Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as “bright spots”, “dark clouds”, and “shadowy circles”, etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.
Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40–50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs.
We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data.
The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium.
The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs.
Dilated cardiomyopathies (DCM) show remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of DCM, either by genetic or epigenetic factors that may interact with environmental stimuli. In the present study, we examined genome-wide cardiac DNA methylation in patients with idiopathic DCM and controls. We detected methylation differences in pathways related to heart disease, but also in genes with yet unknown function in DCM or heart failure, namely Lymphocyte antigen 75 (LY75), Tyrosine kinase-type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A (ADORA2A). Mass-spectrometric analysis and bisulphite-sequencing enabled confirmation of the observed DNA methylation changes in independent cohorts. Aberrant DNA methylation in DCM patients was associated with significant changes in LY75 and ADORA2A mRNA expression, but not in ERBB3 and HOXB13. In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate a functional role of these genes in adaptive or maladaptive pathways in heart failure.
biomarker; dilated cardiomyopathy; DNA methylation; epigenetics; heart failure
We designed a study to investigate genetic relationships between primary tumors of oral squamous cell carcinoma (OSCC) and their lymph node metastases, and to identify genomic copy number aberrations (CNAs) related to lymph node metastasis. For this purpose, we collected a total of 42 tumor samples from 25 patients and analyzed their genomic profiles by array-based comparative genomic hybridization. We then compared the genetic profiles of metastatic primary tumors (MPTs) with their paired lymph node metastases (LNMs), and also those of LNMs with non-metastatic primary tumors (NMPTs). Firstly, we found that although there were some distinctive differences in the patterns of genomic profiles between MPTs and their paired LNMs, the paired samples shared similar genomic aberration patterns in each case. Unsupervised hierarchical clustering analysis grouped together 12 of the 15 MPT-LNM pairs. Furthermore, similarity scores between paired samples were significantly higher than those between non-paired samples. These results suggested that MPTs and their paired LNMs are composed predominantly of genetically clonal tumor cells, while minor populations with different CNAs may also exist in metastatic OSCCs. Secondly, to identify CNAs related to lymph node metastasis, we compared CNAs between grouped samples of MPTs and LNMs, but were unable to find any CNAs that were more common in LNMs. Finally, we hypothesized that subpopulations carrying metastasis-related CNAs might be present in both the MPT and LNM. Accordingly, we compared CNAs between NMPTs and LNMs, and found that gains of 7p, 8q and 17q were more common in the latter than in the former, suggesting that these CNAs may be involved in lymph node metastasis of OSCC. In conclusion, our data suggest that in OSCCs showing metastasis, the primary and metastatic tumors share similar genomic profiles, and that cells in the primary tumor may tend to metastasize after acquiring metastasis-associated CNAs.
Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (P<0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21Cip1, p27Kip1, p15INK4B, and p16INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (P<0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (P<0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.
Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.
Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.
Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glyco)protein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008) FASEB J. 22; 3358–3369) enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo) and depleted in phospholipids (lipids forming liquid-disordered phase, Ld), we designed Synthetic Exosome-Like Nanoparticles (SELN) with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.
Anaplastic oligodendrogliomas (AOD) are rare glial tumors in adults with relative homogeneous clinical, radiological and histological features at the time of diagnosis but dramatically various clinical courses. Studies have identified several molecular abnormalities with clinical or biological relevance to AOD (e.g. t(1;19)(q10;p10), IDH1, IDH2, CIC and FUBP1 mutations).
To better characterize the clinical and biological behavior of this tumor type, the creation of a national multicentric network, named “Prise en charge des OLigodendrogliomes Anaplasiques (POLA),” has been supported by the Institut National du Cancer (InCA). Newly diagnosed and centrally validated AOD patients and their related biological material (tumor and blood samples) were prospectively included in the POLA clinical database and tissue bank, respectively.
At the molecular level, we have conducted a high-resolution single nucleotide polymorphism array analysis, which included 83 patients. Despite a careful central pathological review, AOD have been found to exhibit heterogeneous genomic features. A total of 82% of the tumors exhibited a 1p/19q-co-deletion, while 18% harbor a distinct chromosome pattern. Novel focal abnormalities, including homozygously deleted, amplified and disrupted regions, have been identified. Recurring copy neutral losses of heterozygosity (CNLOH) inducing the modulation of gene expression have also been discovered. CNLOH in the CDKN2A locus was associated with protein silencing in 1/3 of the cases. In addition, FUBP1 homozygous deletion was detected in one case suggesting a putative tumor suppressor role of FUBP1 in AOD.
Our study showed that the genomic and pathological analyses of AOD are synergistic in detecting relevant clinical and biological subgroups of AOD.
The study of somatic genetic alterations in tumors contributes to the understanding and management of cancer. Genetic alterations, such us copy number or copy neutral changes, generate allelic imbalances (AIs) that can be determined using polymorphic markers. Here we report the development of a simple set of calculations for analyzing microsatellite multiplex PCR data from control-tumor pairs that allows us to obtain accurate information not only regarding the AI status of tumors, but also the percentage of tumor-infiltrating normal cells, the locus copy-number status and the mechanism involved in AI. We validated this new approach by re-analyzing a set of Neurofibromatosis type 1-associated dermal neurofibromas and comparing newly generated data with results obtained for the same tumors in a previous study using MLPA, Paralog Ratio Analysis and SNP-array techniques.
Microsatellite multiplex PCR analysis (MMPA) should be particularly useful for analyzing specific regions of the genome containing tumor suppressor genes and also for determining the percentage of infiltrating normal cells within tumors allowing them to be sorted before they are analyzed by more expensive techniques.
Pancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer.
Materials and Methods
Total RNA was extracted from feces using Qiagen's miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays.
All selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (p<0.05). Analysis of fecal miRNA expression in controls and patients with chronic pancreatitis and PCA revealed that the expression of miR-216a, -196a, -143 und -155 were highest in controls and lowest in PCA. The expression of the remaining three miRNAs (miR-21, -210 and -375) remained unchanged among controls and the patients with either chronic pancreatitis or PCA.
Our data provide novel evidence for the differential expression of miRNAs in feces of patients with PCA. If successfully validated in large-scale prospective studies, the fecal miRNA biomarkers may offer novel tools for PCA screening research.
Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.
TrkB-T1 is a BDNF receptor lacking a tyrosine kinase domain that is highly expressed in astrocytes and regulates BDNF-evoked calcium transients. Previous studies indicate that downregulation of TrkB-T1 in frontal cortex may be involved in neurobiological processes underlying suicide.
In a microarray screening study (N = 8), we interrogated all known microRNA in the frontal cortex of suicide completers with low expression of TrkB-T1 and normal controls. These findings were validated and followed up in a larger sample of cases and controls (N = 55). Functional analyses included microRNA silencing, microRNA overexpression and luciferase assays to investigate specificity and to validate interactions between differentially expressed microRNA and TrkB-T1.
MicroRNAs Hsa-miR-185* and Hsa-miR-491-3p were upregulated in suicide completers with low expression of TrkB.T1 (Pnominal: 9.10−5 and 1.8.10−4 respectively; FDR-corrected p = 0.031). Bioinformatic analyses revealed five putative binding sites for the DiGeorge syndrome linked microRNA Hsa-miR-185*in the 3′UTR of TrkB-T1, but none for Hsa-miR-491-3P. The increase of Hsa-miR-185* in frontal cortex of suicide completers was validated then confirmed in a larger, randomly selected group of suicide completers, where an inverse correlation between Hsa-miR-185* and TrkB-T1 expression was observed (R = −0.439; p = 0.001). Silencing and overexpression studies performed in human cell lines confirmed the inverse relationship between hsa-mir-185* and trkB-T1 expression. Luciferase assays demonstrated that Hsa-miR-185* binds to sequences in the 3′UTR of TrkB-T1.
These results suggest that an increase of Hsa-miR-185* expression levels regulates, at least in part, the TrkB-T1 decrease observed in the frontal cortex of suicide completers and further implicate the 22q11 region in psychopathology.
Similar to other malignancies, urothelial carcinoma (UC) is characterized by specific recurrent chromosomal aberrations and gene mutations. However, the interconnection between specific genomic alterations, and how patterns of chromosomal alterations adhere to different molecular subgroups of UC, is less clear. We applied tiling resolution array CGH to 146 cases of UC and identified a number of regions harboring recurrent focal genomic amplifications and deletions. Several potential oncogenes were included in the amplified regions, including known oncogenes like E2F3, CCND1, and CCNE1, as well as new candidate genes, such as SETDB1 (1q21), and BCL2L1 (20q11). We next combined genome profiling with global gene expression, gene mutation, and protein expression data and identified two major genomic circuits operating in urothelial carcinoma. The first circuit was characterized by FGFR3 alterations, overexpression of CCND1, and 9q and CDKN2A deletions. The second circuit was defined by E3F3 amplifications and RB1 deletions, as well as gains of 5p, deletions at PTEN and 2q36, 16q, 20q, and elevated CDKN2A levels. TP53/MDM2 alterations were common for advanced tumors within the two circuits. Our data also suggest a possible RAS/RAF circuit. The tumors with worst prognosis showed a gene expression profile that indicated a keratinized phenotype. Taken together, our integrative approach revealed at least two separate networks of genomic alterations linked to the molecular diversity seen in UC, and that these circuits may reflect distinct pathways of tumor development.
In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.
The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes – CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 – in the cell lines EHEB and MEC-1 as well as patient samples.
Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.
For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.
The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.
B cell chronic lymphocytic leukaemia; Wnt/β-catenin pathway; Inhibitor genes; DNA hypermethylation; Epigenetic silencing; β-catenin
With the advent of whole-genome and whole-exome sequencing, high-quality catalogs of recurrently mutated cancer genes are becoming available for many cancer types. Increasing access to sequencing technology, including bench-top sequencers, provide the opportunity to re-sequence a limited set of cancer genes across a patient cohort with limited processing time. Here, we re-sequenced a set of cancer genes in T-cell acute lymphoblastic leukemia (T-ALL) using Nimblegen sequence capture coupled with Roche/454 technology. First, we investigated how a maximal sensitivity and specificity of mutation detection can be achieved through a benchmark study. We tested nine combinations of different mapping and variant-calling methods, varied the variant calling parameters, and compared the predicted mutations with a large independent validation set obtained by capillary re-sequencing. We found that the combination of two mapping algorithms, namely BWA-SW and SSAHA2, coupled with the variant calling algorithm Atlas-SNP2 yields the highest sensitivity (95%) and the highest specificity (93%). Next, we applied this analysis pipeline to identify mutations in a set of 58 cancer genes, in a panel of 18 T-ALL cell lines and 15 T-ALL patient samples. We confirmed mutations in known T-ALL drivers, including PHF6, NF1, FBXW7, NOTCH1, KRAS, NRAS, PIK3CA, and PTEN. Interestingly, we also found mutations in several cancer genes that had not been linked to T-ALL before, including JAK3. Finally, we re-sequenced a small set of 39 candidate genes and identified recurrent mutations in TET1, SPRY3 and SPRY4. In conclusion, we established an optimized analysis pipeline for Roche/454 data that can be applied to accurately detect gene mutations in cancer, which led to the identification of several new candidate T-ALL driver mutations.
In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.
A solid process for diagnosis could have a substantial impact on the successful treatment of pancreatic cancer, for which currently mortality is nearly identical to incidence. Variations in the abundance of all microRNA molecules from peripheral blood cells and pancreas tissues were analyzed on microarrays and in part validated by real-time PCR assays. In total, 245 samples from two clinical centers were studied that were obtained from patients with pancreatic ductal adenocarcinoma or chronic pancreatitis and from healthy donors. Utilizing the minimally invasive blood test, receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analysis demonstrated very high sensitivity and specificity of a distinction between healthy people and patients with either cancer or chronic pancreatitis; respective AUC values of 0.973 and 0.950 were obtained. Confirmative and partly even more discriminative diagnosis could be performed on tissue samples with AUC values of 1.0 and 0.937, respectively. In addition, discrimination between cancer and chronic pancreatitis was achieved (AUC = 0.875). Also, several miRNAs were identified that exhibited abundance variations in both tissue and blood samples. The results could have an immediate diagnostic value for the evaluation of tumor reoccurrence in patients, who have undergone curative surgical resection, and for people with a familial risk of pancreatic cancer.
To estimate, from the perspective of the Chinese healthcare system, the economic outcomes of five different first-line strategies among patients with metastatic renal cell carcinoma (mRCC).
Methods and Findings
A decision-analytic model was developed to simulate the lifetime disease course associated with renal cell carcinoma. The health and economic outcomes of five first-line strategies (interferon-alfa, interleukin-2, interleukin-2 plus interferon-alfa, sunitinib and bevacizumab plus interferon-alfa) were estimated and assessed by indirect comparison. The clinical and utility data were taken from published studies. The cost data were estimated from local charge data and current Chinese practices. Sensitivity analyses were used to explore the impact of uncertainty regarding the results. The impact of the sunitinib patient assistant program (SPAP) was evaluated via scenario analysis. The base-case analysis showed that the sunitinib strategy yielded the maximum health benefits: 2.71 life years and 1.40 quality-adjusted life-years (QALY). The marginal cost-effectiveness (cost per additional QALY) gained via the sunitinib strategy compared with the conventional strategy was $220,384 (without SPAP, interleukin-2 plus interferon-alfa and bevacizumab plus interferon-alfa were dominated) and $16,993 (with SPAP, interferon-alfa, interleukin-2 plus interferon-alfa and bevacizumab plus interferon-alfa were dominated). In general, the results were sensitive to the hazard ratio of progression-free survival. The probabilistic sensitivity analysis demonstrated that the sunitinib strategy with SPAP was the most cost-effective approach when the willingness-to-pay threshold was over $16,000.
Our analysis suggests that traditional cytokine therapy is the cost-effective option in the Chinese healthcare setting. In some relatively developed regions, sunitinib with SPAP may be a favorable cost-effective alternative for mRCC.
Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
The molecular network sustained by different types of interactions among proteins is widely manifested as the fundamental driving force of cellular operations. Many biological functions are determined by the crosstalk between proteins rather than by the characteristics of their individual components. Thus, the searches for protein partners in global networks are imperative when attempting to address the principles of biology.
We have developed a web-based tool “Sequence-based Protein Partners Search” (SPPS) to explore interacting partners of proteins, by searching over a large repertoire of proteins across many species. SPPS provides a database containing more than 60,000 protein sequences with annotations and a protein-partner search engine in two modes (Single Query and Multiple Query). Two interacting proteins of human FBXO6 protein have been found using the service in the study. In addition, users can refine potential protein partner hits by using annotations and possible interactive network in the SPPS web server.
SPPS provides a new type of tool to facilitate the identification of direct or indirect protein partners which may guide scientists on the investigation of new signaling pathways. The SPPS server is available to the public at http://mdl.shsmu.edu.cn/SPPS/.
MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls.
We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718).
Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202.
MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use.
Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.
A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated.
The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.
SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process.
We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel.
High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.