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1.  Cassava genome from a wild ancestor to cultivated varieties 
Nature Communications  2014;5:5110.
Cassava is a major tropical food crop in the Euphorbiaceae family that has high carbohydrate production potential and adaptability to diverse environments. Here we present the draft genome sequences of a wild ancestor and a domesticated variety of cassava and comparative analyses with a partial inbred line. We identify 1,584 and 1,678 gene models specific to the wild and domesticated varieties, respectively, and discover high heterozygosity and millions of single-nucleotide variations. Our analyses reveal that genes involved in photosynthesis, starch accumulation and abiotic stresses have been positively selected, whereas those involved in cell wall biosynthesis and secondary metabolism, including cyanogenic glucoside formation, have been negatively selected in the cultivated varieties, reflecting the result of natural selection and domestication. Differences in microRNA genes and retrotransposon regulation could partly explain an increased carbon flux towards starch accumulation and reduced cyanogenic glucoside accumulation in domesticated cassava. These results may contribute to genetic improvement of cassava through better understanding of its biology.
Cassava is a major source of food in tropical and subtropical regions. Here the authors sequence the genomes of wild and domesticated cassava varieties and identify genes that have been selected for and against during the evolution and domestication of this economically important crop.
doi:10.1038/ncomms6110
PMCID: PMC4214410  PMID: 25300236
2.  Radiotherapy for asymptomatic brain metastasis in epidermal growth factor receptor mutant non-small cell lung cancer without prior tyrosine kinase inhibitors treatment: a retrospective clinical study 
Background
Non-small cell lung cancer (NSCLC) with brain metastasis (BM) harboring an epidermal growth factor receptor (EGFR) mutation shows good response to tyrosine kinase inhibitors (TKIs). This study is to assess the appropriate timing of brain radiotherapy (RT) for asymptomatic BM in EGFR mutant NSCLC patients.
Methods
There were 628 patients diagnosed with EGFR mutant NSCLC between October 2005 and December 2011. Treatment outcomes had been retrospectively evaluated in 96 patients with asymptomatic BM without prior TKI treatment. 39 patients received first-line brain RT, 23 patients received delayed brain RT, and 34 patients did not receive brain RT.
Results
With a median follow-up of 26 months, the 2-year OS was 40.6 %. Univariate analyses revealed that ECOG performance status (p = 0.006), other distant metastases (p = 0.002) and first line systemic treatment (p = 0.032) were significantly associated with overall survival (OS). Multivariate analyses revealed that other sites of distant metastases (p = 0.030) were prognostic factor. The timing of brain RT was not significantly related to OS (p = 0.246). The 2-year BM progression-free survival (PFS) was 26.9 %. Brain RT as first-line therapy failed to demonstrate a significant association with BM PFS (p = 0.643).
Conclusions
First-line brain RT failed to improve long-term survival in TKI-naïve EGFR mutant NSCLC patients with asymptomatic BM. Prospective studies are needed to validate these clinical findings.
doi:10.1186/s13014-015-0421-9
PMCID: PMC4490723  PMID: 26014133
Asymptomatic brain metastasis; Radiotherapy; Chemotherapy; Epidermal growth factor receptor mutation; Tyrosine kinase inhibitor
3.  Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China 
Pseudomonas aeruginosa is a major opportunistic pathogen in hospital-acquired infections and exhibits increasing antibiotic resistance. A rapid and sensitive molecular method for its detection in clinical samples is needed to guide therapeutic treatment and to control P. aeruginosa outbreaks. In this study, we established a polymerase spiral reaction (PSR) method for rapid detection of P. aeruginosa by targeting the toxA gene, which regulates exotoxin A synthesis. Real-time turbidity monitoring and a chromogenic visualization using hydroxynaphthol blue were used to assess the reaction. All 17 non- P. aeruginosa strains tested negative, indicating the high specificity of the PSR primers. The detection limit was 2.3 pg/μl within 60 min at isothermal temperature (65°C), 10-fold more sensitive than conventional PCR. Then, the PSR assay was applied to a clinical surveillance of P. aeruginosa in three top hospitals in Beijing, China. Of the 130 sputum samples collected from ICU patients with suspected multi-resistant infections, 37 P. aeruginosa isolates were identified from the positive samples. All clinical strains belonged to 10 different P. aeruginosa multilocus sequence typing groups and exhibited high resistance to carbapenems, cephalosporins, and aminoglycosides. Interestingly, of the 33 imipenem-resistant isolates, 30 (90.9%) had lost the outer membrane porin oprD gene. Moreover, isolate SY-95, containing multiple antibiotic resistance genes, possessed the ability to hydrolyze all antibiotics used in clinic and was susceptible only to polymyxin B. Our study showed the high level of antibiotic resistance and co-occurrence of resistance genes in the clinical strains, indicating a rapid and continuing evolution of P. aeruginosa. In conclusion, we developed a P. aeruginosa PSR assay, which could be a useful tool for clinical screening, especially in case of poor resources, or for point-of-care testing.
doi:10.3389/fmicb.2015.01100
PMCID: PMC4594016  PMID: 26500639
P. aeruginosa; PSR; toxA; rapid diagnosis; isothermal
4.  Scoring the correlation of genes by their shared properties using OScal, an improved overlap quantification model 
Scientific Reports  2015;5:10583.
Scoring the correlation between two genes by their shared properties is a common and basic work in biological study. A prospective way to score this correlation is to quantify the overlap between the two sets of homogeneous properties of the two genes. However the proper model has not been decided, here we focused on studying the quantification of overlap and proposed a more effective model after theoretically compared 7 existing models. We defined three characteristic parameters (d, R, r) of an overlap, which highlight essential differences among the 7 models and grouped them into two classes. Then the pros and cons of the two groups of model were fully examined by their solution space in the (d, R, r) coordinate system. Finally we proposed a new model called OScal (Overlap Score calculator), which was modified on Poisson distribution (one of 7 models) to avoid its disadvantages. Tested in assessing gene relation using different data, OScal performs better than existing models. In addition, OScal is a basic mathematic model, with very low computation cost and few restrictive conditions, so it can be used in a wide-range of research areas to measure the overlap or similarity of two entities.
doi:10.1038/srep10583
PMCID: PMC4445036  PMID: 26015386
5.  ERIC-PCR genotyping of Pseudomonas aeruginosa isolates from haemorrhagic pneumonia cases in mink 
Veterinary Record Open  2014;1(1):e000043.
Background
Pseudomonas aeruginosa is a significant pathogen of mink and the cause of haemorrhagic pneumonia, an acute fatal disease in farmed mink.
Results
Among 90 P. aeruginosa isolates from haemorrhagic pneumonia in mink from 16 farms in Shandong province, China, 43 genotypes were identified by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), with a diversity index of 0.96. The most prevalent ERIC-PCR types were type 18, found in 16 isolates, and type 39, found in 15 isolates. Four serotypes were detected, with serotype G (55.6 per cent) being the most frequent.
Conclusions
These results showed that there was a high degree of clonal diversity among mink P. aeruginosa clinical isolates in this study.
doi:10.1136/vropen-2014-000043
PMCID: PMC4562448  PMID: 26392878
Bacterial diseases; Clinical practice; Genetics
7.  Construction and Analysis of High-Density Linkage Map Using High-Throughput Sequencing Data 
PLoS ONE  2014;9(6):e98855.
Linkage maps enable the study of important biological questions. The construction of high-density linkage maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. However, the marker number explosion and genotyping errors from NGS data challenge the computational efficiency and linkage map quality of linkage study methods. Here we report the HighMap method for constructing high-density linkage maps from NGS data. HighMap employs an iterative ordering and error correction strategy based on a k-nearest neighbor algorithm and a Monte Carlo multipoint maximum likelihood algorithm. Simulation study shows HighMap can create a linkage map with three times as many markers as ordering-only methods while offering more accurate marker orders and stable genetic distances. Using HighMap, we constructed a common carp linkage map with 10,004 markers. The singleton rate was less than one-ninth of that generated by JoinMap4.1. Its total map distance was 5,908 cM, consistent with reports on low-density maps. HighMap is an efficient method for constructing high-density, high-quality linkage maps from high-throughput population NGS data. It will facilitate genome assembling, comparative genomic analysis, and QTL studies. HighMap is available at http://highmap.biomarker.com.cn/.
doi:10.1371/journal.pone.0098855
PMCID: PMC4048240  PMID: 24905985
8.  Complete Resequencing of 40 Genomes Reveals Domestication Events and Genes in Silkworm (Bombyx) 
Science (New York, N.Y.)  2009;326(5951):433-436.
A single–base pair resolution silkworm genetic variation map was constructed from 40 domesticated and wild silkworms, each sequenced to approximately threefold coverage, representing 99.88% of the genome. We identified ∼16 million single-nucleotide polymorphisms, many indels, and structural variations. We find that the domesticated silkworms are clearly genetically differentiated from the wild ones, but they have maintained large levels of genetic variability, suggesting a short domestication event involving a large number of individuals. We also identified signals of selection at 354 candidate genes that may have been important during domestication, some of which have enriched expression in the silk gland, midgut, and testis. These data add to our understanding of the domestication processes and may have applications in devising pest control strategies and advancing the use of silkworms as efficient bioreactors.
doi:10.1126/science.1176620
PMCID: PMC3951477  PMID: 19713493
9.  Three-dimensional conformal radiotherapy with concurrent chemotherapy for postoperative recurrence of esophageal squamous cell carcinoma: clinical efficacy and failure pattern 
Background
To assess the therapeutic outcome and failure pattern of three-dimensional conformal radiotherapy (3D-CRT)-based concurrent chemoradiotherapy (CCRT) for recurrence of esophageal squamous cell carcinoma (SCC) after radical surgery.
Methods
Treatment outcome and failure pattern were retrospectively evaluated in 83 patients with localized cervical and thoracic recurrences after radical surgery for thoracic esophageal SCC. All patients were treated with 3DCRT-based CCRT (median radiation dose 60 Gy), in which 39 received concurrent cisplatin plus 5-fluorouracil (PF), and 44 received concurrent docetaxel plus cisplatin (TP). Treatment response was evaluated at 1–3 months after CCRT.
Results
With a median follow-up of 34 months (range, 2–116 months), the 3-year overall survival (OS) of all the patients was 51.8% and the median OS time was 43.0 months. The overall tumor response rate was 75.9% (63/83), with a complete remission (CR) rate of 44.6% (37/83). In univariate analysis, tumor response after CCRT (p = 0.000), recurrence site (p = 0.028) and concurrent chemotherapy (p = 0.090) showed a trend favoring better OS. Multivariate analysis revealed that tumor response after CCRT (p = 0.000) and concurrent chemotherapy (p = 0.010) were independent predictors of OS. Forty-seven patients had progressive diseases after CCRT, 27 had local failure (27/47, 57.4%), 18 had distant metastasis (18/47, 38.3%) and 2 had both local and distant failures (2/47, 4.3%).
Conclusions
3DCRT-based CCRT is effective in postoperatively recurrent esophageal SCC. Patients that obtained complete remission after CCRT appeared to achieve long-term OS and might benefit from concurrent TP regimen. Local and distant failures remained high and prospective studies are needed to validate these factors.
doi:10.1186/1748-717X-8-241
PMCID: PMC3816598  PMID: 24139225
Esophageal squamous cell carcinoma; Postoperative recurrence; Concurrent chemoradiotherapy
10.  Identification and Characterization of Long Non-Coding RNAs Related to Mouse Embryonic Brain Development from Available Transcriptomic Data 
PLoS ONE  2013;8(8):e71152.
Long non-coding RNAs (lncRNAs) as a key group of non-coding RNAs have gained widely attention. Though lncRNAs have been functionally annotated and systematic explored in higher mammals, few are under systematical identification and annotation. Owing to the expression specificity, known lncRNAs expressed in embryonic brain tissues remain still limited. Considering a large number of lncRNAs are only transcribed in brain tissues, studies of lncRNAs in developmental brain are therefore of special interest. Here, publicly available RNA-sequencing (RNA-seq) data in embryonic brain are integrated to identify thousands of embryonic brain lncRNAs by a customized pipeline. A significant proportion of novel transcripts have not been annotated by available genomic resources. The putative embryonic brain lncRNAs are shorter in length, less spliced and show less conservation than known genes. The expression of putative lncRNAs is in one tenth on average of known coding genes, while comparable with known lncRNAs. From chromatin data, putative embryonic brain lncRNAs are associated with active chromatin marks, comparable with known lncRNAs. Embryonic brain expressed lncRNAs are also indicated to have expression though not evident in adult brain. Gene Ontology analysis of putative embryonic brain lncRNAs suggests that they are associated with brain development. The putative lncRNAs are shown to be related to possible cis-regulatory roles in imprinting even themselves are deemed to be imprinted lncRNAs. Re-analysis of one knockdown data suggests that four regulators are associated with lncRNAs. Taken together, the identification and systematic analysis of putative lncRNAs would provide novel insights into uncharacterized mouse non-coding regions and the relationships with mammalian embryonic brain development.
doi:10.1371/journal.pone.0071152
PMCID: PMC3743905  PMID: 23967161
11.  Epithelial Mesenchymal Transition Is Required for Acquisition of Anoikis Resistance and Metastatic Potential in Adenoid Cystic Carcinoma 
PLoS ONE  2012;7(12):e51549.
Human adenoid cystic carcinoma (ACC) is characterized by diffused invasion of the tumor into adjacent organs and early distant metastasis. Anoikis resistance and epithelial mesenchymal transition (EMT) are considered prerequisites for cancer cells to metastasize. Exploring the relationship between these processes and their underlying mechanism of action is a promising way to better understand ACC tumors. We initially established anoikis-resistant sublines of ACC cells; the variant cells revealed a mesenchymal phenotype through Slug-mediated EMT-like transformation and displayed enhanced metastatic potential both in vitro and in vivo. Suppression of EMT by knockdown of Slug significantly impaired anoikis resistance, migration, and invasion of the variant cells. With overexpression of Slug and Twist, we determined that induction of EMT in normal ACC cells could prevent anoikis, albeit partially. These findings strongly suggest that EMT is indispensable in anoikis resistance, at least in ACC cells. Furthermore, we found that the EGFR/PI3K/Akt pathway acts as the common regulator for EMT-like transformation and anoikis resistance, as confirmed by their specific inhibitors. Gefitinib and LY294003 restored the sensibilities of anoikis-resistant cells to anoikis and simultaneously impaired their metastatic potential. In addition, the results from our in vivo model of metastasis suggest that pretreatment with gefitinib promotes mouse survival by alleviating pulmonary metastasis. Most importantly, immunohistochemistry of human ACC specimens showed a correlation between the overexpression of Slug and EGFR staining. This study has demonstrated that Slug-mediated EMT-like transformation is required by human ACC cells to achieve anoikis resistance and their metastatic potential. Targeting the EGFR/PI3K/Akt pathway holds potential as a preventive strategy against distant metastasis of ACC.
doi:10.1371/journal.pone.0051549
PMCID: PMC3522696  PMID: 23272116
12.  Optimization of fatty alcohol biosynthesis pathway for selectively enhanced production of C12/14 and C16/18 fatty alcohols in engineered Escherichia coli 
Background
With the increasing stress from oil price and environmental pollution, aroused attention has been paid to the microbial production of chemicals from renewable sources. The C12/14 and C16/18 alcohols are important feedstocks for the production of surfactants and detergents, which are widely used in the most respected consumer detergents, cleaning products and personal care products worldwide. Though bioproduction of fatty alcohols has been carried out in engineered E. coli, several key problems have not been solved in earlier studies, such as the quite low production of C16/18 alcohol, the lack of optimization of the fatty alcohol biosynthesis pathway, and the uncharacterized performance of the engineered strains in scaled-up system.
Results
We improved the fatty alcohol production by systematically optimizing the fatty alcohol biosynthesis pathway, mainly targeting three key steps from fatty acyl-acyl carrier proteins (ACPs) to fatty alcohols, which are sequentially catalyzed by thioesterase, acyl-coenzyme A (CoA) synthase and fatty acyl-CoA reductase. By coexpression of thioesterase gene BTE, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene acr1, 210.1 mg/L C12/14 alcohol was obtained. A further optimization of expression level of BTE, fadD and acr1 increased the C12/14 alcohol production to 449.2 mg/L, accounting for 75.0% of the total fatty alcohol production (598.6 mg/L). In addition, by coexpression of thioesterase gene ‘tesA, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene FAR, 101.5 mg/L C16/18 alcohol was obtained, with C16/18 alcohol accounting for 89.2% of the total fatty alcohol production.
Conclusions
To our knowledge, this is the first report on selective production of C12/14 and C16/18 alcohols by microbial fermentation. This work achieved high-specificity production of both C12/14 and C16/18 alcohols. The encouraging 598.6 mg/L of fatty alcohols represents the highest titer reported so far. In addition, the 101.5 mg/L 89.2% C16/18 alcohol suggests an important breakthrough in C16/18 alcohol production. A more detailed optimization of the expression level of fatty alcohol biosynthesis pathway may contribute to a further improvement of fatty alcohol production.
doi:10.1186/1475-2859-11-65
PMCID: PMC3439321  PMID: 22607313
Fatty alcohol; Escherichia coli; Pathway optimization; Selective production; Fermentation
13.  First Report on Co-Occurrence Knockdown Resistance Mutations and Susceptibility to Beta-Cypermethrin in Anopheles sinensis from Jiangsu Province, China 
PLoS ONE  2012;7(1):e29242.
The increasing prevalence of insecticide resistance in Anopheles sinensis, a major vector of malaria in Jiangsu province in eastern China, threatens to compromise the successful use of insecticides in malaria control strategies. It is therefore vital to understand the insecticide resistance status of An. sinensis in the region. This study examined the nucleotide diversity of the para-sodium channel and knockdown resistance (kdr) in five field populations of adult An. sinensis mosquitoes collected in Jiangsu province, identifying the L1014F and L1014C substitutions for the first time. Competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) for resistance diagnosis were developed and validated. Comparing the results with direct sequencing revealed that the PCR-RFLP method was more sensitive and specific whereas the cPASA method was more convenient and suitable. The significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014C substitutions in the kdr gene provides a useful molecular marker for monitoring beta-cypermethrin resistance in natural populations of An. sinensis. Our results point to the L1014F substitution as the key mutation associated with beta-cypermethrin resistance. The high resistance and mutation frequency detected in the five populations also suggest cross-resistance with other pyrethroids may occur in An. sinensis, highlighting the need for further surveys to map insecticide resistance in China and the adoption of a rational management of insecticide application for resistance management and mosquito vector control.
doi:10.1371/journal.pone.0029242
PMCID: PMC3260143  PMID: 22272229
14.  Chinese Culture, Homosexuality Stigma, Social Support and Condom Use: A Path Analytic Model 
Stigma research and action  2011;1(1):27-35.
Purpose
The objective of this study was to examine the interrelationships among individualism, collectivism, homosexuality-related stigma, social support, and condom use among Chinese homosexual men.
Methods
A cross-sectional study using the respondent-driven sampling approach was conducted among 351 participants in Shenzhen, China. Path analytic modeling was used to analyze the interrelationships.
Results
The results of path analytic modeling document the following statistically significant associations with regard to homosexuality: (1) higher levels of vertical collectivism were associated with higher levels of public stigma [β (standardized coefficient) = 0.12] and self stigma (β = 0.12); (2) higher levels of vertical individualism were associated with higher levels self stigma (β = 0.18); (3) higher levels of horizontal individualism were associated with higher levels of public stigma (β = 0.12); (4) higher levels of self stigma were associated with higher levels of social support from sexual partners (β = 0.12); and (5) lower levels of public stigma were associated with consistent condom use (β = −0.19).
Conclusions
The findings enhance our understanding of how individualist and collectivist cultures influence the development of homosexuality-related stigma, which in turn may affect individuals’ decisions to engage in HIV-protective practices and seek social support. Accordingly, the development of HIV interventions for homosexual men in China should take the characteristics of Chinese culture into consideration.
doi:10.5463/sra.v1i1.16
PMCID: PMC3124705  PMID: 21731850
collectivism; HIV/AIDS; homosexuality; individualism
15.  CADgene: a comprehensive database for coronary artery disease genes 
Nucleic Acids Research  2010;39(Database issue):D991-D996.
Coronary artery disease (CAD) is a complex, multifactorial disease and a leading cause of mortality world wide. Over the past decades, great efforts have been made to elucidate the underlying genetic basis of CAD and massive data have been accumulated. To integrate these data together and to provide a useful resource for researchers, we developed the CADgene, a comprehensive database for CAD genes. We manually extracted CAD-related evidence for more than 300 candidate genes for CAD from over 1300 publications of genetic studies. We classified these candidate genes into 12 functional categories based on their roles in CAD. For each gene, we extracted detailed information from related studies (e.g. the size of case–control, population, SNP, odds ratio, P-value, etc.) and made useful annotations, which include general gene information, Gene Ontology annotations, KEGG pathways, protein–protein interactions and others. Besides the statistical number of studies for each gene, CADgene also provides tools to search and show the most frequently studied candidate genes. In addition, CADgene provides cumulative data from 11 publications of CAD-related genome-wide association studies. CADgene has a user-friendly web interface with multiple browse and search functions. It is freely available at http://www.bioguo.org/CADgene/.
doi:10.1093/nar/gkq1106
PMCID: PMC3013698  PMID: 21045063
16.  LPS-induced down-regulation of signal regulatory protein α contributes to innate immune activation in macrophages 
The Journal of Experimental Medicine  2007;204(11):2719-2731.
Activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) cascades after Toll-like receptor (TLR) stimulation contributes to innate immune responses. Signal regulatory protein (SIRP) α, a member of the SIRP family that is abundantly expressed in macrophages, has been implicated in regulating MAPK and NF-κB signaling pathways. In addition, SIRPα can negatively regulate the phagocytosis of host cells by macrophages, indicating an inhibitory role of SIRPα in innate immunity. We provide evidences that SIRPα is an essential endogenous regulator of the innate immune activation upon lipopolysaccharide (LPS) exposure. SIRPα expression was promptly reduced in macrophages after LPS stimulation. The decrease in SIRPα expression levels was required for initiation of LPS-induced innate immune responses because overexpression of SIRPα reduced macrophage responses to LPS. Knockdown of SIRPα caused prolonged activation of MAPKs and NF-κB pathways and augmented production of proinflammatory cytokines and type I interferon (IFN). Mice transferred with SIRPα-depleted macrophages were highly susceptible to endotoxic shock, developing multiple organ failure and exhibiting a remarkable increase in mortality. SIRPα may accomplish this mainly through its association and sequestration of the LPS signal transducer SHP-2. Thus, SIRPα functions as a biologically important modulator of TLR signaling and innate immunity.
doi:10.1084/jem.20062611
PMCID: PMC2118489  PMID: 17954568
17.  A novel MVA-mediated pathway for isoprene production in engineered E. coli 
BMC Biotechnology  2016;16:5.
Background
To deal with the increasingly severe energy crisis and environmental consequences, biofuels and biochemicals generated from renewable resources could serve as a promising alternative for replacing petroleum as a source of fuel and chemicals, among which isoprene (2-methyl-1,3-butadiene) in particular is of great significance in that it is an important platform chemical, which has been used in industrial production of synthetic rubber for tires and coatings or aviation fuel.
Results
We firstly introduced fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species into E. coli to directly convert MVA(mevalonate) into 3-methy-3-buten-1-ol. And then to transform 3-methy-3-buten-1-ol to isoprene, oleate hydratase (OhyAEM) from Elizabethkingia meningoseptica was overexpressed in E. coli. A novel biosynthetic pathway of isoprene in E. coli was established by co-expressing the heterologous mvaE gene encoding acetyl-CoA acetyltransferase/HMG-CoA reductase and mvaS gene encoding HMG-CoA synthase from Enterococcus faecalis, fatty acid decarboxylase (OleTJE) and oleate hydratase (OhyAEM). Furthermore, to enhance isoprene production, a further optimization of expression level of OleTJE, OhyAEM was carried out by using different promoters and copy numbers of plasmids. Thereafter, the fermentation process was also optimized to improve the production of isoprene. The final engineered strain, YJM33, bearing the innovative biosynthetic pathway of isoprene, was found to produce isoprene up to 2.2 mg/L and 620 mg/L under flask and fed-batch fermentation conditions, respectively.
Conclusions
In this study, by using metabolic engineering techniques, the novel MVA-mediated biosynthetic pathway of isoprene was successfully assembled in E. coli BL21(DE3) with the heterologous MVA upper pathway, OleTJE from Jeotgalicoccus species and OhyAEM from Elizabethkingia meningoseptica. Compared with traditional MVA pathway, the novel pathway is shortened by 3 steps. In addition, this is the first report on the reaction of converting MVA into 3-methy-3-buten-1-ol by fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species. In brief, this study provided an alternative method for isoprene biosynthesis, which is largely different from the well-developed MEP pathway or MVA pathway.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-016-0236-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12896-016-0236-2
PMCID: PMC4719670  PMID: 26786050
Isoprene; MVA-mediated pathway; OleTJE; OhyAEM; E. coli
18.  Decomposition of RNA methylome reveals co-methylation patterns induced by latent enzymatic regulators of epitranscriptome 
Molecular bioSystems  2014;11(1):262-274.
Biochemical modifications to mRNA, especially N6-methyladenosine (m6A) and 5-methylcytosine (m5C), are recently shown to be associated with crucial biological functions. Despite the intriguing advancements, little is known so far about the dynamic landscape of RNA methylome across different cell types and how the epitranscriptome is regulated at system level by enzymes, i.e., RNA methyltransferases and demethylases. To investigate this issue, a meta-analysis of m6A MeRIP-Seq datasets from 10 different experimental conditions (cell type/tissue or treatment) are collected, and the combinatorial epitranscriptome, which consists of 42758 m6A sites, is extracted and divided into 3 clusters, in which the methylation sites are likely to be hyper- or hypo-methylated simultaneously (or co-methylated), indicating the sharing of a common methylation regulator. Four different clustering approaches are used, including, K-means, hierarchical clustering (HC), Bayesian factor regression model (BFRM) and nonnegative matrix factorization (NMF) to unveil the co-methylation patterns.
To validate whether the patterns are corresponding to enzymatic regulators, i.e., RNA methyltransferases or demethylases, the target sites of a known m6A regulator, fat mass and obesity-associated protein (FTO), are identified from an independent mouse MeRIP-Seq dataset and lifted to human. Our study shows that, 3 out of the 4 clustering approaches used can successfully identify a group of methylation sites overlapping with FTO target sites at significance level 0.05 (after multiple hypothesis adjustment), among which, the result of NMF is the most significant (p-value 2.81e-06). We defined a new approach evaluating the consistency between two clustering results and shows that clustering results of different methods are highly correlated indicating strongly the existence of co-methylation patterns. Consistent with recent studies, a number of cancer and neuronal disease-related bimolecular functions are enriched in the identified clusters, which are biological functions can be regulated at epitranscriptional level, indicating the pharmaceutical prospect of RNA N6-methyladenosine-related studies.
This result successfully reveals the linkage between the global RNA co-methylation patterns embedded in the epitranscriptomic data under multiple experimental conditions and the latent enzymatic regulators, suggesting a promising direction towards more comprehensive understanding of the epitranscriptome.
doi:10.1039/c4mb00604f
PMCID: PMC4253022  PMID: 25370990
19.  Autophagy induction contributes to the resistance to methotrexate treatment in rheumatoid arthritis fibroblast-like synovial cells through high mobility group box chromosomal protein 1 
Background
Rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) show resistance to methotrexate (MTX) treatment. To better understand the mechanisms of this resistance, RA-FLS and osteoarthritis fibroblast-like synovial cells (OA-FLS) were isolated and exposed to MTX. We analyzed the autophagy induced by MTX in vitro and its relationship to apoptosis.
Methods
Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was detected by flow cytometry and Western blot analysis. Autophagy was determined by transmission electron microscopy as well as Western blot analysis. The expression levels of Beclin-1, LC3, Akt, p-Akt, mammalian target of rapamycin (mTOR), p-mTOR, high mobility group box chromosomal protein 1 (HMGB1), and an 85 kDa caspase cleaved fragment of poly(ADP-ribose) polymerase were measured by Western blotting.
Results
MTX-induced apoptosis was increased in OA-FLS compared with RA-FLS. However, MTX stimulated the autophagy response in RA-FLS by inducing autophagosome formation, but not in OA-FLS. In RA-FLS, transfection with Beclin-1 small interfering RNA inhibited autophagy and increased susceptibility to MTX, which induces cell death. MTX upregulated autophagy through its ability to enhance the expression of HMGB1 and Beclin-1 rather than through the Akt/mTOR pathway.
Conclusions
Autophagy induction contributes to resistance to MTX treatment in fibroblasts from patients with rheumatoid arthritis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0892-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-015-0892-y
PMCID: PMC4718027  PMID: 26702616
Rheumatoid arthritis; Fibroblast-like synovial cells; Methotrexate; Autophagy; Apoptosis
20.  The association between osteopontin and survival in non-small-cell lung cancer patients: a meta-analysis of 13 cohorts 
OncoTargets and therapy  2015;8:3513-3521.
In the last decade, osteopontin (OPN) was identified as one of the important proteins that promote the metastasis of tumor. However, the association between OPN overexpression and clinical outcome of non-small-cell lung cancer (NSCLC) was unclear. The purpose of this study is to investigate the role of OPN in NSCLC patients. A total of 13 studies are included to explore the relationship between the OPN elevation and the overall survival (OS) and disease-free survival (DFS) in NSCLC patients. We searched for related articles in PubMed, Web of Science, Google Scholar, and Cochrane Library databases, which were published before January 31, 2015. Hazard ratio (HR), odds ratio (OR), and 95% confidence interval (CI) in the high OPN expression group compared with the low OPN expression group were calculated and analyzed. Primary results were summarized by using a fixed-effects model or a random-effects model. The stratified analyses in subgroups were also performed. Thirteen cohort studies, which involved 1,630 patients, were included. Subgroup analyses were performed by area and test method of OPN. We found that OPN was significantly associated with poor OS (HR =2.20, 95% CI 1.71–2.83, P<0.001) and DFS (HR =2.11, 95% CI 1.62–2.74, P<0.001) in NSCLC patients. OPN overexpression tended to be associated with the presence of advanced tumor TNM stage (III and IV) (OR =2.57, 95% CI 1.61–4.11, P<0.001). The Egger’s test suggested that there was no publication bias in OS studies (P=0.062) and DFS studies (P=0.740). These data indicate that OPN seems to have a significant predictive potential in estimating survival in NSCLC.
doi:10.2147/OTT.S94082
PMCID: PMC4664515  PMID: 26648743
metastasis; overall survival; disease-free survival; tumor stage
21.  Growth and metabolism of Beauveria bassiana spores and mycelia 
BMC Microbiology  2015;15:267.
Background
Fungi are ubiquitous in nature and have evolved over time to colonize a wide range of ecosystems including pest control. To date, most research has focused on the hypocrealean genera Beauveria bassiana, which is a typical filamentous fungus with a high potential for insect control. The morphology and components of fungi are important during the spores germination and outgrow to mycelia. However, to the best of our knowledge, there is no report on the morphology and components of B. bassiana spores and mycelia. In the work, the growth and metabolism of Beauveria bassiana spores and mycelia were studied. High performance liquid chromatography-mass spectrometry (HPLC-MS) was employed to study the metabolism of B. bassiana spores and mycelia. Principal component analysis (PCA) based on HPLC-MS was conducted to study the different components of the spores and mycelia of the fungus. Metabolic network was established based on HPLC-MS and KEGG database.
Results
Through Gompertz model based on macroscopic and microscopic techniques, spore elongation length was found to increase exponentially until approximately 23.1 h after cultivation, and then growth became linear. In the metabolic network, the decrease of glyoxylate, pyruvate, fumarate, alanine, succinate, oxaloacetate, dihydrothymine, ribulose, acetylcarnitine, fructose-1, 6-bisphosphate, mycosporin glutamicol, and the increase of betaine, carnitine, ergothioneine, sphingosine, dimethyl guanosine, glycerophospholipids, and in spores indicated that the change of the metabolin can keep spores in inactive conditions, protect spores against harmful effects and survive longer.
Conclusions
Analysis of the metabolic pathway in which these components participate can reveal the metabolic difference between spores and mycelia, which provide the tools for understand and control the process of of spores germination and outgrow to mycelia.
doi:10.1186/s12866-015-0592-4
PMCID: PMC4652391  PMID: 26581712
Beauveria bassiana; Gompertz model; PCA; HPLC-MC; Metabolism
22.  Expression of MICA in oral squamous carcinoma cells and its effect on NK cells 
Objective: This study aims to observe the expression of MHC-class I chain related protein A (MICA) in oral squamous carcinoma cell and explore its effects on NK cells. Methods: Normal oral mucosa epithelial cell line NOK and oral squamous carcinoma cell lines OEC-M1, SAS and SCC-25 were used in this study. MICA expression in the cells was detected by western blotting and RT-PCR methods, sMICA was detected by ELISA method. The cells were transfected by pEGFP-MICA and pEGFP-NC respectively using Lipofectamine 2000 kit. The transfected cells were co-cultured with NK92 cells. Killing activity of NK92 cells was detected by LDH release method and NKG2D was detected by Flow cytometry. ADAM10 and ADAM17 were detected by ELISA method. Results: MICA expression in OEC-M1, SAS and SCC-25 cells were lower than that of NOK cells (P<0.01), sMICA levels in OEC-M1, SAS and SCC-25 cells were higher than that of NOK cells (P<0.01). Over-expression of MICA in SCC-25 cells could significantly increase the killing activity of NK92 cells (P<0.01), up-regulate NKG2D (P<0.01) and decrease ADAM10 and ADAM17 contents (P<0.01). Conclusions: MICA expressed lowly in oral squamous cell carcinoma cells, over-expression of MICA could significantly increase the killing activity of NK92 cells, which could be related with the regulation of ADAM.
PMCID: PMC4694322  PMID: 26770422
MHC-class I chain related protein A (MICA); oral squamous carcinoma cell lines; NK cells
23.  Applying Side-chain Flexibility in Motifs for Protein Docking 
Genomics Insights  2015;8:1-10.
Conventional rigid docking algorithms have been unsatisfactory in their computational results, largely due to the fact that protein structures are flexible in live environments. In response, we propose to introduce the side-chain flexibility in protein motif into the docking. First, the Morse theory is applied to curvature labeling and surface region growing, for segmentation of the protein surface into smaller patches. Then, the protein is described by an ensemble of conformations that incorporate the flexibility of interface side chains and are sampled using rotamers. Next, a 3D rotation invariant shape descriptor is proposed to deal with the flexible motifs and surface patches; thus, pairwise complementarity matching is needed only between the convex patches of ligand and the concave patches of receptor. The iterative closest point (ICP) algorithm is implemented for geometric alignment of the two 3D protein surface patches. Compared with the fast Fourier transform-based global geometric matching algorithm and other methods, our FlexDock system generates much less false-positive docking results, which benefits identification of the complementary candidates. Our computational experiments show the advantages of the proposed flexible docking algorithm over its counterparts.
doi:10.4137/GEI.S29821
PMCID: PMC4610727  PMID: 26508871
protein docking; motif; side-chain; flexibility; spherical harmonic descriptor
24.  Preparation, characterization, in vivo pharmacokinetics, and biodistribution of polymeric micellar dimethoxycurcumin for tumor targeting 
Dimethoxycurcumin (DMC) is an analog of curcumin with superior efficacy in various disease models. Currently, drug delivery system research on DMC is very limited, and it has become a huge challenge to realize further developments and clinical applications. In the present study, a kind of amphiphilic block copolymer, N-t-butoxycarbonyl-phenylalanine terminated monomethoxyl poly (ethylene glycol)-b-poly (ε-caprolactone), or mPEG-PCL-Phe(Boc), was prepared from monomethoxyl poly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL) with its hydroxyl terminal chemically converted into N-t-butoxycarbonyl-phenylalanine (Boc-Phe). This copolymer was determined to have a fairly low critical micelle concentration (2.56×10−3 mg/mL) and passive targeting potential to tumor tissue, and thus was applied to develop a polymeric micellar formulation of DMC for the first time. The DMC-loaded micelles prepared by thin-film hydration method had typical shell–core structure, with an average particle size of 17.9±0.4 nm and a polydispersity index of 0.045±0.011. The drug loading capacity and entrapment efficiency were 9.94%±0.15% and 97.22%±0.18%, respectively, indicating a high-affinity interaction between DMC and the copolymer. At a concentration of 2 mg/mL, the reconstituted micelle solution could be maintained for at least 10 days at room temperature, and displayed a low initial burst release followed by a sustained release in vitro. Pharmacokinetic study in rats revealed that in vivo drug exposure of DMC was significantly increased and prolonged by intravenously administering DMC-loaded micelles when compared with the same dose of free DMC dissolved in dimethyl sulfoxide. Furthermore, in vivo distribution results from tumor-bearing nude mice demonstrated that this micellar formulation significantly changed the biodistribution profile of DMC and increased drug accumulation in tumors. Therefore, the polymeric micellar formulation of DMC, based on the amphiphilic block copolymer, mPEG-PCL-Phe(Boc), could provide a desirable method for delivering DMC, especially for applications in cancer therapy.
doi:10.2147/IJN.S91961
PMCID: PMC4605235  PMID: 26504386
dimethoxycurcumin; polymeric micelles; characterization; pharmacokinetic profile; biodistribution; tumor targeting
25.  Multidetector Computed Tomography Features in Differentiating Exophytic Renal Angiomyolipoma from Retroperitoneal Liposarcoma 
Medicine  2015;94(37):e1521.
Abstract
This study aims to evaluate the multidetector computed tomography (CT) imaging features in differentiating exophytic renal angiomyolipoma (AML) from retroperitoneal liposarcoma.
We retrospectively enrolled 42 patients with confirmed exophytic renal AML (31 patients) or retroperitoneal liposarcoma (11 patients) during 8 years period to assess: renal parenchymal defect at site of tumor contact, supply from branches of renal artery, tumoral vessel extending through the renal parenchyma, dilated intratumoral vessels, hemorrhage, non–fat-containing intratumoral nodules with postcontrast enhancement, calcification, renal sinus enlargement, anterior displacement of kidneys, and other associated AML.
Renal parenchymal defect, renal arterial blood supply, tumoral vessel through the renal parenchyma, dilated intratumoral vessels, intratumoral/perirenal hemorrhage, renal sinus enlargement, and associated AML were seen only or mainly in exophytic renal AML (all P value < 0.05); however, non–fat-attenuating enhancing intratumoral nodules, intratumoral calcification, and anterior displacement of the kidney were more common in liposarcoma (all P value < 0.05).
AMLs reveal renal parenchymal defect at the site of tumor contact, supply from renal artery, tumoral vessel extending through the renal parenchyma, dilated intratumoral vessels, intratumoral and/or perirenal hemorrhage, renal sinus enlargement, and associated AML. Non–fat-attenuating enhancing intratumoral nodules, intratumoral calcifications, and anterior displacement of kidney were more commonly seen in liposarcoma.
doi:10.1097/MD.0000000000001521
PMCID: PMC4635812  PMID: 26376398

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