Phytogenic compounds with anti-oxidant and anti-inflammatory properties, such as ginsenoside metabolite compound K (CK) or berberine (BBR), are currently discussed as promising complementary agents in the prevention and treatment of cancer and inflammation. The latest study showed that ginsenoside Rb1 and its metabolites could inhibit TNBS-induced colitis injury. However, the functional mechanisms of anti-inflammation effects of ginsenoside, particularly its metabolite CK are still not clear. Here, using dextran sulfate sodium (DSS)-induced colitis in mice, clinical parameters, intestinal integrity, pro-inflammatory cytokines production, and signaling pathways in colonic tissues were determined. In mild and sever colitis mice, CK and BBR (as a positive agent) alleviated colitis histopathology injury, ameliorated myeloperoxidase (MPO) activity, reduced pro-inflammatory cytokines production, such as, IL-6, IL-1β, TNF-α, and increased anti-inflammatory cytokine IL-10 production in both mice colon tissues and blood. Nevertheless, the results revealed that CK and BBR inhibited NF-κB p65 nuclear translocation, downregulated p-IκBα and upregulated IκBα, indicating that CK, as well as BBR, suppressed the activation of the NF-κB pathway in the progression of colitis with immunofluorescence, immunohistochemical and western blotting analysis. Furthermore, CK inhibited pro-inflammatory cytokines production in LPS-activated macrophages via down-regulation of NF-κB signaling pathway. Taken together, our results not only reveal that CK promotes the recovery of the progression of colitis and inhibits the inflammatory responses by suppressing NF-κB activation, but also suggest that CK downregulates intestinal inflammation through regulating the activation of macrophages and pro-inflammatory cytokines production.
Although it is clear that there are short-term effects of sodium intake on blood pressure, little is known about the most relevant timing of sodium exposure for the onset of hypertension. This question can only be addressed in cohorts with repeated measures of sodium intake.
Using up to 7 measures of dietary sodium intake and blood pressure between 1991 and 2009, we compared baseline, the mean of all measures, and the most recent sodium intake in association with incident hypertension, in 6578 adults enrolled in the China Health and Nutrition Survey aged 18 to 65 free of hypertension at baseline. We used survival methods that account for the interval-censored nature of this study, and inverse probability weights to generate adjusted survival curves and time-specific cumulative risk differences; hazard ratios were also estimated.
For mean and most recent measures, the probability of hypertension-free survival was the lowest among those in the highest intake sodium group compared to all other intake groups across the entire follow-up. In addition, the most recent sodium intake measure had a positive dose-response association with incident hypertension [Risk Difference at 11 years of follow-up = 0.04 (95%CI −0.01, 0.09), 0.06 (0.00, 0.13), 0.18 (0.12, 0.24) and 0.20 (0.12, 0.27) for the second to fifth sodium intake groups compared to the lowest group respectively]. Baseline sodium intake was not associated with incident hypertension.
These results suggest caution when using baseline sodium intake measures with long-term follow up.
China; sodium intake; incident hypertension; interval-censored; adjusted survival curves
Poly (ADP-ribose) polymerase-1 (PARP-1) plays critical roles in the detection and repair of damaged DNA, as well as cell proliferation and death. Numerous studies have examined the associations between PARP1 Val762Ala (rs1136410 T>C) polymorphism and cancer susceptibility; nevertheless, the findings from different research groups remain controversial.
We searched literatures from MEDLINE, EMBASE and CBM pertaining to such associations, and then calculated pooled odds ratio (OR) and 95% confidence interval (CI) by using random-effects model. The false-positive report probability (FPRP) analysis was used to confirm the validity of significant findings. Moreover, potential effects of rs1136410 variants on PARP1 mRNA expression were analyzed for three ethnicities by combining data from HapMap (genotype) and SNPexp (mRNA expression).
The final meta-analysis incorporated 43 studies, consisting of 17,351 cases and 22,401 controls. Overall, our results did not suggest significant associations between Ala variant (Ala/Ala or Ala/Val genotype) and cancer risk. However, further stratification analysis showed significantly increased risk for gastric cancer (Ala/Ala vs. Val/Val: OR = 1.56, 95% CI = 1.01–2.42, Ala/Val vs. Val/Val: OR = 1.34, 95% CI = 1.14–1.58, dominant model: OR = 1.41, 95% CI = 1.21–1.65 and Ala vs. Val: OR = 1.29, 95% CI = 1.07–1.55). On the contrary, decreased risk for brain tumor (Ala/Val vs. Val/Val: OR = 0.77, 95% CI = 0.68–0.87, dominant model: OR = 0.77, 95% CI = 0.68–0.87 and Ala vs. Val: OR = 0.82, 95% CI = 0.74–0.91). Additionally, we found that the Ala carriers had a significantly increased risk in all models for Asians. Our mRNA expression data provided further biological evidence to consolidate this finding.
Despite some limitations, this meta-analysis found evidence for an association between the PAPR1 Val762Ala and cancer susceptibility within gastric cancer, brain tumor and Asian subgroups.
During our efforts to characterize regulatory properties of human liver pyruvate kinase (L-PYK), we have noted that the affinity of the protein for PEP becomes reduced after several days post cell lysis. A 1.8Å crystallographic structure of L-PYK with the S12D mimic of phosphorylation indicates that Cys436 is oxidized, the first potential insight into explaining the effect of “aging”. Interestingly, the oxidation is only to sulfenic acid despite the two-week time of crystal growth. Mutagenesis confirms that the 436 side-chain is energetically coupled to PEP binding. Mass spectrometry confirms that the oxidation is present in solution and is not an artifact due to X-ray exposure. Exposure to the L-PYK mutations to H2O2 also confirms that PEP affinity is sensitive to the nature of the side-chain at position 436. A 1.95Å structure of the C436M mutation of L-PYK, the only mutation at position 436 identified to strengthen PEP affinity, revealed that the methionine substitution results in the ordering of several N-terminal residues that have not been ordered in previous structures. This result allowed a speculation that oxidation of Cys436 and phosphorylation of the N-terminus at Ser12 may function through a similar mechanism, namely the interruption of an activating interaction between the non-phosphorylated N-terminus with the non-oxidized main body of the protein. Mutant cycles were used to provide evidence that mutations of Cys436 are energetically synergistic with N-terminal modifications, a result that is consistent with phosphorylation of the N-terminus and oxidation of Cys436 functioning through mechanisms with common features. Alanine-scanning mutagenesis was used to confirm that the newly ordered N-terminal residues were important to the regulation of enzyme function by the N-terminus of the enzyme (i.e. not an artifact due to the introduced methionine substitution) and to further define which residues in the N-terminus are energetically coupled to PEP affinity. Collectively, these studies indicate energetic coupling (and potentially mechanistic similarities) between the oxidation of Cys436 and phosphorylation of Ser12 in the N-terminus of L-PYK.
The fish gill, as one of the mucosal barriers, plays an important role in mucosal immune response. The fish swimbladder functions for regulating buoyancy. The fish swimbladder has long been postulated as a homologous organ of the tetrapod lung, but the molecular evidence is scarce. In order to provide new information that is complementary to gill immune genes, initiate new research directions concerning the genetic basis of the gill immune response and understand the molecular function of swimbladder as well as its relationship with lungs, transcriptome analysis of the fugu Takifugu rubripes gill and swimbladder was carried out by RNA-Seq. Approximately 55,061,524 and 44,736,850 raw sequence reads from gill and swimbladder were generated, respectively. Gene ontology (GO) and KEGG pathway analysis revealed diverse biological functions and processes. Transcriptome comparison between gill and swimbladder resulted in 3,790 differentially expressed genes, of which 1,520 were up-regulated in the swimbladder while 2,270 were down-regulated. In addition, 406 up regulated isoforms and 296 down regulated isoforms were observed in swimbladder in comparison to gill. By the gene enrichment analysis, the three immune-related pathways and 32 immune-related genes in gill were identified. In swimbladder, five pathways including 43 swimbladder-enriched genes were identified. This work should set the foundation for studying immune-related genes for the mucosal immunity and provide genomic resources to study the relatedness of the fish swimbladder and mammalian lung.
Previous studies have linked work, home production, travel activities, and inactivity with weight and health outcomes. However, these focused on average physical activity over time rather than changes in physical activity and associated socio-demographic and economic factors and urbanicity. Using the 1991–2011 China Health and Nutrition Survey data, we estimated the metabolic equivalent of task hours per week for individuals in occupational, domestic, travel, and active leisure domains and sedentary hours per week. We present the distributions among adult men and women (aged 18–60) and use quantile regression models to explore factors associated with these trends. Trend analyses on the distribution of physical activity show declines along the whole distribution of occupational physical activity for men and women and domestic physical activity for women in China. These patterns remain consistent after adjusting for individual- and household-level factors. Controlling for urbanicity mitigated the decrease in occupational physical activity, particularly for men, but not the decrease in domestic physical activity. Given China's rapid urbanization and its association with occupational physical activity declines and the strong time trend in domestic physical activity, there is a need to invest in interventions and policies that promote physical activity during leisure and travel times.
physical activity; adults; quantile regression; occupation; domestic; China
Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.
MiRNAs are small, noncoding RNA molecules that act as posttranscriptional regulators of gene expression and function as important regulators in cancer-related processes. The miR-19a is overexpressed in various cancers and has been causally related to cellular proliferation and growth. To determine whether miR-19a plays a role in laryngeal squamous cell carcinoma (LSCC), we used quantitative real time PCR to detect miR-19a expression in LSCC tissues. We found that miR-19a is overexpressed in LSCC and correlated with neck nodal metastasis, poor differentiation and advanced stage. Statistical analysis suggests that higher level of miR-19a was associated with reduced overall survival. In vitro functional study showed that inhibition of miR-19a by antisense oligonucleotides (ASO) led to apoptosis and reduction of cell proliferation in LSCC cells. Furthermore, growth of LSCC xenograft tumors was significantly suppressed by repeated injection of ASO-miR-19a lentivirus. The TUNEL stain and transmission electron microscopy also detected increased apoptotic cells in ASO-miR-19a treated LSCC xenografts. In addition, both realtime PCR and western blot showed ASO-miR-19a can upregulate TIMP-2 expression and this suggests miR-19a is related with TIMP-2 pathway in LSCC cells. Taken together, these data suggest that miR-19a plays an oncogenic role in the progression of LSCC, and may serve as a biomarker or therapeutic target for patients with LSCC.
miR-19a; LSCC; TIMP-2; apoptosis
Recent US work identifies “metabolically healthy overweight” and “metabolically at risk normal weight” individuals. Less is known for modernizing countries with recent increased obesity. Fasting blood samples, anthropometry and blood pressure from 8,233 adults aged 18–98 in the 2009 nationwide China Health and Nutrition Survey, were used to determine prevalence of overweight (Asian cut point, BMI≥23 kg/m2) and five risk factors [pre-diabetes/diabetes (HbA1c≥5.7%) inflammation (hsCRP ≥3 mg/L), pre-hypertension/hypertension (SBP/DBP≥130/85 mmHg), high triglycerides (≥150 mg/dL), low high-density lipoprotein cholesterol (<40 (men)/<50 mg/dL (women)]. Sex-stratified, logistic and multinomial logistic regression models estimated concurrent obesity and cardiometabolic risk, with and without abdominal obesity, adjusting for age, smoking, alcohol consumption, physical activity, urbanicity, and income. Irrespective of urbanicity, 78.3% of the sample had ≥1 elevated cardiometabolic risk factor (normal weight: 33.2% had ≥1 elevated risk factor; overweight: 5.7% had none). At age 18–30, 47.4% had no elevated risk factors, which dropped to 6% by age 70, largely due to age-related increase in hypertension risk (18–30 years: 11%, >70 years: 73%). Abdominal obesity was highly predictive of metabolic risk, irrespective of overweight (e.g., “metabolically at risk overweight” relative to “metabolically healthy normal weight” [men: Relative Risk Ratio (RRR) =39.06; 95% Confidence Interval (CI): 23.47, 65.00; women: RRR=22.26; 95% CI: 17.49, 28.33]). To conclude, a large proportion of Chinese adults have metabolic abnormalities. High hypertension risk with age, irrespective of obesity underlies the low prevalence of metabolically healthy overweight. Screening for cardiometabolic-related outcomes dependent upon overweight will likely miss a large portion of the Chinese at-risk population.
Both transcriptional subtype and signaling network analyses have proved useful in cancer genomics research. However, these two approaches are usually applied in isolation in existing studies. We reason that deciphering genomic alterations based on cancer transcriptional subtypes may help reveal subtype-specific driver networks and provide insights for the development of personalized therapeutic strategies. In this study, we defined transcriptional subtypes for colorectal cancer (CRC) and identified driver networks/pathways for each subtype. Applying consensus clustering to a patient cohort with 1173 samples identified three transcriptional subtypes, which were validated in an independent cohort with 485 samples. The three subtypes were characterized by different transcriptional programs related to normal adult colon, early colon embryonic development, and epithelial mesenchymal transition, respectively. They also showed statistically different clinical outcomes. For each subtype, we mapped somatic mutation and copy number variation data onto an integrated signaling network and identified subtype-specific driver networks using a random walk-based strategy. We found that genomic alterations in the Wnt signaling pathway were common among all three subtypes; however, unique combinations of pathway alterations including Wnt, VEGF and Notch drove distinct molecular and clinical phenotypes in different CRC subtypes. Our results provide a coherent and integrated picture of human CRC that links genomic alterations to molecular and clinical consequences, and which provides insights for the development of personalized therapeutic strategies for different CRC subtypes.
The ferritin is an important participant of iron-storage but its regulation and related factors were not well defined. The present objective was to explore the potential association between serum ferritin levels and sex hormones.
1999 Chinese men in the Fangchenggang Area Male Health and Examination Survey (FAMHES) were recruited in this cross-sectional study. Levels of serum ferritin, total testosterone (free testosterone was calculated from the total one), estradiol and sex hormone-binding protein were detected in venous blood samples. The effects of age, BMI, smoking as well as alcohol consumption were analyzed on ferritin levels, respectively, and then the Pearson’s correlation analysis was used to evaluate the association between ferritin levels and sex hormones adjusting for the above factors.
The age, BMI and alcohol consumption significantly affected serum ferritin levels, but there was no significant difference between smokers and nonsmokers. Ferritin levels were significantly and negatively associated with total testosterone (R = −0.205, P< 0.001), sex hormone-binding protein (R = −0.161, P<0.001) and free testosterone (R = −0.097, P<0.001). After age and alcohol consumption were adjusted, the above associations were still significant (R = −0.200, −0.181 and −0.083, respectively, all P<0.001). However, there was only borderline negative association between ferritin levels and estradiol (adjusted R = −0.039, P = 0.083).
The large scale of epidemic results showed the significantly negative associations between serum ferritin levels and sex hormones, which may provide more clues to explore the potential regulation and biological mechanism of ferritin.
In the study of complex diseases, a major challenge is disease heterogeneity, where the dysregulation of different pathways often lead to similar disease phenotypes. As a result, a given pathway could be differentially expressed with respect to controls for some patients, but not for others. Therefore, to develop successful personalized treatment regime, in addition to identifying disease relevant pathways for the entire patient group, it’s also important to test if a particular pathway is dysregulated for an individual patient. To this end, we compare pathway gene expression profile for a particular individual in the patient group to the “norm” (or standard) established by a group of control patients. We studied statistical analysis of patient-specific pathway activities under the mixed models framework. Using gene expression dataset with realistic correlation patterns, we showed the proposed hypothesis testing procedure had false positive rate (type I error) as expected. In addition, we illustrated the proposed methodology using a Type 2 Diabetes dataset. Our results showed a previously diabetes associated pathway was only differentially expressed (relative to the control group) in less than 30% of the diabetes patients, which provided an explanation for the moderate group level statistical significance seen in a previous study. This result also suggested targeting this particular pathway would likely be beneficial for only 30% of the patients. In addition to the case-control study we have illustrated, this model can be easily extended to handle more complex designs with additional covariates and multiple sources of variations. Moreover, the proposed model operates within a well-established statistical framework and can be implemented in common statistical packages.
Microarray; Gene expression; Mixed models; Pathway analysis; Gene set analysis; Statistical significance
Summary: Database search is the most widely used approach for peptide and protein identification in mass spectrometry-based proteomics studies. Our previous study showed that sample-specific protein databases derived from RNA-Seq data can better approximate the real protein pools in the samples and thus improve protein identification. More importantly, single nucleotide variations, short insertion and deletions and novel junctions identified from RNA-Seq data make protein database more complete and sample-specific. Here, we report an R package customProDB that enables the easy generation of customized databases from RNA-Seq data for proteomics search. This work bridges genomics and proteomics studies and facilitates cross-omics data integration.
Availability and implementation:
customProDB and related documents are freely available at http://bioconductor.org/packages/2.13/bioc/html/customProDB.html.
Supplementary data are available at Bioinformatics online.
Both Ang-2 and VEGFR-3 are major regulators of angiogenesis and lymphangiogenesis, respectively, and thus may affect prognosis of OSCC. We sought to determine the associations between Ang-2 and VEGFR-3 expression and survival of OSCC.
Ang-2 and VEGFR-3 expression was determined immunohistochemically in tumor tissues from 112 patients with OSCC; OSCC-adjacent noncancerous oral tissue from 85 OSCC patients; and normal oral mucosa from 37 cancer-free individuals. A log-rank test and Cox proportional hazard models were used to compare survival among different groups with expression of Ang-2 and VEGFR-3.
Ang-2 and VEGFR-3 expression was upregulated in OSCC compared to nontumor tissue (all P<0.05). High Ang-2 expression positively correlated with microvessel density (MVD) (P<0.01), and high VEGFR-3 expression positively correlated with lymph node metastasis (P<0.01) and lymphatic vessel density (LVD) (P<0.01). The patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly worse survival than in patients with low expression of Ang-2 or any other co-expression status (all P<0.05), respectively. Furthermore, multivariable analysis showed that patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly increased risk of death compared with those with low expression of Ang-2 or any other co-expression status (HR, 2.7, 95% CI, 1.1–6.2 and 5.0, 1.3–15.4, respectively).
These results suggest that increased expression in tumors of Ang-2 may individually, or in combination with VEGFR-3, predict poor prognosis of OSCC.
The standard shotgun proteomics data analysis strategy relies on searching MS/MS spectra against a context-independent protein sequence database derived from the complete genome sequence of an organism. Because transcriptome sequence analysis (RNA-Seq) promises an unbiased and comprehensive picture of the transcriptome, we reason that a sample-specific protein database derived from RNA-Seq data can better approximate the real protein pool in the sample and thus improve protein identification. In this study, we have developed a two-step strategy for building sample-specific protein databases from RNA-Seq data. First, the database size is reduced by eliminating unexpressed or lowly expressed genes according to transcript quantification. Secondly, high-quality nonsynonymous coding single nucleotide variations (SNVs) are identified based on RNA-Seq data, and corresponding protein variants are added to the database. Using RNA-Seq and shotgun proteomics data from two colorectal cancer cell lines SW480 and RKO, we demonstrated that customized protein sequence databases could significantly increase the sensitivity of peptide identification, reduce ambiguity in protein assembly, and enable the detection of known and novel peptide variants. Thus, sample-specific databases from RNA-Seq data can enable more sensitive and comprehensive protein discovery in shotgun proteomics studies.
RNA-Seq; Shotgun Proteomics; Peptide Identification; Single Nucleotide Variations; data integration
Angiogenesis, the recruitment and re-configuration of pre-existing vasculature, is essential for tumor growth and metastasis. Increased tumor vascularization often correlates with poor patient outcomes in a broad spectrum of carcinomas. We identified four jointed box 1 (FJX1) as a candidate regulator of tumor angiogenesis in colorectal cancer. FJX1 mRNA and protein are upregulated in human colorectal tumor epithelium as compared with normal epithelium and colorectal adenomas, and high expression of FJX1 is associated with poor patient prognosis. FJX1 mRNA expression in colorectal cancer tissues is significantly correlated with changes in known angiogenesis genes. Augmented expression of FJX1 in colon cancer cells promotes growth of xenografts in athymic mice and is associated with increased tumor cell proliferation and vascularization. Furthermore, FJX1 null mice develop significantly fewer colonic polyps than wild-type littermates after combined dextran sodium sulfate (DSS) and azoxymethane (AOM) treatment. In vitro, conditioned media from FJX1 expressing cells promoted endothelial cell capillary tube formation in a HIF1-α dependent manner. Taken together our results support the conclusion that FJX1 is a novel regulator of tumor progression, due in part, to its effect on tumor vascularization.
Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.
Paris polyphylla var. yunnanensis is an important medicinal plant. Seed dormancy is one of the main factors restricting artificial cultivation. The molecular mechanisms of seed dormancy remain unclear, and little genomic or transcriptome data are available for this plant.
In this study, massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform was used to generate a substantial sequence dataset for the P. polyphylla embryo. 369,496 high quality reads were obtained, ranging from 50 to 1146 bp, with a mean of 219 bp. These reads were assembled into 47,768 unigenes, which included 16,069 contigs and 31,699 singletons. Using BLASTX searches of public databases, 15,757 (32.3%) unique transcripts were identified. Gene Ontology and Cluster of Orthologous Groups of proteins annotations revealed that these transcripts were broadly representative of the P. polyphylla embryo transcriptome. The Kyoto Encyclopedia of Genes and Genomes assigned 5961 of the unique sequences to specific metabolic pathways. Relative expression levels analysis showed that eleven phytohormone-related genes and five other genes have different expression patterns in the embryo and endosperm in the seed stratification process.
Gene annotation and quantitative RT-PCR expression analysis identified 464 transcripts that may be involved in phytohormone catabolism and biosynthesis, hormone signal, seed dormancy, seed maturation, cell wall growth and circadian rhythms. In particular, the relative expression analysis of sixteen genes (CYP707A, NCED, GA20ox2, GA20ox3, ABI2, PP2C, ARP3, ARP7, IAAH, IAAS, BRRK, DRM, ELF1, ELF2, SFR6, and SUS) in embryo and endosperm and at two temperatures indicated that these related genes may be candidates for clarifying the molecular basis of seed dormancy in P. polyphlla var. yunnanensis.
Embryo; Stratification; Seed dormancy; High-throughput sequencing; Paris polyphylla
The presence of homoeologous sequences and absence of a reference genome sequence make discovery and genotyping of single nucleotide polymorphisms (SNPs) more challenging in polyploid crops.
To address this challenge, we constructed reduced representation libraries (RRLs) for two Brassica napus inbred lines and their 91 doubled haploid (DH) progenies using a modified ddRADseq technique. A bioinformatics pipeline termed RFAPtools was developed to discover and genotype SNPs and presence/absence variations (PAVs). Using this pipeline, a pseudo-reference sequence (PRF) containing 180,991 sequence tags was constructed. By aligning sequence reads to the pseudo-reference sequence, allelic SNPs as well as PAVs were identified and genotyped with RFAPtools. Two parallel linkage maps, one SNP bin map containing 8,780 SNP loci and one PAV linkage map containing 12,423 dominant loci, were constructed. By aligning marker sequences to B. rapa sequence scaffolds, whose genome is available, we assigned 44 unassembled sequence scaffolds comprising 8.15 Mb onto the B. rapa chromosomes, and also identified 14 instances of misassembly and eight instances of mis-ordering sequence scaffolds.
These results indicate that the modified ddRADseq approach is a cost-effective and simple method to genotype tens of thousands SNPs and PAV markers in a polyploidy plant species. The results also demonstrated that RFAPtools developed in this study are powerful to mine allelic SNPs from homoeologous sequences in polyploids, therefore they are generally applicable in either diploid or polyploid species with or without a reference genome sequence.
Polyploid crops; Brassica napus; Pseudo-reference sequence; Single nucleotide polymorphism; Presence/absence variation
Functional enrichment analysis is an essential task for the interpretation of gene lists derived from large-scale genetic, transcriptomic and proteomic studies. WebGestalt (WEB-based GEne SeT AnaLysis Toolkit) has become one of the popular software tools in this field since its publication in 2005. For the last 7 years, WebGestalt data holdings have grown substantially to satisfy the requirements of users from different research areas. The current version of WebGestalt supports 8 organisms and 201 gene identifiers from various databases and different technology platforms, making it directly available to the fast growing omics community. Meanwhile, by integrating functional categories derived from centrally and publicly curated databases as well as computational analyses, WebGestalt has significantly increased the coverage of functional categories in various biological contexts including Gene Ontology, pathway, network module, gene–phenotype association, gene–disease association, gene–drug association and chromosomal location, leading to a total of 78 612 functional categories. Finally, new interactive features, such as pathway map, hierarchical network visualization and phenotype ontology visualization have been added to WebGestalt to help users better understand the enrichment results. WebGestalt can be freely accessed through http://www.webgestalt.org or http://bioinfo.vanderbilt.edu/webgestalt/.
Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data.
Plant-derived active constituents and their semi-synthetic or synthetic analogs have served as major sources of anticancer drugs. 20(S)-protopanaxadiol (PPD) is a metabolite of ginseng saponin of both American ginseng (Panax quinquefolius L.) and Asian ginseng (Panax ginseng C.A. Meyer). We previously demonstrated that ginsenoside Rg3, a glucoside precursor of PPD, exhibits anti-proliferative effects on HCT116 cells and reduces tumor size in a xenograft model. Our subsequent study indicated that PPD has more potent antitumor activity than that of Rg3 in vitro although the mechanism underlying the anticancer activity of PPD remains to be defined. Here, we investigated the mechanism underlying the anticancer activity of PPD in human cancer cells in vitro and in vivo. PPD was shown to inhibit growth and induce cell cycle arrest in HCT116 cells. The in vivo studies indicate that PPD inhibits xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced when the animals were treated with PPD (30 mg/kg body weight) for 3 weeks. When the expression of previously identified Rg3 targets, A kinase (PRKA) anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein α (PITPNA), was analyzed, PPD was shown to inhibit the expression of PITPNA while upregulating AKAP8L expression in HCT116 cells. Pathway-specific reporter assays indicated that PPD effectively suppressed the NF-κB, JNK and MAPK/ERK signaling pathways. Taken together, our results suggest that the anticancer activity of PPD in colon cancer cells may be mediated through targeting NF-κB, JNK and MAPK/ERK signaling pathways, although the detailed mechanisms underlying the anticancer mode of PPD action need to be fully elucidated.
ginseng; ginseng metabolites; 20(S)-protopanaxadiol; colorectal cancer; signaling pathway; natural products
The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene was originally identified as a transformation suppressor gene that is widely expressed in normal tissues. In tumor tissues, RECK expression levels are significantly reduced, and the downregulation of RECK has been implicated in tumors that are more aggressive with a poor prognosis. In the present study, RECK expression in peripheral T-cell lymphoma (PTCL; n=82) was examined using immunohistochemistry, and its correlation with clinicopathological factors was analyzed. According to the proportion of positively-stained cells and the staining intensity (SI), the patients were categorized into RECK-negative or RECK-positive groups. RECK expression was observed in 30 of the 82 patients (36.6%). The 3-year survival rate of the patients with RECK-positive tumors (65.5%) was significantly high compared with that of the patients with RECK-negative tumors (20.3%; P=0.046). Reduced RECK expression was found to be significantly correlated with extranodal lymphomatous involvement (P=0.012). The survival analysis showed that RECK-negative expression was an independent and significant factor for predicting a poor prognosis. RECK status is a useful prognostic factor for assessing the biological behavior in PTCL.
reversion-inducing cysteine-rich protein with Kazal motifs; prognosis; peripheral T-cell lymphoma