Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) has been demonstrated to be an important player in various human malignancies; it is thought to promote tumor growth by cell cycle regulating. However, the roles of MALAT1 in esophageal squamous cell carcinoma(ESCC), and the mechanisms involved in cell cycle regulation remain poorly understood. Moreover, the factors contributing to its up-regulation in tumor tissues are still largely unclear.
Expression of MALAT1 was determined from cell lines and clinical samples by qRT-PCR. The effects of MALAT1 knockdown on cell proliferation, cell cycle, apoptosis, migration, and invasion were evaluated by in vitro and in vivo assays. The potential protein expression changes were investigated by Western-blotting. The methylation status of the CpG island in the MALAT1 promoter was explored by bisulfite sequencing, while the copy numbers in tumor tissues and blood samples were detected by a well-established AccuCopyTM method.
MALAT1 was over-expressed in 46.3% of ESCC tissues, mostly in the high-stage tumor samples. Enhanced MALAT1 expression levels were positively correlated with clinical stages, primary tumor size, and lymph node metastasis. Inhibition of MALAT1 suppressed tumor proliferation in vitro and in vivo, as well as the migratory and invasive capacity. MALAT1 depletion also induced G2/M phase arrest and increased the percentage of apoptotic cells. Western-blotting results implicated that the ATM-CHK2 pathway which is associated with G2/M arrest was phosphorylated by MALAT1 knockdown. No effects of CpG island methylation status on MALAT1 expression were found, whereas amplification of MALAT1 was found in 22.2% of tumor tissues, which correlated significantly with its over-expression. However, neither association between tissue copy number amplification and germline copy number variation, nor correlation between germline copy number variation and ESCC risk were identified in the case–control study.
Our data suggest that MALAT1 serves as an oncogene in ESCC, and it regulates ESCC growth by modifying the ATM-CHK2 pathway. Moreover, amplification of MALAT1 in tumor tissues may play an important role for its up-regulation, and it seems that the gene amplification in tumor tissues emerges during ESCC progression, but is not derived from germline origins.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-015-0123-z) contains supplementary material, which is available to authorized users.
Long noncoding RNA; MALAT1; Esophageal cancer; Copy number; Cell cycle arrest
High-throughput expression data, such as gene expression and metabolomics data, exhibit modular structures. Groups of features in each module follow a latent factor model, while between modules, the latent factors are quasi-independent. Recovering the latent factors can shed light on the hidden regulation patterns of the expression. The difficulty in detecting such modules and recovering the latent factors lies in the high dimensionality of the data, and the lack of knowledge in module membership.
Here we describe a method based on community detection in the co-expression network. It consists of inference-based network construction, module detection, and interacting latent factor detection from modules.
In simulations, the method outperformed projection-based modular latent factor discovery when the input signals were not Gaussian. We also demonstrate the method's value in real data analysis.
The new method nMLSA (network-based modular latent structure analysis) is effective in detecting latent structures, and is easy to extend to non-linear cases. The method is available as R code at http://web1.sph.emory.edu/users/tyu8/nMLSA/.
matrix decomposition; modularity; latent factors; network; community detection
Nanotechnology-based near-infrared quantum dots (NIR QDs) have many excellent optical properties, such as high fluorescence intensity, good fluorescence stability, and strong tissue-penetrating ability. Integrin αvβ3 is highly and specifically expressed in tumor angiogenic vessel endothelial cells of almost all carcinomas. Recent studies have shown that NIR QDs linked to peptides containing the arginine–glycine–aspartic acid (RGD) sequence (NIR QDs-RGD) can specifically target integrin αvβ3 expressed in endothelial cells of tumor angiogenic vessels in vivo, and they offer great potential for early cancer diagnosis, in vivo tumor imaging, and tumor individualized therapy. However, the toxicity profile of NIR QDs-RGD has not been reported. This study was conducted to investigate the toxicity of NIR QDs-RGD when intravenously administered to mice singly and repeatedly at the dose required for successful tumor imaging in vivo.
Materials and methods
A NIR QDs-RGD probe was prepared by linking NIR QDs with the maximum emission wavelength of 800 nm (QD800) to the RGD peptide (QD800-RGD). QD800-RGD was intravenously injected to BALB/C mice once or twice (200 pmol equivalent of QD800 for each injection). Phosphate-buffered saline solution was used as control. Fourteen days postinjection, toxicity tests were performed, including complete blood count (white blood cell, red blood cell, hemoglobin, platelets, lymphocytes, and neutrophils) and serum biochemical analysis (total protein, albumin, albumin/globulin, aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen). The coefficients of liver, spleen, kidney, and lung weight to body weight were measured, as well as their oxidation and antioxidation indicators, including superoxide dismutase, glutathione, and malondialdehyde. The organs were also examined histopathologically.
After one or two intravenous injections of QD800-RGD, as compared with control, no significant differences were observed in the complete blood count; biochemical indicators of blood serum, organ coefficient, and oxidation and antioxidation indicators; and no cell necrosis or inflammation were seen in the liver, spleen, kidney, or lung through histopathological examination.
Our data demonstrate that the single and repeated intravenous injection of QD800-RGD at a dose needed for successful tumor imaging in vivo is not toxic to mice. Our work lays a solid foundation for further biomedical applications of NIR QDs-RGD.
nanotechnology; RGD peptides; integrin αvβ3; quantum dots; intravenous injection; toxicity
MicroRNAs (miRNAs) are important post-transcriptional regulators which control growth and development in eukaryotes. The cestode Echinococcus granulosus has a complex life-cycle involving different development stages but the mechanisms underpinning this development, including the involvement of miRNAs, remain unknown.
Using Illumina next generation sequencing technology, we sequenced at the genome-wide level three small RNA populations from the adult, protoscolex and cyst membrane of E. granulosus. A total of 94 pre-miRNA candidates (coding 91 mature miRNAs and 39 miRNA stars) were in silico predicted. Through comparison of expression profiles, we found 42 mature miRNAs and 23 miRNA stars expressed with different patterns in the three life stages examined. Furthermore, considering both the previously reported and newly predicted miRNAs, 25 conserved miRNAs families were identified in the E. granulosus genome. Comparing the presence or absence of these miRNA families with the free-living Schmidtea mediterranea, we found 13 conserved miRNAs are lost in E. granulosus, most of which are tissue-specific and involved in the development of ciliated cells, the gut and sensory organs. Finally, GO enrichment analysis of the differentially expressed miRNAs and their potential targets indicated that they may be involved in bi-directional development, nutrient metabolism and nervous system development in E. granulosus.
This study has, for the first time, provided a comprehensive description of the different expression patterns of miRNAs in three distinct life cycle stages of E. granulosus. The analysis supports earlier suggestions that the loss of miRNAs in the Platyhelminths might be related to morphological simplification. These results may help in the exploration of the mechanism of interaction between this parasitic worm and its definitive and intermediate hosts, providing information that can be used to develop new interventions and therapeutics for the control of cystic echinococcosis.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-736) contains supplementary material, which is available to authorized users.
Echinococcus granulosus; microRNA; Deep sequencing; Differential expression; Life cycle stage development
HIV replication requires nuclear export of unspliced and singly spliced viral transcripts. Although a unique RNA structure has been proposed for the Rev-response element (RRE) responsible for viral mRNA export, how it recruits multiple HIV Rev proteins to form an export complex has been unclear. We show here that initial binding of Rev to the RRE triggers RNA tertiary structural changes, enabling further Rev binding and the rapid formation of a viral export complex. Analysis of the Rev-RRE assembly pathway using SHAPE-Seq and small-angle X-ray scattering (SAXS) reveals two major steps of Rev-RRE complex formation, beginning with rapid Rev binding to a pre-organized region presenting multiple Rev binding sites. This step induces long-range remodeling of the RNA to expose a cryptic Rev binding site, enabling rapid assembly of additional Rev proteins into the RNA export complex. This kinetic pathway may help maintain the balance between viral replication and maturation.
HIV is a virus that causes the immune system of an infected person to gradually fail, which can eventually result in AIDS. The virus consists of an RNA molecule—which encodes its genetic information—surrounded by coats of proteins. Once HIV enters a host cell, its RNA genome is converted into a DNA molecule, which travels to the nucleus and becomes part of the host's genome. The integrated viral genome can remain dormant for an extended period before the virus starts to replicate.
HIV replication begins with the production of RNA copies of the viral genome. For certain types of viral RNA molecules to be translated and packaged into new virus particles they need to be exported from the nucleus as part of the ‘nuclear–export complex’. This is made up of: a HIV RNA molecule, a HIV protein called Rev, and two host proteins.
Formation of the nuclear–export complex begins with multiple copies of the Rev protein attaching to specific stretches of the viral RNA, but how the Rev proteins assemble on the RNA molecule was previously unclear. Bai et al. have now used both structural and biochemical techniques to dissect the individual steps in this process. First, Rev proteins rapidly bind to a pre-formed region of the RNA molecule where multiple binding sites are compactly organized. This causes the overall shape of the RNA to change, and exposes a previously hidden extra binding site for Rev proteins. More Rev proteins then quickly bind to the newly exposed site, before finally the two host proteins bind and the whole complex is exported from the nucleus.
Bai et al. propose that checkpoints during this two-step assembly process are required to ensure that Rev proteins specifically bind to viral RNAs, and that such checkpoints may be important for controlling viral replication. The findings of Bai et al. may, in future, help to develop new drugs that treat HIV infection by blocking the export of the virus from the nucleus and thus inhibiting HIV replication.
SHAPE-Seq; HIV RRE-Rev complex; RNA nuclear export; viruses
The 2009 influenza A(H1N1) pandemic strain was for the first time included in the 2010–2011 seasonal trivalent influenza vaccine (TIV). We conducted a double-blind, randomized trial in Chinese population to assess the immunogenicity and safety of the 2010–2011 TIV manufactured by GlaxoSmithKline and compared it with the counterpart vaccines manufactured by Sanofi Pasteur and Sinovac Biotech. Healthy toddlers (6–36 mo), children (6–12 y) and older adults (≥60 y) with 300 participants in each age group were enrolled to randomly receive two doses (toddlers, 28 d apart) or one dose (children and older adults). The immunogenicity was assessed by hemagglutination-inhibition (HI) assay. The solicited injection-site and systemic adverse events (AEs) were collected within 7 d after vaccination. All the three TIVs were well-tolerated with 15.1% of participants reporting AEs, most of which were mild. No serious AEs and unusual AEs were reported. Fever and pain were the most common systemic and injection-site AEs, respectively. The three TIVs showed good immunogenicity. The seroprotection rates against both H1N1 and H3N2 strains were more than 87% in toddlers after two doses and more than 95% in children and more than 86% in older adults after one dose. The seroprotection rates against B strain were 68–71% in toddlers after two doses, 70–74% in children and 69–72% in older adults after one dose. In conclusion, the three 2010–2011 TIVs had good immunogenicity and safety in Chinese toddlers, children and older adults and were generally comparable in immunogenicity and reactogenicity.
influenza; vaccine; seasonal trivalent influenza vaccine; influenza A (H1N1); immunogenicity; safety
To elucidate further from the biomechanical aspect whether microgravity-induced cerebral vascular mal-adaptation might be a contributing factor to postflight orthostatic intolerance and the underlying mechanism accounting for the potential effectiveness of intermittent artificial gravity (IAG) in preventing this adverse effect.
Middle cerebral arteries (MCAs) were isolated from 28-day SUS (tail-suspended, head-down tilt rats to simulate microgravity effect), S+D (SUS plus 1-h/d −Gx gravitation by normal standing to simulate IAG), and CON (control) rats. Vascular myogenic reactivity and circumferential stress-strain and axial force-pressure relationships and overall stiffness were examined using pressure arteriography and calculated. Acellular matrix components were quantified by electron microscopy. The results demonstrate that myogenic reactivity is susceptible to previous pressure-induced, serial constrictions. During the first-run of pressure increments, active MCAs from SUS rats can strongly stiffen their wall and maintain the vessels at very low strains, which can be prevented by the simulated IAG countermeasure. The strains are 0.03 and 0.14 respectively for SUS and S+D, while circumferential stress being kept at 0.5 (106 dyn/cm2). During the second-run pressure steps, both the myogenic reactivity and active stiffness of the three groups declined. The distensibility of passive MCAs from S+D is significantly higher than CON and SUS, which may help to attenuate the vasodilatation impairment at low levels of pressure. Collagen and elastin percentages were increased and decreased, respectively, in MCAs from SUS and S+D as compared with CON; however, elastin was higher in S+D than SUS rats.
Susceptibility to previous myogenic constrictions seems to be a self-limiting protective mechanism in cerebral small resistance arteries to prevent undue cerebral vasoconstriction during orthostasis at 1-G environment. Alleviating of active stiffening and increasing of distensibility of cerebral resistance arteries may underlie the countermeasure effectiveness of IAG.
Syndactyly type 1 (SD1) is an autosomal dominant limb malformation characterized in its classical form by complete or partial webbing between the third and fourth fingers and/or the second and third toes. Its four subtypes (a, b, c, and d) are defined based on variable phenotypes, but the responsible gene is yet to be identified. SD1-a has been mapped to chromosome 3p21.31 and SD1-b to 2q34–q36. SD1-c and SD1-d are very rare and, to our knowledge, no gene loci have been identified.
Methods and Results
In two Chinese families with SD1-c, linkage and haplotype analyses mapped the disease locus to 2q31-2q32. Copy number variation (CNV) analysis, using array-based comparative genomic hybridization (array CGH), excluded the possibility of microdeletion or microduplication. Sequence analyses of related syndactyly genes in this region identified c.917G>A (p.R306Q) in the homeodomain of HOXD13 in family A. Analysis on family B identified the mutation c.916C>G (p.R306G) and therefore confirmed the genetic homogeneity. Luciferase assays indicated that these two mutations affected the transcriptional activation ability of HOXD13. The spectrum of HOXD13 mutations suggested a close genotype-phenotype correlation between the different types of HOXD13-Syndactyly. Overlaps of the various phenotypes were found both among and within families carrying the HOXD13 mutation.
Mutations (p.R306Q and p.R306G) in the homeodomain of HOXD13 cause SD1-c. There are affinities between SD1-c and synpolydactyly. Different limb malformations due to distinct classes of HOXD13 mutations should be considered as a continuum of phenotypes and further classification of syndactyly should be done based on phenotype and genotype.
Mining novel biomarkers from gene expression profiles for accurate disease classification is challenging due to small sample size and high noise in gene expression measurements. Several studies have proposed integrated analyses of microarray data and protein-protein interaction (PPI) networks to find diagnostic subnetwork markers. However, the neighborhood relationship among network member genes has not been fully considered by those methods, leaving many potential gene markers unidentified. The main idea of this study is to take full advantage of the biological observation that genes associated with the same or similar diseases commonly reside in the same neighborhood of molecular networks.
We present EgoNet, a novel method based on egocentric network-analysis techniques, to exhaustively search and prioritize disease subnetworks and gene markers from a large-scale biological network. When applied to a triple-negative breast cancer (TNBC) microarray dataset, the top selected modules contain both known gene markers in TNBC and novel candidates, such as RAD51 and DOK1, which play a central role in their respective ego-networks by connecting many differentially expressed genes.
Our results suggest that EgoNet, which is based on the ego network concept, allows the identification of novel biomarkers and provides a deeper understanding of their roles in complex diseases.
Gene expression; Network medicine; Machine learning; Cancer biology; Biological networks; Microarray
Since morbidity and mortality rates of anaphylaxis diseases have been increasing year by year, how to prevent and manage these diseases effectively has become an important issue. Mast cells play a central regulatory role in allergic diseases. Angiopoietin1 (Ang-1) exhibits anti-inflammatory properties by inhibiting vascular permeability, leukocyte migration and cytokine production. However, Ang-1's function in mast cell activation and anaphylaxis diseases is unknown. The results of our study suggest that Ang-1 decreased lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production of mast cells by suppressing IκB phosphorylation and NF-κB nuclear translocation. Ang-1 also strongly inhibited compound 48/80 induced and FcεRI-mediated mast cells degranulation by decreasing intracellular calcium levels in vitro. In vivo lentivirus-mediated delivery of Ang-1 in mice exhibited alleviated leakage in IgE-dependent passive cutaneous anaphylaxis (PCA). Furthermore, exogenous Ang-1 intervention treatment prevented mice from compound 48/80-induced mesentery mast cell degranulation, attenuated increases in pro-inflammatory cytokines, relieved lung injury, and improved survival in anaphylaxis shock. The results of our study reveal, for the first time, the important role of Ang-1 in the activation of mast cells, and identify a therapeutic effect of Ang-1 on anaphylaxis diseases.
In the current study, microRNA (miRNA) microarrays were used to detect differentially expressed miRNAs in the myocardial tissues of rat models under stress, to screen target miRNA candidates for miRNA therapy of stress-induced myocardial injury. Rats were bound and suspended in order to induce acute stress (AS) and chronic stress (CS) models. miRNA microarrays were used to detect differentially expressed miRNA in the myocardial tissues of the stressed and control groups. In comparison to the normal control, there were 68 differentially expressed miRNAs in the AS model, of which 32 were upregulated and 36 were downregulated. There were 55 differentially expressed miRNAs in the CS model, of which 20 were upregulated and 35 were downregulated. Of the 123 miRNAs, 15 miRNAs were differentially expressed between the AS and CS groups, of which four were significantly upregulated (rno-miR-296, rno-miR-141, rno-miR-382 and rno-miR-219-5p) and 11 were downregulated (significantly downregulated, rno-miR-135a and rno-miR-466b). The stress of being bound and suspended induces myocardial injury in the rats. Myocardial injury may cause the differential expression of certain miRNAs. Psychological stress may lead to the significant upregulation of rno-miR-296, rno-miR-141, rno-miR-382 and rno-miR-219-5p in addition to the significant downregulation of miR-135a and miR-466b.
stress; microRNAs; myocardium; rat; microarray
To characterize the spectrum of CYP4V2 gene mutations in 92 unrelated Chinese probands with Bietti’s crystalline dystrophy (BCD) and to describe the molecular and clinical characteristics of four novel CYP4V2 mutations associated with BCD.
All study participants underwent a complete ophthalmological examination. Mutational screening of CYP4V2 coding regions and flanking intron sequences was examined via directional Sanger sequencing, with allele separation confirmed by screening other family members. Subsequent in silico analysis of the mutational consequence on protein function was undertaken, with the impact of the novel mutation on pre-mRNA splicing examined via RT–PCR.
Fifteen disease-causing variants were identified in 92 probands with BCD, including four novel mutations and eleven previously reported mutations. The most prevalent mutation was c.802_810del17insGC, which was detected in 69 unrelated families, with an allele frequency of 52.7% (97/184). Homozygosity was revealed in 35 unrelated families, and compound heterozygosity was observed in 43 subjects. Four patients harbored four novel variants, with these mutations cosegregated within all affected individuals and were not found in unaffected family members and 100 unrelated controls. Transcriptional analysis of a novel splice mutation revealed altered RNA splicing. In silico analysis predicted that the missense variant, p.Tyr343Asp, disrupted the CYP4V2 surface electrostatic potential distribution and spatial conformation. Among the patients with four novel mutations, genotype did not always correlate with age at onset, disease course, or electroretinogram (ERG) changes, with phenotypic variations even noted within the same genotype.
The c.802_810del17insCG mutation was the most common mutation in the 92 Chinese probands with BCD examined. Four novel mutations were identified, contributing to the spectrum of CYP4V2 mutations associated with BCD, with no clear link established between disease phenotype and genotype.
The paired box gene 6 (PAX6) is an essential transcription factor for eye formation. Genetic alterations in PAX6 can lead to various ocular malformations including aniridia. The purpose of this study was to identify genetic defects as the underlying cause of familial ocular coloboma in a large Chinese family.
After linkage analysis was carried out in this family, all exons of PAX6 in the proband were sequenced by the Sanger sequencing technique. Then the genome of the proband was evaluated by a microarray-based comparative genomic hybridization (aCGH). Quantitative real-time PCR was applied to verify the abnormal aCGH findings.
All patients presented bilateral partial coloboma of iris, severe congenital nystagmus, hyperpresbyopia and congenital posterior polar cataracts. Two-point linkage analysis in the autosomal dominant family showed loss of heterozygosity at the D11S914 locus. There was no pathogenic mutation in the exons of PAX6. The aCGH analysis revealed a 681 kb heterozygous deletion on chromosome 11p13. Quantitative real-time PCR verified the deletion in the patients and further confirmed this deletion cosegregation with the ocular coloboma phenotype in the family.
The 681 kb large deletion of chromosome 11p13 downstream of PAX6 is the genetic cause of the familial ocular coloboma in this large Chinese family. aCGH should be applied if there is a negative result for the mutation detection of PAX6 in patients with ocular coloboma.
Molecular imaging plays a key role in personalized medicine and tumor diagnosis. Quantum dots with near-infrared emission spectra demonstrate excellent tissue penetration and photostability, and have recently emerged as important tools for in vivo tumor imaging. Integrin αvβ3 has been shown to be highly and specifically expressed in endothelial cells of tumor angiogenic vessels in almost all types of tumors, and specifically binds to the peptide containing arginine-glycine-aspartic acid (RGD). In this study, we conjugated RGD with quantum dots with emission wavelength of 800 nm (QD800) to generate QD800-RGD, and used it via intravenous injection as a probe to image tumors in nude mice bearing head and neck squamous cell carcinoma (HNSCC). Twelve hours after the injection, the mice were still alive and were sacrificed to isolate tumors and ten major organs for ex vivo analysis to localize the probe in these tissues. The results showed that QD800-RGD was specifically targeted to integrin αvβ3 in vitro and in vivo, producing clear tumor fluorescence images after the intravenous injection. The tumor-to-background ratio and size of tumor image were highest within 6 hours of the injection and declined significantly at 9 hours after the injection, but there was still a clearly visible tumor image at 12 hours. The greatest amount of QD800-RGD was found in liver and spleen, followed by tumor and lung, and a weak fluorescence signal was seen in tibia. No detectable signal of QD800-RGD was found in brain, heart, kidney, testis, stomach, or intestine. Our study demonstrated that using integrin αvβ3 as target, it is possible to use intravenously injected QD800-RGD to generate high quality images of HNSCC, and the technique offers great potential in the diagnosis and personalized therapy for HNSCC.
nanotechnology; near-infrared fluorescence; tumor angiogenic vessel; head and neck cancer; in vivo imaging
Neither HBV DNA nor HBsAg positivity at birth is an accurate marker for HBV infection of infants. No data is available for continuous changes of HBV markers in newborns to HBsAg(+) mothers. This prospective, multi-centers study aims at observing the dynamic changes of HBV markers and exploring an early diagnostic marker for mother-infant infection.
One hundred forty-eight HBsAg(+) mothers and their newborns were enrolled after mothers signed the informed consent forms. Those infants were received combination immunoprophylaxis (hepatitis B immunoglobulin [HBIG] and hepatitis B vaccine) at birth, and then followed up to 12 months. Venous blood of the infants (0, 1, 7, and 12 months of age) was collected to test for HBV DNA and HBV markers.
Of the 148 infants enrolled in our study, 41 and 24 infants were detected as HBsAg(+) and HBV DNA(+) at birth, respectively. Nine were diagnosed with HBV infection after 7 mo follow-up. Dynamic observation of the HBV markers showed that HBV DNA and HBsAg decreased gradually and eventually sero-converted to negativity in the non-infected infants, whereas in the infected infants, HBV DNA and HBsAg were persistently positive, or higher at the end of follow-up. At 1 mo, the infants with anti-HBs(+), despite positivity for HBsAg or HBV DNA at birth, were resolved after 12 mo follow-up, whereas all the nine infants with anti-HBs(−) were diagnosed with HBV infection. Anti-HBs(−) at 1 mo showed a higher positive likelihood ratio for HBV mother-infant infection than HBV DNA and/or HBsAg at birth.
Negativity for anti-HBs at 1 mo can be considered as a sensitive and early diagnostic indictor for HBV infection in the infants with positive HBV DNA and HBsAg at birth, especially for those infants with low levels of HBV DNA load and HBsAg titer.
A ‘lung cancer'-specific mutant EGFR interactome was generated by a global analysis of protein–protein interactions and phosphorylation. After functional screening, nine proteins were identified as essential for the viability of EGFR-mutant lung cancer cells.
The interactome of lung cancer-associated mutant forms of epidermal growth factor receptor (EGFR), consisting of 263 proteins, was built by integrating protein–protein interactions and tyrosine phosphorylation.Systematic perturbations of the network nodes revealed a core network of 14 proteins, 9 of which were shown to be specifically associated with survival of EGFR-mutant lung cancer cells.Cells with acquired resistance to EGFR tyrosine kinase inhibitors showed differential dependence on the core network proteins.A drug network associated with the core network proteins led to the identification of two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib.
We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.
epidermal growth factor receptor; interactome; lung cancer; proteomics; tyrosine kinase inhibitor
Stem cell factor–dependent KIT activation is an essential process for mast cell homeostasis. The two major splice variants of KIT differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differs in systemic mastocytosis and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK− and GNNK+ transcripts from bone marrow mononuclear cells was developed. The GNNK−/GNNK+ copy number ratio showed a trend toward a positive correlation with the percentage of neoplastic mast cell involvement, and KIT D816V containing transcripts displayed a significantly elevated GNNK−/GNNK+ copy number ratio. Relative expression of only the GNNK− variant correlated with increasing percentage of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK− isoform of wild type KIT was associated with increased granule formation, histamine content, and growth. When accompanying the KIT D816V mutation, the GNNK− isoform enhanced cytokine-free metabolism and moderately reduced sensitivity to the tyrosine kinase inhibitor, PKC412. These data suggest that neoplastic mast cells favor a GNNK− variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus could influence therapeutic sensitivity in systemic mastocytosis.
We selected 180 clinical isolates of the Mycobacterium tuberculosis complex (MTBC) from patients in China and performed comparative sequence analysis of the mpt64 gene after amplification. From the results, we found that polymorphisms of the mpt64 gene in the MTBC may be the reason for changes in the antigen produced, which may in turn cause alterations of related functions, thereby allowing immune evasion.
Metabolic profiling is the unbiased detection and quantification of low molecular-weight metabolites in a living system. It is rapidly developing in biological and translational research, contributing to disease mechanism elucidation, environmental chemical surveillance, biomarker detection, and health outcome prediction. Recent developments in experimental and computational technology allow more and more known metabolites to be detected and quantified from complex samples. As the coverage of the metabolic network improves, it has become feasible to examine metabolic profiling data from a systems perspective, i.e. interpreting the data and performing statistical inference in the context of pathways and genome-scale metabolic networks. Recently a number of methods have been developed in this area, and much improvement in algorithms and databases are still needed. In this review, we survey some methods for the analysis of metabolic profiling data based on metabolic networks.
Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild type (WT) and, where also examined, KIT D816V was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines; and in CD34+-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcεRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF enhanced mast cell activation may provide significant therapeutic benefits.
mastocytosis; switch pocket; KIT; KIT D816V; tyrosine kinase inhibitor; mast cell
The 3′ untranslated region (3′UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3′UTR and the host ribosome. The 3′UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3′UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3′UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.
To investigate the effects of genetic factors on idiopathic scoliosis (IS) and genetic modes through genetic epidemiological survey on IS in Chongqing City, China, and to determine whether SH3GL1, GADD45B, and FGF22 in the chromosome 19p13.3 are the pathogenic genes of IS through genetic sequence analysis.
214 nuclear families were investigated to analyse the age incidence, familial aggregation, and heritability. SH3GL1, GADD45B, and FGF22 were chosen as candidate genes for mutation screening in 56 IS patients of 214 families. The sequence alignment analysis was performed to determine mutations and predict the protein structure.
The average age of onset of 10.8 years suggests that IS is a early onset disease. Incidences of IS in first-, second-, third-degree relatives and the overall incidence in families (5.68%) were also significantly higher than that of the general population (1.04%). The U test indicated a significant difference, suggesting that IS has a familial aggregation. The heritability of first-degree relatives (77.68 ±10.39%), second-degree relatives (69.89 ±3.14%), and third-degree relatives (62.14 ±11.92%) illustrated that genetic factors play an important role in IS pathogenesis. The incidence of first-degree relatives (10.01%), second-degree relatives (2.55%) and third-degree relatives (1.76%) illustrated that IS is not in simple accord with monogenic Mendel’s law but manifests as traits of multifactorial hereditary diseases. Sequence alignment of exons of SH3GL1, GADD45B, and FGF22 showed 17 base mutations, of which 16 mutations do not induce open reading frame (ORF) shift or amino acid changes whereas one mutation (C→T)occurred in SH3GL1 results in formation of the termination codon, which induces variation of protein reading frame. Prediction analysis of protein sequence showed that the SH3GL1 mutant encoded a truncated protein, thus affecting the protein structure.
IS is a multifactorial genetic disease and SH3GL1 may be one of the pathogenic genes for IS.
Zymomonas mobilis ZM401 is a flocculating strain which can be self-immobilized within fermentors for a high-cell-density culture to improve ethanol productivity, as well as high-gravity fermentation to increase ethanol titer, due to its improved ethanol tolerance associated with the morphological change. Here, we report its draft genome sequence.
CD274, one of two co-stimulatory ligands for programmed death 1 and widely expressed in the mononuclear phagocyte system (MPS), may co-stimulate T cells and regulates inflammatory responses. However, changes in CD274 gene expression and the underlying molecular mechanism are poorly understood during inflammatory responses. Therefore, delineation of the complex mechanisms regulating CD274 expression is critical to understand this immunoregulatory system during inflammatory responses. The purpose of this study was to assess the molecular mechanisms regulating CD274 expression in an in vitro monocyte model of inflammatory response. Firstly, CD274 expression levels in human primary monocytes after lipopolysaccharide (LPS) treatment were observed and correlated with NF-κB activation. Secondly, based on the distribution of putative NF-κB binding sites, 5′ truncated human CD274 promoter reporters were constructed, transfected into U937 cells and critical promoter regions for basal (nt −570 to +94) and LPS-induced (nt −1735 to −570) transcription were identified by dual luciferase assays. Finally, a key NF-κB binding site (nt −610 to −601) for LPS-inducible CD274 transcriptional activity was characterized by point mutation analysis and chromatin immunoprecipitation analysis assays (ChIP). Thus, the present study establishes a molecular basis to understand the mechanisms governing CD274 expression in certain infections and inflammatory disorders.
The recent development of antibodies specific for the major hotspot mutations in the epidermal growth factor receptor (EGFR), L858R and E746_A750del, may provide an opportunity to use immunohistochemistry (IHC) as a screening test for EGFR gene mutations. This study was designed to optimize the IHC protocol and the criteria for interpretation of the results using DNA sequencing as the gold-standard.
Tumor sections from fifty lung adenocarcinoma specimens from Chinese patients were immunostained using L858R and E746_A750del-specific antibodies using three different antigen retrieval solutions, and the results were evaluated using three different sets of criteria. The same specimens were used for DNA purification and analysis of EGFR gene mutations.
In this study the optimal buffer for antigen retrieval was EDTA (pH 8.0), and the optimal scoring method was to call positive results when there was moderate to strong staining of membrane and/or cytoplasm in >10% of the tumor cells. Using the optimized protocol, L858R-specific IHC showed a sensitivity of 81% and a specificity of 97%, and E746_A750del-specific IHC showed a sensitivity of 59% and a specificity of 100%, both compared with direct DNA analysis. Additionally, the mutant proteins as assessed by IHC showed a more homogeneous than heterogeneous pattern of expression.
Our data demonstrate that mutation-specific IHC, using optimized procedures, is a reliable prescreening test for detecting EGFR mutations in lung adenocarcinoma.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2059012601872392
Lung adenocarcinoma; Epidermal growth factor receptor; Mutation; Immunohistochemistry