Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-x_L, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.     

Low-temperature time-resolved spectroscopic study of the major light-harvesting complex of Amphidinium carterae

The major light-harvesting complex of Amphidinium (A.) carterae, chlorophyll-a–chlorophyll-c ₂–peridinin–protein complex (acpPC), was studied using ultrafast pump-probe spectroscopy at low temperature (60 K). An efficient peridinin–chlorophyll-a energy transfer was observed. The stimulated emission signal monitored in the near-infrared spectral region was stronger when redder part of peridinin pool was excited, indicating that these peridinins have the S₁/ICT (intramolecular charge-transfer) state with significant charge-transfer character. This may lead to enhanced energy transfer efficiency from “red” peridinins to chlorophyll-a. Contrary to the water-soluble antenna of A. carterae, peridinin–chlorophyll-a protein, the energy transfer rates in acpPC were slower under low-temperature conditions. This fact underscores the influence of the protein environment on the excited-state dynamics of pigments and/or the specificity of organization of the two pigment–protein complexes.     

Feeding behaviour and performance of different populations of the black currant‐lettuce aphid,Nasonovia ribisnigri,on resistant and susceptible lettuce

When crops are bred for resistance to herbivores, these herbivores are under strong selection pressure to overcome this resistance, which may result in the emergence of virulent biotypes. This is a growing problem for crop species attacked by aphids. The Nr‐gene in lettuce confers near‐complete resistance against the black currant‐lettuce aphid, Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). Since 2007, populations of N. ribisnigri have been reported in several locations in Europe to infest resistant lettuce varieties that possess the Nr‐gene. The objective of this study was to analyse the behaviour and level of virulence of several N. ribisnigri populations observed to have colonized Nr‐locus‐containing lettuce lines. We analysed the stylet penetration and feeding behaviour, and the performance of these N. ribisnigri populations on resistant and susceptible lettuce lines. Large variation in the degree of virulence to the Nr‐locus‐containing lettuce lines was found among populations of the Nr:1 biotype. The German population was highly virulent on the Nr‐containing resistant lettuce lines, and showed similar feeding behaviour and performance on both the susceptible and resistant lettuces. The French population from Paris was the second most virulent, though reproduction on the resistant lines was reduced. The French population from Perpignan and a population from Belgium, however, showed reduced performance and feeding rate on the resistant compared to the susceptible lettuces. The lettuce background in which the Nr‐gene is expressed influences the level of resistance to the various Nr:1 aphid populations, because the performance and feeding behaviour differed between the aphids on the cultivars (romaine lettuce) compared to the near‐isogenic lines (butterhead/iceberg lettuce). This study also shows that being able to feed on a plant not automatically implies that a population can successfully develop on that plant, because aphids showed phloem ingestion during the 8‐h recording period on resistant lettuce, but were not able to survive and reproduce on the same lettuce line.     

Body size as an important factor determining trophic niche partitioning in three syntopic rhinolophid bat species

We investigated a community of syntopically occurring horseshoe bats (Rhinolophus hipposideros, R. euryale, R. ferrumequinum) in southern Slovakia. The faecal pellets of these bat species were collected in the field and later analysed under a dissecting microscope. The three species studied are known to be very similar as far as their ecology, echolocation and preferred habitats are concerned, but they diverge significantly in their body sizes. In this study, all three species fed predominantly on moths [59–80 percentage frequency (%f); 87–95 percentage volume (%vol)], but their diet compositions differed in the size of individuals consumed. The smallest bat species (R. hipposideros) fed only on the smallest moths (%f = 59; %vol = 87), the medium-sized species (R. euryale) mainly on medium-sized moths (%f = 60; %vol = 74) and the largest one (R. ferrumequinum) especially on the largest moths (%f = 54; %vol = 89). Despite similar preferred habitat and the main prey category, the rates of trophic niche overlap were surprisingly low. The trophic niche percentage overlap was 7–31% (computed from %f data) and 1–20% (computed from %vol data), respectively and suggests an extraordinary importance of mere divergences of bats in their body sizes for trophic niche partitioning and stable species coexistence.     

Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei—the causative agent of Human African Trypanosomiasis—by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)₂), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)₂ redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC–MS/MS analysis was established to quantify intra-cellular CYN, T(SH)₂, GSH, as well as GS-CYN and T(S-CYN)₂ adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)₂ pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)₂ redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)₂ redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.     

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.     

Variation in eggshell traits between geographically distant populations of pied flycatchers Ficedula hypoleuca

The expression and impact of maternal effects may vary greatly between populations and environments. However, little is known about large‐scale geographical patterns of variation in maternal deposition to eggs. In birds, as in other oviparous animals, the outermost maternal component of an egg is the shell, which protects the embryo, provides essential mineral resources and allows its interaction with the environment in the form of gas exchange. In this study, we explored variation of eggshell traits (mass, thickness, pore density and pigmentation) across 15 pied flycatcher populations at a large geographic scale. We found significant between‐population variation in all eggshell traits, except in pore density, suggesting spatial variation in their adaptive benefits or in the females’ physiological limitations during egg laying. Between‐ population variation in shell structure was not due to geographic location (latitude and longitude) or habitat type. However, eggshells were thicker in populations that experienced higher ambient temperature during egg laying. This could be a result of maternal resource allocation to the shell being constrained under low temperatures or of an adaptation to reduce egg water loss under high temperatures. We also found that eggshell colour intensity was positively associated with biliverdin pigment concentration, shell thickness and pore density. To conclude, our findings reveal large‐ scale between‐population variation of eggshell traits, although we found little environmental dependency in their expression. Our findings call for further studies that explore other environmental factors (e.g. calcium availability and pollution levels) and social factors like sexual selection intensity that may account for differences in shell structure between populations.     

Great tits provided with ad libitum food lay larger eggs when exposed to colder temperatures

The amount of nutrients deposited into a bird egg varies both between and within clutches of the same female. Larger eggs enhance offspring traits, but as a tradeoff, laying large eggs also infers energetic costs to the female. Income breeders usually lay larger eggs later in the season, when temperatures and food availability are higher. Egg size is thus affected by the daily amount of energy available to produce an egg under cold conditions, but it is less well known in how far temperature exerts direct effects on egg size. We show that great tit females Parus major with access to ad libitum food and breeding in climate‐controlled aviaries varied their egg investments. The size of an individual egg was best predicted by mean temperatures one week pre‐laying, with females laying larger, rather than smaller, eggs under colder conditions. Eggs increased in size over the season, but not significantly over the laying sequence. The degree of daily temperature fluctuation did not influence egg size. In addition to a substantial between‐female variation, sisters were more similar to each other than unrelated females, showing that egg size does also reflect heritable intrinsic female properties. Natural variation in egg size is thus not only determined by energy‐limitation, but also due to females allocating more resources to eggs laid in colder environments, thus increasing early survival of the chicks. That the positive correlation between temperature and egg investments that is found in a natural population is reversed under ad libitum food conditions demonstrates that wild great tits tradeoff own condition with survival prospects of their chicks as a function of available food, not ambient temperature.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

Spontaneous Atopic Dermatitis-Like Symptoms in a/a ma ft/ma ft/J Flaky Tail Mice Appear Early after Birth

Loss-of-function mutations in human profilaggrin gene have been identified as the cause of ichthyosis vulgaris (IV), and as a major predisposition factor for atopic dermatitis (AD). Similarly, flaky tail (a/a ma ft/ma ft/J) mice were described as a model for IV, and shown to be predisposed to eczema. The aim of this study was to correlate the flaky tail mouse phenotype with human IV and AD, in order to dissect early molecular events leading to atopic dermatitis in mice and men, suffering from filaggrin deficiency. Thus, 5-days old flaky tail pups were analyzed histologically, expression of cytokines was measured in skin and signaling pathways were investigated by protein analysis. Human biopsies of IV and AD patients were analyzed histologically and by real time PCR assays. Our data show acanthosis and hyperproliferation in flaky tail epidermis, associated with increased IL1β and thymic stromal lymphopoietin (TSLP) expression, and Th2-polarization. Consequently, NFκB and Stat pathways were activated, and IL6 mRNA levels were increased. Further, quantitative analysis of late epidermal differentiation markers revealed increased Small proline-rich protein 2A (Sprr2a) synthesis. Th2-polarization and Sprr2a increase may result from high TSLP expression, as shown after analysis of 5-days old K14-TSLP tg mouse skin biopsies. Our findings in the flaky tail mouse correlate with data obtained from patient biopsies of AD, but not IV. We propose that proinflammatory cytokines are responsible for acanthosis in flaky tail epidermis, and together with the Th2-derived cytokines lead to morphological changes. Accordingly, the a/a ma ft/ma ft/J mouse model can be used as an appropriate model to study early AD onset associated with profilaggrin deficiency.