Recapitulation of Tumor Heterogeneity and Molecular Signatures in a 3D Brain Cancer Model with Decreased Sensitivity to Histone Deacetylase Inhibition

Introduction

Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS).

Methods

CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.

Results

Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches.

Conclusions

Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.     

The taxonomic name resolution service: an online tool for automated standardization of plant names

Background

The digitization of biodiversity data is leading to the widespread application of taxon names that are superfluous, ambiguous or incorrect, resulting in mismatched records and inflated species numbers. The ultimate consequences of misspelled names and bad taxonomy are erroneous scientific conclusions and faulty policy decisions. The lack of tools for correcting this ‘names problem’ has become a fundamental obstacle to integrating disparate data sources and advancing the progress of biodiversity science.

Results

The TNRS, or Taxonomic Name Resolution Service, is an online application for automated and user-supervised standardization of plant scientific names. The TNRS builds upon and extends existing open-source applications for name parsing and fuzzy matching. Names are standardized against multiple reference taxonomies, including the Missouri Botanical Garden's Tropicos database. Capable of processing thousands of names in a single operation, the TNRS parses and corrects misspelled names and authorities, standardizes variant spellings, and converts nomenclatural synonyms to accepted names. Family names can be included to increase match accuracy and resolve many types of homonyms. Partial matching of higher taxa combined with extraction of annotations, accession numbers and morphospecies allows the TNRS to standardize taxonomy across a broad range of active and legacy datasets.

Conclusions

We show how the TNRS can resolve many forms of taxonomic semantic heterogeneity, correct spelling errors and eliminate spurious names. As a result, the TNRS can aid the integration of disparate biological datasets. Although the TNRS was developed to aid in standardizing plant names, its underlying algorithms and design can be extended to all organisms and nomenclatural codes. The TNRS is accessible via a web interface at http://tnrs.iplantcollaborative.org/ and as a RESTful web service and application programming interface. Source code is available at https://github.com/iPlantCollaborativeOpenSource/TNRS/.     

The Emerging Role of Copeptin

Direct measurement of the nonapeptide vasopressin has been limited by analyte instability ex vivo and in vivo rapid degradation, low serum concentrations requiring a sensitive assay and inherent secretory pulsatility. Copeptin is a 39 amino acid glycopeptide cleavage product of vasopressin synthesis with high stability, providing a marker of vasopressin secretion. Copeptin measurement has applications in diagnosis of diabetes insipidus and other diseases with altered vasopressin secretion. This review summarises our current understanding of serum copeptin measurement in diabetes insipidus and possible future applications of copeptin assays. As vasopressin is a stress hormone, there is emerging evidence on the use of copeptin for diagnosis and prognostication of disorders such as syndrome of inappropriate anti-diuretic hormone secretion, diabetes mellitus, critical illness, stroke, cardiovascular disease, respiratory disease, renal disease and thermal stress. Copeptin concentration measurement is likely to improve the diagnostic reliability of diabetes insipidus and, as a marker of stress, may have diagnostic or prognostic utility in specific clinical circumstances. Further studies are needed to determine if goal-directed therapy using plasma copeptin concentrations may improve patient outcomes.     

Does spatial aggregation of total nectar production lead to genetic structure?

Spatial aggregation of plants of high nectar production, receiving an enhanced pollinator service is known to occur in Echium vulgare. Moreover, an emanating effect of nectar production on pollinator visits may occur, i.e. many pollinator visits may be observed around high nectar patches. Consequently, gene flow within patches of plants of high nectar production and their close neighbours may result in genetic structure. In this study, we investigated whether aggregation of total nectar production (nectar production per flower×number of flowers) and its emanating effect resulted in genetic structure in a natural E. vulgare population. We compared the spatial structure of total nectar production, pollinator visits and microsatellite markers using spatial autocorrelation analysis. Increased geitonogamy, caused by longer boutlengths in plants of high nectar production may generate genetic structure. We estimated selfing rates of plants of the highest and lowest total nectar production. Spatial aggregation of total nectar production occurred on a relatively small scale up to 2.83 m. A significant emanating correlation between total nectar production and pollinator visits was observed on a relatively large scale up to a 4.24 m. Thus, around patches of high nectar production numbers of pollinator visits were relatively high, while few visits were observed around patches of low nectar production. Weak genetic structure was present on a small scale up to 2.20 m. This corresponded with the scale of aggregation of total nectar production. High gene flow around the patches of high nectar production seems to weaken genetic structure. This is supported by the relatively low selfing rates. The average selfing rate of the plants of highest nectar production was 8.8% and that of the plants of lowest nectar production 5.0%. Low gene flow within and around low nectar patches sustain a weak genetic structure or, conversely, may have caused it in the first instance. Results indicate the importance of spatial structure of nectar production for pollinator movement.     

Microbial Growth under Supercritical CO2

Growth of microorganisms in environments containing CO₂ above its critical point is unexpected due to a combination of deleterious effects, including cytoplasmic acidification and membrane destabilization. Thus, supercritical CO₂ (scCO₂) is generally regarded as a sterilizing agent. We report isolation of bacteria from three sites targeted for geologic carbon dioxide sequestration (GCS) that are capable of growth in pressurized bioreactors containing scCO₂. Analysis of 16S rRNA genes from scCO₂ enrichment cultures revealed microbial assemblages of varied complexity, including representatives of the genus Bacillus. Propagation of enrichment cultures under scCO₂ headspace led to isolation of six strains corresponding to Bacillus cereus, Bacillus subterraneus, Bacillus amyloliquefaciens, Bacillus safensis, and Bacillus megaterium. Isolates are spore-forming, facultative anaerobes and capable of germination and growth under an scCO₂ headspace. In addition to these isolates, several Bacillus type strains grew under scCO₂, suggesting that this may be a shared feature of spore-forming Bacillus spp. Our results provide direct evidence of microbial activity at the interface between scCO₂ and an aqueous phase. Since microbial activity can influence the key mechanisms for permanent storage of sequestered CO₂ (i.e., structural, residual, solubility, and mineral trapping), our work suggests that during GCS microorganisms may grow and catalyze biological reactions that influence the fate and transport of CO₂ in the deep subsurface.     

Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants

Loss of the ability of Pseudomonas syringae pv. "phaseolicola" NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola" genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

CO2 enrichment of soybeans. Effects of leaf/pod ratio

The effect of varying leaf number on response of soybean ( Glycine max (L.) Merr. cv. Fiskeby V) to CO₂ enrichment was studied. Plants were trimmed at pod set to 15 pods and 1 or 3 leaves (15:1 and 5:1 pod/leaf ratio) and placed in 350 or 1000 μl/l CO₂ growth chambers. Photosynthetic rates and dry weights were measured 6 times in all plants at each CO₂ concentration over a period of 39 days. Measured at treatment CO₂ concentration, photosynthetic rates deelined rapidly in enriched plants, but remained higher than those of non-enriched plants. When all plants were measured at the same CO₂ concentration, for most sampling dates, neither growth, CO₂ concentration or pod/leaf ratio significantly affected rates of photosynthesis per unit area of comparable leaves. CO₂ enrichment significantly increased total weights and pod weights in 15:1 but not 5:1 pod/leaf ratio plants. Plants with a 5:1 pod/leaf ratio had significantly higher total and pod weights than 15:1 ratio plants. Both the photosynthesis and dry weight data suggest that plants in the 5:1 ratio enriched treatment were sink-limited, but plants in all other treatments were source limited.