The epidermal growth factor receptor (EGFR) is a validated target in head and neck squamous cell carcinoma (HNSCC). In recurrent and/or metastatic (R/M) HNSCC, resistance to anti-EGFR therapy inevitably occurs. Downstream activation of the PI3K/Akt/mTOR pathway is an established resistance mechanism. Concurrent mTOR blockade may improve efficacy of anti-EGFR therapy.
Materials and methods
Erlotinib 150 mg daily and temsirolimus 15 mg weekly were administered to patients with platinum-refractory R/M HNSCC and ECOG performance status 0–2. The primary endpoint was progression-free survival (PFS). Correlative studies determined PIK3CA and HRAS mutation status; p16, EGFR, pS6K, pAkt and PTEN expression; and pre- and post-treatment plasma levels of 20 immunomodulatory cytokines.
Twelve patients enrolled; six withdrew within 6 weeks due to toxicity or death, prompting early closure of the trial. Grade ≥3 toxicities included fatigue, diarrhea, gastrostomy tube infection, peritonitis, pneumonia, dyspnea, and HN edema. Median PFS was 1.9 months. Median overall survival was 4.0 months. Six/12 tumors were p16(+), 9/11 lacked measurable PTEN expression, and 1/12 harbored a PIK3CA mutation. On exploratory analysis, high baseline plasma VEGF and interferon-gamma levels marginally associated with tumor progression.
The combination of erlotinib and temsirolimus was poorly tolerated. Low prevalence of PTEN expression and 8% incidence of PIK3CA mutations indicate biological relevance of this pathway in R/M disease. Investigation of more tolerable combinations of EGFR and PI3K/Akt/mTOR pathway inhibitors in selected HNSCC patients is warranted.
Temsirolimus; Erlotinib; Platinum-refractory; Head and neck squamous cell carcinoma; mTOR; EGFR; PIK3CA
Spatial organization of G-protein coupled receptors (GPCRs) into dimers and higher order oligomers has been demonstrated in vitro and in vivo. The pharmacological readout was shown to depend on the specific interfaces, but why particular regions of the GPCR structure are involved, and how ligand-determined states change them remains unknown. Here we show why protein-membrane hydrophobic matching is attained upon oligomerization at specific interfaces from an analysis of coarse-grained molecular dynamics simulations of the spontaneous diffusion-interaction of the prototypical beta2-adrenergic (β2AR) receptors in a POPC lipid bilayer. The energy penalty from mismatch is significantly reduced in the spontaneously emerging oligomeric arrays, making the spatial organization of the GPCRs dependent on the pattern of mismatch in the monomer. This mismatch pattern is very different for β2AR compared to the highly homologous and structurally similar β1AR, consonant with experimentally observed oligomerization patterns of β2AR and β1AR. The results provide a mechanistic understanding of the structural context of oligomerization.
Activation of adenosine A2A receptor aggravates lung damage in a neurogenic mouse model of acute lung injury (ALI) but protects against nonneurogenic ALI.
The bone marrow–derived cell (BMDC)–associated inflammatory response plays a key role in the development of acute lung injury (ALI). Activation of adenosine A2A receptor (A2AR) is generally considered to be antiinflammatory, inhibiting BMDC activities to protect against ALI. However, in the present study, we found that in a mouse model of neurogenic ALI induced by severe traumatic brain injury (TBI), BMDC A2AR exerted a proinflammatory effect, aggravating lung damage. This is in contrast to the antiinflammatory effect observed in the mouse oleic acid–induced ALI model (a nonneurogenic ALI model.) Moreover, the A2AR agonist CGS21680 aggravated, whereas the antagonist ZM241385 attenuated, the severe TBI-induced lung inflammatory damage in mice. Further investigation of white blood cells isolated from patients or mouse TBI models and of cultured human or mouse neutrophils demonstrated that elevated plasma glutamate after severe TBI induced interaction between A2AR and the metabotropic glutamate receptor 5 (mGluR5) to increase phospholipase C–protein kinase C signaling, which mediated the proinflammatory effect of A2AR. These results are in striking contrast to the well-known antiinflammatory and protective role of A2AR in nonneurogenic ALI and indicate different therapeutic strategies should be used for nonneurogenic and neurogenic ALI treatment when targeting A2AR.
Biomarkers capable of discriminating the patients who are likely to respond to certain chemotherapeutic agents could improve the clinical efficiency. The sulfatases(SULFs) play a critical role in the pathogenesis of a variety of human cancers. Here, we focused our investigation on the prognostic and predictive impact of SULF2 methylation in gastric cancer.
Promoter CpG island methylation of SULF2 was analyzed in 100 gastric cancer samples. The in vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan and pemetrexed were determined by histoculture drug response assay(HDRA). Additionally, 56 gastric cancer patients treated with a modified FOLFOX regimen(biweekly oxaliplatin plus 5-FU and folinic acid) were retrospectively analyzed to further evaluate the prognostic and predictive impact of SULF2 methylation in gastric cancer.
Methylated SULF2(SULF2M) was detected in 28 patients, while the remaining 72 patients showed unmethylated SULF2(SULF2U, methylation rate: 28%). Samples with SULF2U were more sensitive to cisplatin than those with SULF2M(inhibition rate: 48.80% vs. 38.15%, P = 0.02), while samples with SULF2M were more sensitive to irinotecan than SULF2U(inhibition rate: 53.61% vs. 40.92%, P = 0.01). There were no association between SULF2 methylation and in vitro sensitivity to docetaxel, gemcitabine and pemetrexed. SULF2 methylation was found to have a significant association with cisplatin efficacy(SULF2M: 57.14%, SULF2U: 80.56%, P = 0.02) and irinotecan efficacy(SULF2M: 89.29%, SULF2U: 62.50%, P = 0.01). Among the 56 patients receiving the modified FOLFOX regimen, a significant association was observed between survival and SULF2 methylation status(SULF2M: 309 days, 95% CI = 236 to 382 days; SULF2U: 481 days, 95% CI = 418 to 490 days; P = 0.02). Multivariate analysis revealed that SULF2 methylation was an independent prognostic factor of overall survival in gastric cancer patients treated with platinum-based chemotherapy.
SULF2 methylation is negatively associated with cisplatin sensitivity in vitro. SULF2 methylation may be a novel prognostic biomarker for gastric cancer patients treated with platinum-based chemotherapy.
The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective. In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Using this system, we screened candidate sequences that were initially identified by chromatin profiling for binding by CTCF and for DNase hypersensitivity. All 12 analyzed candidates blocked enhancer-promoter activity. The enhancer-blocking activity of the top two candidates was confirmed in two complementary plasmid-based assays. Studies in a gammaretroviral reporter vector indicated these two candidates have little to no effect on vector titers, and do not diminish vector expression in primary mouse bone marrow cultures. Subsequent assessment in a mouse in vivo tumor formation model demonstrated that both candidates reduced the rate of gammaretroviral vector-mediated genotoxicity as effectively as the cHS4 insulator. In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression. These findings expand the limited arsenal of insulators functionally validated to reduce the rate of retroviral vector-mediated genotoxicity.
The structure of a complex of an endoglucanase with cellotriose was determined with a hexagonal unit cell and showed how the substrate interacts with the enzyme.
The endoglucanase EglA from Piromyces rhizinflata found in cattle stomach belongs to the GH5 family of glycoside hydrolases. The crystal structure of the catalytic domain of EglA shows the (β/α)8-barrel fold typical of GH5 enzymes. Adjacent to the active site of EglA, a loop containing a disulfide bond not found in other similar structures may participate in substrate binding. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme–substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the −3, −2 and −1 subsites shows an extensive hydrogen-bonding network between the enzyme and the substrate, along with a stacking interaction between Trp44 and the −3 sugar. A possible dimer was observed in the crystal structure, but retention of activity in the E242A mutant suggested that the enzyme probably does not function as a dimer in solution. On the other hand, the first 100 amino acids encoded by the original cDNA fragment are very similar to those in the last third of the (β/α)8-barrel fold, indicating that EglA comprises at least two catalytic domains acting in tandem.
carbohydrate utilization; catalytic domain; cellulase; molecular interactions
Nanomaterials have been increasingly employed as drug(s)-incorporated vectors for drug delivery due to their potential of maximizing therapeutic efficacy while minimizing systemic side effects. However, there have been two main challenges for these vectors: (i) the existing synthetic approaches are cumbersome and incapable of achieving precise control of their structural properties, which will affect their biodistribution and therapeutic efficacies, and (ii) lack of an early checkpoint to quickly predict which drug(s)-incorporated vectors exhibit optimal therapeutic outcomes. In this work, we utilized a new rational developmental approach to rapidly screen nanoparticle (NP)-based cancer therapeutic agents containing a built-in companion diagnostic utility for optimal therapeutic efficacy. The approach leverages the advantages of a self-assembly synthetic method for preparation of two different sizes of drug-incorporated supramolecular nanoparticles (SNPs), and a positron emission tomography (PET) imaging-based biodistribution study to quickly evaluate the accumulation of SNPs at a tumor site in vivo and select the favorable SNPs for in vivo therapeutic study. Finally, the enhanced in vivo anti-tumor efficacy of the selected SNPs was validated by tumor reduction/inhibition studies. We foresee our rational developmental approach providing a general strategy in the search of optimal therapeutic agents among the diversity of NP-based therapeutic agents.
Supramolecular assembly; Nanoparticles; Drug delivery; Positron emission tomography; Cancer therapeutics
Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Though it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. We demonstrate using the zebrafish model that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions.
Stereoselectivities of the dihydroxylations of cis-bicyclo[3.3.0]octene intermediates for a projected total synthesis of chromodorolide A have been explored experimentally. The reaction occurs unexpectedly on the apparently more hindered (concave) face; this result has been explained through computational studies using B3LYP and B3LYP-D3 methods. Torsional effects are largely responsible for the stereoselectivity encountered in the chromodorolide A synthesis. Many literature examples have been reported on related cases. QM calculations show that the stereoselectivities of dihydroxylations of fused cyclopentenes are influenced by the conformational rigidity or flexibility of the substrate. Torsional, electrostatic, and steric effects can all influence stereoselectivity, and the rigidity or flexibility of conformations of reactants provides a predictive guide to stereoselectivity.
Hepatitis A virus (HAV) is the most common cause of infectious hepatitis throughout the world, spread largely by the fecal-oral route. To characterize the genetic diversity of the virus circulating in China where HAV in endemic, we selected the outbreak cases with identical sequences in VP1-2A junction region and compiled a panel of 42 isolates. The VP3-VP1-2A regions of the HAV capsid-coding genes were further sequenced and analyzed. The quasispecies distribution was evaluated by cloning the VP3 and VP1-2A genes in three clinical samples. Phylogenetic analysis demonstrated that the same genotyping results could be obtained whether using the complete VP3, VP1, or partial VP1-2A genes for analysis in this study, although some differences did exist. Most isolates clustered in sub-genotype IA, and fewer in sub-genotype IB. No amino acid mutations were found at the published neutralizing epitope sites, however, several unique amino acid substitutions in the VP3 or VP1 region were identified, with two amino acid variants closely located to the immunodominant site. Quasispecies analysis showed the mutation frequencies were in the range of 7.22x10-4 -2.33x10-3 substitutions per nucleotide for VP3, VP1, or VP1-2A. When compared with the consensus sequences, mutated nucleotide sites represented the minority of all the analyzed sequences sites. HAV replicated as a complex distribution of closely genetically related variants referred to as quasispecies, and were under negative selection. The results indicate that diverse HAV strains and quasispecies inside the viral populations are presented in China, with unique amino acid substitutions detected close to the immunodominant site, and that the possibility of antigenic escaping mutants cannot be ruled out and needs to be further analyzed.
Aims: In this study we identified viral gene targets of the important redox regulator thioredoxin (Trx), and explored in depth how Trx interacts with the immediate early gene #1 (IE1) of the white spot syndrome virus (WSSV). Results: In a pull-down assay, we found that recombinant Trx bound to IE1 under oxidizing conditions, and a coimmunoprecipitation assay showed that Trx bound to WSSV IE1 when the transfected cells were subjected to oxidative stress. A pull-down assay with Trx mutants showed that no IE1 binding occurred when cysteine 62 was replaced by serine. Electrophoretic mobility shift assay (EMSA) showed that the DNA binding activity of WSSV IE1 was downregulated under oxidative conditions, and that Penaeus monodon Trx (PmTrx) restored the DNA binding activity of the inactivated, oxidized WSSV IE1. Another EMSA experiment showed that IE1's Cys-X-X-Cys motif and cysteine residue 55 were necessary for DNA binding. Measurement of the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in WSSV-infected shrimp showed that oxidative stress was significantly increased at 48 h postinfection. The biological significance of Trx was also demonstrated in a double-strand RNA Trx knockdown experiment where suppression of shrimp Trx led to significant decreases in mortality and viral copy numbers. Innovation and Conclusion: WSSV's pathogenicity is enhanced by the virus' use of host Trx to rescue the DNA binding activity of WSSV IE1 under oxidizing conditions. Antioxid. Redox Signal. 17, 914–926.
Genome-wide association studies (GWAS) have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by DNase I hypersensitive sites (DHSs). 88% of such DHSs are active during fetal development, and are enriched for gestational exposure-related phenotypes. We identify distant gene targets for hundreds of DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrate tissue-selective enrichment of more weakly disease-associated variants within DHSs, and the de novo identification of pathogenic cell types for Crohn’s disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease, and provide pathogenic insights into diverse disorders.
Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG.
The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9–11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided.
FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC50 = 3.5nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure.
Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206mm3, 95% CI = 0.068 to 0.344mm3; control: mean = 0.580mm3, 95% CI = 0.267 to 0.893mm3) and 0.24 (treated: mean = 0.131mm3, 95% CI = 0.09 to 0.180mm3; control: mean = 0.543mm3, 95% CI = 0.173 to 0.913mm3), respectively, on day 21 after therapy.
This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.
Long-term systemic blockade of the renin–angiotensin system (RAS) with either an angiotensin (Ang) II type 1 receptor antagonist or an angiotensin-converting enzyme inhibitor attenuates age-related cardiac remodeling and oxidative damage, and improves myocardial relaxation. However, the role of the brain RAS in mediating the development of diastolic dysfunction during aging is not known. We hypothesized that low brain RAS protects against the development of age-related diastolic dysfunction and left ventricular remodeling.
Sixty-week-old transgenic male ASrAOGEN rats (n =9), with normal circulating Ang II and functionally low brain Ang II, because of a GFAP promoter-linked angiotensinogen antisense targeted to glia, and age-matched and sex-matched Hannover Sprague–Dawley (SD; n= 9) rats, with normal levels of both circulating and brain Ang II, underwent echocardiograms to evaluate cardiac structure and function. Postmortem hearts were further compared for histological, molecular, and biochemical changes consistent with cardiac aging.
ASrAOGEN rats showed preserved systolic and diastolic function at mid-life and this was associated with a lower, more favorable ratio of the phospholamban–SERCA2 ratio, reduced incidence of histological changes in the left ventricle, and increased cardiac Ang-(1–7) when compared with the in-vivo functional, and ex-vivo structural and biochemical indices from age-matched SD rats. Moreover, ASrAOGEN rats had lower percent body fat and a superior exercise tolerance when compared with SD rats of the same age.
Our data indicate that the central RAS plays a role in the maintenance of diastolic function and exercise tolerance in mid-life and this may be related to effects on body habitus.
brain; cardiac aging; diastolic dysfunction; renin–angiotensin system; SERCA2; tissue Doppler
To investigate the G protein-coupled receptor kinase 2 (GRK 2) level in peripheral blood lymphocytes with cardiac function in elderly patients with acute myocardial infarction.
This study enrolled 40 patients with acute ST-segment elevation myocardial infarction (STEMI) and 40 patients with unstable angina. All patients were 65 years or older. Cardiac function was evaluated by echocardiography, and the GRK 2 level in peripheral blood lymphocytes was measured. Patients with STEMI were followed up for 2 years.
The GRK 2 level in peripheral blood lymphocytes was significantly higher in patients with STEMI than in patients with unstable angina, and was negatively correlated with left ventricular ejection fraction, cardiac output, stroke volume, and left ventricular fractional shortening. The GRK 2 level was significantly elevated in some patients with acute STEMI and poor cardiac function.
Increased GRK 2 level in patients with acute STEMI may contribute to poor myocardial systolic function and myocardial remodeling. Measurement of the GRK 2 level in peripheral blood lymphocytes may assist in the evaluation of cardiac function and myocardial remodeling in elderly patients with acute STEMI.
ST-segment elevation myocardial infarction; Heart function; G protein-coupled receptor kinase 2
When releasing data to the public, data stewards are ethically and often legally obligated to protect the confidentiality of data subjects’ identities and sensitive attributes. They also strive to release data that are informative for a wide range of secondary analyses. Achieving both objectives is particularly challenging when data stewards seek to release highly resolved geographical information. We present an approach for protecting the confidentiality of data with geographic identifiers based on multiple imputation. The basic idea is to convert geography to latitude and longitude, estimate a bivariate response model conditional on attributes, and simulate new latitude and longitude values from these models. We illustrate the proposed methods using data describing causes of death in Durham, North Carolina. In the context of the application, we present a straightforward tool for generating simulated geographies and attributes based on regression trees, and we present methods for assessing disclosure risks with such simulated data.
Confidentiality; disclosure; dissemination; spatial; synthetic; tree
Controlling the size distribution of polymer-based nanoparticles is a challenging task due to their flexible core and surface structures. To accomplish such as task requires a very precise control at the molecular level. Here, we demonstrate a new approach whereby uniform-sized supramolecular nanoparticles (SNPs) can be reliably generated using a digital microfluidic droplet generator (DMDG) chip. A microfluidic environment enabled precise control over the processing parameters and, therefore, high batch-to-batch reproducibility and robust production of SNPs with a very narrow size distribution could be realized. Digitally adjusting the mixing ratios of the building blocks on the DMDG chip allowed us to rapidly scan a variety of synthesis conditions without consuming significant amounts of reagents. Nearly uniform SNPs with sizes ranging from 35 to 350 nm were obtained and characterized by transmission electron microscopy and dynamic light scattering. In addition, we could fine-tune the surface chemistry of the SNPs by incorporating an additional building block functionalized with specific ligands for targeting cells. The sizes and surface properties of these SNPs correlated strongly with their cell uptake efficiencies. This study showed a feasible microfluidic-assisted SNP production and provided a great means for preparing size-controlled SNP with desired surface ligand coverage.
To determine the biological effectiveness of single, fractionated and continuous low dose rate irradiation on the human colorectal cancer cell line CL187 in vitro and explore the cellular mechanisms.
Materials and methods
The CL187 cells were exposed to radiation of 6 MV X-ray at a high dose rate of 4Gy/min and 125I seed at a low dose rate of 2.77 cGy/h. Three groups were employed: single dose radiation group (SDR), fractionated dose radiation group (FDR) by 2Gy/f and continuous low dose rate radiation group (CLDR). Four radiation doses 2, 4, 6 and 8Gy were chosen and cells without irradiation as the control. The responses of CL187 cells to distinct modes of radiation were evaluated by the colony-forming assay, cell cycle progression as well as apoptosis analysis. In addition, we detected the expression patterns of DNA-PKcs, Ku70 and Ku80 by Western blotting.
The relative biological effect for 125I seeds compared with 6 MV X-ray was 1.42. 48 hrs after 4Gy irradiation, the difference between proportions of cells at G2/M phase of SDR and CLDR groups were statistically significant (p = 0.026), so as the FDR and CLDR groups (p = 0.005). 48 hrs after 4Gy irradiation, the early apoptotic rate of CLDR group was remarkably higher than SDR and FDR groups (CLDR vs. SDR, p = 0.001; CLDR vs. FDR, p = 0.02), whereas the late apoptotic rate of CLDR group increased significantly compared with SDR and FDR group (CLDR vs. SDR, p = 0.004; CLDR vs. FDR, p = 0.007). Moreover, DNA-PKcs and Ku70 expression levels in CLDR-treated cells decreased compared with SDR and FDR groups.
Compared with the X-ray high dose rate irradiation, 125I seeds CLDR showed more effective induction of cell apoptosis and G2/M cell cycle arrest. Furthermore, 125I seeds CLDR could impair the DNA repair capability by down-regulating DNA-PKcs and Ku70 expression.
125I seeds; CL187 cell; DNA repair
Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNaseI, leaving nucleotide-resolution footprints. Using genomic DNaseI footprinting across 41 diverse cell and tissue types, we detected 45 million factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNaseI cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50 base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation, and pluripotency.
chromatin; protein occupancy; DNaseI footprinting; ENCODE; regulation
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world,and the identification of biomarkers for the early detection is a relevant target. The purpose of the study is to discover specific low molecular weight (LMW) serum peptidome biomarkers and establish a diagnostic pattern for HCC.
We undertook this pilot study using a combined application of magnetic beads with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique and ClinPro Tools v2.2 to detect 32 patients with HCC, 16 patients with chronic hepatitis (CH), 16 patients with liver cirrhosis (LC) and 16 healthy volunteers.
The results showed 49, 33 and 37 differential peptide peaks respectively appeared in HCC, LC and CH groups. A Supervised Neural Network (SNN) algorithm was used to set up the classification model. Eleven of the identified peaks at m/z 5247.62, 7637.05, 1450.87, 4054.21, 1073.37, 3883.64, 5064.37, 4644.96, 5805.51, 1866.47 and 6579.6 were used to construct the peptides patterns. According to the model, we could clearly distinguish between HCC patients and healthy controls as well as between LC or CH patients and healthy controls.
The study demonstrated that a combined application of magnetic beads with MALDI-TOF MB technique was suitable for identification of potential serum biomarkers for HCC and it is a promising way to establish a diagnostic pattern.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1503629821958720.
Hepatocellular carcinoma; Chronic hepatitis; Liver cirrhosis; Magnetic beads; Matri-assisted laser desorption/ionization time-of-flight mass spectrometry; Serum biomarkers
This study investigated the efficacy of a combination gene therapy to repress IL-1 and RANKL for the treatment of particulate debris-induced aseptic loosening, and tried to explore the molecular mechanism the exogenous gene modifications on osteoclastogenesis. RAW cells activated by titanium particles were transduced with DFG-IL-1Ra and AAV-OPG individually or in combination for 4 weeks. Pro-inflammatory cytokines in culture media were determined by ELISA, and gene expressions of RANK, IL-1β, c-Fos, TRAF6, JNK1, and CPK were examined using real-time PCR. An established knee-implant-failure mouse model was employed to evaluate the efficacy of the in vivo double-gene therapy. The surgical implantation of a titanium alloy pin into the proximal tibia was followed by monthly challenge with titanium debris. Peri-implant gene transfers of IL-1Ra and OPG (respectively or in combination) were given three weeks after surgery. The combination of OPG and IL-1Ra gene transfer exhibited strong synergetic effects in blockage of inflammation and osteoclastogenesis at 8-weeks after gene modification. The combination therapy reversed peri-implant bone resorption and restored implant stability when compared with either single gene transduction. Real-time PCR data indicated that the action of IL-1Ra gene therapy may be mediated via the JNK1 pathway, while the reduction of osteoclastogenesis by OPG gene modification may be regulated by c-Fos expression. In addition, both gene modifications resulted in significantly diminishment of TRAF6 expression.
periprosthetic osteolysis; osteoclastogenesis; synergetic effects
In this paper, we present a data mining approach to address challenges in the matching of heterogeneous datasets. In particular, we propose solutions to two problems that arise in integrating information from different results of scientific research. The first problem, attribute matching, involves discovery of correspondences among distinct numeric features (attributes) that are used to characterize datasets that have been collected and analyzed in different research labs. The second problem, cluster matching, involves discovery of matchings between patterns (clusters) across datasets. We treat both of these problems together as a multi-objective optimization problem. A multi-objective metaheuristics algorithm is described to find the optimal solution and compared with the genetic algorithm. The utility of this approach is demonstrated in a series of experiments using synthetic and realistic datasets that are designed to simulate heterogeneous data from different sources.
Multi-Objective Optimization; Cluster Matching; Attribute Matching; Metaheuristics
Active and passive immunizations with Aβ and Aβ antibodies successfully reduced AD pathology and improved cognitive functions in an AD mouse model. However, human clinical trials of vaccination with synthetic Aβ(AN1792), were halted due to brain inflammation, presumably induced by T cell-mediated immune response. In this study, we used Picha pastoris to produce a recombinant peptide vaccine, r4 × Aβ15(recombinant 4 × Aβ15), four tandem repeats of Aβ(1-15) interlinked by spacers . Wild-type mice were injected subcutaneously with CFA/IFA as adjuvant. r4 × Aβ15 vaccine elicited high titer anti-Aβ antibodies which bound to Aβ plaque in brain tissue from Tg2576 mouse. The antibody isotype was mainly IgG(1), indicating anti-inflammatory Th2 type. There was no splenocyte proliferation against Aβ peptide, which indicates that the r4 × Aβ15 vaccine does not induce Aβ-specific T cellular immune response. Thus, r4 × Aβ15 vaccine may be a safe and efficient vaccine for AD.
Alzheimer disease (AD); C57BL/6 mice; P.pastoris; T cellular immune response; r4×Aβ15 vaccine
No proven pharmacological therapies to delay or reverse age-related diastolic dysfunction exist. We hypothesized that late-life low-dose (non-blood-pressure-lowering) angiotensin-converting enzyme inhibition vs. angiotensin II receptor blockade would be equally efficacious at mitigating diastolic dysfunction in the senescent Fischer 344 × Brown Norway rat. Enalapril (10 mg/kg/day; n = 9) initiated at 24 months of age and continued for 6 months, increased myocardial relaxation (e'), reduced Doppler-derived indices of filling pressure (E/e'), favorably lowered the ratio of phospholamban–SERCA2 and reduced oxidative stress markers, Rac1 and nitrotyrosine, in aged hearts. Treatment with losartan (15 mg/kg/day; n = 9) similarly mitigated signs of cardiac oxidative stress, but impairments in diastolic function persisted when compared with untreated rats (n = 7). Our findings favor the idea that the lusitropic benefit of low-dose angiotensin-converting enzyme inhibitor initiated late in life may be related to an antioxidant-mediated modulation of SERCA2, resulting in improved relaxation rather than via overt effects on cardiac structure or blood pressure.
Angiotensin-converting enzyme inhibitor; Angiotensin II receptor blocker; Diastolic dysfunction; Oxidative stress; SERCA2; Tissue Doppler
In this study, we sought to investigate the expression of the transcription factor E2F1 in chicken pulmonary arterial smooth muscle cells upon hypoxia exposure, as well as the role that E2F1 played in the regulation of cell proliferation. Isolated chicken pulmonary arterial smooth muscle cells were subjected to hypoxia or normoxia for indicated time points. Cell viability, DNA synthesis, cell cycle profile, and expression of E2F1 were analyzed. The results showed that hypoxia promoted cell proliferation and DNA synthesis which was accompanied by an increased S phase entry and upregulation of E2F1 at mRNA and protein levels. Using siRNA technology, we demonstrated that gene inactivation of endogenous E2F1 abolished hypoxia-induced cell proliferation, DNA synthesis, and S phase entry compared with negative siRNA transfected cells. These results suggest that hypoxia-induced proliferation is mediated by inducing E2F1 in chicken pulmonary arterial smooth muscle cells.
E2F1; Hypoxia; Proliferation; Pulmonary arterial smooth muscle cells