass index; obesity; elderly
4.  Ph+ ALL patients in first complete remission have similar survival after reduced intensity and myeloablative allogeneic transplantation: Impact of tyrosine kinase inhibitor and minimal residual disease 
Leukemia  2013;28(3):658-665.
The efficacy of reduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT) for Ph+ acute lymphoblastic leukemia (ALL) is uncertain. We analyzed 197 adults with Ph+ ALL in first complete remission; 67 patients receiving RIC were matched with 130 receiving myeloablative conditioning (MAC) for age, donor type, and HCT year. Over 75% received pre-HCT tyrosine kinase inhibitors (TKI), mostly imatinib; 39% (RIC) and 49% (MAC) were MRDneg pre-HCT. At a median 4.5 years follow-up, 1-year transplant-related mortality (TRM) was lower in RIC (13%) than MAC (36%;p=0.001) while the 3-year relapse rate was 49% in RIC and 28% in MAC (p=0.058). Overall survival was similar (RIC 39% [95% CI:27–52] vs. 35% [95% CI:270–44];p=0.62). Patients MRDpos pre-HCT had higher risk of relapse with RIC versus MAC (HR 1.97;p=0.026). However, patients receiving pre-HCT TKI in combination with MRD negativity pre-RIC HCT had superior OS (55%) compared to a similar MRDneg population after MAC (33%; p=0.0042). In multivariate analysis, RIC lowered TRM (HR 0.6; p=0.057), but absence of pre-HCT TKI (HR 1.88;p=0.018), RIC (HR 1.891;p=0.054) and pre-HCT MRDpos (HR 1.6; p=0.070) increased relapse risk. RIC is a valid alternative strategy for Ph+ ALL patients ineligible for MAC and MRDneg status is preferred pre-HCT.
PMCID: PMC3951192  PMID: 23989431
Acute lymphoblastic leukemia; Philadelphia chromosome; reduced intensity conditioning; allograft; minimal residual disease; tyrosine-kinase inhibitor
5.  RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes 
BMC Genomics  2014;15(1):571.
Stevia (Stevia rebaudiana) is an important medicinal plant that yields diterpenoid steviol glycosides (SGs). SGs are currently used in the preparation of medicines, food products and neutraceuticals because of its sweetening property (zero calories and about 300 times sweeter than sugar). Recently, some progress has been made in understanding the biosynthesis of SGs in Stevia, but little is known about the molecular mechanisms underlying this process. Additionally, the genomics of Stevia, a non-model species, remains uncharacterized. The recent advent of RNA-Seq, a next generation sequencing technology, provides an opportunity to expand the identification of Stevia genes through in-depth transcript profiling.
We present a comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent SG compositions characterized using RNA-seq. 191,590,282 high-quality reads were generated and then assembled into 171,837 transcripts with an average sequence length of 969 base pairs. A total of 80,160 unigenes were annotated, and 14,211 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Gene sequences of all enzymes known to be involved in SG synthesis were examined. A total of 143 UDP-glucosyltransferase (UGT) unigenes were identified, some of which might be involved in SG biosynthesis. The expression patterns of eight of these genes were further confirmed by RT-QPCR.
RNA-seq analysis identified candidate genes encoding enzymes responsible for the biosynthesis of SGs in Stevia, a non-model plant without a reference genome. The transcriptome data from this study yielded new insights into the process of SG accumulation in Stevia. Our results demonstrate that RNA-Seq can be successfully used for gene identification and transcript profiling in a non-model species.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-571) contains supplementary material, which is available to authorized users.
PMCID: PMC4108789  PMID: 25001368
Stevia rebaudiana; Transcriptome; RNA-seq
6.  Nuclear NF-κB p65 in Peripheral Blood Mononuclear Cells Correlates with Urinary MCP-1, RANTES and the Severity of Type 2 Diabetic Nephropathy 
PLoS ONE  2014;9(6):e99633.
To investigate if nuclear NF-κB p65 expression in ex vivo isolated peripheral blood mononuclear cells correlates with urinary MCP-1 or RANTES and the severity of type 2 diabetic nephropathy.
According to their urinary albumin-to-creatinine ratio (uACR), 107 patients with type 2 diabetes (eGFR >60 ml/min) were divided into normal albuminuria group (DN0 group, 38 cases), microalbuminuria group (DN1 group, 38 cases), and macroalbuminuria group (DN2 group, 31 cases), compared with matched healthy normal control group (NC group, 30 cases). Nuclear NF-κB p65 protein expression levels in peripheral blood mononuclear cells were detected by western blotting. Real-time quantitative polymerase chain reaction was used to detect NF-κB p65 mRNA expression and ELISA assay was used to detect the levels of urinary MCP-1 and RANTES.
Nuclear NF-κB p65 protein and NF-κB p65 mRNA expression levels in peripheral blood mononuclear cells, urinary MCP-1/Cr and RANTES/Cr were all significantly higher in all diabetes groups as compared with NC group. In particular, the increase of nuclear NF-κB p65 protein and NF-κB p65 mRNA expressions, urinary MCP-1/Cr and RANTES/Cr all correlated with the severity of type 2 diabetic nephropathy as indicated by the increase in uACR. Pearson correlation analysis indicated that both urinary MCP-1/Cr and RANTES/Cr were positively correlated with nuclear NF-κB p65 protein or NF-κB p65 mRNA levels. Stepwise multiple regression analysis showed that nuclear NF-κB p65 protein or NF-κB p65 mRNA was an independent variable for urinary MCP-1/Cr, and MCP-1/Cr and RANTES/Cr were two independent variables for uACR.
Our research demonstrates that nuclear NF-κB p65 protein and mRNA expressions in ex vivo isolated peripheral blood mononuclear cells well correlate with urinary MCP-1/Cr, RANTES/Cr and the severity of type 2 diabetic nephropathy.
PMCID: PMC4061032  PMID: 24936866
7.  The Interaction between Stent(s) Implantation, PICA Involvement, and Immediate Occlusion Degree Affect Symptomatic Intracranial Spontaneous Vertebral Artery Dissection Aneurysm (sis-VADA) Recurrence after Reconstructive Treatment with Stent(s)-Assisted Coiling 
European Radiology  2014;24(9):2088-2096.
We aimed to evaluate risk factors of sis-VADAs recurrence after reconstructive treatment based on 113 reconstructed lesions.
A total of 111 patients (M:F = 68:43; median age, 47 years) with 113 sis-VADAs underwent reconstruction from October 2000 to March 2011, using stent(s) and coils. Treatments and predictors of recurrence were retrospectively analysed.
Fifty-eight sis-VADAs underwent single-stent treatment, and the remaining 55 sis-VADAs underwent treatment with 2-4 overlapping stents. Follow-up angiography was available for 94 sis-VADAs 12 -78 months, with recurrence in ten patients, including seven angiographic recurrences and three post-treatment haemorrhagic recurrences. A higher rate of post-treatment recurrence was observed in the single stent group than in the multiple stents group (p = 0.010). The interaction between stent (s) implantation and immediate occlusion degree (odds ratio [OR] =3.152; 95 % confidence interval [CI], 1.293-7.686; p = 0.012), between stent (s) implantation and the posterior inferior cerebellar artery (PICA) involvement (OR = 4.607; 95 % CI, 1.172-18.113; p = 0.029), and between PICA involvement and immediate occlusion degree (OR = 5.018; 95 % CI, 1.263-19.933; p = 0.022) affect recurrence in the reconstructed sis-VADAs.
This single centre cohort study indicated that the interaction effect between stent (s) implantation, PICA involvement, and immediate occlusion degree were closely associated with recurrence after reconstructive treatment of sis-VADA.
Key Points
• The interaction between stent (s) and immediate occlusion degree affect aneurysmal recurrence
• The interaction between stent and PICA involvement affect aneurysmal recurrence
• The interaction between PICA involvement and immediate occlusion degree affect aneurysmal recurrence
Electronic supplementary material
The online version of this article (doi:10.1007/s00330-014-3225-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4127004  PMID: 24895032
Vertebral Artery Dissection; Aneurysm; Recurrence; Stent; Coiling
8.  Risk factors of intra-abdominal bacterial infection after liver transplantation in patients with hepatocellular carcinoma 
To explore the risk factors of intra-abdominal bacterial infection (IAI) after liver transplantation (LT) in patients with hepatocellular carcinoma (HCC).
A series of 82 HCC patients who received LT surgeries in our department between March 2004 and April 2010 was recruited in this study. Then we collected and analyzed the clinical data retrospectively. Statistical analysis system (SPSS) software was adopted to perform statistical analysis. Chi-square test, t-test and Wilcoxon rank sum test were used to analyze the clinical data and compute the significance of the incidences of early-stage IAI after LT for HCC patients. Binary logistic regression was performed to screen out the risk factors, and multiple logistic regression analyses were performed to compute the independent risk factors.
A series of 13 patients (13/82, 15.9%) had postoperative IAI. The independent risk factors of postoperative intra-abdominal bacterial infections after LT for HCC patients were preoperative anemia [Hemoglobin (HGB) <90 g/L] and postoperative abdominal hemorrhage (72 hours >400 mL), with the odds ratios at 8.121 (95% CI, 1.417 to 46.550, P=0.019) and 5.911 (95% CI, 1.112 to 31.432, P=0.037).
Postoperative IAI after LT in patients with HCC was a common complication. Preoperative moderate to severe anemia, as well as postoperative intra-abdominal hemorrhage more than 400 mL within the first 72 hours might independently indicate high risk of IAI for these patients.
PMCID: PMC4076729  PMID: 25035658
Liver transplantation (LT); intra-abdominal bacterial infections (IAI); hepatocellular carcinoma (HCC)
9.  Identification of boron-deficiency-responsive microRNAs in Citrus sinensis roots by Illumina sequencing 
BMC Plant Biology  2014;14:123.
Boron (B)-deficiency is a widespread problem in many crops, including Citrus. MicroRNAs (miRNAs) play important roles in nutrient deficiencies. However, little is known on B-deficiency-responsive miRNAs in plants. In this study, we first identified miRNAs and their expression pattern in B-deficient Citrus sinensis roots by Illumina sequencing in order to identify miRNAs that might be involved in the tolerance of plants to B-deficiency.
We isolated 52 (40 known and 12 novel) up-regulated and 82 (72 known and 10 novel) down-regulated miRNAs from B-deficient roots, demonstrating remarkable metabolic flexibility of roots, which might contribute to the tolerance of plants to B-deficiency. A model for the possible roles of miRNAs in the tolerance of roots to B-deficiency was proposed. miRNAs might regulate the adaptations of roots to B-deficiency through following several aspects: (a) inactivating reactive oxygen species (ROS) signaling and scavenging through up-regulating miR474 and down-regulating miR782 and miR843; (b) increasing lateral root number by lowering miR5023 expression and maintaining a certain phenotype favorable for B-deficiency-tolerance by increasing miR394 expression; (c) enhancing cell transport by decreasing the transcripts of miR830, miR5266 and miR3465; (d) improving osmoprotection (miR474) and regulating other metabolic reactions (miR5023 and miR821). Other miRNAs such as miR472 and miR2118 in roots increased in response to B-deficiency, thus decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots.
Our work demonstrates the possible roles of miRNAs and related mechanisms in the response of plant roots to B-deficiency.
PMCID: PMC4041134  PMID: 24885979
Boron-deficiency; Boron-tolerance; Citrus sinensis; Illumina sequencing; microRNA; Reactive oxygen species
10.  Parent artery reconstruction for large or giant cerebral aneurysms using a Tubridge flow diverter (PARAT): study protocol for a multicenter, randomized, controlled clinical trial 
BMC Neurology  2014;14:97.
The treatment of large (10-25 mm) or giant (≥25 mm) cerebral aneurysms remains technically challenging, with a much higher complication and recanalization rate than that is observed for smaller aneurysms. The use of a flow diverter seems to facilitate the treatment of this special entity. In a previous single-center prospective study approved by the Ethics Committee and China Food and Drug Administration (CFDA), we obtained promising results, showing remarkable safety and effectiveness for the Tubridge flow diverter. Nevertheless, the previous study may have been limited by biases due to its single-center design and limited number of subjects. Furthermore, although various articles have reported durable results from treating aneurysms using flow diverters, increasing questions have arisen about this form of treatment. Thus, prospective, multiple-center, randomized trials containing more subjects are needed.
This study is a multicenter, randomized, controlled clinical trial comparing clinical outcomes for patients with unruptured large/giant intracranial aneurysms treated with either conventional stent-assisted coiling or flow diverter implantation. A total of 124 patients who fulfill the inclusion and exclusion criteria will be randomized into either a treatment group or a control group in the ratio of 1:1. The treatment group will receive Tubridge implantation alone or combined with bared coils, and the control group will be treated with stent-assisted coiling (bare coils). The primary endpoint will be the complete occlusion rate at 6-month follow-up. Secondary endpoints include the immediate technique success rate, overall mortality, adverse events (ischemic stroke or intracranial bleeding) within 30 days, 90 days and 1 year post-operation, and the rate of intra-stent stenosis and thrombosis 6 months post-operation.
This prospective trial may provide more information on the safety and efficacy of the Tubridge flow diverter and may potentially change the strategy for treatment of large or giant aneurysms.
Trial registration
The trial is registered on the Chinese Clinical Trial Registry: ChiCTR-TRC-13003127
PMCID: PMC4016793  PMID: 24885396
Large; Giant; Aneurysm; Tubridge; Flow diverter; Trail
11.  MicroRNA and Cancer Chemoprevention 
MicroRNAs (miRNAs) are a group of naturally occurring, small, non-coding, and single-strand RNA molecules that regulate gene expression at the post-transcriptional and translational levels. By controlling the expression of oncogenic and tumor suppressor proteins, miRNAs are believed to play an important role in pathological processes associated with malignant progression including tumor cell proliferation, apoptosis, differentiation, angiogenesis, invasion and metastasis. However, relatively few studies have investigated the influence of chemopreventive agents on miRNA expression and their regulation of target genes. Given the significance of miRNAs in modulating gene expression, such research can provide insight into the pleiotropic biological effects that chemopreventive agents often display and a deeper understanding of their mechanism of action to inhibit carcinogenesis. Additionally, miRNAs can provide useful biomarkers for assessing antineoplastic activity of these agents in preclinical and clinical observations. In this review, we summarize recent publications that highlight a potentially important role of miRNAs in cancer chemoprevention research.
PMCID: PMC3644360  PMID: 23531448
microRNA; chemoprevention; cancer
12.  Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288 
BMC Genomics  2014;15(1):322.
Cytoplasmic male sterility (CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. CMS may be caused by mutations, rearrangement or recombination in the mitochondrial genome. Understanding the mitochondrial genome is often the first and key step in unraveling the molecular and genetic basis of CMS in plants. Comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line in B. juneca (Brassica juncea) may help show the origin of the CMS-associated gene orf288.
Through next-generation sequencing, the B. juncea hau CMS mitochondrial genome was assembled into a single, circular-mapping molecule that is 247,903 bp in size and 45.08% in GC content. In addition to the CMS associated gene orf288, the genome contains 35 protein-encoding genes, 3 rRNAs, 25 tRNA genes and 29 ORFs of unknown function. The mitochondrial genome sizes of the maintainer line and another normal type line “J163-4” are both 219,863 bp and with GC content at 45.23%. The maintainer line has 36 genes with protein products, 3 rRNAs, 22 tRNA genes and 31 unidentified ORFs. Comparative analysis the mitochondrial genomes of the hau CMS line and its maintainer line allowed us to develop specific markers to separate the two lines at the seedling stage. We also confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its maintainer lines in B. juncea. The number of repeats larger than 100 bp in the hau CMS line (16 repeats) are nearly twice of those found in the maintainer line (9 repeats). Phylogenetic analysis of the CMS-associated gene orf288 and four other homologous sequences in Brassicaceae show that orf288 was clearly different from orf263 in Brassica tournefortii despite of strong similarity.
The hau CMS mitochondrial genome was highly rearranged when compared with its iso-nuclear maintainer line mitochondrial genome. This study may be useful for studying the mechanism of natural CMS in B. juncea, performing comparative analysis on sequenced mitochondrial genomes in Brassicas, and uncovering the origin of the hau CMS mitotype and structural and evolutionary differences between different mitotypes.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-322) contains supplementary material, which is available to authorized users.
PMCID: PMC4035054  PMID: 24884490
Brassica juncea; Mitochondrial; Cytoplasmic male sterility; orf288; Mitotype
13.  Tumors of the angle of Treitz: A single-center experience 
AIM: To explore the feasibility and oncologic outcomes of segmental jejunal resection on the left side of the mesenteric vessels in patients with tumors of the angle of Treitz using data from a single center.
METHODS: Thirteen patients with tumors of the angle of Treitz who underwent surgery at our institution were prospectively followed. A segmental jejunal resection on the left side of the mesenteric vessels was performed in all patients. Formalin-fixed and paraffin-embedded tumor samples were examined. The primary end point of this analysis was disease-free survival.
RESULTS: In this study, there were 8 males and 5 females (mean age, 50.1 years; range, 36-74 years). The mean tumor size was 8.1 cm (range, 3.2-15 cm). Histologic examination showed 11 gastrointestinal stromal tumors (GISTs) and 2 adenocarcinomas. Five of the GIST patients presented with potential low risk, and 6 presented with intermediate and high risk, according to the National Institutes of Health criteria. One potentially high-risk patient showed tumor progression at 46 mo and died 52 mo after surgery. One patient with locally advanced adenocarcinoma received neoadjuvant chemotherapy and adjuvant radiotherapy, but the disease progressed, and the patient died 9 mo after surgery. One GIST patient without progression died 16 mo after surgery because of a postoperative intestinal obstruction. The median overall survival rate was 84.6 mo, and the median disease-free survival rate was 94.5 mo.
CONCLUSION: The overall survival of patients with tumors of the angle of Treitz was encouraging even when the tumor size was relatively large. A segmental resection on the left side of the mesenteric vessels is considered to be a reliable and curative option for tumors of the angle of Treitz.
PMCID: PMC3974531  PMID: 24707147
Gastrointestinal stromal tumor; Adenocarcinoma; Angle of Treitz; Surgical treatment; Prognosis
14.  Comparative transcript profiling of the fertile and sterile flower buds of pol CMS in B. napus 
BMC Genomics  2014;15:258.
The Polima (pol) system of cytoplasmic male sterility (CMS) and its fertility restoration gene Rfp have been used in hybrid breeding in Brassica napus, which has greatly improved the yield of rapeseed. However, the mechanism of the male sterility transition in pol CMS remains to be determined.
To investigate the transcriptome during the male sterility transition in pol CMS, a near-isogenic line (NIL) of pol CMS was constructed. The phenotypic features and sterility stage were confirmed by anatomical analysis. Subsequently, we compared the genomic expression profiles of fertile and sterile young flower buds by RNA-Seq. A total of 105,481,136 sequences were successfully obtained. These reads were assembled into 112,770 unigenes, which composed the transcriptome of the bud. Among these unigenes, 72,408 (64.21%) were annotated using public protein databases and classified into functional clusters. In addition, we investigated the changes in expression of the fertile and sterile buds; the RNA-seq data showed 1,148 unigenes had significantly different expression and they were mainly distributed in metabolic and protein synthesis pathways. Additionally, some unigenes controlling anther development were dramatically down-regulated in sterile buds.
These results suggested that an energy deficiency caused by orf224/atp6 may inhibit a series of genes that regulate pollen development through nuclear-mitochondrial interaction. This results in the sterility of pol CMS by leading to the failure of sporogenous cell differentiation. This study may provide assistance for detailed molecular analysis and a better understanding of pol CMS in B. napus.
PMCID: PMC4051170  PMID: 24707970
Brassica napus; pol CMS; Anther; Transcriptome
15.  Correlation of CTGF gene promoter methylation with CTGF expression in type 2 diabetes mellitus with or without nephropathy 
Molecular Medicine Reports  2014;9(6):2138-2144.
Increasing evidence shows that DNA methylation is involved in the development and progression of diabetes mellitus (DM) and its complications. Previous studies conducted by our group have indicated that high glucose levels may induce the demethylation process of the connective tissue growth factor (CTGF) gene promoter and increase the expression of CTGF in human glomerular mesangial cells. Based on these findings, the aim of the present study was to investigate the methylation level of genomic DNA and the CTGF promoter in patients with type 2 DM and to analyze its possible correlation with CTGF expression. Methylation levels of the whole genomic DNA were detected by high-performance liquid chromatography in a non-diabetes control (NDM) group (n=29), a diabetes without nephropathy (NDN) group (n=37) and a diabetes with nephropathy (DN) group (n=38). CTGF promoter methylation levels were detected by methylation-specific polymerase chain reaction and bisulfite sequencing. The levels of serum CTGF were assessed using the enzyme-linked immunosorbent assay. The methylation levels of the whole genomic DNA were not significantly different among the three groups. However, the CTGF methylation levels in the two diabetes groups were significantly lower than those in the NDM group (P<0.05), with the lowest methylation level in the DN group (P<0.05). The CTGF protein levels in the DN group were significantly higher than those in the NDM and NDN groups (P<0.05). Levels of CTGF were negatively correlated with the estimated glomerular filtration rate (eGFR) and the methylation level of the promoter, while they were positively correlated with age, urinary albumin-to-creatinine ratio (UACR), blood urea nitrogen, creatinine, fasting blood sugar and postprandial blood glucose. Multiple stepwise regression analysis showed that CTGF expression was associated with the UACR, CTGF methylation level and eGFR. DNA methylation is a regulatory mechanism of CTGF expression, which is decreased in patients with DM, particularly in those with DN, and may contribute to the pathogenesis of nephropathy.
PMCID: PMC4055476  PMID: 24676352
connective tissue growth factor; DNA methylation; diabetic nephropathy
16.  A pivotal role of the vascular endothelial growth factor signaling pathway in the formation of venous hypertension-induced dural arteriovenous fistulas 
Molecular Medicine Reports  2014;9(5):1551-1558.
Dural arteriovenous fistulas (DAVFs) are associated with venous hypertension. Numerous studies have revealed high expression levels of vascular endothelial growth factor (VEGF) in human DAVF specimens, as well as in animal models of experimental venous hypertension. The objective of the present study was to clarify whether the VEGF signaling pathway is important in the development of DAVFs. Rats (n=216) were randomly divided into six groups. In the rats from five groups (groups A and C-E, n=45 in each group; group B, n=12), experimental venous hypertension was induced by right common carotid artery (CCA)-external jugular vein (EJV) anastomosis, superior sinus occlusion and left transver sinus occlusion, while the remaining group (group F, n=24) underwent sham surgery. The rats in group A received a VEGF recombinant adenovirus injection into the distal section of the right EJV 30 min prior to anastomosis of the CCA and EJV. An equivalent control adenovirus was injected into the right EJV of group B rats prior to anastomosis. The rats in group C received no virus prior to anastomosis and no medicine subsequent to surgery. The group D rats were lavaged with Vatalanib, a VEGF receptor (VEGFR) inhibitor, and the group E rats were lavaged with an equal quantity of saline weekly following surgery. Six rats from groups A-E and one rat from group F were sacrificed in the first, second, fourth and twelfth weeks after surgery for immunohistochemical analysis of VEGF expression and analysis of microvessel density. Cerebral angiography was performed on the remaining rats in each group on the twelfth week after surgery. The results revealed that following transfection with VEGF recombinant adenovirus, angiogenesis in the dura mater of venous hypertensive rats was increased subsequent to the increase in the VEGF expression levels of the brain and dura mater. The rate of DAVF induction by venous hypertension was significantly reduced by the VEGFR antagonist due to reduced angiogenesis in the dura mater. In conclusion, VEGF and its receptor may be important in the formation of venous hypertension-induced DAVFs.
PMCID: PMC4020488  PMID: 24626343
dural arteriovenous fistula; vascular endothelial growth factor; angiogenesis; venous hypertension; local hypoferfusion
17.  Ambra1 Is an Essential Regulator of Autophagy and Apoptosis in SW620 Cells: Pro-Survival Role of Ambra1 
PLoS ONE  2014;9(2):e90151.
Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis.
PMCID: PMC3936000  PMID: 24587252
18.  Sequence-Based Discovery of Bradyrhizobium enterica in Cord Colitis Syndrome 
The New England journal of medicine  2013;369(6):517-528.
Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin.
We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls.
DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease.
We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen. (Funded by the National Cancer Institute and others.)
PMCID: PMC3889161  PMID: 23924002
19.  Malignant extra-gastrointestinal stromal tumor of the pancreas: Report of two cases and review of the literature 
Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that arise from the gastrointestinal tract. In rare cases, these tumors are found in intra-abdominal sites unrelated to the gastrointestinal tract, such as the mesentery, omentum and retroperitoneum. However, pancreatic extra-gastrointestinal stromal tumors are extremely rare, with only 14 previous cases reported. A 61-year-old man with no clinical symptoms had a routine check-up, during which an abdominal mass located in the pancreas tail was detected. Abdominal surgery was performed with resection of the pancreas tail and the spleen, and he was diagnosed with low-risk GISTs. Another 60-year-old man with no clinical symptoms underwent Computed tomography which revealed a well-demarcated tumor, 6 cm in diameter, in the head of the pancreas. He was diagnosed with pancreatic GISTs. Here, we describe two rare cases of pancreatic GISTs and review the cases previously reported in the literature.
PMCID: PMC3921496  PMID: 24574760
Gastrointestinal Stromal tumors; Extra-gastrointestinal Stromal Tumors; Pancreatic gastrointestinal stromal tumors
20.  GRIN2B Gene and Associated Brain Cortical White Matter Changes in Bipolar Disorder: A Preliminary Combined Platform Investigation 
BioMed Research International  2013;2013:635131.
Abnormalities in glutamate signaling and glutamate toxicity are thought to be important in the pathophysiology of bipolar disorder (BD). Whilst previous studies have found brain white matter changes in BD, there is paucity of data about how glutamatergic genes affect brain white matter integrity in BD. Based on extant neuroimaging data, we hypothesized that GRIN2B risk allele is associated with reductions of brain white matter integrity in the frontal, parietal, temporal, and occipital regions and cingulate gyrus in BD. Fourteen patients with BD and 22 healthy controls matched in terms of age, gender and handedness were genotyped using blood samples and underwent diffusion tensor imaging. Compared to G allele, brain FA values were significantly lower in BD patients with risk T allele in left frontal region (P = 0.001), right frontal region (P = 0.002), left parietal region (P = 0.001), left occipital region (P = 0.001), right occipital region (P < 0.001), and left cingulate gyrus (P = 0.001). Further elucidation of the interactions between different glutamate genes and their relationships with such structural, functional brain substrates will enhance our understanding of the link between dysregulated glutamatergic neurotransmission and neuroimaging endophenotypes in BD.
PMCID: PMC3893811  PMID: 24490167
21.  A High-Density SNP Map for Accurate Mapping of Seed Fibre QTL in Brassica napus L 
PLoS ONE  2013;8(12):e83052.
A high density genetic linkage map for the complex allotetraploid crop species Brassica napus (oilseed rape) was constructed in a late-generation recombinant inbred line (RIL) population, using genome-wide single nucleotide polymorphism (SNP) markers assayed by the Brassica 60 K Infinium BeadChip Array. The linkage map contains 9164 SNP markers covering 1832.9 cM. 1232 bins account for 7648 of the markers. A subset of 2795 SNP markers, with an average distance of 0.66 cM between adjacent markers, was applied for QTL mapping of seed colour and the cell wall fiber components acid detergent lignin (ADL), cellulose and hemicellulose. After phenotypic analyses across four different environments a total of 11 QTL were detected for seed colour and fiber traits. The high-density map considerably improved QTL resolution compared to the previous low-density maps. A previously identified major QTL with very high effects on seed colour and ADL was pinpointed to a narrow genome interval on chromosome A09, while a minor QTL explaining 8.1% to 14.1% of variation for ADL was detected on chromosome C05. Five and three QTL accounting for 4.7% to 21.9% and 7.3% to 16.9% of the phenotypic variation for cellulose and hemicellulose, respectively, were also detected. To our knowledge this is the first description of QTL for seed cellulose and hemicellulose in B. napus, representing interesting new targets for improving oil content. The high density SNP genetic map enables navigation from interesting B. napus QTL to Brassica genome sequences, giving useful new information for understanding the genetics of key seed quality traits in rapeseed.
PMCID: PMC3873396  PMID: 24386142
22.  A novel dominant glossy mutation causes suppression of wax biosynthesis pathway and deficiency of cuticular wax in Brassica napus 
BMC Plant Biology  2013;13:215.
The aerial parts of land plants are covered with cuticular waxes that limit non-stomatal water loss and gaseous exchange, and protect plants from ultraviolet radiation and pathogen attack. This is the first report on the characterization and genetic mapping of a novel dominant glossy mutant (BnaA.GL) in Brassica napus.
Transmission electron microscopy revealed that the cuticle ultrastructure of GL mutant leaf and stem were altered dramatically compared with that of wide type (WT). Scanning electron microscopy corroborated the reduction of wax on the leaf and stem surface. A cuticular wax analysis of the GL mutant leaves further confirmed the drastic decrease in the total wax content, and a wax compositional analysis revealed an increase in aldehydes but a severe decrease in alkanes, ketones and secondary alcohols. These results suggested a likely blockage of the decarbonylation step in the wax biosynthesis pathway. Genetic mapping narrowed the location of the BnaA.GL gene to the end of A9 chromosome. A single-nucleotide polymorphism (SNP) chip assay in combination with bulk segregant analysis (BSA) also located SNPs in the same region. Two SNPs, two single sequence repeat (SSR) markers and one IP marker were located on the flanking region of the BnaA.GL gene at a distance of 0.6 cM. A gene homologous to ECERIFERUM1 (CER1) was located in the mapped region. A cDNA microarray chip assay revealed coordinated down regulation of genes encoding enzymes of the cuticular wax biosynthetic pathway in the glossy mutant, with BnCER1 being one of the most severely suppressed genes.
Our results indicated that surface wax biosynthesis is broadly affected in the glossy mutant due to the suppression of the BnCER1 and other wax-related genes. These findings offer novel clues for elucidating the molecular basis of the glossy phenotype.
PMCID: PMC3881019  PMID: 24330756
Brassica napus; Glossy mutant; Genetic mapping; Wax biosynthesis; Microarray assays; Candidate genes
23.  Exploring and exploiting the fundamental role of microRNAs in tumor pathogenesis 
OncoTargets and therapy  2013;6:1675-1684.
miRNAs (miRs) are short RNA molecules that are involved in the posttranscriptional regulation of mRNA. The roles of miRs in tumor pathogenesis have only recently become a focus of research. It is becoming increasingly clear that miRs are important regulators of apoptosis, proliferation, invasion, and metastasis in cancer cells during cancer genesis and progression, furthering our understanding of cancer. In the present review, we summarize and evaluate the recent advances in our understanding of the characteristics of miRs as well as their regulated functions in cancer stem cells (CSCs), the epithelial-mesenchymal transition (EMT), and the tumor microenvironment (TM), describing their roles in tumor pathogenesis and their possible use as new therapeutic targets and biomarkers.
PMCID: PMC3836659  PMID: 24273410
miRNA; cancer stem cell; epithelial-mesenchymal transition; tumor microenvironment
24.  Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma 
AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor (HSVGM-CSF) in pancreatic carcinoma.
METHODS: Tumor blocks were homogenized in a sterile grinder in saline. The homogenate was injected into the right armpit of each mouse. After vaccination, the mice were randomly assigned into four groups: a control group, a high dose HSVGM-CSF group [1 × 107 plaque forming units (pfu)/tumor], a medium dose HSVGM-CSF group (5 × 106 pfu/tumor) and a low dose HSVGM-CSF group (5 × 105 pfu/tumor). After initiation of drug administration, body weights and tumor diameters were measured every 3 d. Fifteen days later, after decapitation of the animal by cervical dislocation, each tumor was isolated, weighed and stored in 10% formaldehyde solution. The drug effectiveness was evaluated according to the weight, volume and relative volume change of each tumor. Furthermore, GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1, 2, 3 and 4 d after injection of HSVGM-CSF.
RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group, and dose-dependent effects were observed: the relative tumor proliferation rates of the low dose, medium dose and high dose groups on 15 d after injection were 45.5%, 55.2% and 65.5%, respectively. The inhibition rates of the tumor weights of the low, middle, and high dose groups were 41.4%, 46.7% and 50.5%, respectively. Furthermore, the production of GM-CSF was significantly increased in the mice infected with HSVGM-CSF. The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups.
CONCLUSION: Our study provides the first evidence that HSVGM-CSF could inhibit the growth of pancreatic cancer. The enhanced GM-CSF expression might be responsible for the phenomenon.
PMCID: PMC3746387  PMID: 23964149
Pancreatic carcinoma; Gene therapy; Animal test; Herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor
25.  Reconstructive Treatment of Ruptured Intracranial Spontaneous Vertebral Artery Dissection Aneurysms: Long-Term Results and Predictors of Unfavorable Outcomes 
PLoS ONE  2013;8(6):e67169.
Few studies focused on predictors of unfavorable outcomes (modified Rankin Scale, 2–6) after reconstructive treatment of the ruptured intracranial spontaneous vertebral artery dissection aneurysms (ris-VADAs), which was evaluated based on 57 reconstructed lesions in this study.
Results of 57 consecutive patients (M:F = 29∶28; median age, 48 years; range, 27 to 69 years) harboring 57 ris-VADAs, which were treated with coils combined with single stent(n = 32), double overlapping stents (n = 16), and triple overlapping stents (n = 9) between October 2000 to March 2011, were retrospectively reviewed and analyzed.
The available (n = 54) mean durations of angiographic and clinical follow-ups were 27 months (range, 12 to 78) and 62 months (range, 12 to 132), respectively. The involvement of PICA (p = 0.004), size of lesions (p = 0.000), quantity of stent (p = 0.001), and coil type (p = 0.002) affected the immediate obliteration grade, which was only risk factor for angiographic recurrences (p = 0.031). Although the post-treatment outcomes did not differ between single stent and multiple stents (p = 0.434), 5 angiographic recurrences, 1 rebleeding and 1 suspected rebleeding, all occurred in partial obliteration after single-stent-assisted coiling. Progressive thrombosis and in-stent obliteration were not detected on follow-up angiograms. Older age (odds ratio [OR] = 1.090; 95% confidence interval [CI], 1.004–1.184; p = 0.040) and unfavorable Hunt-Hess scale (OR = 4.289; 95%CI, 1.232–14.933; p = 0.022) were independent predictors of unfavorable outcomes in the reconstructed ris-VADAs.
Immediate obliteration grade was only risk factor for angiographic recurrence after reconstructive treatment. Unfavorable Hunt-Hess grade and older age were independent predictors of unfavorable outcomes in ris-VADAs.
PMCID: PMC3693966  PMID: 23840616

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