Pancreatic carcinoma, pancreatitis, and healthy controls: metabolite models in a three-class diagnostic dilemma

Metabolomics as one of the most rapidly growing technologies in the “-omics” field denotes the comprehensive analysis of low molecular-weight compounds and their pathways. Cancer-specific alterations of the metabolome can be detected by high-throughput mass-spectrometric metabolite profiling and serve as a considerable source of new markers for the early differentiation of malignant diseases as well as their distinction from benign states. However, a comprehensive framework for the statistical evaluation of marker panels in a multi-class setting has not yet been established. We collected serum samples of 40 pancreatic carcinoma patients, 40 controls, and 23 pancreatitis patients according to standard protocols and generated amino acid profiles by routine mass-spectrometry. In an intrinsic three-class bioinformatic approach we compared these profiles, evaluated their selectivity and computed multi-marker panels combined with the conventional tumor marker CA 19-9. Additionally, we tested for non-inferiority and superiority to determine the diagnostic surplus value of our multi-metabolite marker panels. Compared to CA 19-9 alone, the combined amino acid-based metabolite panel had a superior selectivity for the discrimination of healthy controls, pancreatitis, and pancreatic carcinoma patients $ [ {\text{volume under ROC surface}}\;\left( {\text{VUS}} \right) = 0. 8 9 1 { }\left( { 9 5\,\% {\text{ CI }}0. 7 9 4- 0. 9 6 8} \right)]. $ We combined highly standardized samples, a three-class study design, a high-throughput mass-spectrometric technique, and a comprehensive bioinformatic framework to identify metabolite panels selective for all three groups in a single approach. Our results suggest that metabolomic profiling necessitates appropriate evaluation strategies and—despite all its current limitations—can deliver marker panels with high selectivity even in multi-class settings.     

Fertilizer application decreases insect abundance on Plantago lanceolata: a large-scale experiment in three geographic regions

Humans have substantially altered the nitrogen cycle of ecosystems through the application of agricultural fertilizer. Fertilization may not only affect plant species diversity, but also insect dynamics by altering plant nitrogen supplies. We investigated the effect of experimental fertilization on the vegetation, with the ribwort plantain as the focal plant, and on higher trophic levels on differently managed grasslands throughout Germany. Over a period of 2 years, we examined two specialist herbivores and their parasitoid on Plantago lanceolata L., and the composition and structure of the surrounding vegetation. Over 70 sites in three geographic regions, within the large-scale project “German Biodiversity Exploratories”, were included in the study. The model system consisted of the host plant P. lanceolata L., the monophagous weevils Mecinus labilis Herbst and M. pascuorum Gyllenhal, and their parasitoid Mesopolobus incultus Walker. Fertilization decreased plant species richness and host plant abundance, whereas it enhanced the total vegetation growth. The increased size and heigher leaf nitrogen content did not improve herbivore performance. On the contrary, the abundance of the two herbivores was decreased by fertilization. The parasitoid depended on the abundance of one of its hosts, M. pascuorum (positively density-dependent). Reduced herbivore abundance due to fertilization might be explained by a lower abundance of the host plant, a lower stalk number, and by changed patterns of host localization within higher vegetation. Fertilization negatively affected the third trophic level by cascading up via host abundance. The relationships between fertilization, surrounding vegetation and the tritrophic system were measured throughout the three regions and over the 2-year period. Our findings present consequences of intensification for a plant–herbivore–parasitoid system, and may have significant implications for the conservation of multitrophic systems in managed grasslands.     

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

Simulating effects of climate change under direct and diapause development in a butterfly

Temperature is one of the most important ecological factors affecting species survival and distributions. Therefore, global climate change, involving increases in mean surface temperature and the occurrence of extreme weather events, may pose a substantial challenge to biodiversity. Whereas tropical ectotherms are believed to be very sensitive to climate change, temperate‐zone species may actually benefit from higher temperatures. However, as in temperate zones large parts of the year are unsuitable for growth and reproduction, seasonal time constraints may complicate matters. Against this background we here investigate the impact of simulated climate change, involving increased mean temperatures and heat waves, across developmental pathways of the butterfly Lycaena tityrus (Poda) (Lepidoptera: Lycaenidae). Increased temperatures speeded up development but decreased pupal mass as expected. However, we found no evidence for detrimental effects of increased temperatures or even simulated heat waves. Furthermore, patterns did not differ between indirectly and directly developing individuals, which are assumed to be more time constrained. Our findings support the notion that not all species will be detrimentally affected by climate change, and suggest that species attributes may be more important than potential time constraints imposed by different developmental pathways.     

TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N‐terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy‐specific TRAPP subunit, forms part of a mammalian TRAPPIII‐like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.     

RNA interference silencing of the adaptor molecules ShcC and Fe65 differentially affect amyloid precursor protein processing and Abeta generation

The amyloid precursor protein (APP) and its pathogenic by-product amyloid-beta protein (Abeta) play central roles in Alzheimer disease (AD) neuropathogenesis. APP can be cleaved by beta-secretase (BACE) and alpha-secretase to produce APP-C99 and APP-C83. These C-terminal fragments can then be cleaved by gamma-secretase to produce Abeta and p3, respectively. p3 has been reported to promote apoptosis, and Abeta is the key component of senile plaques in AD brain. APP adaptor proteins with phosphotyrosine-binding domains, including ShcA (SHC1), ShcC (SHC3), and Fe65 (APBB1), can bind to and interact with the conserved YENPTY motif in the APP-C terminus. Here we have described for the first time the effects of RNA interference (RNAi) silencing of ShcA, ShcC, and Fe65 expression on APP processing and Abeta production. RNAi silencing of ShcC led to reductions in the levels of APP-C-terminal fragments (APP-CTFs) and Abeta in H4 human neuroglioma cells stably overexpressing full-length APP (H4-FL-APP cells) but not in those expressing APP-C99 (H4-APP-C99 cells). RNAi silencing of ShcC also led to reductions in BACE levels in H4-FL-APP cells. In contrast, RNAi silencing of the homologue ShcA had no effect on APP processing or Abeta levels. RNAi silencing of Fe65 increased APP-CTF levels, although also decreasing Abeta levels in H4-FL-APP cells. These findings suggest that pharmacologically blocking interaction of APP with ShcC and Fe65 may provide novel therapeutic strategies against AD.