Dendritic cell targeting of survivin protein in a xenogeneic form elicits strong CD4+ T cell immunity to mouse survivin

To determine whether strong CD4+ T cell immunity could be induced to a nonmutated self protein that is important for tumorigenesis, we selectively targeted the xenogeneic form of survivin, a survival protein overexpressed in tumors, to maturing dendritic cells in lymphoid tissues. Dendritic cell targeting via the DEC205 receptor in the presence of anti-CD40 and poly(I:C) as maturation stimuli, induced strong human and mouse survivin-specific CD4+ T cell responses, as determined by IFN-gamma, TNF-alpha, and IL-2 production, as well as the development of lytic MHC class II-restricted T cells and memory. Immunity was enhanced further by depletion of CD25+foxp3+ cells before vaccination. anti-DEC205-human survivin was superior in inducing CD4+ T cell responses relative to other approaches involving survivin plasmid DNA or survivin peptides with adjuvants. However, we were unable to induce CD8+ T cell immunity to survivin by two doses of DEC205-targeted survivin or the other strategies. Therefore, significant CD4+ T cell immunity to a self protein that is overexpressed in most human cancers can be induced by DEC205 targeting of the Ag in its xenogeneic form to maturing DCs.     

Retention of 45Ca in rats and lambs associated with the onset of nutritional muscular dystrophy.

Nutritional myopathy has been induced in both rats and lambs by feeding diets low in selenium. The distribution of 45Ca, administered as 45CaCl2, has been examined firstly by autoradiography, and secondly by measuring the excretion of 45Ca in urine and faeces. Autoradiographs of skeletal muscle from unsupplemented animals showed radioactivity over discrete muscle fibres at a stage when no abnormalities were apparent using conventional staining techniques. Similar retention of 45Ca was found in some of the tubules in the kidneys of selenium-deficient rats. Total excretion in urine and faeces of lambs, examined for 48 h after intravenous administration of 45CaCl2, showed that in normal animals 18-6% of the dose was excreted, whereas in dystrophic lambs 12-0% was lost. The difference was significant at the 2% level. The respiratory rates of isolated skeletal muscle mitochondria, measured polarographically in the presence of glutamate and pyruvate as substrates, were low for dystrophic rats. Respiratory control indices were 1-0 for the same preparations but for supplemented rats they were all above 1-0. The differences in respiratory rates were significant at the 1% level. The major conclusion drawn from the results of these experiments is that one of the first effects of sleenium deficiency which can be visualized is the abnormal retention of calcium by individual muscle fibres.     

Role of gamma-synuclein in microtubule regulation

Gamma-synuclein is a neuronal protein found in peripheral and motor nerve systems. It becomes highly expressed in metastatic but not in primary tumor or normal tissues. The close association between gamma-synuclein expression and cancer spreading has been demonstrated in a broad range of malignancies. Our previous study showed that exogenous expression of gamma-synuclein in ovarian and breast cancer cells significantly enhanced cell migration and resistance to paclitaxel-induced apoptotic death. In our current research, we found that gamma-synuclein can affect microtubule properties and act as a functional microtubule associated protein. In vitro assays revealed that gamma-synuclein can bind and promote tubulin polymerization, induce the microtubule bundling and alter microtubule morphology developed in the presence of microtubule associated protein 2 (MAP2). Using cancer cell lysate, gamma-synuclein protein was found to be localized in both cytosolic compartment and extracted cytoskeleton portion. Immunofluorescence staining demonstrated that gamma-synuclein can colocalize with microtubule in HeLa cells and decrease rigidity of microtubule bundles caused by paclitaxel. In human ovarian cancer epithelial A2780 cells, gamma-synuclein overexpression improved cell adhesion and microtubule structure upon paclitaxel treatment. Importantly, it led to microtubule-dependent mitochondria clustering at perinuclear area. These observations suggest that overexpression of gamma-synuclein may reduce cell chemo-sensitivity of tumor cells through decreasing microtubule rigidity. In summary, our studies suggested that gamma-synuclein can directly participate in microtubule regulation.     

IGF1R signaling in Ewing sarcoma is shaped by clathrin-/caveolin-dependent endocytosis

Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling.     

Multiple statistical analysis techniques corroborate intratumor heterogeneity in imaging mass spectrometry datasets of myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information.     

Large‐scale GWAS in sorghum reveals common genetic control of grain size among cereals

Grain size is a key yield component of cereal crops and a major quality attribute. It is determined by a genotype’s genetic potential and its capacity to fill the grains. This study aims to dissect the genetic architecture of grain size in sorghum. An integrated genome‐wide association study (GWAS) was conducted using a diversity panel (n = 837) and a BC‐NAM population (n = 1421). To isolate genetic effects associated with genetic potential of grain size, rather than the genotype’s capacity to fill the grains, a treatment of removing half of the panicle was imposed during flowering. Extensive and highly heritable variation in grain size was observed in both populations in 5 field trials, and 81 grain size QTL were identified in subsequent GWAS. These QTL were enriched for orthologues of known grain size genes in rice and maize, and had significant overlap with SNPs associated with grain size in rice and maize, supporting common genetic control of this trait among cereals. Grain size genes with opposite effect on grain number were less likely to overlap with the grain size QTL from this study, indicating the treatment facilitated identification of genetic regions related to the genetic potential of grain size. These results enhance understanding of the genetic architecture of grain size in cereal, and pave the way for exploration of underlying molecular mechanisms and manipulation of this trait in breeding practices.     

IRAP,a retrotransposon-based marker system for the detection of somaclonal variation in barley

The retrotransposon-based marker system, inter-retrotransposon amplified polymorphism (IRAP), and inter-simple sequence repeats (ISSRs) were used to detect somaclonal variation induced by tissue culture. IRAPs use a single primer designed to amplify out from the 5′ LTR sequence of the BARE-1 retrotransposon combined with a degenerate 3′ anchor, similar to that of ISSR primers. We analysed DNA polymorphisms in 147 primary regenerants and parental controls from three cultivars of barley (Hordeum vulgare). The ISSR marker system generated an average of 218 bands per primer, with 29 polymorphisms of which 12 were novel non-parental bands. In comparison, the IRAP system generated an average of 121 bands per primer, with 15 polymorphisms of which nine were novel non-parental bands. Polymorphism detected for IRAP and ISSR markers was more than twofold higher in Golden Promise than Mackay and Tallon cultivars. However, there was no significant difference in the frequency of novel non-parental bands. Cluster analysis revealed that the level of polymorphism and genetic variability detected was comparable between IRAP and ISSR markers. This suggests that retrotransposon-based marker systems, such as IRAP, based on retrotransposons such as BARE-1, are valuable tools for the detailed characterisation of mutation profiles that arise during tissue culture. Their use should improve our understanding of processes influencing mutation and somaclonal variation and allow for the design of methods that yield fewer genome changes in applications where maintaining clonal integrity is important.     

Discovery and gram-scale synthesis of BMS-593214, a potent,selective FVIIa inhibitor

A 6-amidinotetrahydroquinoline screening hit was driven to a structurally novel, potent, and selective FVIIa inhibitor through a combination of library synthesis and rational design. An efficient gram-scale synthesis of the active enantiomer BMS-593214 was developed, which required significant optimization of the key Povarov annulation. Importantly, BMS-593214 showed antithrombotic efficacy in a rabbit arterial thrombosis model. A crystal structure of BMS-593214 bound to FVIIa highlights key contacts with Asp 189, Lys 192, and the S2 pocket.