Efficient Application of De Novo RNA Assemblers for Proteomics Informed by Transcriptomics

RNA sequencing is a powerful method to build reference transcriptome assemblies and eventually sample-specific protein databases for mass-spectrometry-based analyses. This novel proteomics informed by transcriptomics (PIT) workflow improves proteome characterization of dynamic and especially nonmodel organism proteomes and moreover helps to identify novel gene products. With increasing popularity of such proteogenomics applications a growing number of researchers demand qualitative but resource-friendly and easy to use analysis strategies. Most PIT applications so far rely on the initially introduced Trinity de novo assembly tool. To aid potential users to start off with PIT, we compared main performance criteria of Trinity and other alternative RNA assembly tools known from the transcriptomics field including Oases, SOAPdenovo-Trans, and Trans-ABySS. Using exemplary data sets and software-specific default parameters, Trinity and alternative assemblers produced comparable and high-quality reference data for vertebrate transcriptomes/proteomes of varying complexity. However, Trinity required large computational resources and time. We found that alternative de novo assemblers, in particular, SOAPdenovo-Trans but also Oases and Trans-ABySS, rapidly produced protein databases with far lower computational requirements. By making choice among various RNA assembly tools, proteomics researchers new to transcriptome assembly and with future projects with high sample numbers can benefit from alternative approaches to efficiently apply PIT

Site-Specific Retention of Colloids at Rough Rock Surfaces

The spatial deposition of polystyrene latex colloids (<i>d</i> = 1 μm) at rough mineral and rock surfaces was investigated quantitatively as a function of Eu­(III) concentration. Granodiorite samples from Grimsel test site (GTS), Switzerland, were used as collector surfaces for sorption experiments. At a scan area of 300 × 300 μm², the surface roughness (rms roughness, <i>Rq</i>) range was 100–2000 nm, including roughness contribution from asperities of several tens of nanometers in height to the sample topography. Although, an increase in both roughness and [Eu­(III)] resulted in enhanced colloid deposition on granodiorite surfaces, surface roughness governs colloid deposition mainly at low Eu­(III) concentrations (≤5 × 10^–7 M). Highest deposition efficiency on granodiorite has been found at walls of intergranular pores at surface sections with roughness <i>Rq</i> = 500–2000 nm. An about 2 orders of magnitude lower colloid deposition has been observed at granodiorite sections with low surface roughness (<i>Rq</i> < 500 nm), such as large and smooth feldspar or quartz crystal surface sections as well as intragranular pores. The site-specific deposition of colloids at intergranular pores is induced by small scale protrusions (mean height = 0.5 ± 0.3 μm). These protrusions diminish locally the overall DLVO interaction energy at the interface. The protrusions prevent further rolling over the surface by increasing the hydrodynamic drag required for detachment. Moreover, colloid sorption is favored at surface sections with high density of small protrusions (density (<i>D</i>) = 2.6 ± 0.55 μm^–1, asperity diameter (ϕ) = 0.6 ± 0.2 μm, height (<i>h</i>) = 0.4 ± 0.1 μm) in contrast to surface sections with larger asperities and lower asperity density (<i>D</i> = 1.2 ± 0.6 μm^–1, ϕ = 1.4 ± 0.4 μm, <i>h</i> = 0.6 ± 0.2 μm). The study elucidates the importance to include surface roughness parameters into predictive colloid-borne contaminant migration calculations

Variability of Zinc Oxide Dissolution Rates

Zinc oxide (ZnO) is of widespread use for numerous applications, including many in the cosmetic industry. Thus, ZnO particles are quite likely to enter the environment. ZnO may be harmful because of the release of cytotoxic Zn²⁺ ions during dissolution reactions. Here, we analyze the dissolution kinetics of the polar zinc-terminated (000–1) and nonpolar (10–10) crystal surfaces in ultrapure water to examine the impact of the crystal defects on dissolution. By using a complementary approach of atomic force microscopy and vertical scanning interferometry, we quantify the difference in reaction rate between the crystal faces, the overall range of rate variability, and the rate components that combine to an overall rate. The mean dissolution rate of the (000–1) crystal surface is more than 4 times that of the (10–10) surface. By using the rate spectrum analysis, we observed an overall dissolution rate variability of more than 1 order of magnitude. The rate components and the range of dissolution rate are important input parameters in reactive transport models for the prediction of potential release of Zn²⁺ into the environment

Cell cycle analysis of HT-29 colon carcinoma cells after treatment with 30 μg/ml IAE for 24 h.

<p>A, Cell cycle was analyzed by flow cytometry of propidium iodide stained cells. Histograms (top) show one representative experiment for each treatment condition. Bar plots (bottom) show percent of cell population in apoptotic SubG1, G0/G1, S and G2/M phases of the cell cycle and are expressed as mean ± SEM (n = 3). *p≤0.05, ***p≤0.001 vs. control. B, Whole cell lysates were analyzed for the expression of cyclin A2, cyclin D3, CDK2, CDK4, CDK6 and GAPDH proteins by immunoblotting using specific antibodies.</p

Cytotoxicity test and evaluation of inhibition of tumor growth by treating HT-29 tumor xenograft mouse models with IAE.

<p>Athymic nude mice were ectopically implanted with 5 million HT-29 cells in the flank and orally gavaged bidaily by 50 mg/kg IAE or vehicle for 4 weeks. A, Cytotoxicity was tested for colon cancer and primary colon cells (IC₅₀ = 25.82 μg/mL (HT-29); IC₅₀ = 36.12 μg/mL (CCD 841 CoN)). Data are expressed as mean ± SD (n = 4). B, Body weight during entire experiment. C, Tumor volume during entire experiment and images of exemplary xenografts of untreated and IAE-treated mice at end point. D, Tumor weight at the end of the study. Data are expressed as mean ± SEM (n = 18). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control (one-tailed t test).</p

Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.

<p>Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.</p

Extra- and intracellular formation of reactive oxygen species (ROS) and of lipid peroxides in HT-29 cells after treatment with IAE.

<p>A, Extracellular formation of ROS in full cell culture medium was kinetically detected using the ROS-sensitive CellROX Orange fluorogenic probe. Data are expressed as mean ± SEM (n = 8). B, Intracellular ROS was detected by flow cytometry of HT-29 cells stained with CellROX Orange after treatment for 24 h. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). C, Intracellular lipid peroxidation was detected by flow cytometry of cells treated for 24 h using the Click-iT technology. Increasing fluorescence intensities are a result of enhanced lipid peroxidation upon treatment. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). D, Intracellular lipid peroxidation was visualized by fluorescence microscopy (green, lipid peroxides; blue, nucleus). Scale bars, 25 μm. n.s. not significant, **p≤0.01, ***p≤0.001 vs. control.</p

Activation of apoptosis signaling pathway in colon carcinoma cells after treatment with IAE.

<p>A, HT-29 and T84 cells were treated for 24 h. Enzymatic activation of caspases 2, 3/7, 6, 8 and 9 was determined by use of luminescence-based assays. Data are normalized to control treatment and are expressed as mean ± SEM (n = 4). B, Whole cell lysates from HT-29 cells treated for 24 h were analyzed for the expression of total and cleaved proteins of caspase 3, caspase 9 and PARP by immunoblotting. Numbers indicate densitometric ratios of the cleaved to total proteins normalized to control treatments. C, Fluorescence microscopy of HT-29 cells treated for 24 h. Cleaved caspase 3 was labeled green, F-actin red and the nucleus blue. Scale bars, 25 μm. D, HT-29 cells were treated for 6 h. DNA fragmentation was detected through accumulation of cytoplasmic BrdU-labeled DNA by ELISA. Data are normalized to control treatment and are expressed as mean ± SEM (n = 5). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control.</p

Amorfrutin C Induces Apoptosis and Inhibits Proliferation in Colon Cancer Cells through Targeting Mitochondria

A known (<b>1</b>) and a structurally related new natural product (<b>2</b>), both belonging to the amorfrutin benzoic acid class, were isolated from the roots of <i>Glycyrrhiza foetida</i>. Compound <b>1</b> (amorfrutin B) is an efficient agonist of the nuclear peroxisome proliferator activated receptor (PPAR) gamma and of other PPAR subtypes. Compound <b>2</b> (amorfrutin C) showed comparably lower PPAR activation potential. Amorfrutin C exhibited striking antiproliferative effects for human colorectal cancer cells (HT-29 and T84), prostate cancer (PC-3), and breast cancer (MCF7) cells (IC₅₀ values ranging from 8 to 16 μM in these cancer cell lines). Notably, amorfrutin C (<b>2</b>) showed less potent antiproliferative effects in primary colon cells. For HT-29 cells, compound <b>2</b> induced G0/G1 cell cycle arrest and modulated protein expression of key cell cycle modulators. Amorfrutin C further induced apoptotic events in HT-29 cells, including caspase activation, DNA fragmentation, PARP cleavage, phosphatidylserine externalization, and formation of reactive oxygen species. Mechanistic studies revealed that <b>2</b> disrupts the mitochondrial integrity by depolarization of the mitochondrial membrane (IC₅₀ 0.6 μM) and permanent opening of the mitochondrial permeability transition pore, leading to increased mitochondrial oxygen consumption and extracellular acidification. Structure–activity-relationship experiments revealed the carboxylic acid and the hydroxy group residues of <b>2</b> as fundamental structural requirements for inducing these apoptotic effects. Synergy analyses demonstrated stimulation of the death receptor signaling pathway. Taken together, amorfrutin C (<b>2</b>) represents a promising lead for the development of anticancer drugs