Quantitative proteomic analysis of oligodendrogliomas with and without 1p/19q deletion.

Approximately 50-80% of oligodendrogliomas demonstrate a combined loss of chromosome 1p and 19q. Chromosome 1p/19q deletion, appearing early in tumorigenesis, is associated with improved clinical outcomes, including response to chemotherapy and radiation. Although many hypotheses have been proposed, the molecular mechanisms underlying improved clinical outcomes with 1p/19q deletion in oligodendrogliomas have not been characterized fully. To investigate the molecular differences between oligodendrogliomas, we employed an unbiased proteomic approach using microcapillary liquid chromatography mass spectrometry, along with a quantitative technique called isotope-coded affinity tags, on patient samples of grade II oligodendrogliomas. Following conventional biochemical separation of pooled tumor tissue from five samples of undeleted and 1p/19q deleted grade II oligodendrogliomas into nuclei-, mitochondria-, and cytosol-enriched fractions, relative changes in protein abundance were quantified. Among the 442 total proteins identified, 163 nonredundant proteins displayed significant changes in relative abundance in at least one of the three fractions between oligodendroglioma with and without 1p/19q deletion. Bioinformatic analyses of differentially regulated proteins supported the potential importance of metabolism and invasion/migration to the codeleted phenotype. A subset of altered proteins, including the pro-invasive extracellular matrix protein BCAN, was further validated by Western blotting as candidate markers for the more aggressive undeleted phenotype. These studies demonstrate the utility of proteomic analysis to identify candidate biological motifs and molecular mechanisms that drive differential malignancy related to 1p19q phenotypes. Future analysis of larger patient samples are warranted to further refine biomarker panels to predict biological behavior and assist in the identification of deleted gene products that define the 1p/19q phenotype.

Residence time and drift patterns of larval June sucker Chasmistes liorus in the lower Provo River as determined by otolith microstructure.

Estimates of age derived from daily ring counts from otoliths and capture rates of larval June sucker Chasmistes liorus were used to determine the relationship between discharge rates of the Provo River and residence time and patterns of larval drift. During 1997, larval drift occurred over a 22 day period when discharge rates were low (mean +/-s.d. 3.2 +/- 0.0 m(3) s(-1)). In 1998, larval drift occurred in two separate events over a 40 day period. Discharge was higher during the first larval drift period (19 days; 24.8 +/- 1.3 m(3) s(-1)) and lower during the second larval drift period (17 days; 7.0 +/- 0.9 m(3) s(-1)). In 1997, no larval fish were collected at the lowermost transect on the Provo River (nearest Utah Lake), and few larvae >21 days of age were found. During the first drift period of 1998, larval C. liorus were collected at all transects, and mean age of larvae collected between upstream and downstream transects increased by c. 7 days. During the second drift period of 1998, only a few were collected in the lowermost transects, and age did not increase with proximity to the lake. Patterns in catch and age distribution of larval C. liorus in the lower Provo River suggest that recruitment failure occurs during the larval drift period in years with insufficient discharge to transport larvae into the lake.

The evolution of mathematical modeling of glioma proliferation and invasion.

Gliomas are well known for their potential for aggressive proliferation as well as their diffuse invasion of the normal-appearing parenchyma peripheral to the bulk lesion. This review presents a history of the use of mathematical modeling in the study of the proliferative-invasive growth of gliomas, illustrating the progress made in understanding the in vivo dynamics of invasion and proliferation of tumor cells. Mathematical modeling is based on a sequence of observation, speculation, development of hypotheses to be tested, and comparisons between theory and reality. These mathematical investigations, iteratively compared with experimental and clinical work, demonstrate the essential relationship between experimental and theoretical approaches. Together, these efforts have extended our knowledge and insight into in vivo brain tumor growth dynamics that should enhance current diagnoses and treatments.

A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium.

BACKGROUND: Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. RESULTS: PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. CONCLUSIONS: Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development.

The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis.

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.

"Just look," he said.

Five personal examples are given illustrating the general principle that one can see if one looks or, conversely, that one cannot see anything that one believes not to exist.

March 2004: a 24-year-old woman with bifrontal headaches.

A 24-year-old woman with bifrontal headaches was found to have a well-circumscribed lesion in the frontal lobe subcortical white matter. Microscopic examination showed clusters of small round cells separated by hypocellular neuropil-like areas, and a distinct border between tumor and surrounding white matter. Synaptophysin was diffusely positive in neuropil-like areas, and many tumor cells expressed NeuN. Based on these findings, a diagnosis of "extraventricular neurocytoma" was made. A double-label immunofluorescence stain was performed with NeuN and Ki-67 antibodies to determine if NeuN+ cells remained in the mitotic cycle. No colocalization of these markers was found, thus supporting the hypothesis that neuronal differentiation (as marked by NeuN expression) is incompatible with continued proliferation of tumor cells, as well as normal neurons.

Widespread tissue distribution of mitochondrial DNA deletions in Kearns-Sayre syndrome.

We performed Southern analysis of mitochondrial DNA (mtDNA) in 6 tissues from a patient with Kearns-Sayre syndrome and found a single deletion of 4.9 kb in all tissues. The percentage of deleted mtDNAs varied widely between tissues, from only 4% in smooth muscle to approximately 50% in skeletal muscle. Samples of DNA obtained from 3 different skeletal muscles and from separate areas of individual tissues showed little variation in percentage of deleted mtDNA. Biochemical analysis showed no clear correlation between mitochondrial enzyme activity and deleted mtDNAs.

Virtual and real brain tumors: using mathematical modeling to quantify glioma growth and invasion.

Over the last 10 years increasingly complex mathematical models of cancerous growths have been developed, especially on solid tumors, in which growth primarily comes from cellular proliferation. The invasiveness of gliomas, however, requires a change in the concept to include cellular motility in addition to proliferative growth. In this article we review some of the recent developments in mathematical modeling of gliomas. We begin with a model of untreated gliomas and continue with models of polyclonal gliomas following chemotherapy or surgical resection. From relatively simple assumptions involving homogeneous brain tissue bounded by a few gross anatomical landmarks (ventricles and skull) the models have recently been expanded to include heterogeneous brain tissue with different motilities of glioma cells in grey and white matter on a geometrically complex brain domain, including sulcal boundaries, with a resolution of 1 mm(3) voxels. We conclude that the velocity of expansion is linear with time and varies about 10-fold, from about 4 mm/year for low-grade gliomas to about 3 mm/month for high-grade ones.

Vocal learning in the Grey parrot (Psittacus erithacus): effects of species identity and number of trainers.

Grey parrots (Psittacus erithacus) learn referential English labels when they view and interact with 2 humans who model vocal labeling and who demonstrate referentiality and functionality of a label (I.M. Pepperberg, 1990a). To test if both trainers are necessary, the authors contrasted 2-trainer modeling with training by 1 human who presented targeted labels to a bird in concert with appropriate items, who asked questions, and who would reward attempts at the label with the item. The bird was also tutored by either 1 or 2 interactive humans in conjunction with a conspecific who already used referential labels. Referential labels were learned from multiple live tutors but not a single trainer. Presence of a conspecific enhanced learning compared with single-trainer sessions but did not affect acquisition in 2-human sessions. Specific aspects of paired tutoring seem critical for acquiring referential vocal labels.

Spinal arachnoiditis. What is the clinical spectrum? II. Arachnoiditis induced by Pantopaque/autologous blood in dogs, a possible model for human disease.

Experimentally induced chronic arachnoiditis was studied over a one-year period in beagle dogs. All animals demonstrated severe extensive changes at postmortem which were strikingly similar to those reported in man. Arachnoid inflammation, fibrosis, and adhesions were often associated with nerve roots embedded in collagen. These changes were present in all four experimental dogs, and were absent in four controls. In spite of having pathologically severe changes of arachnoiditis, two of four dogs appeared clinically normal. We suggest that the clinical appearance of chronic arachnoiditis in this animal model may reflect the human experience, wherein presentations may vary from merely complaints of back pain to severe sensory-motor impairment.

Language development in low birth weight infants: the first two years of life.

The four main aims of this cohort study were to (1) determine the number of consecutively referred low birth weight (LBW) infants presenting with delayed language at 12 and 24 months of age, (2) examine language profiles by measuring both functional and spontaneous language ability in 24-month-olds, (3) examine the relationship between perinatal medical history and language status at 12 and 24 months, and (4) examine the clinical validity of the Early Language Milestone (ELM) scale, a brief language screening instrument. Only infants without serious sensory impairment or mental handicap were included in the final sample. Seventy-one LBW infants (36 12-month-olds, 35 24-month-olds) were seen for developmental and language assessments. The findings suggest that within the first 2 years of life, low to moderate rates of language delay are evident in LBW infants who have already been screened for serious sensory or mental handicap. At 12 months of age, 8.3% of the infants had delayed expressive language; none had delayed receptive language. At 24 months of age, 28% of the infants had delayed expressive language; 5.7% had delayed receptive language. Furthermore, only 32% of those with normal expressive language and sufficient language sample had a mean length of response within the normal range. Language status was not related to a specific perinatal medical variable. Additional study into the clinical validity of the ELM as a screening measure for the LBW population is warranted. The ELM specificity for both receptive and expressive language domains was good at both ages (80 to 100%), but sensitivity was low to moderate (0 to 68%).

Applying a patient-specific bio-mathematical model of glioma growth to develop virtual [18F]-FMISO-PET images.

Glioblastoma multiforme (GBM) is a class of primary brain tumours characterized by their ability to rapidly proliferate and diffusely infiltrate surrounding brain tissue. The aggressive growth of GBM leads to the development of regions of low oxygenation (hypoxia), which can be clinically assessed through [18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) imaging. Building upon the success of our previous mathematical modelling efforts, we have expanded our model to include the tumour microenvironment, specifically incorporating hypoxia, necrosis and angiogenesis. A pharmacokinetic model for the FMISO-PET tracer is applied at each spatial location throughout the brain and an analytical simulator for the image acquisition and reconstruction methods is applied to the resultant tracer activity map. The combination of our anatomical model with one for FMISO tracer dynamics and PET image reconstruction is able to produce a patient-specific virtual PET image that reproduces the image characteristics of the clinical PET scan as well as shows no statistical difference in the distribution of hypoxia within the tumour. This work establishes proof of principle for a link between anatomical (magnetic resonance image [MRI]) and molecular (PET) imaging on a patient-specific basis as well as address otherwise untenable questions in molecular imaging, such as determining the effect on tracer activity from cellular density. Although further investigation is necessary to establish the predicitve value of this technique, this unique tool provides a better dynamic understanding of the biological connection between anatomical changes seen on MRI and biochemical activity seen on PET of GBM in vivo.

Quantifying the role of angiogenesis in malignant progression of gliomas: in silico modeling integrates imaging and histology.

Gliomas are uniformly fatal forms of primary brain neoplasms that vary from low- to high-grade (glioblastoma). Whereas low-grade gliomas are weakly angiogenic, glioblastomas are among the most angiogenic tumors. Thus, interactions between glioma cells and their tissue microenvironment may play an important role in aggressive tumor formation and progression. To quantitatively explore how tumor cells interact with their tissue microenvironment, we incorporated the interactions of normoxic glioma cells, hypoxic glioma cells, vascular endothelial cells, diffusible angiogenic factors, and necrosis formation into a first-generation, biologically based mathematical model for glioma growth and invasion. Model simulations quantitatively described the spectrum of in vivo dynamics of gliomas visualized with medical imaging. Furthermore, we investigated how proliferation and dispersal of glioma cells combine to induce increasing degrees of cellularity, mitoses, hypoxia-induced neoangiogenesis and necrosis, features that characterize increasing degrees of "malignancy," and we found that changes in the net rates of proliferation (ρ) and invasion (D) are not always necessary for malignant progression. Thus, although other factors, including the accumulation of genetic mutations, can change cellular phenotype (e.g., proliferation and invasion rates), this study suggests that these are not required for malignant progression. Simulated results are placed in the context of the current clinical World Health Organization grading scheme for studying specific patient examples. This study suggests that through the application of the proposed model for tumor-microenvironment interactions, predictable patterns of dynamic changes in glioma histology distinct from changes in cellular phenotype (e.g., proliferation and invasion rates) may be identified, thus providing a powerful clinical tool.

Quantitative metrics of net proliferation and invasion link biological aggressiveness assessed by MRI with hypoxia assessed by FMISO-PET in newly diagnosed glioblastomas.

Glioblastoma multiforme (GBM) are aggressive and uniformly fatal primary brain tumors characterized by their diffuse invasion of the normal-appearing parenchyma peripheral to the clinical imaging abnormality. Hypoxia, a hallmark of aggressive tumor behavior often noted in GBMs, has been associated with resistance to therapy, poorer survival, and more malignant tumor phenotypes. Based on the existence of a set of novel imaging techniques and modeling tools, our objective was to assess a hypothesized quantitative link between tumor growth kinetics [assessed via mathematical models and routine magnetic resonance imaging (MRI)] and the hypoxic burden of the tumor [assessed via positron emission tomography (PET) imaging]. Our biomathematical model for glioma kinetics describes the spatial and temporal evolution of a glioma in terms of concentration of malignant tumor cells. This model has already been proven useful as a novel tool to dynamically quantify the net rates of proliferation (rho) and invasion (D) of the glioma cells in individual patients. Estimates of these kinetic rates can be calculated from routinely available pretreatment MRI in vivo. Eleven adults with GBM were imaged preoperatively with (18)F-fluoromisonidazole (FMISO)-PET and serial gadolinium-enhanced T1- and T2-weighted MRIs to allow the estimation of patient-specific net rates of proliferation (rho) and invasion (D). Hypoxic volumes were quantified from each FMISO-PET scan following standard techniques. To control for tumor size variability, two measures of hypoxic burden were considered: relative hypoxia (RH), defined as the ratio of the hypoxic volume to the T2-defined tumor volume, and the mean intensity on FMISO-PET scaled to the blood activity of the tracer (mean T/B). Pearson correlations between RH and the net rate of cell proliferation (rho) reached significance (P < 0.04). Moreover, highly significant positive correlations were found between biological aggressiveness ratio (rho/D) and both RH (P < 0.00003) and the mean T/B (P < 0.0007).

Prognostic significance of growth kinetics in newly diagnosed glioblastomas revealed by combining serial imaging with a novel biomathematical model.

Glioblastomas are the most aggressive primary brain tumors, characterized by their rapid proliferation and diffuse infiltration of the brain tissue. Survival patterns in patients with glioblastoma have been associated with a number of clinicopathologic factors including age and neurologic status, yet a significant quantitative link to in vivo growth kinetics of each glioma has remained elusive. Exploiting a recently developed tool for quantifying glioma net proliferation and invasion rates in individual patients using routinely available magnetic resonance images (MRI), we propose to link these patient-specific kinetic rates of biological aggressiveness to prognostic significance. Using our biologically based mathematical model for glioma growth and invasion, examination of serial pretreatment MRIs of 32 glioblastoma patients allowed quantification of these rates for each patient's tumor. Survival analyses revealed that even when controlling for standard clinical parameters (e.g., age and Karnofsky performance status), these model-defined parameters quantifying biological aggressiveness (net proliferation and invasion rates) were significantly associated with prognosis. One hypothesis generated was that the ratio of the actual survival time after whatever therapies were used to the duration of survival predicted (by the model) without any therapy would provide a therapeutic response index (TRI) of the overall effectiveness of the therapies. The TRI may provide important information, not otherwise available, about the effectiveness of the treatments in individual patients. To our knowledge, this is the first report indicating that dynamic insight from routinely obtained pretreatment imaging may be quantitatively useful in characterizing the survival of individual patients with glioblastoma. Such a hybrid tool bridging mathematical modeling and clinical imaging may allow for stratifying patients for clinical studies relative to their pretreatment biological aggressiveness.

Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells.

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B(61L) or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B(61L) and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B(17N) do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.

Cefazolin-induced pseudomembranous colitis resulting in perforation of the sigmoid colon.

The seventh case of probable cefazolin-induced pseudomembranous colitis is reported. Perforation of the colon necessitated sigmoid resection. The postoperative course was protracted, and illustrates the difficulty of managing advanced pseudomembranous colitis when the oral route of antibiotic administration is not available. Although rare, pseudomembranous colitis related to cefazolin administration is a potentially fatal complication. The routine use of prophylactic antibiotics must be weighed against this possibility.