A GFP-Tagged Gross Deletion on Chromosome 1 Causes Malignant Peripheral Nerve Sheath Tumors and Carcinomas in Zebrafish

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas, characterized by complex karyotypes. The molecular bases of such malignancy are poorly understood and efficient targeted molecular therapies are currently lacking. Here we describe a novel zebrafish model of MPNSTs, represented by the transgenic mutant line Tg(-8.5nkx2.2a:GFP) ^ia2. ia2 homozygous animals displayed embryonic lethality by 72 hpf, while the heterozygotes develop visible tumor masses with high frequency in adulthood. Histological and immunohistochemical examination revealed aggressive tumors with either mesenchymal or epithelial features. The former (54% of the cases) arose either in the abdominal cavity, or as intrathecal/intraspinal lesions and is composed of cytokeratin-negative spindle cells with fascicular/storiform growth pattern consistent with zebrafish MPNSTs. The second histotype was composed by polygonal or elongated cells, immunohistochemically positive for the pan-cytokeratin AE1/AE3. The overall histologic and immunohistochemical features were consistent with a malignant epithelial neoplasm of possible gastrointestinal/pancreatic origin. With an integrated approach, based on microsatellite (VNTR) and STS markers, we showed that ia2 insertion, in Tg(-8.5nkx2.2a:GFP) ^ia2 embryos, is associated with a deletion of 15.2 Mb in the telomeric portion of chromosome 1. Interestingly, among ia2 deleted genes we identified the presence of the 40S ribosomal protein S6 gene that may be one of the possible drivers for the MPNSTs in ia2 mutants. Thanks to the peculiar features of zebrafish as animal model of human cancer (cellular and genomic similarity, transparency and prolificacy) and the GFP tag, the Tg(-8.5nkx2.2a:GFP) ^ia2 line provides a manageable tool to study in vivo with high frequency MPNST biology and genetics, and to identify, in concert with the existing zebrafish MPNST models, conserved relevant mechanisms in zebrafish and human cancer development.     

Infant Growth after Preterm Birth and Mental Health in Young Adulthood

Objectives

Faster growth after preterm birth benefits long-term cognitive functioning. Whether these benefits extend to mental health remains largely unknown. We examined if faster growth in infancy is associated with better self-reported mental health in young adults born preterm at very low birth weight (VLBW) (<1500g).

Study Design

As young adults, participants of the Helsinki Study of Very Low Birth Weight Adults self-reported symptoms of depression and attention deficit/hyperactivity disorder (ADHD) (n = 157) and other psychiatric problems (n = 104). As main predictors of mental health outcomes in linear regression models, we used infant weight, length, and head circumference at birth, term, and 12 months of corrected age, and growth between these time points. Growth data were collected from records and measures at term and at 12 months of corrected age were interpolated. Additionally, we examined the moderating effects of intrauterine growth restriction.

Results

Size at birth, term, or 12 months of corrected age, or growth between these time points were not associated with mental health outcomes (p-values >0.05). Intrauterine growth restriction did not systematically moderate any associations.

Conclusions

Despite the high variability in early growth of VLBW infants, the previously described association between slow growth in infancy and poorer cognitive functioning in later life is not reflected in symptoms of depression, ADHD, and other psychiatric problems. This suggests that the development of cognitive and psychiatric problems may have dissimilar critical periods in VLBW infants.     

Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing’s Sarcoma

Ewing’s sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing’s sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing’s sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing’s sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing’s sarcoma patients with PARP inhibitors.     

Fostering Formal Commutativity Knowledge with Approximate Arithmetic

How can we enhance the understanding of abstract mathematical principles in elementary school? Different studies found out that nonsymbolic estimation could foster subsequent exact number processing and simple arithmetic. Taking the commutativity principle as a test case, we investigated if the approximate calculation of symbolic commutative quantities can also alter the access to procedural and conceptual knowledge of a more abstract arithmetic principle. Experiment 1 tested first graders who had not been instructed about commutativity in school yet. Approximate calculation with symbolic quantities positively influenced the use of commutativity-based shortcuts in formal arithmetic. We replicated this finding with older first graders (Experiment 2) and third graders (Experiment 3). Despite the positive effect of approximation on the spontaneous application of commutativity-based shortcuts in arithmetic problems, we found no comparable impact on the application of conceptual knowledge of the commutativity principle. Overall, our results show that the usage of a specific arithmetic principle can benefit from approximation. However, the findings also suggest that the correct use of certain procedures does not always imply conceptual understanding. Rather, the conceptual understanding of commutativity seems to lag behind procedural proficiency during elementary school.     

Evaluation of coumarin-based fluorogenic P450 BM3 substrates and prospects for competitive inhibition screenings

Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid. Particularly high enzyme-mediated O-dealkylation yielding the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin was observed with mutants containing the F87V substitution, with A74G/F87V showing the highest catalytic efficiency (0.458 min⁻¹ μM⁻¹). To simplify the assay procedure and show its versatility, different modes of application were successfully demonstrated, including (i) the direct use of NADPH or its oxidized form NADP⁺ along with diverse NADPH recycling systems for electron supply, (ii) the use of cell-free lysates and whole-cell preparations as the biocatalyst source, and (iii) its use for competitive inhibition screens to identify or characterize substrates and inhibitors. A detailed comparison with known, fluorescence-based P450 BM3 assays finally emphasizes the relevance of our contribution to the ongoing research.     

Comparative biocenological analysis of Gastropoda on the Macedonian part of Lake Ohrid and its watershed

Lake Ohrid represents a refugial ecosystem which harbors a great number of endemic and relics living forms. Though the whole Lake’s fauna characterizes high biodiversity and endemism, this is most obvious in the class of Gastropoda. Unlike the Lake, the fauna of the adjacent waters fairly differs from the Lake’s fauna, i.e., it is poorer both in term of diversity and endemism. The main goal of our study was to perform comparative biocenological researches on the gastropod fauna from Lake Ohrid and the adjacent waters in the watershed of the Lake. Based on the results we have obtained, it could be clearly noted that different habitats are characterized with different qualitative composition of the gastropod fauna not only when compared the gastropod settlement between the Lake and its surrounding waters, but also in the Lake for itself. Total of 50 species of gastropods have been recorded in the littoral regions of the lake and its coastal waters during 2009/2010. They belong to subclass Orthogastropoda (50 taxa). 21 species out of 50 are recorded in the adjacent waters: 13 of them settle both the adjacent waters and the Lake, while 8 strictly inhabit the adjacent waters. In terms of endemism, 17 are endemic and 4 cosmopolitan. The remainig 29 (out of 50 recorded) settle up only the littoral zone of the Lake: 25 are endemic and 4 are cosmopolitan. The percentage of endemism based on the recorded species for the class Gastropoda is 84%.     

Short-term responses of ecosystem carbon fluxes to experimental soil warming at the Swiss alpine treeline

Climatic warming will probably have particularly large impacts on carbon fluxes in high altitude and latitude ecosystems due to their great stocks of labile soil C and high temperature sensitivity. At the alpine treeline, we experimentally warmed undisturbed soils by 4 K for one growing season with heating cables at the soil surface and measured the response of net C uptake by plants, of soil respiration, and of leaching of dissolved organic carbon (DOC). Soil warming increased soil CO₂ effluxes instantaneously and throughout the whole vegetation period (+45%; +120 g C m y^?1). In contrast, DOC leaching showed a negligible response of a 5% increase (NS). Annual C uptake of new shoots was not significantly affected by elevated soil temperatures, with a 17, 12, and 14% increase for larch, pine, and dwarf shrubs, respectively, resulting in an overall increase in net C uptake by plants of 20–40 g C m^?2y^?1. The Q ₁₀ of 3.0 measured for soil respiration did not change compared to a 3-year period before the warming treatment started, suggesting little impact of warming-induced lower soil moisture (?15% relative decrease) or increased soil C losses. The fraction of recent plant-derived C in soil respired CO₂ from warmed soils was smaller than that from control soils (25 vs. 40% of total C respired), which implies that the warming-induced increase in soil CO₂ efflux resulted mainly from mineralization of older SOM rather than from stimulated root respiration. In summary, one season of 4 K soil warming, representative of hot years, led to C losses from the studied alpine treeline ecosystem by increasing SOM decomposition more than C gains through plant growth.     

Estimating the contribution of assembly activity to cortical dynamics from spike and population measures

The hypothesis that cortical networks employ the coordinated activity of groups of neurons, termed assemblies, to process information is debated. Results from multiple single-unit recordings are not conclusive because of the dramatic undersampling of the system. However, the local field potential (LFP) is a mesoscopic signal reflecting synchronized network activity. This raises the question whether the LFP can be employed to overcome the problem of undersampling. In a recent study in the motor cortex of the awake behaving monkey based on the locking of coincidences to the LFP we determined a lower bound for the fraction of spike coincidences originating from assembly activation. This quantity together with the locking of single spikes leads to a lower bound for the fraction of spikes originating from any assembly activity. Here we derive a statistical method to estimate the fraction of spike synchrony caused by assemblies—not its lower bound—from the spike data alone. A joint spike and LFP surrogate data model demonstrates consistency of results and the sensitivity of the method. Combining spike and LFP signals, we obtain an estimate of the fraction of spikes resulting from assemblies in the experimental data.     

Modulating neuronal activity produces specific and long-lasting changes in numerical competence

Around 20% of the population exhibits moderate to severe numerical disabilities [1-3], and a further percentage loses its numerical competence during the lifespan as a result of stroke or degenerative diseases [4]. In this work, we investigated the feasibility of using noninvasive stimulation to the parietal lobe during numerical learning to selectively improve numerical abilities. We used transcranial direct current stimulation (TDCS), a method that can selectively inhibit or excitate neuronal populations by modulating GABAergic (anodal stimulation) and glutamatergic (cathodal stimulation) activity [5, 6]. We trained subjects for 6 days with artificial numerical symbols, during which we applied concurrent TDCS to the parietal lobes. The polarity of the brain stimulation specifically enhanced or impaired the acquisition of automatic number processing and the mapping of number into space, both important indices of numerical proficiency [7-9]. The improvement was still present 6 months after the training. Control tasks revealed that the effect of brain stimulation was specific to the representation of artificial numerical symbols. The specificity and longevity of TDCS on numerical abilities establishes TDCS as a realistic tool for intervention in cases of atypical numerical development or loss of numerical abilities because of stroke or degenerative illnesses.     

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N‐linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence‐activated cell sorting (FACS) and site‐specific gene excision to establish high‐yield glycoprotein expression for structural studies with stable clones derived from the well‐established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single‐chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome‐associated membrane protein 3 (LAMP3d). In both cases, stable GFP‐expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.