Search

In an effort to develop a new therapy for cancer and to improve antiprogrammed death inhibitor-1 (anti-PD-1) and anticytotoxic T lymphocyte-associated protein (anti-CTLA-4) responses, we have created a telomerase reverse transcriptase promoter-regulated oncolytic adenovirus rAd.sT containing a soluble transforming growth factor receptor II fused with human IgG Fc fragment (sTGFβRIIFc) gene. Infection of breast and renal tumor cells with rAd.sT produced sTGFβRIIFc protein with dose-dependent cytotoxicity. In immunocompetent mouse 4T1 breast tumor model, intratumoral delivery of rAd.sT inhibited both tumor growth and lung metastases. rAd.sT downregulated the expression of several transforming growth factor β (TGFβ) target genes involved in tumor growth and metastases, inhibited Th2 cytokine expression, and induced Th1 cytokines and chemokines, and granzyme B and perforin expression. rAd.sT treatment also increased the percentage of CD8(+) T lymphocytes, promoted the generation of CD4(+) T memory cells, reduced regulatory T lymphocytes (Tregs), and reduced bone marrow-derived suppressor cells. Importantly, rAd.sT treatment increased the percentage of CD4(+) T lymphocytes, and promoted differentiation and maturation of antigen-presenting dendritic cells in the spleen. In the immunocompetent mouse Renca renal tumor model, similar therapeutic effects and immune activation results were observed. In the 4T1 mammary tumor model, rAd.sT improved the inhibition of tumor growth and lung and liver metastases by anti-PD-1 and anti-CTLA-4 antibodies. Analysis of the human breast and kidney tumors showed that a significant number of tumor tissues expressed high levels of TGFβ and TGFβ-inducible genes. Therefore, rAd.sT could be a potential enhancer of anti-PD-1 and anti-CTLA-4 therapy for treating breast and kidney cancers.

The advent of next generation DNA sequencing (NGS) has revolutionized clinical medicine by enabling wide‐spread testing for genomic anomalies and polymorphisms. With that explosion in testing, however, come several informatics challenges including managing large amounts of data, interpreting the results and providing clinical decision support. We present Flype, a web‐based bioinformatics platform built by a small group of bioinformaticians working in a community hospital setting, to address these challenges by allowing us to: (a) securely accept data from a variety of sources, (b) send orders to a variety of destinations, (c) perform secondary analysis and annotation of NGS data, (d) provide a central repository for all genomic variants, (e) assist with tertiary analysis and clinical interpretation, (f) send signed out data to our EHR as both PDF and discrete data elements, (g) allow population frequency analysis and (h) update variant annotation when literature knowledge evolves. We discuss the multiple use cases Flype supports such as (a) in‐house NGS tests, (b) in‐house pharmacogenomics (PGX) tests, (c) dramatic scale‐up of genomic testing using an external lab, (d) consumer genomics using two external partners, and (e) a variety of reporting tools. The source code for Flype is available upon request to the authors.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor β-1 (TGFβ-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFβRIIFc, a TGFβ decoy that can inhibit TGFβ pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFβRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

Germline mutations in BRCA1/2 and ATM have been associated with prostate cancer (PCa) risk.To directly assess whether germline mutations in these three genes distinguish lethal from indolent PCa and whether they confer any effect on age at death.A retrospective case-case study of 313 patients who died of PCa and 486 patients with low-risk localized PCa of European, African, and Chinese descent. Germline DNA of each of the 799 patients was sequenced for these three genes.Mutation carrier rates and their effect on lethal PCa were analyzed using the Fisher's exact test and Cox regression analysis, respectively.The combined BRCA1/2 and ATM mutation carrier rate was significantly higher in lethal PCa patients (6.07%) than localized PCa patients (1.44%), p=0.0007. The rate also differed significantly among lethal PCa patients as a function of age at death (10.00%, 9.08%, 8.33%, 4.94%, and 2.97% in patients who died ≤ 60 yr, 61-65 yr, 66-70 yr, 71-75 yr, and over 75 yr, respectively, p=0.046) and time to death after diagnosis (12.26%, 4.76%, and 0.98% in patients who died ≤ 5 yr, 6-10 yr, and10 yr after a PCa diagnosis, respectively, p=0.0006). Survival analysis in the entire cohort revealed mutation carriers remained an independent predictor of lethal PCa after adjusting for race and age, prostate-specific antigen, and Gleason score at the time of diagnosis (hazard ratio=2.13, 95% confidence interval: 1.24-3.66, p=0.004). A limitation of this study is that other DNA repair genes were not analyzed.Mutation status of BRCA1/2 and ATM distinguishes risk for lethal and indolent PCa and is associated with earlier age at death and shorter survival time.Prostate cancer patients with inherited mutations in BRCA1/2 and ATM are more likely to die of prostate cancer and do so at an earlier age.

Myocardial physiology in the aftermath of myocardial infarction (MI) before remodeling is an under-explored area of investigation. Here, we describe the effects of MI on the cardiac sarcomere with focus on the possible contributions of reactive oxygen species (ROS). We surgically induced MI in 6–7 month old female CD1 mice by ligation of the left anterior descending coronary artery. Data were collected 3–4 days after MI or sham surgery (SH). MI hearts demonstrated ventricular dilatation and systolic dysfunction upon echo cardiographic analysis. Sub-maximum Ca-activated tension in detergent extracted fiber bundles from papillary muscles increased significantly in the preparations from MI hearts. Ca++ sensitivity increased after MI, whereas cooperativity of activation decreased. To assess myosin enzymatic integrity we measured splitting of CaATP in myofibrillar preparations, which demonstrated a decline in CaATPase activity of myofilament myosin. Biochemical analysis demonstrated post-translational modification of sarcomeric proteins. Phosphorylation of cardiac troponin I (cTnI) and myosin light chain 2 was reduced after MI in papillary samples, as measured using a phospho-specific stain. Tropomyosin was oxidized after MI, forming disulfide products detectable by diagonal nonreducing-reducing SDS-PAGE. Our analysis of myocardial protein oxidation post-MI also demonstrated increased S-glutathionylation. We functionally linked protein oxidation with sarcomere function by treating skinned fibers with the sulfhydryl reducing agent dithiothreitol, which reduced Ca++ sensitivity in MI, but not SH, samples. Our data indicate important structural and functional alterations to the cardiac sarcomere after MI, and the contribution of protein oxidation to this process.

Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1h without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissues. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of

Background: Genetic interactions play a critical role in cancer development. Existing knowledge about cancer genetic interactions is incomplete, especially lacking evidences derived from large-scale cancer genomics data. The Cancer Genome Atlas (TCGA) produces multimodal measurements across genomics and features of thousands of tumors, which provide an unprecedented opportunity to investigate the interplays of genes in cancer. Methods: We introduce Zodiac, a computational tool and resource to integrate existing knowledge about cancer genetic interactions with new information contained in TCGA data. It is an evolution of existing knowledge by treating it as a prior graph, integrating it with a likelihood model derived by Bayesian graphical model based on TCGA data, and producing a posterior graph as updated and data-enhanced knowledge. In short, Zodiac realizes “Prior interaction map + TCGA data → Posterior interaction map.” Results: Zodiac provides molecular interactions for about 200 million pairs of genes. All the results are generated from a big-data analysis and organized into a comprehensive database allowing customized search. In addition, Zodiac provides data processing and analysis tools that allow users to customize the prior networks and update the genetic pathways of their interest. Zodiac is publicly available at www.compgenome.org/ZODIAC. Conclusions: Zodiac recapitulates and extends existingmore » knowledge of molecular interactions in cancer. It can be used to explore novel gene-gene interactions, transcriptional regulation, and other types of molecular interplays in cancer.« less

Summary Neuronal microtubules support intracellular transport, facilitate axon growth, and form a basis for neuronal morphology. While microtubules in nonneuronal cells are depolymerized by cold, Ca 2+ , or antimitotic drugs, neuronal microtubules are unusually stable. Such stability is important for normal axon growth and maintenance, while hyperstability may compromise neuronal function in aging and degeneration. Though mechanisms for stability are unclear, studies suggest that stable microtubules contain biochemically distinct tubulins that are more basic than conventional tubulins. Transglutaminase-catalyzed posttranslational incorporation of polyamines is one of the few modifications of intracellular proteins that add positive charges. Here we show that neuronal tubulin can be polyaminated by transglutaminase. Endogenous brain transglutaminase-catalyzed polyaminated tubulins have the biochemical characteristics of neuronal stable microtubules. Inhibiting polyamine synthesis or transglutaminase activity significantly decreases microtubule stability in vitro and in vivo. Together, these findings suggest that transglutaminase-catalyzed polyamination of tubulins stabilizes microtubules essential for unique neuronal structures and functions.

The Ets proteins are a family of transcription factors characterized by an evolutionarily conserved DNA binding domain that controls key cellular processes. Prostate-derived Ets transcription factor (PDEF), a member of the Ets family, is reported to be present in tissues with high epithelial content, notably breast and prostate. However, the role of PDEF in cancer development is not fully understood. To gain insight into the molecular mechanisms associated with prostate cancer progression, we employed iTRAQ labeling followed by mass spectrometric (MS) analysis to identify candidate proteins that are differentially expressed in prostate cancer cells with or without PDEF. To this end, we overexpressed PDEF in PC3 human prostate cells using a tetracycline inducible system (Tet-On). Many differentially expressed proteins which play important roles in various cellular and biological processes were identified. Among them, stathmin (STMN), which is a microtubule (MT)-destabilizing protein, was found to be downregulated in multiple analyses. We demonstrated that re-expression of STMN reversed the antitumor properties of PDEF in PDEF-overexpressing PC3 cells. Using in vitro functional assays, we showed that STMN overexpression counteracted PDEF's effects against cell proliferation, colony formation and tumor migration. Similar results were further confirmed with the prostate cancer cell line CWR22rv1. In conclusion, many differentially expressed proteins were identified and STMN was found to be downregulated by PDEF. These results suggest that PDEF may inhibit prostate cancer progression by transcriptional downregulation of oncogenic STMN expression. Analyzing the association among differentially expressed proteins may provide a basis to better understand the molecular mechanisms underlying the process of cancer progression and development and further aid in designing therapeutics in the future.

Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate because of physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5-10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear that this behavior is highly complex and needs to be further explored.

Background It has been an abiding belief among geneticists that multicellular organisms’ genomes can be analyzed under the assumption that a single individual has a uniform genome in all its cells. Despite some evidence to the contrary, this belief has been used as an axiomatic assumption in most genome analysis software packages. In this paper we present observations in human whole genome data, human whole exome data and in mouse whole genome data to challenge this assumption. We show that heterogeneity is in fact ubiquitous and readily observable in ordinary Next Generation Sequencing (NGS) data. Results Starting with the assumption that a single NGS read (or read pair) must come from one haplotype, we built a procedure for directly observing haplotypes at a local level by examining 2 or 3 adjacent single nucleotide polymorphisms (SNPs) which are close enough on the genome to be spanned by individual reads. We applied this procedure to NGS data from three different sources: whole genome of a Central European trio from the 1000 genomes project, whole genome data from laboratory-bred strains of mouse, and whole exome data from a set of patients of head and neck tumors. Thousands of loci were found in each genome where reads spanning 2 or 3 SNPs displayed more than two haplotypes, indicating that the locus is heterogeneous. We show that such loci are ubiquitous in the genome and cannot be explained by segmental duplications. We explain them on the basis of cellular heterogeneity at the genomic level. Such heterogeneous loci were found in all normal and tumor genomes examined. Conclusions Our results highlight the need for new methods to analyze genomic variation because existing ones do not systematically consider local haplotypes. Identification of cancer somatic mutations is complicated because of tumor heterogeneity. It is further complicated if, as we show, normal tissues are also heterogeneous. Methods for biomarker discovery must consider contextual haplotype information rather than just whether a variant “is present”. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-418) contains supplementary material, which is available to authorized users.

Subcellular fractionation of a complex proteome (cardiac tissue) allows for enrichment of a subset of low abundant proteins. This permits more in depth analysis of the proteome by reducing the complexity. Adult rat cardiac tissue was made ischemic by ligating the left anterior descending coronary artery in vivo for 1 hour with no reperfusion, and the healthy remote area was differentiated from the ischemic tissue by staining with Evans Blue dye after harvesting the heart. A series of differential centrifugation steps produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric fractions of rat ischemic and remote healthy tissue. The sarcomeric fractions of the remote vs ischemic cardiac tissue were digested with trypsin, and the peptides were labeled with isobaric tags for relative quantitation (iTRAQ). The labeled peptides were then fractionated with an Agilent 3100 OFFGEL fractionator, which separated the peptides in 12 fractions based on their isoelectric point from pH 3-10. The 12 fractions from the OFFGEL were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode to detect the low mass reporter ions of the isobaric tags on the peptides. The peptides were analyzed with MASCOT (MatrixScience), and Scaffold v2.5.2 Q+ with a minimum of two unique peptides and a level of confidence set at 95%. Five-fold more proteins were identified when the labeled digests were fractionated with OFFGEL compared to no fractionation prior to LC-MS/MS. With a one hour ischemic event, we found approximately 11% of the detected proteins in the sarcomeric fraction had changed at least 1.5 fold. Therefore, this in-solution method incorporating sub-proteomic fractionation in conjunction with OFFGEL separation may be an approach for discovery of relative protein changes in cardiac tissue.

The thermally induced in vitro self-assembly of collagen molecules to form active fibrils illustrates that collagen molecules themselves contain all of the structural information necessary for assembly. The molecule contains three structural domains, the NH2 and carboxyl-terminal extra helical regions (the telopeptides) and the major triple helical rod-like domain. Proteolytic removal of the short telopeptide domains drastically alters the in vitro self-assembly process. We have examined the specific contributions of each telopeptide to the initiation ("nucleation") and growth stages of self-assembly in collagens modified by selective proteinase treatment and by isolating a peptide containing the amino telopeptide and adding this to both normal and proteinase-modified collagen self-assembly systems. Pronase-modified collagen, devoid of both telopeptides, initiated self-assembly very poorly. Addition of small amounts of intact collagen accelerated the rate of nucleation of pronase-modified collagen. Addition of carboxypeptidase-modified collagen also accelerated the nucleation of pronase-modified collagen, suggesting that the remaining amino telopeptide was involved in nucleation. This was confirmed by isolating the cyanogen bromide fragment of the alpha 1(I) subunit containing the amino telopeptide and finding that it specifically accelerated the nucleation of intact pepsin- and pronase-modified to collagens. The amino telopeptide appears to bind to a specific region within the collagen triple helical domain. The isolated peptide requires thermal pretreatment to be active; hence, this interaction must involve a unique telopeptide conformation. This behavior is compatible with the recent model (Helseth, D. L., Jr., Lechner, J. H., and Veis, A. (1979) Biopolymers 18, 3005-3014) proposed for the conformation of the amino telopeptide and its interaction with a helical receptor site as a step in nucleation. Comparison of the behavior of leucine aminopeptidase- and carboxypeptidase-modified collagens suggests that the carboxyl telopeptide has its major role in the growth stages of self-assembly.

The amino-terminal telopeptide of the collagen α1(I) chain has a highly conserved sequence. This sequence was analyzed by the Chou-Fasman criteria, and a folded β-sheet conformation, including a β-turn, was predicted. This folded “hairpin” region favors both ionic and hydrophobic intermolecular interactions with α1(I) chain residues 930–938 on a neighboring, end-overlapped molecule. An end-overlap interaction of this nature could direct the initial step in fibril formation. The predicted structure also places the potential crosslink-forming lysyl residue, 9N, in a unique site at the β-turn end of the telopeptide.

The proteolytic removal of the extension COOH-terminal propeptide from procollagen has been examined in vitro. A crude enzyme activity was identified in a whole-chicken-embryo extract that acted at acid pH and appeared to be similar to one identified previously [Davidson, J. M., McEneany , L. S. G. & Bornstein , P. (1979) Eur. J. Biochem. 100, 551-558]. This activity was inhibitable by pepstatin but not by leupeptin, suggesting that it might be cathepsin D. Cathepsin D was purified 907-fold from chicken livers by affinity chromatography on pepstatin-aminohexyl-Sepharose 4B and was found to remove the COOH propeptides from procollagen. At pH 6.0, the site of cleavage appeared to shift from the COOH telopeptide to the COOH telopeptide/propeptide junction, based upon the difference in electrophoretic migration of the cleavage products, although determining the actual cleavage site will require end-group analysis. A model for the involvement of cathepsin D in the in vivo processing of procollagen is presented.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c : oxygen oxidoreductase (EC 1.9.3.1.), monoamine: oxygen oxidoreductase (deaminating) (EC 1.4.3.4), rotenoneinsensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, ( Na + − K + )- activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.

As part of a study of the suitability of new materials for use as a retrofilling material, we examined the polymerization properties of Cavalite, a light-cured, hydroxyapatite and glass ionomer-containing cavity liner. By varying the time of photopolymerization, it was found that polymerization for 20 to 30 seconds according to the manufacturer's recommendations is not sufficient to ensure complete polymerization. The implications of this incomplete polymerization are discussed in terms of possible cytotoxic effects on tissues exposed to unpolymerized Cavalite, both when used in retrofilling situations and as a deep cavity liner.

Chick brain cortical slices and crude mitochondrial fractions were fixed with glutaraldehyde, stained only with cis-Pt (II)-uracil and processed for electron microscopy. The optimal time of staining was determined to be 10 min. Results show that this platinum-pyrimidine complex is a relatively specific stain for the nucleic acids of brain slices. However, staining of crude mitochondrial fractions apparently resulted in some protein staining and other artifacts. The method should be helpful identifying ribosomal contamination of subcellular preparations and if its specificity can be increased it may prove a useful addition to staining methods of the electron microscopist.