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Fanconi anemia (FA) is a genetically heterogeneous disorder with 22 disease-causing genes reported to date. In some FA genes, monoallelic mutations have been found to be associated with breast cancer risk, while the risk associations of others remain unknown. The gene for FA type C, FANCC, has been proposed as a breast cancer susceptibility gene based on epidemiological and sequencing studies. We used the Oncoarray project to genotype two truncating FANCC variants (p.R185X and p.R548X) in 64,760 breast cancer cases and 49,793 controls of European descent. FANCC mutations were observed in 25 cases (14 with p.R185X, 11 with p.R548X) and 26 controls (18 with p.R185X, 8 with p.R548X). There was no evidence of an association with the risk of breast cancer, neither overall (odds ratio 0.77, 95%CI 0.44–1.33, p = 0.4) nor by histology, hormone receptor status, age or family history. We conclude that the breast cancer risk association of these two FANCC variants, if any, is much smaller than for BRCA1, BRCA2 or PALB2 mutations. If this applies to all truncating variants in FANCC it would suggest there are differences between FA genes in their roles on breast cancer risk and demonstrates the merit of large consortia for clarifying risk associations of rare variants.
Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.
Upon oncogenic stress, the tumor suppressor Arf can induce irreversible cell cycle arrest or apoptosis, depending on the oncogenic insult. In this study, it could be shown that Arf interacts with Myc and the Myc-associated zinc-finger protein Miz1 to facilitate repression of genes involved in cell adhesion. Formation of a DNA-binding Arf/Myc/Miz1 complex disrupts interaction of Miz1 with its coactivator nucleophosmin and induces local heterochromatinisation, causing cells to lose attachment and undergo anoikis. The assembly of the complex relies on Myc, which might explain why high Myc levels trigger apoptosis and not cell cycle arrest in the Arf response. This mechanism could play an important role in eliminating cells harboring an oncogenic mutation. Arf furthermore induces sumoylation of Miz1 at a specific lysine by repressing the desumoylating enzyme Senp3. A sumoylation-deficient mutant of Miz1 however does not show phenotypic differences under the chosen experimental conditions. Myc can also be modified by Sumo by multisumoylation at many different lysines, which is unaffected by Arf. The exact mechanism and effect of this modification however stays unsolved.
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.
The most commonly used assay to quantitate the response of peripheral T~cells upon stimulation with growth factors is determination of incorporated (JH]TdR. We compared thls test to three other methods: 1. direct countlog of cells with a Coulter type counter as reference assay, 2. a colorimetric assay using the tetrazolium dye 3-[ 4,S-dimethylthiazol-l-yl]-2,5diphenyl tetrazolium (MTT), which is a cheap and increasingly popular non-radioactive method and 3. incorporation of the thymidine analog 5-bromo-2'-deoxyuridine detection with a monoclonal antibody on cytospins. Primary human PHA-blasts from >30 healthy individuals were stimulated with IL-2, IL-4 aod IL-7 and assayed with up to four different methods. We discuss the advantages and disadvantages of the assays used and tbe effects of differences between cell preparations. We observed no significant variations between individuals for the dose dependence, but the relative emctency of IL4 compared to IL-2 and IL-7 was variable. This was probably due to the slower response observed upon stimulation with this factor.
Protein phosphatases can be classified into at least three major families based on amino acid sequences at their active sites. A newly emerging phosphatase family contains the active site sequence DXDX(T/V), and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases, a ubiquitous and evolutionarily conserved enzyme family. Although the existence of 58 human HAD enzymes has been predicted by database analysis, our understanding of their biological functions remains rudimentary.By database mining amd phylogenetic analysis of human HAD phosphatases, we have found a marked increase in cell area of spreading cells, as well as accelerated cell spreading onfibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. a previously unidentified enzyme with homology to Chronophin, a cytoskeletal regulatory HAD phosphatase. We have cloned and characterized this novel enzyme and named it AUM,for actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase. By Northern blot, real-time PCR and Western blot analysis, we show that AUM is broadly expressed in all major human and mouse tissues with highest levels found in testis. Using immunohistochemistry, we can show that AUM is specifically expressed in maturing germ cells and that its expression peaks during spermiogenesis. To characterize the substrate preference of AUM, we have conducted an in vitro phosphatase substrate screen with 720 phosphopeptides derived from human phosphorylation sites. AUM exclusively dephosphorylates phosphotyrosine (pTyr)-containing peptides. Furthermore, only 17 pTyr peptides (~2% of all pTyr peptides investigated) acted as AUM substrates, indicating a high degree of substrate specificity. Putative AUM substrates include proteins involved in cytoskeletal dynamics and tyrosine kinase signaling.In accordance with the phosphopeptide screen, phosphatase overlay assays employing whole-cell extracts of pervanadate-treated HeLa cells show that AUM dephosphorylates only a limited number of tyrosyl-phosphorylated proteins.The role of AUM for cellular signaling was investigated in response to epidermal growth factor (EGF) stimulation in a spermatogonial cell line (GC-1 spg). The overexpression of AUM reduces, whereas the RNAi-mediated depletion of endogenous AUM increases EGF inducedtyrosine phosphorylation, including changes in the phosphorylation of the EGF receptor itself. Interestingly, in vitro kinase/phosphatase assays with purified Src and AUM indicate that AUM can activate Src, which in turn phosphorylates and inactivates AUM. Although it is at present unclear how Src and AUM regulate each other, our initial findings suggests that AUM enhances Src kinase activity independently of its phosphatase activity, whereas Src diminishes AUM phosphatase activity in a kinase dependent manner. On a cellular level, AUM-depleted cells are characterized by altered actin cytoskeletal dynamics and adhesion, as indicated by stabilized actin filaments, enlarged focal adhesions,a marked increase in cell area of spreading cells, as well as accelerated cell spreading on fibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration.
Abiotic stress by elevated tropospheric ozone and temperature can alter plants’ metabolism, growth, and nutritional value and modify the life cycle of their herbivores. We investigated how the duration of exposure of Sinapis arvensis plants to high ozone and temperature levels affect the life cycle of the large cabbage white, Pieris brassicae. Plants were exposed to ozone-clean (control) or ozone-enriched conditions (120 ppb) for either 1 or 5 days and were afterwards kept in a greenhouse with variable temperature conditions. When given the choice, P. brassicae butterflies laid 49% fewer eggs on ozone-exposed than on control plants when the exposure lasted for 5 days, but showed no preference when exposure lasted for 1 day. The caterpillars took longer to hatch on ozone-exposed plants and at lower ambient temperatures. The ozone treatment had a positive effect on the survival of the eggs. Ozone decreased the growth of caterpillars reared at higher temperatures on plants exposed for 5 days, but not on plants exposed for 1 day. Overall, longer exposure of the plants to ozone and higher temperatures affected the life cycle of the herbivore more strongly. With global warming, the indirect impacts of ozone on herbivores are likely to become more common.
Exposure of plants to environmental stressors can modify their metabolism, interactions with other organisms and reproductive success. Tropospheric ozone is a source of plant stress. We investigated how an acute exposure to ozone at different times of plant development affects reproductive performance, as well as the flowering patterns and the interactions with pollinators and herbivores, of wild mustard plants. The number of open flowers was higher on plants exposed to ozone at earlier ages than on the respective controls, while plants exposed at later ages showed a tendency for decreased number of open flowers. The changes in the number of flowers provided a good explanation for the ozone-induced effects on reproductive performance and on pollinator visitation. Ozone exposure at earlier ages also led to either earlier or extended flowering periods. Moreover, ozone tended to increase herbivore abundance, with responses depending on herbivore taxa and the plant age at the time of ozone exposure. These results suggest that the effects of ozone exposure depend on the developmental stage of the plant, affecting the flowering patterns in different directions, with consequences for pollination and reproduction of annual crops and wild species.
Anthropogenic activities are causing air pollution. Amongst air pollutants, tropospheric ozone is a major threat to human health and ecosystem functioning. In this dissertation, I present three studies that aimed at increasing our knowledge on how plant exposure to ozone affects its reproduction and its interactions with insect herbivores and pollinators.
For this purpose, a new fumigation system was built and placed in a greenhouse. The annual plant Sinapis arvensis (wild mustard) was used as the model plant.
Plants were exposed to either 0 ppb (control) or 120 ppb of ozone, for variable amounts of time and at different points of their life cycle. After fumigation, plants were exposed to herbivores or pollinators in the greenhouse, or to both groups of insects in the field.
My research shows that ozone affected reproductive performance differently, depending on the timing of exposure: plants exposed at earlier ages had their reproductive fitness increased, while plants exposed later in their life cycle showed a tendency for reduced reproductive fitness. Plant phenology was a key factor influencing reproductive fitness: ozone accelerated flowering and increased the number of flowers produced by plants exposed at early ages, while plants exposed to ozone at later ages tended to have fewer flowers. On the other hand, the ozone-mediated changes in plant-insect interactions had little impact on plant reproductive success.
The strongest effect of ozone on plant-pollinator interactions was the change in the number of flower visits received per plant, which was strongly linked to the number of open flowers. This means that, as a rule, exposure of plants to ozone early in the life cycle resulted in a higher number of pollinator visits, while exposure later in the life cycle resulted in fewer flower visits by potential pollinators. An exception was observed: the higher number of visits performed by large syrphid flies to young ozone-exposed plants than to the respective control plants went beyond the increase in the number of open flowers in those plants. Also, honeybees spent more time per flower in plants exposed to ozone than on control plants, while other pollinators spent similar amounts of time in control and ozone-exposed plants. This guild-dependent preference for ozone-exposed plants may be due to species-specific preferences related to changes in the quality and quantity of floral rewards.
In the field, ozone-exposed plants showed only a tendency for increased colonization by sucking herbivores and slightly more damage by chewing herbivores than control plants. On the other hand, in the greenhouse experiment, Pieris brassicae butterflies preferred control plants over ozone-exposed plants as oviposition sites. Eggs laid on ozone-exposed plants took longer to hatch, but the chances of survival were higher. Caterpillars performed better in control plants than in ozone-exposed plants, particularly when the temperature was high.
Most of the described effects were dependent on the duration and timing of the ozone exposure and the observed temperature, with the strongest effects being observed for longer exposures and higher temperatures. Furthermore, the timing of exposure altered the direction of the effects.
The expected climate change provides ideal conditions for further increases in tropospheric ozone concentrations, therefore for stronger effects on plants and plant-insect interactions. Acceleration of flowering caused by plant exposure to ozone may put plant-pollinator interactions at risk by promoting desynchronization between plant and pollinator activities. Reduced performance of caterpillars feeding on ozone-exposed plants may weaken herbivore populations. On the other hand, the increased plant reproduction that results from exposing young plants to ozone may be a source of good news in the field of horticulture, when similar results would be achieved in high-value crops. However, plant response to ozone is highly species-specific. In fact, Sinapis arvensis is considered a weed and the advantage conferred by ozone exposure may increase its competitiveness, with negative consequences for crops or plant communities in general. Overall, plant exposure to ozone might constitute a threat for the balance of natural and agro-ecosystems.
Meiotic chromosomes undergo rapid prophase movements, which are thought to facilitate the formation of inter-homologue recombination intermediates that underlie synapsis, crossing over and segregation. The meiotic telomere complex (MAJIN, TERB1, TERB2) tethers telomere ends to the nuclear envelope and transmits cytoskeletal forces via the LINC complex to drive these rapid movements. Here, we report the molecular architecture of the meiotic telomere complex through the crystal structure of MAJIN-TERB2, together with light and X-ray scattering studies of wider complexes. The MAJIN-TERB2 2:2 hetero-tetramer binds strongly to DNA and is tethered through long flexible linkers to the inner nuclear membrane and two TRF1-binding 1:1 TERB2-TERB1 complexes. Our complementary structured illumination microscopy studies and biochemical findings reveal a telomere attachment mechanism in which MAJIN-TERB2-TERB1 recruits telomere-bound TRF1, which is then displaced during pachytene, allowing MAJIN-TERB2-TERB1 to bind telomeric DNA and form a mature attachment plate.