Knockdown of Na,K-ATPase ß-subunits in Oncopeltus fasciatus induces molting problems and alterations in tracheal morphology.

The ubiquitously expressed transmembrane enzyme Na,K-ATPase (NKA) is vital in maintaining functionality of cells. The association of α- and ß-subunits is believed to be essential for forming a functional enzyme. In the large milkweed bug Oncopeltus fasciatus four α1-paralogs and four ß-subunits exist that can associate into NKA complexes. This diversity raises the question of possible tissue-specific distribution and function. While the α1-subunits are known to modulate cardenolide-resistance and ion-transport efficiency, the functional importance of the ß-subunits needed further investigation. We here characterize all four different ß-subunits at the cellular, tissue, and whole organismal scales. A knockdown of different ß-subunits heavily interferes with molting success resulting in strongly hampered phenotypes. The failure of ecdysis might be related to disrupted septate junction (SJ) formation, also reflected in ß2-suppression-induced alteration in tracheal morphology. Our data further suggest the existence of isolated ß-subunits forming homomeric or ß-heteromeric complexes. This possible standalone and structure-specific distribution of the ß-subunits predicts further, yet unknown pump-independent functions. The different effects caused by ß knockdowns highlight the importance of the various ß-subunits to fulfill tissue-specific requirements.

Parasite-driven replacement of a sexual by a closely related asexual taxon in nature.

Asexual species are thought to suffer more from coevolving parasites than related sexuals. Yet a variety of studies do not find the patterns predicted by theory. Here, to shine light on this conundrum, we investigate one such case of an asexual advantage in the presence of parasites. We follow the frequency dynamics of sexual and asexual Daphnia pulex in a natural pond that was initially dominated by sexuals. Coinciding with an epidemic of a microsporidian parasite infecting both sexuals and asexuals, the pond was rapidly taken over by the initially rare asexuals. With experiments comparing multiple sexual and asexual clones from across the local metapopulation, we confirm that asexuals are less susceptible and also suffer less from the parasite once infected. These results are consistent with the parasite-driven, ecological replacement of dominant sexuals by closely related, but more resistant asexuals, ultimately leading to the extinction of the formerly superior sexual competitor. Our study is one of the clearest examples from nature, backed up by experimental verification, showing a parasite-mediated reversal of competition dynamics. The experiments show that, across the metapopulation, asexuals have an advantage in the presence of parasites. In this metapopulation, asexuals are relatively rare, likely due to their recent invasion. While we cannot rule out other reasons for the observed patterns, the results are consistent with a temporary parasite-mediated advantage of asexuals due to the fact that they are rare, which is an underappreciated aspect of the Red Queen Hypothesis.

Robust reference gene design and validation for expression studies in the large milkweed bug, Oncopeltus fasciatus, upon cardiac glycoside stress.

Despite its history as a developmental and evolutionary model organism, gene expression analysis in the large milkweed bug, Oncopeltus fasciatus, has rarely been explored using quantitative real-time PCR. The strength of this method depends greatly on the endogenous controls used for normalization, which are lacking for the milkweed bug system. Here, to fill in this gap in our knowledge, we validated the stability of a set of ten candidate reference genes identified from the O. fasciatus transcriptome, and did so upon exposure to a dietary toxin, a cardiac glycoside, and across four different exposure periods. To increase robustness against gDNA contaminants, genome resources were used to design intron-bridging primers. A comprehensive stability validation by the Bestkeeper, Normfinder, geNorm and comparative ΔCt methods identified ef1a and tubulin as the most stable genes across treatments and time points, whereas 18S rRNA was the most unstable. However, accounting for the temporal scale indicated that time point confined normalizers might enable higher quantification accuracy for treatment comparison. Overall this study demonstrates: (i) a robust RT-qPCR primer design approach is possible for non-model organisms where genome annotation is often incomplete, and (ii) the importance of detailed reference gene stability exploration in multifactorial experimental designs.

Does senescence promote fitness in Caenorhabditis elegans by causing death?

A widely appreciated conclusion from evolutionary theory is that senescence (aging) is of no adaptive value to the individual that it afflicts. Yet studies of Caenorhabditis elegans and Saccharomyces cerevisiae are increasingly revealing the presence of processes which actively cause senescence and death, leading some biogerontologists to wonder about the established theory. Here we argue that programmed death that increases fitness could occur in C. elegans and S. cerevisiae, and that this is consistent with the classic evolutionary theory of aging. This is because of the special conditions under which these organisms have evolved, particularly the existence of clonal populations with limited dispersal and, in the case of C. elegans, the brevity of the reproductive period caused by protandrous hermaphroditism. Under these conditions, death-promoting mechanisms could promote worm fitness by enhancing inclusive fitness, or worm colony fitness through group selection. Such altruistic, adaptive death is not expected to evolve in organisms with outbred, dispersed populations (e.g. most vertebrate species). The plausibility of adaptive death in C. elegans is supported by computer modelling studies, and new knowledge about the ecology of this species. To support these arguments we also review the biology of adaptive death, and distinguish three forms: consumer sacrifice, biomass sacrifice and defensive sacrifice.

The function and evolutionary significance of a triplicated Na,K-ATPase gene in a toxin-specialized insect.

BACKGROUND: The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell's resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing cardenolides. Here we test the function of these duplicated gene copies in the cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C. RESULTS: Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion, and when we injected either α1A or α1B knockdowns with cardenolides this proved fatal (whereas not in controls). CONCLUSIONS: These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge.

Toad toxin-resistant snake (Thamnophis elegans) expresses high levels of mutant Na+/K+-ATPase mRNA in cardiac muscle.

Toads are chemically defended by bufadienolides, which are lethal to most predators. These toxins exert their lethal effects by binding to and disabling the Na+/K+-ATPases of cell membranes. Many species of snakes exhibit resistance to the effects of bufadienolides due to target-site insensitivity of the Na+/K+-ATPase. Mutations that confer resistance have previously been identified in ATP1a3, the gene that codes for the Na+/K+-ATPase α-3 paralog. We have found that this mutant gene is expressed at a significantly elevated level in heart tissue compared to gut, kidney, and liver of the bufadienolide-resistant snake, Thamnophis elegans. Furthermore, we found that exposure to bufadienolides elicits a significant increase in the expression levels of ATP1a3 in the heart, but not in the kidneys, liver, or gut 1h after exposure. We suggest that upregulation of ATP1a3 in the heart plays an important role in the physiological processes involved in tolerance of bufadienolides among genetically resistant snakes.

Genetic load, inbreeding depression, and hybrid vigor covary with population size: An empirical evaluation of theoretical predictions.

Reduced population size is thought to have strong consequences for evolutionary processes as it enhances the strength of genetic drift. In its interaction with selection, this is predicted to increase the genetic load, reduce inbreeding depression, and increase hybrid vigor, and in turn affect phenotypic evolution. Several of these predictions have been tested, but comprehensive studies controlling for confounding factors are scarce. Here, we show that populations of Daphnia magna, which vary strongly in genetic diversity, also differ in genetic load, inbreeding depression, and hybrid vigor in a way that strongly supports theoretical predictions. Inbreeding depression is positively correlated with genetic diversity (a proxy for Ne ), and genetic load and hybrid vigor are negatively correlated with genetic diversity. These patterns remain significant after accounting for potential confounding factors and indicate that, in small populations, a large proportion of the segregation load is converted into fixed load. Overall, the results suggest that the nature of genetic variation for fitness-related traits differs strongly between large and small populations. This has large consequences for evolutionary processes in natural populations, such as selection on dispersal, breeding systems, ageing, and local adaptation.

Endothelial Cells Derived from Non-malignant Tissues Are of Limited Value as Models for Brain Tumor Vasculature.

BACKGROUND: Human umbilical cord vein endothelial cells (HUVECs) are commonly chosen over freshly isolated endothelial cells from glioblastomas (GECs) due to accessibility and costs. MATERIALS AND METHODS: To test their suitability for in vitro studies, we comprehensively compared the transcriptomes and responses to major angiogenic cytokines of HUVECs (n=2) and GECs (n=5). Purity of GEC cultures was confirmed by uptake of acetylated low-density protein and immunostaining. RESULTS: Unsupervised analysis revealed a distinct grouping. We identified 854 differentially expressed genes. Pathway and gene ontology enrichment analyses pointed to clear differences in angiogenesis and leukocyte transmigration. Comparing the expression of cell adhesion molecules in five major angiogenic cytokines revealed that HUVECs in contrast to GECs did not exhibit a previously described down-regulation of cell adhesion molecules upon incubation with transforming growth factor betas, but rather with basic fibroblast growth factor. CONCLUSION: Given our findings, we strongly recommend the use of GECs as model cells for brain tumor endothelium for experiments investigating angiogenesis and immunobiology.

Reduced lifespan and increased ageing driven by genetic drift in small populations.

Explaining the strong variation in lifespan among organisms remains a major challenge in evolutionary biology. Whereas previous work has concentrated mainly on differences in selection regimes and selection pressures, we hypothesize that differences in genetic drift may explain some of this variation. We develop a model to formalize this idea and show that the strong positive relationship between lifespan and genetic diversity predicted by this model indeed exists among populations of Daphnia magna, and that ageing is accelerated in small populations. Additional results suggest that this is due to increased drift in small populations rather than adaptation to environments favoring faster life histories. First, the correlation between genetic diversity and lifespan remains significant after statistical correction for potential environmental covariates. Second, no trade-offs are observed; rather, all investigated traits show clear signs of increased genetic load in the small populations. Third, hybrid vigor with respect to lifespan is observed in crosses between small but not between large populations. Together, these results suggest that the evolution of lifespan and ageing can be strongly affected by genetic drift, especially in small populations, and that variation in lifespan and ageing may often be nonadaptive, due to a strong contribution from mutation accumulation.

Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy.

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02(+) glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8(+) T cells in vitro. In vivo, glioblastoma-specific CD8(+) T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma.

Microglia isolated from patients with glioma gain antitumor activities on poly (I:C) stimulation.

The role of microglia, the brain-resident macrophages, in glioma biology is still a matter of debate. Clinical observations and in vitro studies in the mouse model indicate that microglia and macrophages that infiltrate the brain tumor tissue in high numbers play a tumor-supportive role. Here, we provide evidence that human microglia isolated from brain tumors indeed support tumor cell growth, migration, and invasion. However, after stimulation with the Toll-like receptor 3 agonist poly (I:C), microglia secrete factors that exerted toxic and suppressive effects on different glioblastoma cell lines, as assessed in cytotoxicity, migration, and tumor cell spheroid invasion assays. Remarkably, these effects were tumor-specific because the microglial factors impaired neither growth nor viability of astrocytes and neurons. Culture supernatants of tumor cells inhibited the poly (I:C) induction of this microglial M1-like, oncotoxic profile. Microglia stimulation before coculture with tumor cells circumvented the tumor-mediated suppression, as demonstrated by the ability to kill and phagocytose glioma cells. Our results show, for the first time to our knowledge, that human microglia exert tumor-supporting functions that are overridden by tumor-suppressing activities gained after poly (I:C) stimulation.

Effector T-cell infiltration positively impacts survival of glioblastoma patients and is impaired by tumor-derived TGF-ß.

PURPOSE: In glioma-in contrast to various other cancers-the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma. EXPERIMENTAL DESIGN: T-cell subpopulations from entire sections of 93 WHO°II-IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II-IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells. RESULTS: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (T(eff), cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of T(eff) cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-ß molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-ß receptor signaling increased transmigration. CONCLUSIONS: This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-ß1 and TGF-ß2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches.

Predator-induced defense makes Daphnia more vulnerable to parasites.

Inducible defensive traits against herbivores or predators are widespread in plants and animals. Theory predicts that defended morphs have greater fitness in the presence of predators, but lower fitness than undefended morphs in the absence of predators. If such costs did not exist, then a constitutively defended morph would be favored by natural selection; yet, evidence for such costs has been elusive. Our current work reveals a significant cost to inducible defenses. Using the waterflea (Daphnia) model system, we show that induced defended morphs are significantly more vulnerable to infection by a virulent yeast parasite than undefended morphs. In two independent experiments, the proportion of successful infections and the number of parasite spores were higher among defended versus undefended Daphnia. Thus, by demonstrating a previously unknown and environmentally relevant cost to inducible defenses, this study enhances our understanding of adaptive phenotypic plasticity and its evolution.

A Daphnia parasite (Caullerya mesnili) constitutes a new member of the Ichthyosporea, a group of protists near the animal-fungi divergence.

Caullerya mesnili is a protozoan endoparasite in the gut epithelium of Daphnia, which causes regular epidemics in lakes throughout Europe. Its classification has remained unchanged for over a century, leaving it placed with the Haplosporidia, despite speculation that this position is incorrect. The difficulty in classifying C. mesnili stems from its few known morphological and ecological characteristics, as well as a lack of genetic markers. Here we sequenced the nuclear small subunit (SSU) and internal transcribed spacer rDNA regions of C. mesnili samples from 10 locations. Based on sequence similarities, we suggest the re-classification of C. mesnili to the Ichthyosporea, a class of protists near the animal-fungi divergence. We report average intragenomic variation of 0.75% and 2.27% in the SSU and internal transcribed spacer regions, respectively. From electron micrographs and light microscopy of histological sections we determined that C. mesnili spores grow within the intestinal epithelium where they establish themselves intercellularly. In addition, we confirmed previous accounts regarding the high virulence of this parasite. Caullerya mesnili reduces host lifespan, the number of clutches, and the total number of offspring. This high selection pressure placed on hosts supports the importance of C. mesnili as a model parasite for the study of host-parasite biology in permanent lakes.

Prior residency does not always pay off--co-infections in Daphnia.

The epidemiological and ecological processes which govern the success of multiple-species co-infections are as yet unresolved. Here we investigated prior versus late residency within hosts, meaning which parasite contacts the host first, to determine if the outcomes of intra-host competition are altered. We infected a single genotype of the waterflea Daphnia galeata with both the intestinal protozoan Caullerya mesnili and the haemolymph fungus Metschnikowia sp. (single genotype of each parasite species), as single infections, simultaneous co-infections and as sequential co-infections, with each parasite given 4 days prior residency. Simultaneous co-infections were significantly more virulent than both single infections and sequential co-infections, as measured by a decreased host life span and fecundity. Further, in addition to the Daphnia host, the parasites also suffered fitness decreases in simultaneous co-infections, as measured by spore production. The sequential co-infections, however, had mixed effects: C. mesnili benefited from prior residency, whereas Metschnikowia sp. experienced a decline in fitness. Our results show that multiple-species co-infections of Daphnia may be more virulent than single infections, and that prior residency does not always provide a competitive advantage.

Differentiation therapy exerts antitumor effects on stem-like glioma cells.

PURPOSE: Stem-like tumor cells comprise a highly tumorigenic and therapy-resistant tumor subpopulation, which is believed to substantially influence tumor initiation and therapy resistance in glioma. Currently, therapeutic, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population; retinoic acid is well known as a potent modulator of differentiation and proliferation in normal stem cells. In glioma, knowledge about the efficacy of retinoic acid-induced differentiation to target the stem-like tumor cell pool could have therapeutic implications. EXPERIMENTAL DESIGN: Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid-containing medium to study the effect of differentiation on angiogenesis, invasive growth, as well as radioresistance and chemoresistance of SLGCs. In vivo effects were studied using live microscopy in a cranial window model. RESULTS: Our data suggest that in vitro differentiation of SLGCs induces therapy-sensitizing effects, impairs the secretion of angiogenic cytokines, and disrupts SLGCs motility. Further, ex vivo differentiation reduces tumorigenicity of SLGCs. Finally, we show that all-trans retinoic acid treatment alone can induce antitumor effects in vivo. CONCLUSIONS: Altogether, these results highlight the potential of differentiation treatment to target the stem-like cell population in glioblastoma.

Angiogenic growth factors in tissue homogenates of HNSCC: expression pattern, prognostic relevance, and interrelationships.

Head and neck squamous cell carcinoma has still a poor prognosis. Since angiogenesis is crucial for tumor growth, a better understanding of the potential clinical relevance as well as the interactions between the numerous proangiogenic growth factors is essential to develop improved therapeutic strategies in these tumors. Expression levels of eight growth factors known to induce angiogenesis (HGF, bFGF, VEGF-A, VEGF-D, PDGF-AB, PDGF-BB, G-CSF, and GM-CSF) were quantitatively measured by ELISA in homogenates of 41 head and neck squamous cell carcinomas. In addition, microvessel density and protein localization of growth factors were assessed by immunohistochemistry. Statistical analysis was performed to assess interrelationships between growth factors analyzed and to correlate protein levels with patient outcome. In 90% of the tissues at least 4/8 growth factors analyzed were detectable. Highest amounts and most frequent expression were found for HGF, bFGF and VEGF-A while PDGF-AB and PDGF-BB were present in two-thirds and G-CSF and GM-CSF in approximately half of the cases. Although there was no significant relation to microvessel density, we identified significant associations for bFGF with HGF and G-CSF as well as of PDGF-AB with those of VEGF-A and PDGF-BB. For the first time we demonstrate that expression levels of HGF as well as that of bFGF and G-CSF in head and neck squamous tumors are negative prognostic factors for patient survival. Our data indicate a network of interrelated and prognostically relevant growth factors in these tumors that have to be taken into consideration when planning an antiangiogenic and antitumor therapy.

Combination of thalidomide and cisplatin in an head and neck squamous cell carcinomas model results in an enhanced antiangiogenic activity in vitro and in vivo.

Thalidomide is an immunomodulatory, antiangiogenic drug. Although there is evidence that it might be more effective in combination with chemotherapy the exact mechanism of action is unclear. Therefore, we investigated its effect in combination with metronomically applied cisplatin in a xenotransplant mouse model characteristic for advanced head and neck squamous cell carcinomas, its possible synergistic action in vitro, and which tumor-derived factors might be targeted by thalidomide. Although thalidomide alone was ineffective, a combined treatment with low-dose cisplatin inhibited significant tumor growth, proliferation and angiogenesis in vivo as well as migration and tube formation of endothelial cells in vitro. Noteworthy, the latter effect was enhanced after coapplication of cisplatin in nontoxic doses. An inhibitory effect on tumor cell migration was also observed suggesting a direct antitumor effect. Although thalidomide alone did not influence cell proliferation, it augmented antiproliferative response after cisplatin application emphasizing the idea of a potentiated effect when both drugs are combined. Furthermore, we could show that antiangiogenic effects of thalidomide are related to tumor-cell derived factors including vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and Il-8 some known and with, granulocyte colony stimulating growth factor and granulocyte macrophage colony stimulating growth factor, some new target molecules of thalidomide. Altogether, our findings reveal new insights into thalidomide-mediated antitumor and antiangiogenic effects and its interaction with cytostatic drugs.

Development of adrenal chromaffin cells in Sf1 heterozygous mice.

Adrenal medullary chromaffin cells are derivatives of the neural crest and are widely believed to share a common sympathoadrenal (SA) progenitor with sympathetic neurons. For decades, the adrenal cortical environment was assumed to be essential for channelling SA progenitors towards an endocrine chromaffin cell fate. Our recent analysis of steroidogenic factor 1(Sf1) -/- mice, which lack an adrenal cortex, has challenged this view: in Sf1 -/- mice chromaffin cells migrate to the correct "adrenal" location and undergo largely normal differentiation. In contrast to Sf1 homozygous mutants, heterozygous animals have an adrenal cortex, which, however, is smaller than in wildtype littermates. We show here that the Sf1 +/- adrenal cortical anlagen attract normal numbers of chromaffin progenitor cells into their vicinity by embryonic day 13.5 (E13.5). Two days later, however, only a few scattered cells with highly immature features have immigrated into the adrenal cortex, whereas the remainder form a coherent cell assembly ectopically located at the medial surface of the gland. These cells appear more mature than the scattered intracortical chromaffin progenitors and express the adrenaline synthesizing enzyme PNMT with a delay of 1 day in comparison with wildtype littermates. Nevertheless, chromaffin progenitor cells undergo a numerical reduction of approximately 30% by E17.5. Together, our data suggest that normal adrenocortical development is critical for the correct immigration of chromaffin progenitors into the cortical anlagen, for the timing of PNMT expression and for the regulation of chromaffin cell numbers.