Identification of peanut oleosins using peptide mass fingerprinting and N-terminal sequencing.

<p>Peptides resulting from tryptic digestion were searched against the NCBInr database using the Mascot search engine. Bold letters indicate identified sequences by MALDI-TOF-MS, whereas underlined letters mark sequences obtained after N-terminal sequencing. The hydrophobic domains, not accessible to tryptic cleavage, are written in italics. The methionine residue, highlighted in grey, was not observed.</p

Western blot of oleosin prototypes isolated from roasted peanuts and purified by preparative electrophoresis.

<p>Fractions obtained by preparative electrophoresis containing the prototypes 1/2 (A) and prototypes 3/4 (B) were pooled, subjected to SDS-PAGE and subsequent immunoblotting. M, molecular mass marker; G, Gold staining; CS, Coomassie staining; A, polyclonal anti-oleosin antibody; P1–P4, allergic patients’ sera; P°, allergic individual (not to peanut) with respiratory symptoms; P*, healthy control; C1, secondary antibody control (mouse anti-human IgE antibody); C2, secondary antibody control (goat anti-rabbit IgG antibody).</p

Consensus Micro RNAs Governing the Switch of Dormant Tumors to the Fast-Growing Angiogenic Phenotype

<div><p>Tumor dormancy refers to a critical stage in cancer development in which tumor cells remain occult for a prolonged period of time until they eventually progress and become clinically apparent. We previously showed that the switch of dormant tumors to fast-growth is angiogenesis dependent and requires a stable transcriptional reprogramming in tumor cells. Considering microRNAs (miRs) as master regulators of transcriptome, we sought to investigate their role in the control of tumor dormancy. We report here the identification of a consensus set of 19 miRs that govern the phenotypic switch of human dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors to fast-growth. Loss of expression of dormancy-associated miRs (DmiRs, 16/19) was the prevailing regulation pattern correlating with the switch of dormant tumors to fast-growth. The expression pattern of two DmiRs (miR-580 and 190) was confirmed to correlate with disease stage in human glioma specimens. Reconstitution of a single DmiR (miR-580, 588 or 190) led to phenotypic reversal of fast-growing angiogenic tumors towards prolonged tumor dormancy. Of note, 60% of angiogenic glioblastoma and 100% of angiogenic osteosarcoma over-expressing miR190 remained dormant during the entire observation period of ∼ 120 days. Next, the ability of DmiRs to regulate angiogenesis and dormancy-associated genes was evaluated. Transcriptional reprogramming of tumors via DmiR-580, 588 or 190 over-expression resulted in downregulation of pro-angiogenic factors such as TIMP-3, bFGF and TGFalpha. In addition, a G-CSF independent downregulation of Bv8 was found as a common target of all three DmiRs and correlated with decreased tumor recruitment of bone marrow-derived CD11b+ Gr-1+ myeloid cells. In contrast, antiangiogenic and dormancy promoting pathways such as EphA5 and Angiomotin were upregulated in DmiR over-expressing tumors. This work suggests novel means to reverse the malignant tumor phenotype into an asymptomatic dormant state and may provide promising targets for early detection or prevention of cancer.</p> </div

Differential expression of angiogenesis and dormancy related genes in DmiR expressing tumors.

<p>qRT-PCR analysis of gene expression in glioblastoma cells. Expression levels of genes in miR-580, miR-588 and miR-190 over-expressing A-GBM tumors were normalized to GFP-vector control. DmiR expression induced a transcriptional switch towards antiangiogenesis by down-regulation of multiple angiogenesis related genes (e.g., bFGF (FGF2), TIMP-3, HIF1A and TGFalpha). In contrast antiangiogenic and dormancy promoting pathways were upregulated after DmiR-expression (e.g., Amot-1 or EphA5). These experiments were performed in triplicates and repeated at least twice to confirm similar pattern of differential expression. Significant differential expression as compared to GFP-control is indicated with p<0.001, # p≤0.01 and § p<0.05.</p

MiR-190 expression in osteosarcoma model.

<p>Tumor growth kinetic of vector control (GFP; n = 5) vs. miR-190 (n = 5) expressing angiogenic fast-growing “A-Osteosarcoma” (A). MiR-190 expression led to complete (i.e., 100% inhibition, 5/5) phenotypic reversal of fast-growing osteosarcoma resulting in significant increase of overall survival (p<0.005 by log-rank test) (B). Death event: mouse sacrificed based on large tumor size.</p

Consensus signature of dormancy associated miRs.

<p>A schematic overview of the <i>in-vivo</i> experimental vascular tumor dormancy models employed (A). Dormant tumors are formed after s.c. injection of tumor cells in immunocompromised mice and remain undetectable by gross examination for a prolonged period of time. This dormancy period is characterized by impaired tumor angiogenesis and high levels of cell turnover, i.e., balanced tumor cell proliferation and apoptosis. Tumors spontaneously exit the dormancy phase and switch to a rapid “angiogenic” growth. Differential regulation of miRs in dormant vs. fast-growing tumors (B). 19 miRs were significantly differentially regulated during the switch of dormant tumors to fast-growth in all four dormancy models (p<0.03). The prevailing regulation pattern was the loss of dormancy associated miRs (DmiRs) after the switch of dormant tumors. The heatmap represents fold expression values of miRs in dormant vs. fast-growing tumors according to the scale bar. Red: miRs upregulated in dormant tumors. Green: miRs down-regulated in dormant tumors. MiRs are sorted by hierarchical clustering. The expression level of miR-580, miR-588 and miR-190 was detected in human glioma specimens (C–E). In support of the experimental data, the expression of miR-580 and miR-190 was significantly decreased with advanced tumor grade in glioma specimens. WHO-I (n:3), WHO-III (n:4) and WHO-IV (n:8). *p<0.01, #p = 0.05.</p

Over-expression of DmiRs reversed tumor phenotype.

<p>Tumor growth kinetics of angiogenic fast-growing glioblastoma cells (A-GBM) was monitored <i>in-vivo</i> (A). Average tumor volume of parental tumor (labeled in red line) n = 11, cells infected with GFP only control vector (n = 9), miR-580 (n = 5), miR-588 (n = 4), and miR-190 (n = 5) expressing A-GBM tumors. Kaplan-Meier analysis of tumor free survival (B). The criterion for “tumor free” was defined as mice bearing no detectable tumors by gross examination or tumors with volume smaller than 50 mm³. The median tumor free survival was markedly increased in miR-580 and miR-588 expressing tumors as compared to the GFP control. 60% of miR-190 expressing tumors remained dormant for the whole observation period. Statistical significance was reached for miR-190 (p<0.03) and miR-580 (p = 0.05), respectively, by log-rank test. The dormancy promoting effect of DmiRs was not attributed to impaired proliferation kinetic or enhanced apoptosis of DmiR expressing tumor cells <i>in-vitro</i> (C). In contrast, there was a trend towards higher proliferation rates and reduced apoptosis in miR-580 expressing cells (C). Proliferating tumor cells were also detected by Ki67 immunohistochemistry and representative photomicrographs are shown (D) in dormant miR-588 expressing GBMs (107days post implantation) and fast-growing GFP expressing control tumors (day 113 after implantation). <i>In-vivo</i> proliferation index was determined by counting the fraction of Ki67+ tumor cells. This analysis revealed no significant difference between the dormant miR-588 and the fast growing control (GFP) tumors (p = 0.12). Enhanced apoptosis was detected <i>in-vivo</i> in miR-588 vs. control (GFP) tumors by TUNEL staining (E). A threefold increase in the fraction of TUNEL+ apoptotic tumor cells was found in dormant miR-588 expressing- vs. control tumors (* p<0.0001).</p

Reduced recruitment of myeloid BMDCs in DmiR expressing tumors.

<p>RT-PCR analysis of Bv8 level in DmiR over-expressing cells (A). All three DmiRs potently inhibit the expression of Bv8. Evaluation of Gr1 and CD11b positive bone marrow-derived myeloid cells in DmiR vs. control (GFP) expressing A-GBM tumors (B). MiR-588-GFP or vector-control- GFP expressing A-GBM tumors were stained for Gr1/CD11b myeloid cell- or CD31 vascular endothelial marker. Images of the same plain with detectors for GFP or antibody staining are shown. Scale bar represent 20 µm. Tumors were collected 73 days following injection to mice. Images were taken as representatives of at least three different tumors per group. Impaired mobilization of Gr1 and CD11b positive myeloid cells in circulation of miR-588 tumor bearing mice (C). Percentage of Gr1 or CD11b expressing cells among CD45 positive cells in circulation of mice bearing fast-growing glioblastoma expressing either GFP (black bars) or miR-588 (dotted bars) was determined using FACS analysis. Error bars represent SEM, ** p<0.02.</p

Additional file 1: of Quantitative assessment of radiation dose and fractionation effects on normal tissue by utilizing a novel lung fibrosis index model

Table S1. A list of experimentally derived mouse lung α/β ratios from the literatures with special reference to late lung damage. Data is presented as Mean ± SE. (B.R. = breathing rate, F = female, BPM = breath per minute, M = male, d = day, wk. = week). Figure S1. Reciprocal total isoeffect dose for ED 50 as a function of dose per fraction. The data points were simulated using eq. (3). The α/β was obtained as the ratio of the intercept and the slope of the line using the conventional Fe plot. Appendix. CT histogram profiling in differential diagnosis of emphysema or pleural effusions. (DOCX 350 kb

Image_4_The impact of tumor metabolic activity assessed by 18F-FET amino acid PET imaging in particle radiotherapy of high-grade glioma patients.jpeg

BackgroundSelective uptake of (18)F-fluoro-ethyl-tyrosine (18F-FET) is used in high-grade glioma (HGG) to assess tumor metabolic activity via positron emission tomography (PET). We aim to investigate its value for target volume definition, as a prognosticator, and associations with whole-blood transcriptome liquid biopsy (WBT lbx) for which we recently reported feasibility to mirror tumor characteristics and response to particle irradiation in recurrent HGG (rHGG).Methods18F-FET-PET data from n = 43 patients with primary glioblastoma (pGBM) and n = 33 patients with rHGG were assessed. pGBM patients were irradiated with photons and sequential proton/carbon boost, and rHGG patients were treated with carbon re-irradiation (CIR). WBT (Illumina HumanHT-12 Expression BeadChips) lbx was available for n = 9 patients from the rHGG cohort. PET isocontours (40%–70% SUVmax, 10% steps) and MRI-based treatment volumes (MRIvol) were compared using the conformity index (CI) (pGBM, n = 16; rHGG, n = 27). Associations with WBT lbx data were tested on gene expression level and inferred pathways activity scores (PROGENy) and from transcriptome estimated cell fractions (CIBERSORT, xCell).ResultsIn pGBM, median SUVmax was higher in PET acquired pre-radiotherapy (4.1, range (R) 1.5–7.8; n = 20) vs. during radiotherapy (3.3, R 1.5–5.7, n = 23; p = 0.03) and in non-resected (4.7, R 2.9–7.9; n = 11) vs. resected tumors (3.3, R 1.5–7.8, n = 32; p = 0.01). In rHGG, a trend toward higher SUVmax values in grade IV tumors was observed (p = 0.13). Median MRIvol was 32.34 (R 8.75–108.77) cm3 in pGBM (n = 16) and 20.77 (R 0.63–128.44) cm3 in rHGG patients (n = 27). The highest median CI was observed for 40% (pGBM, 0.31) and 50% (rHGG, 0.43, all tumors) isodose, with 70% (40%) isodose in grade III (IV) rHGG tumors (median CI, 0.38 and 0.49). High SUVmax was linked to shorter survival in pGBM (>3.3, p = 0.001, OR 6.0 [2.1–17.4]) and rHGG (>2.8, p = 0.02, OR 4.1 [1.2–13.9]). SUVmax showed associations with inferred monocyte fractions, hypoxia, and TGFbeta pathway activity and links to immune checkpoint gene expression from WBT lbx.ConclusionThe benefits of 18F-FET-PET imaging on gross tumor volume (GTV) definition for particle radiotherapy warrant further evaluation. SUVmax might assist in prognostic stratification of HGG patients for particle radiotherapy, highlights heterogeneity in rHGG, and is positively associated with unfavorable signatures in peripheral whole-blood transcriptomes.</p