Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed. 2014 American Chemical Societ

Crustose Coralline Algae and a Cnidarian Neuropeptide Trigger Larval Settlement in Two Coral Reef Sponges

In sessile marine invertebrates, larval settlement is fundamental to population maintenance and persistence. Cues contributing to the settlement choices and metamorphosis of larvae have important implications for the success of individuals and populations, but cues mediating larval settlement for many marine invertebrates are largely unknown. This study assessed larval settlement in two common Great Barrier Reef sponges, Coscinoderma matthewsi and Rhopaloeides odorabile, to cues that enhance settlement and metamorphosis in various species of scleractinian coral larvae. Methanol extracts of the crustose coralline algae (CCA), Porolithon onkodes, corresponding to a range of concentrations, were used to determine the settlement responses of sponge larvae. Cnidarian neuropeptides (GLW-amide neuropeptides) were also tested as a settlement cue. Settlement in both sponge species was approximately two-fold higher in response to live chips of CCA and optimum concentrations of CCA extract compared to 0.2 µm filtered sea water controls. Metamorphosis also increased when larvae were exposed to GLW-amide neuropeptides; R. odorabile mean metamorphosis reached 42.0±5.8% compared to 16.0±2.4% in seawater controls and in C. matthewsi mean metamorphosis reached 68.3±5.4% compared to 36.7±3.3% in seawater controls. These results demonstrate the contributing role chemosensory communication plays in the ability of sponge larvae to identify suitable habitat for successful recruitment. It also raises the possibility that larvae from distinct phyla may share signal transduction pathways involved in metamorphosis

Modeling the TNFα-Induced Apoptosis Pathway in Hepatocytes

The proinflammatory cytokine TNFα fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Recently, we showed that TNFα is able to sensitize primary murine hepatocytes cultured on collagen to Fas ligand-induced apoptosis and presented a mathematical model of the sensitizing effect. Here, we analyze how TNFα induces apoptosis in combination with the transcriptional inhibitor actinomycin D (ActD). Accumulation of reactive oxygen species (ROS) in response to TNFR activation turns out to be critical for sustained activation of JNK which then triggers mitochondrial pathway-dependent apoptosis. In addition, the amount of JNK is strongly upregulated in a ROS-dependent way. In contrast to TNFα plus cycloheximide no cFLIP degradation is observed suggesting a different apoptosis pathway in which the Itch-mediated cFLIP degradation and predominantly caspase-8 activation is not involved. Time-resolved data of the respective pro- and antiapoptotic factors are obtained and subjected to mathematical modeling. On the basis of these data we developed a mathematical model which reproduces the complex interplay regulating the phosphorylation status of JNK and generation of ROS. This model was fully integrated with our model of TNFα/Fas ligand sensitizing as well as with a published NF-κB-model. The resulting comprehensive model delivers insight in the dynamical interplay between the TNFα and FasL pathways, NF-κB and ROS and gives an example for successful model integration

Development of the rhopalial nervous system in Aurelia sp.1 (Cnidaria, Scyphozoa)

We examined the development of the nervous system in the rhopalium, a medusa-specific sensory structure, in Aurelia sp.1 (Cnidaria, Scyphozoa) using confocal microscopy. The rhopalial nervous system appears primarily ectodermal and contains neurons immunoreactive to antibodies against tyrosinated tubulin, taurine, GLWamide, and FMRFamide. The rhopalial nervous system develops in an ordered manner: the presumptive gravity-sensing organ, consisting of the lithocyst and the touch plate, differentiates first; the “marginal center,” which controls swimming activity, second; and finally, the ocelli, the presumptive photoreceptors. At least seven bilaterally arranged neuronal clusters consisting of sensory and ganglion cells and their neuronal processes became evident in the rhopalium during metamorphosis to the medusa stage. Our analysis provides an anatomical framework for future gene expression and experimental studies of development and functions of scyphozoan rhopalia

Population-based food consumption survey as an additional tool for foodborne outbreak investigations, Germany, 2017

We conducted a food consumption survey in the general adult population of 18 years and older in Germany to obtain data on the frequency of consumption of food items that caused foodborne disease outbreaks in the past. A total of 1010 telephone interviews were completed that queried the consumption of 95 food items in the 7-day period before the interview. Survey results were weighted to be representative. Six exemplary ‘high risk’ food items were consumed by 6% to 16% of the general population. These were raw ground pork: 6.5%; ‘Teewurst’ (=spreadable sausage-containing raw pork): 15.7%; unpasteurised milk consumed without prior heating: 9.0%; food items prepared with raw eggs: 9.8%; unheated sprouts or seedlings: 8.8% and frozen berries consumed without prior heating: 6.2%. Data from our food consumption survey were comparable to data obtained from control persons in case-control studies conducted during past foodborne disease outbreak investigations. We consider our survey an additional helpful tool that will allow comparison with food consumption data from case-patients obtained in exploratory, hypothesis-generating interviews early on in outbreak investigations, and which may assist in forming hypotheses regarding associations of illnesses with suspected food vehicles. This may facilitate and accelerate investigations of future foodborne disease outbreaks.Peer Reviewe