Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy ¹. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM ². This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments ^3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.     

Double Knockout Mutants of Arabidopsis Grown under Normal Conditions Reveal that the Plastidial Phosphorylase Isozyme Participates in Transitory Starch Metabolism

In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1 × phs1a and mex1 × phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1 × phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.During the last two decades, biochemical analyses of starch metabolism in higher plants have been favored by the availability of large sets of insertion mutants deficient in a single starch-related gene product. Based on phenotypical characterization of these mutants followed by the identification of the respective locus in the genome, novel starch-related proteins were discovered that reside inside the plastid, in the cytosol, in the nucleus, and in the plastidial envelope membranes. Taken together, these results have largely altered the current view on starch metabolism (Zeeman et al., 2010; Fettke et al., 2012a; Smith, 2012).Despite this progress, phenotypical analyses of starch-related mutants are complex and, under certain circumstances, yield misleading conclusions. Loss of function of metabolic steps may cause the entire starch synthesizing or degrading process to become nonfunctional. In this case, mutants are expected to have starch levels that are significantly altered. If, however, single knockout mutants are capable of partially or fully compensating the loss of function by other routes, the resulting phenotypes are less obvious and more difficult to predict. Carbon fluxes through existing paths may be enhanced, or novel metabolic routes may be established that compensate the lost function. As an example, leaves of Arabidopsis (Arabidopsis thaliana) mutants constitutively lacking the plastidial hexose-phosphate isomerase strongly express a distinct plastidial Glc-6-P/orthophosphate antiporter isoform that in wild-type plants is found only in heterotrophic tissues (Kunz et al., 2010). In mesophyll cells of the mutant, the reductive pentose phosphate cycle cannot drive assimilatory starch biosynthesis, as chloroplasts are unable to convert Fru-6-P to Glc-6-P. However, their capacity of transporting Glc-6-P between the cytosolic and the chloroplastic compartment is strongly increased. Furthermore, nonfunctionality of some starch-related proteins can lead to enlarged or diminished metabolite pools that via sensing processes, lead to cellular alterations distant from central carbon metabolism. This complexity is evidenced by several starch-related Arabidopsis mutants that possess a largely altered plastidial ultrastructure and exhibit premature degradation of the entire chloroplast (Stettler et al., 2009; Cho et al., 2011).Furthermore, several starch-related enzymes are capable of forming homomeric or heteromeric complexes that are functionally relevant but, to some extent, variable (Delatte et al., 2005; Utsumi and Nakamura, 2006; Kubo et al., 2010; Emes and Tetlow, 2012; Nakamura et al., 2012; Streb et al., 2012).In starch or glycogen storing prokaryotic and eukaryotic cells, α-glucan phosphorylase (EC 2.4.1.1) is common. Initially, this enzyme was considered to be the main starch synthesizing activity (Hanes, 1940). Later, both starch and glycogen synthases have been discovered that utilize either ADPglucose or UDPglucose (or both; Deschamps et al., 2006) as hexosyl donor. Ample evidence has been presented that these enzymes are essential biosynthetic enzymes (Ballicora et al., 2003; Zeeman et al., 2010; Roach et al., 2012; Palm et al., 2013). Furthermore, it is widely accepted that in glycogen-storing cells, phosphorylase is indispensible for the degradation of the storage polysaccharide (Hwang et al., 1989; Alonso-Casajús et al., 2006; Wilson et al., 2010; Roach et al., 2012; Gazzerro et al., 2013).In plant cells, the metabolic function of phosphorylase is more complex and far from being clear. In lower and higher plants, two distinct phosphorylase types exist as plastid- and cytosol-specific isozymes and are designated as Pho1 (or, in Arabidopsis, PHS1) and Pho2 (PHS2), respectively. Based on the large differences in the affinities for glycogen, the plastidial and the cytosolic phosphorylases are also named as low-affinity (L-type) and high-affinity (H-type) isozymes, respectively. As starch is restricted to the plastids, only the Pho1 (PHS1) type appears to possess direct access to native starch and/or plastidial starch-derived α-glucans.Conflicting phenotypical features have been reported for several mutants possessing altered levels of the plastidial phosphorylase isozyme(s). In the starch-related mutant4 of the unicellular green alga Chlamydomonas reinhardtii, the lack of one plastidial Pho1 isozyme (designated as PhoB) was associated with a lower cellular starch content, abnormally shaped granules, a modified amylopectin structure, and an elevated amylose-to-amylopectin ratio when the cells were kept under nitrogen limitation (Dauvillée et al., 2006). These phenotypical features suggest an involvement of the plastidial phosphorylase PhoB in the biosynthesis of a storage polysaccharide resembling the reserve starch of higher plants. Similarly, a rapid incorporation of ¹⁴C into starch was observed when tuber discs from various transgenic potato lines were incubated with [U-¹⁴C]Glc-1-P. The rate of starch labeling was found to reflect the activity of the plastidial phosphorylase isozyme Pho1 (Fettke et al., 2010, 2012b). By contrast, transgenic potato (Solanum tuberosum) lines have been generated that due to expression of an antisense construct, possess a largely diminished total Pho1 activity in leaves. Leaf starch content is essentially unchanged compared with that of the wild-type plants, suggesting that under normal growth conditions, the plastidial phosphorylase is not necessarily involved in starch metabolism or, alternatively, can easily be replaced by other enzymes (Sonnewald et al., 1995). Likewise, the phenotype (including leaf starch content) of an Arabidopsis mutant lacking functional PHS1 has been reported not to differ from the wild type when the plants were grown under normal conditions. However, under water stress conditions, significantly more local leaf lesions have been reported to occur (Zeeman et al., 2004).When leaf discs from bean (Phaseolus vulgaris) or Arabidopsis plants were exposed to conditions favoring photorespiration (i.e. an atmosphere consisting of 30% [v/v] O₂ and 70% [v/v] N₂ but lacking CO₂), transitory starch was degraded in the light at a high rate and the plastidial Glc-6-P pool increased. In Arabidopsis mutants deficient in PHS1, the Glc monophosphate pool did not respond to photorespiratory conditions (Weise et al., 2006). These data lead to the conclusion that in illuminated leaves with very high rates of photorespiration, PHS1 is involved in the conversion of starch to Glc monophosphates but does not to participate in the nocturnal starch degradation.When studying several starch-related Arabidopsis mutants, we noticed that two single knockout mutations that both affect the maltose metabolism but differ in the subcellular location of the target protein possess a significantly increased PHS1 activity (Malinova et al., 2011a, 2011b). One mutant constitutively lacks the functional cytosolic transglucosidase (also designated as disproportionating enzyme2; DPE2) and, therefore, the cytosolic route of starch-derived maltose metabolism is impaired (Chia et al., 2004; Lu and Sharkey, 2004). The other mutant does not express the plastidial maltose transporter MEX1, resulting in a massively enlarged maltose pool (Niittylä et al., 2004). Thus, in the two mutants, the metabolism of starch-derived maltose is blocked at different subcellular sites, i.e. the cytosol and the chloroplast. The enhanced PHS1 activity as observed for the two mutants is difficult to explain unless a more general function of the phosphorylase isozyme in starch metabolism is assumed.For a detailed functional analysis of PHS1-related processes, we generated two types of constitutive PHS1-deficient double knockout mutants (DPE2 plus PHS1 or MEX1 plus PHS1) and studied their phenotypes in more detail under various experimental conditions. Shoot growth and leaf chlorophyll content are reduced when the plants are grown under a light-dark regime, but under continuous illumination, both effects are far less pronounced. Based on these data, we propose that the plastidial phosphorylase participates in both the turnover of transitory starch and in the maintenance of intact chloroplasts.     

The Epstein-Barr virus lytic program is controlled by the co-operative functions of two transactivators

The propagation of herpesviruses has long been viewed as a temporally regulated sequential process that results from the consecutive expression of specific viral transactivators. As a key step in this process, lytic viral DNA replication is considered as a checkpoint that controls the expression of the late structural viral genes. In a novel genetic approach, we show that both hypotheses do not hold true for the Epstein-Barr virus (EBV). The study of viral mutants of EBV in which the early genes BZLF1 and BRLF1 are deleted allowed a precise assignment of the function of these proteins. Both transactivators were absolutely essential for viral DNA replication. Both BZLF1 and BRLF1 were required for full expression of the EBV proteins expressed during the lytic program, although the respective influence of these molecules on the expression of various viral target genes varied greatly. In replication-defective viral mutants, neither early gene expression nor DNA replication was a prerequisite for late gene expression. This work shows that BRLF1 and BZLF1 harbor distinct but complementary functions that influence all stages of viral production.     

The cumulative impact of annual coral bleaching can turn some coral species winners into losers

Mass coral bleaching events caused by elevated seawater temperatures result in extensive coral loss throughout the tropics, and are projected to increase in frequency and severity. If bleaching becomes an annual event later in this century, more than 90% of coral reefs worldwide may be at risk of long‐term degradation. While corals can recover from single isolated bleaching and can acclimate to recurring bleaching events that are separated by multiple years, it is currently unknown if and how they will survive and possibly acclimatize to annual coral bleaching. Here, we demonstrate for the first time that annual coral bleaching can dramatically alter thermal tolerance in Caribbean corals. We found that high coral energy reserves and changes in the dominant algal endosymbiont type (Symbiodinium spp.) facilitated rapid acclimation in Porites divaricata, whereas low energy reserves and a lack of algal phenotypic plasticity significantly increased susceptibility in Porites astreoides to bleaching the following year. Phenotypic plasticity in the dominant endosymbiont type of Orbicella faveolata did not prevent repeat bleaching, but may have facilitated rapid recovery. Thus, coral holobiont response to an isolated single bleaching event is not an accurate predictor of its response to bleaching the following year. Rather, the cumulative impact of annual coral bleaching can turn some coral species ‘winners’ into ‘losers’, and can also facilitate acclimation and turn some coral species ‘losers’ into ‘winners’. Overall, these findings indicate that cumulative impact of annual coral bleaching could result in some species becoming increasingly susceptible to bleaching and face a long‐term decline, while phenotypically plastic coral species will acclimatize and persist. Thus, annual coral bleaching and recovery could contribute to the selective loss of coral diversity as well as the overall decline of coral reefs in the Caribbean.     

Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [L?bermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.     

Response of human squamous cell carcinoma xenografts of different sizes to irradiation: relationship of clonogenic cells, cellular radiation sensitivity in vivo, and tumor rescuing units

The relationship of clonogenic cells, cellular radiation sensitivity at tumor control does in vivo, and tumor rescuing units at different tumor sizes was investigated in the human squamous cell carcinoma FaDu growing in NCr/Sed nude mice. The composition of the tumors was determined in single cell suspensions and compared to tumor control data after single-dose irradiation. To avoid the influence of varying oxygen concentrations in the tumors, all irradiations were performed under clamp hypoxia. Nude mice and animals further immunosuppressed by 6-Gy whole-body irradiation were used to assess the immunological effects. The numbers of total cells, cells excluding trypan blue, host cells, and colony-forming cells increased linearly with the weight of FaDu tumors. Comparable results were obtained for cell suspensions prepared from tumors growing in nude of pretreated nude mice. The radiation dose required to control 50% of tumors (TCD50) of different sizes between 36 and 470 mm3 increased from 52.1 to 60.1 Gy when the tumors were maintained in normal nude mice and from 50.8 to 61.3 Gy in whole-body-irradiated mice. The D0 of FaDu cells in vivo was calculated by regression analysis of TCD50 vs the logarithm of the clonogenic cell number, assuming an oxygen enhancement ratio of 3.0. The resultant D0S of 1.1 and 1.2 Gy in vivo correspond well to the radiosensitivity of FaDu cells in vitro determined previously. Assuming the single-hit multitarget model of cell killing and extrapolation numbers between 2 and 20, the mean number of tumor rescuing units would be 10(5) to 10(6) for a 100-mm3 tumor growing in whole-body-irradiated nude mice. Comparison of the number of tumor rescuing units to the estimated number of clonogenic cells does not conflict with the assumption that every surviving clonogenic cell is able to repopulate FaDu tumors after irradiation; however, it seems more likely that more than one clonogenic cells is necessary. The proportion of tumor rescuing units in the clonogenic cell population is independent of tumor size.     

Homothorax switches function of Drosophila photoreceptors from color to polarized light sensors

Different classes of photoreceptors (PRs) allow animals to perceive various types of visual information. In the Drosophila eye, the outer PRs of each ommatidium are involved in motion detection while the inner PRs mediate color vision. In addition, flies use a specialized class of inner PRs in the "dorsal rim area" of the eye (DRA) to detect the e-vector of polarized light, allowing them to exploit skylight polarization for orientation. We show that homothorax is both necessary and sufficient for inner PRs to adopt the polarization-sensitive DRA fate instead of the color-sensitive default state. Homothorax increases rhabdomere size and uncouples R7-R8 communication to allow both cells to express the same opsin rather than different ones as required for color vision. Homothorax expression is induced by the iroquois complex and the wingless (wg) pathway. However, crucial wg pathway components are not required, suggesting that additional signals are involved.     

Differences in oscillatory whistles produced by spinner (Stenella longirostris) and pantropical spotted (Stenella attenuata) dolphins

Acoustic recordings of two closely related species, spinner dolphin (Stenella longirostris) and pantropical spotted dolphin (Stenella attenuata), were investigated from four different geographic locations: two in the Central Tropical Pacific, one in the Eastern Tropical Pacific and one in the Indian Ocean. The two delphinid species occur in tropical and warm temperate waters, with overlapping ranges. They produce very similar vocalizations, but at the same time their calls exhibit a certain degree of intraspecific variation among different geographic locations as has been observed in other delphinid species. Oscillatory whistles (whistles with at least two oscillations in their frequency contours) were identified and manually extracted from the recordings. Whistles with four or more maxima (oscillations) occurred only in spinner dolphins and they were present in all geographic regions investigated. In addition, the oscillatory whistles with two and three maxima were significantly more frequent in spinner than in spotted dolphins. The differences in oscillatory whistles for these two species seem to be consistent across study areas and therefore, could be used in addition to other whistle features to help distinguish between them.     

Odontocete occurrence in relation to changes in oceanography at a remote equatorial Pacific seamount

Seamounts are considered hot spots of biodiversity and can aggregate pelagic predators and their prey. Passive acoustic monitoring was conducted over 3 mo in 2012 to document the occurrence of odontocetes near a seamount chain in the central equatorial Pacific in relation to oceanographic changes over time. Beaked whale echolocation signals were most frequently encountered. The main beaked whale signal was an unknown type, BW38, which resembled signals produced by Blainville's beaked whales. It had high occurrence during high sea surface temperature and low sea surface salinity. Cuvier's beaked whales were the second most detected. They had an opposite pattern and were encountered more often when sea surface temperature was low and net primary productivity was high. Risso's dolphins and short‐finned pilot whales had high acoustic densities, and echolocated predominantly at night. Risso's dolphins occurred more often during low sea surface height deviation. False killer whales were less frequently detected and mostly occurred during the day. Sperm whale detections were fewer than expected and associated with high chlorophyll a. Short duration Kogiidae encounters occurred on average every third day. These types of long‐term site studies are an informative tool to comparatively assess species composition, relative abundance, and relationship to oceanographic changes.