Enhancing the cytotoxicity of chemoradiation with radiation-guided delivery of anti-MGMT morpholino oligonucleotides in non-methylated solid tumors.

The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is epigenetically silenced in some tumors by MGMT gene promoter methylation. MGMT-hypermethylated solid tumors have enhanced susceptibility to the cytotoxic effects of alkylating chemotherapy such as temozolomide, compared with non-methylated tumors. In glioblastoma, subjects with MGMT hypermethylation have significantly longer survival rates after chemoradiotherapy. We report the first successful use of a non-ablative dose of ionizing radiation to prime human cancer cells to enhance the uptake of unmodified anti-MGMT morpholino oligonucleotide (AMON) sequences. We demonstrate >40% reduction in the in vitro proliferation index and cell viability in radiation-primed MGMT-expressing human solid tumor cells treated with a single dose of AMONs and temozolomide. We further demonstrate the feasibility of using a non-ablative dose of radiation in vivo to guide and enhance the delivery of intravenously administered AMONs to achieve 50% MGMT knockdown only at radiation-primed tumor sites in a subcutaneous tumor model. Local upregulation of physiological endocytosis after radiation may have a role in radiation-guided uptake of AMONs. This approach holds direct translational significance in glioblastoma and brain metastases where radiation is part of the standard of care; our approach to silence MGMT could overcome the significant problem of MGMT-mediated chemoresistance.

Peptides@mica: from affinity to adhesion mechanism.

Investigating the adsorption of peptides on inorganic surfaces, on the molecular level, is fundamental for medicinal and analytical applications. Peptides can be potent as linkers between surfaces and living cells in biochips or in implantation medicine. Here, we studied the adsorption process of the positively charged pentapeptide RTHRK, a recently identified binding sequence for surface oxidized silicon, and novel analogues thereof to negatively charged mica surfaces. Homogeneous formation of monolayers in the nano- and low micromolar peptide concentration range was observed. We propose an alternative and efficient method to both quantify binding affinity and follow adhesion behavior. This method makes use of the thermodynamic relationship between surface coverage, measured by atomic force microscopy (AFM), and the concomitant free energy of adhesion. A knowledge-based fit to the autocorrelation of the AFM images was used to correct for a biased surface coverage introduced by the finite lateral resolution of the AFM. Binding affinities and mechanisms were further explored by large scale molecular dynamics (MD) simulations. The combination of well validated MD simulations with topological data from AFM revealed a better understanding of peptide adsorption processes on the atomistic scale. We demonstrate that binding affinity is strongly determined by a peptide's ability to form salt bridges and hydrogen bonds with the surface lattice. Consequently, differences in hydrogen bond formation lead to substantial differences in binding affinity despite conservation of the peptide's overall charge. Further, MD simulations give access to relative changes in binding energy of peptide variations in comparison to a lead compound.

CEST-FISP: a novel technique for rapid chemical exchange saturation transfer MRI at 7 T.

Chemical exchange saturation transfer (CEST) and magnetization transfer techniques provide unique and potentially quantitative contrast mechanisms in multiple MRI applications. However, the in vivo implementation of these techniques has been limited by the relatively slow MRI acquisition techniques, especially on high-field MRI scanners. A new, rapid CEST-fast imaging with steady-state free precession technique was developed to provide sensitive CEST contrast in ∼20 sec. In this study at 7 T with in vitro bovine glycogen samples and initial in vivo results in a rat liver, the CEST-fast imaging with steady-state free precession technique was shown to provide equivalent CEST sensitivity in comparison to a conventional CEST-spin echo acquisition with a 50-fold reduction in acquisition time. The sensitivity of the CEST-fast imaging with steady-state free precession technique was also shown to be dependent on k-space encoding with centric k-space encoding providing a 30-40% increase in CEST sensitivity relative to linear encoding for 256 or more k-space lines. Overall, the CEST-fast imaging with steady-state free precession acquisition technique provides a rapid and sensitive imaging platform with the potential to provide quantitative CEST and magnetization transfer imaging data.

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

The origins of sexual dimorphism in body size in ungulates.

Jarman (1974) proposed a series of relationships between habitat use, food dispersion, and social behavior and hypothesized a series of evolutionary steps leading to sexual dimorphism in body size through sexual selection in African antelope species. The hypothesis states that sexual size dimorphism evolved in a three-step process. Initially, ancestral monomorphic and monogamous ungulate species occupying closed habitats radiated into open grassland habitats. Polygynous mating systems then rapidly evolved in response to the aggregation of males and females, perhaps in relation to the clumped distribution of food resources in open habitats. Subsequently, size dimorphism evolved in those species occupying open habitats, but not in species that remained in closed habitats or retained monogamy. This hypothesis has played an important role in explaining the origins of sexual dimorphism in mammals. However, the temporal sequence of the events that Jarman proposed has never been demonstrated. Here we use a phylogeny of extant ungulate species, along with maximum-likelihood statistical techniques, to provide a test of Jarman's hypothesis.

Phylogenetic analysis and comparative data: a test and review of evidence.

The question is often raised whether it is statistically necessary to control for phylogenetic associations in comparative studies. To investigate this question, we explore the use of a measure of phylogenetic correlation, lambda, introduced by Pagel (1999), that normally varies between 0 (phylogenetic independence) and 1 (species' traits covary in direct proportion to their shared evolutionary history). Simulations show lambda to be a statistically powerful index for measuring whether data exhibit phylogenetic dependence or not and whether it has low rates of Type I error. Moreover, lambda is robust to incomplete phylogenetic information, which demonstrates that even partial information on phylogeny will improve the accuracy of phylogenetic analyses. To assess whether traits generally show phylogenetic associations, we present a quantitative review of 26 published phylogenetic comparative data sets. The data sets include 103 traits and were chosen from the ecological literature in which debate about the need for phylogenetic correction has been most acute. Eighty-eight percent of data sets contained at least one character that displayed significant phylogenetic dependence, and 60% of characters overall (pooled across studies) showed significant evidence of phylogenetic association. In 16% of tests, phylogenetic correlation could be neither supported nor rejected. However, most of these equivocal results were found in small phylogenies and probably reflect a lack of power. We suggest that the parameter lambda be routinely estimated when analyzing comparative data, since it can also be used simultaneously to adjust the phylogenetic correction in a manner that is optimal for the data set, and we present an example of how this may be done.

Therapeutic efficacy of aortic administration of N-acetylcysteine as a chemoprotectant against bone marrow toxicity after intracarotid administration of alkylators, with or without glutathione depletion in a rat model.

Modulation of thiol levels may alter both the efficacy and toxicity of chemotherapeutic agents. We investigated cytoenhancement, using L-buthionine-[S,R]-sulfoximine (BSO) to reduce cellular glutathione levels prior to intracarotid alkylator administration. We also evaluated chemoprotection against chemotherapy-induced systemic toxicity when the thiol agents N-acetylcysteine (NAC) and sodium thiosulfate were administered into the descending aorta to limit brain delivery. BSO treatment reduced rat brain and intracerebral tumor glutathione levels by 50-65%, equivalent to the reduction in liver and s.c. tumor. BSO treatment significantly enhanced the toxicity of chemotherapy with carboplatin, melphalan, and etoposide phosphate against granulocytes, total white cells, and platelets. Intracarotid administration of NAC resulted in high delivery to the brain, whereas infusion via the descending aorta minimized brain delivery. When NAC, with or without sodium thiosulfate, was administered via aortic infusion prior to chemotherapy, the magnitude of the bone marrow toxicity nadir was minimized, even with BSO-enhanced myelosuppression. Thus, BSO depleted brain and brain tumor glutathione but thereby increased chemotherapy-induced myelosuppression. Surprisingly, although NAC was found to readily cross the blood-brain barrier when given into the carotid artery, aortic infusion of NAC resulted in minimal exposure to the central nervous system (CNS) vasculature because of rapid clearance. As a result, aortic infusion of NAC to perfuse bone marrow and minimize myelosuppression and toxicity to visceral organs could be performed without interfering with the CNS cytotoxicity of intracarotid alkylators, even after BSO depletion of CNS glutathione.

Major fungal lineages are derived from lichen symbiotic ancestors.

About one-fifth of all known extant fungal species form obligate symbiotic associations with green algae, cyanobacteria or with both photobionts. These symbioses, known as lichens, are one way for fungi to meet their requirement for carbohydrates. Lichens are widely believed to have arisen independently on several occasions, accounting for the high diversity and mixed occurrence of lichenized and non-lichenized (42 and 58%, respectively) fungal species within the Ascomycota. Depending on the taxonomic classification chosen, 15-18 orders of the Ascomycota include lichen-forming taxa, and 8-11 of these orders (representing about 60% of the Ascomycota species) contain both lichenized and non-lichenized species. Here we report a phylogenetic comparative analysis of the Ascomycota, a phylum that includes greater than 98% of known lichenized fungal species. Using a Bayesian phylogenetic tree sampling methodology combined with a statistical model of trait evolution, we take into account uncertainty about the phylogenetic tree and ancestral state reconstructions. Our results show that lichens evolved earlier than believed, and that gains of lichenization have been infrequent during Ascomycota evolution, but have been followed by multiple independent losses of the lichen symbiosis. As a consequence, major Ascomycota lineages of exclusively non-lichen-forming species are derived from lichen-forming ancestors. These species include taxa with important benefits and detriments to humans, such as Penicillium and Aspergillus.

Evolution of base composition and codon usage bias in the genus Flavivirus.

The extent to which base composition and codon usage vary among RNA viruses, and the possible causes of this bias, is undetermined in most cases. A maximum-likelihood statistical method was used to test whether base composition and codon usage bias covary with arthropod association in the genus Flavivirus, a major source of disease in humans and animals. Flaviviruses are transmitted by mosquitoes, by ticks, or directly between vertebrate hosts. Those viruses associated with ticks were found to have a significantly lower G+C content than non-vector-borne flaviviruses and this difference was present throughout the genome at all amino acids and codon positions. In contrast, mosquito-borne viruses had an intermediate G+C content which was not significantly different from those of the other two groups. In addition, biases in dinucleotide and codon usage that were independent of base composition were detected in all flaviviruses, but these did not covary with arthropod association. However, the overall effect of these biases was slight, suggesting only weak selection at synonymous sites. A preliminary analysis of base composition, codon usage, and vector specificity in other RNA virus families also revealed a possible association between base composition and vector specificity, although with biases different from those seen in the Flavivirus genus.

Sexual swellings advertise female quality in wild baboons.

The females of many Old World primate species produce prominent and conspicuous swellings of the perineal skin around the time of ovulation. These sexual swellings have been proposed to increase competition among males for females or to increase the likelihood of a female getting fertilized, by signalling either a female's general reproductive status, or the timing of her ovulation. Here we show that sexual swellings in wild baboons reliably advertise a female's reproductive value over her lifetime, in accordance with a theoretical model of honest signalling. Females with larger swellings attained sexual maturity earlier, produced both more offspring and more surviving offspring per year than females with smaller swellings, and had a higher overall proportion of their offspring survive. Male baboons use the size of the sexual swelling to determine their mating effort, fighting more aggressively to consort females with larger swellings, and spending more time grooming these females. Our results document an unusual case of a sexually selected ornament in females, and show how males, by mating selectively on the basis of the size of the sexual swelling, increase their probability of mating with females more likely to produce surviving offspring.

Protein sequence signatures support the African clade of mammals.

DNA sequence evidence supports a superordinal clade of mammals that comprises elephants, sea cows, hyraxes, aardvarks, elephant shrews, golden moles, and tenrecs, which all have their origins in Africa, and therefore are dubbed Afrotheria. Morphologically, this appears an unlikely assemblage, which challenges-by including golden moles and tenrecs-the monophyly of the order Lipotyphla (Insectivora). We here identify in three proteins unique combinations of apomorphous amino acid replacements that support this clade. The statistical support for such "sequence signatures" as unambiguous synapomorphic evidence for the naturalness of the Afrotherian clade is reported. Using likelihood, combinatorial, and Bayesian methods we show that the posterior probability of the mammalian tree containing the Afrotherian clade is effectively 1.0, based on conservative assumptions. Presenting sequence data for another African insectivore, the otter shrew Micropotamogale lamottei, we demonstrate that such signatures are diagnostic for including newly investigated species in the Afrotheria. Sequence signatures provide "protein-morphological" synapomorphies that may aid in visualizing monophyletic groupings.

Inferring the origins of state-dependent courtship traits.

We examine a simple model of state-dependent (indicator) traits that focuses on their evolutionary origins as courtship signals. A necessary condition for the initial evolution of signals was found: the marginal female preference for minimal traits must exceed a certain threshold, where that threshold is proportional to the marginal male fitness costs for minimal traits. We interpret a positive threshold as implying a need for preexisting sensory bias in order to overcome the threshold if indicator signals are to start to evolve. We extend the model to allow for the possibility that signal costs and female preferences may vary over evolutionary time. If there is independent information on the way that signaling costs have evolved, then one may use measurements of contemporary female preferences to make inferences concerning the presence of the ancestral threshold. It is the marginal female preferences for minimal male traits that are important, whereas reconstructing ancestral origins from measurement of average size signals is not informative. Our analyses suggest two foci for future studies: measurement of the marginal response of contemporary females to minimal male signals and reconstruction of how signaling costs have changed over evolutionary time.

Is mate fidelity related to site fidelity? A comparative analysis in Ciconiiforms.

We tested for an association between divorce rate and site fidelity in 42 avian species belonging to the order Ciconiiforms, using comparative methods that account for the influences of phylogenetic relationships on the data. Our methods enabled us to detect evidence of correlated evolution and provided information on the temporal ordering of evolutionary changes in these two variables. We found a significant correlation between divorce rate and site fidelity, indicating that species with little or no site fidelity are more likely to divorce. Our data suggest that the coupled evolution of divorce and site fidelity can be summarized by three major events. The first event corresponds to a transition from species showing high divorce rate and low or no site fidelity to species that tended to reuse the same nests over consecutive breeding seasons. This was followed by a transition towards higher mate fidelity, with the preservation of pair bonds over consecutive breeding attempts. In a third stage, divorce rate and the rate of site fidelity varied, independently of each other. We discuss our results within the context of the ancestor species and the past environments in which the traits originated, and address the importance of the potential for individual recognition in shaping the observed patterns of covariation between mate fidelity and site fidelity in Ciconiiforms. Copyright 2000 The Association for the Study of Animal Behaviour.

Relationships between protein structure and dynamics from a database of NMR-derived backbone order parameters.

The amplitude of protein backbone NH group motions on a time-scale faster than molecular tumbling may be determined by analysis of (15)N NMR relaxation data according to the Lipari-Szabo model free formalism. An internet-accessible database has been compiled containing 1855 order parameters from 20 independent NMR relaxation studies on proteins whose three-dimensional structures are known. A series of statistical analyses has been performed to identify relationships between the structural features and backbone dynamics of these proteins. Comparison of average order parameters for different amino acid types indicates that amino acids with small side-chains tend to have greater backbone flexibility than those with large side-chains. In addition, the motions of a given NH group are also related to the sizes of the neighboring amino acids in the primary sequence. The secondary structural environment appears to influence backbone dynamics relatively weakly, with only subtle differences between the order parameter distributions of loop structures and regular hydrogen bonded secondary structure elements. However, NH groups near helix termini are more mobile on average than those in the central regions of helices. Tertiary structure influences are also relatively weak but in the expected direction, with more exposed residues being more flexible on average than residues that are relatively inaccessible to solvent.

Delayed administration of sodium thiosulfate in animal models reduces platinum ototoxicity without reduction of antitumor activity.

Platinum-based chemotherapeutic agents, such as carboplatin and cisplatin, are effective against many human tumors, but their use may be limited by a high incidence of ototoxicity. Delayed administration of the chemoprotective agent sodium thiosulfate (STS) reduces the ototoxicity of carboplatin in a guinea-pig model, when given up to 8 h after the chemotherapy, and also reduces hearing loss in patients given carboplatin with osmotic blood-brain barrier opening for treatment of brain tumors. We tested whether STS, given at times that achieved otoprotection, could impact the chemotherapeutic efficacy of carboplatin. The impact of STS was evaluated by measuring the onset of growth of LX-1 human small cell lung carcinoma s.c. xenografts in the nude rat. When STS was administered as two boluses, 2 and 6 h after treatment with carboplatin and etoposide, there was a decrease in the time to tumor progression. In contrast, when STS administration was delayed until 8 h after carboplatin/etoposide, there was no reduction in the antitumor cytotoxicity of the chemotherapy. STS infusion did not significantly affect ultrafilterable platinum pharmacokinetics in the guinea pig. To explore the potential wider applicability of STS, in a pilot study we tested its efficacy against cisplatin ototoxicity. Delayed administration of STS, 2 h after cisplatin, was protective against cisplatin-induced ototoxicity in the guinea pig model, as determined by electrophysiological measures. On the basis of these data, we suggest that delayed administration of STS may provide a mechanism to reduce the ototoxicity caused by administration of carboplatin or cisplatin for both central nervous system and systemic cancer chemotherapy.

Phylogenetic--evolutionary approaches to bioinformatics.

Phylogenies of organisms are essential to investigating a range of evolutionary questions of interest to researchers in the field of bioinformatics. Phylogenies not only help to define how to study many evolutionary questions, they must also be taken into account when conducting statistical analyses. Here it is shown how phylogenies can be used to investigate variability along the sites of a gene, reconstruct ancestral states of ancient genes and proteins, identify and characterise events of parallel and convergent evolution, find events of gene duplication, analyse predictions from molecular clocks, seek evidence for correlated changes among different parts of the same gene or genome, and test theories of molecular evolution. A table of statistical and phylogenetic methods is presented.

Retaliatory cuckoos and the evolution of host resistance to brood parasites.

We present a dynamic model of the evolution of host resistance to avian brood parasites, when the latter can retaliate against hosts that reject parasitic eggs. In a verbal model, Zahavi (1979, American Naturalist, 113, 157-159) suggested that retaliatory cuckoos might prevent the evolution of host resistance by reducing the reproductive success of rejecter hosts (i.e. by destroying their eggs or nestlings). Here we develop a model based on the association between the great spotted cuckoo, Clamator glandarius, and its main host, the European magpie, Pica pica, because this is the only system that has provided supportive evidence, to date, for the existence of retaliatory behaviour. Our aims were (1) to derive the conditions for invasion of the retaliation strategy in a nonretaliatory parasite population and (2) to investigate the consequences of retaliation for the evolution of host defence. If we assume a cost of discrimination for rejecter hosts in the absence of parasitism, and a cost paid by a retaliator for monitoring nests, our model shows cyclical dynamics. There is no evolutionarily stable strategy, and populations of both hosts and parasites will cycle indefinitely, the period of the cycles depending on mutation and/or migration rate. A stable polymorphism of acceptors and rejecters occurs only when parasites are nonretaliators. The spread of retaliator parasites drives rejecter hosts to extinction. Copyright 1999 The Association for the Study of Animal Behaviour.

Mass spectrometric analysis of benzoylated sialooligosaccharides and differentiation of terminal alpha 2-->3 and alpha 2-->6 sialogalactosylated linkages at subpicomole levels.

Perbenzoylated sialooligosaccharides were found to be stable derivatives, giving intense signals during the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis in the positive-ion mode. Terminal Neu5NAc alpha 2-->3 and alpha 2-->6Gal units of oligosaccharides undergo characteristic structural changes during benzoylation, yielding easily recognizable mass spectral patterns. Subpicomole carbohydrate samples were successfully benzoylated and analyzed through MALDI mass spectrometry.

Inferring the historical patterns of biological evolution.

Phylogenetic trees describe the pattern of descent amongst a group of species. With the rapid accumulation of DNA sequence data, more and more phylogenies are being constructed based upon sequence comparisons. The combination of these phylogenies with powerful new statistical approaches for the analysis of biological evolution is challenging widely held beliefs about the history and evolution of life on Earth.

NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.