DRR regulates AKT activation to drive brain cancer invasion.

Glioblastoma (GBM) is the most common and invasive adult brain cancer. The rapid invasion of cancer cells into the normal brain is a major cause of treatment failure, yet the mechanisms that regulate this process are poorly understood. We have identified a novel mechanism of brain cancer invasion. We show that downregulated in renal cell carcinoma (DRR), which is newly expressed in invasive gliomas, recruits AKT to focal adhesions. This DRR- induced pathological relocalization of AKT bypasses commonly altered upstream signaling events and leads to AKT activation and invasion. We also developed an oligonucleotide therapeutic that reduces DRR expression and prevents glioma invasion in an in vivo preclinical model of the disease. Our findings identify DRR as a novel GBM target and show that oligonucleotides targeting DRR is a novel therapeutic approach for the treatment of DRR-positive GBMs.

TLN-4601 peripheral benzodiazepine receptor (PBR/TSPO) binding properties do not mediate apoptosis but confer tumor-specific accumulation.

TLN-4601 is a farnesylated dibenzodiazepinone isolated from Micromonospora sp. with an antiproliferative effect on several human cancer cell lines. Although the mechanism of action of TLN-4601 is unknown, our earlier work indicated that TLN-4601 binds the PBR (peripheral benzodiazepine receptor; more recently known as the translocator protein or TSPO), an 18 kDa protein associated with the mitochondrial permeability transition (mPT) pore. While the exact function of the PBR remains a matter of debate, it has been implicated in heme and steroid synthesis, cellular growth and differentiation, oxygen consumption and apoptosis. Using the Jurkat immortalized T-lymphocyte cell line, documented to have negligible PBR expression, and Jurkat cells stably transfected with a human PBR cDNA, the present study demonstrates that TLN-4601 induces apoptosis independently of PBR expression. As PBRs are overexpressed in brain tumors compared to normal brain, we examined if TLN-4601 would preferentially accumulate in tumors using an intra-cerebral tumor model. Our results demonstrate the ability of TLN-4601 to effectively bind the PBR in vivo as determined by competitive binding assay and receptor occupancy. Analysis of TLN-4601 tissue and plasma indicated that TLN-4601 preferentially accumulates in the tumor. Indeed, drug levels were 200-fold higher in the tumor compared to the normal brain. TLN-4601 accumulation in the tumor (176 µg/g) was also significant compared to liver (24.8 µg/g; 7-fold) and plasma (16.2 µg/mL; 11-fold). Taken together our data indicate that while PBR binding does not mediate cell growth inhibition and apoptosis, PBR binding may allow for the specific accumulation of TLN-4601 in PBR positive tumors.

DRR drives brain cancer invasion by regulating cytoskeletal-focal adhesion dynamics.

Malignant glioma invasion is a primary cause of brain cancer treatment failure, yet the molecular mechanisms underlying its regulation remain elusive. We developed a novel functional-screening strategy and identified downregulated in renal cell carcinoma (DRR) as a regulator of invasion. We show that DRR drives invasion in vitro and in vivo. We found that while DRR is not expressed in normal glial cells, it is highly expressed in the invasive component of gliomas. Exploring underlying mechanisms, we show that DRR associates with and organizes the actin and microtubular cytoskeletons and that these associations are essential for focal adhesion (FA) disassembly and cell invasion. These findings identify DRR as a new cytoskeletal crosslinker that regulates FA dynamics and cell movement.

Message framing and sunscreen use: gain-framed messages motivate beach-goers.

Prospect theory suggests that people respond differentially to factually equivalent messages depending on how these messages are framed (A. Tversky & D. Kahneman, 1981). A. J. Rothman and P. Salovey (1997) relied on prospect theory to predict that messages highlighting potential "gains" should promote prevention behaviors such as sunscreen use best. This experiment compared the effectiveness of 4 differently framed messages (2 highlighting gains, 2 highlighting losses) to persuade 217 beach-goers to obtain and use sunscreen. Attitudes and intentions were measured before and immediately following the delivery of the framed information, and after completing the questionnaire participants were given a coupon redeemable for a small bottle of sunscreen later that same day. People who read either of the 2 gain-framed brochures, compared with those who read either of the 2 loss-framed brochures, were significantly more likely to (a) request sunscreen, (b) intend to repeatedly apply sunscreen while at the beach, and (c) intend to use sunscreen with a sun protection factor of 15 or higher.

Implementation and evaluation of a new pulse sequence for rapid acquisition of double inversion recovery images for simultaneous suppression of white matter and CSF.

We describe a fast double inversion recovery (DIR) imaging sequence that effectively attenuates signal from both white matter and cerebrospinal fluid (CSF). The pulse sequence uses a novel inversion/excitation scheme and fast spin-echo readout to maximize scan efficiency. The white matter/CSF suppressed images can be acquired from the entire brain in approximately 6 minutes. Evaluation of the fast DIR sequence on patients with multiple sclerosis (MS) demonstrates high lesion conspicuity.

Automatic segmentation of gadolinium-enhanced multiple sclerosis lesions.

Automatic detection and quantitation of contrast-enhanced lesions on MRI is expected to be useful in characterizing the disease state in multiple sclerosis (MS). The enhancing structures such as cerebral vasculature and regions with no blood-brain barrier complicate automated analysis of lesion enhancement. A pulse sequence that incorporates both stationary and marching saturation bands and gradient dephasing is described for suppressing the enhancements within the cerebral vasculature. A postprocessing technique based on automatic image segmentation is implemented for eliminating enhancing structures such as choroid plexus. All MS lesions larger than 5 mm3 have been successfully identified. The automated analysis produced no false-positives or false-negative lesions above this volume in 13 patients with MS who were evaluated using the acquisition and evaluation techniques described.

Volumetric analysis of white matter, gray matter, and CSF using fractional volume analysis.

Quantitative cerebral tissue volumes may be useful for an objective assessment of pathological changes in brain. Accurate determination of tissue volumes is complicated, however, by the partial volume averaging (PVA) effect. We have, therefore, developed a new pulse sequence that minimizes the PVA through the use of inversion-recovery (IR) and double inversion-recovery (DIR) techniques. This pulse sequence simultaneously acquires four different sets of images to provide the necessary information for volumetric analysis and reduces potential spatial misregistration of images due to patient motion. The image sets acquired from the proposed pulse sequence are 1) gray matter visible, 2) white matter visible, 3) FLAIR, and 4) fast spin-echo proton-density weighted images. An algorithm has been implemented to correct for differential T1-weighting and for tissue quantitation.

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents.

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.

A dual approach for minimizing false lesion classifications on magnetic resonance images.

Segmentation methods based on dual-echo MR images are generally prone to significant false lesion classifications. We have minimized these false classifications by (1) improving the lesion-to-tissue contrast on MR images by developing a fast spin-echo sequence that incorporates both cerebrospinal fluid signal attenuation and magnetization transfer contrast and (2) including information from MR flow images. Studies on patients with multiple sclerosis indicate that this dual approach to tissue segmentation reduces the volume of false lesion classifications by an average of 87%.

Automatic removal of extrameningeal tissues from MR images of human brain.

An automatic technique for removal of extrameningeal tissues from MR images of human brain is described. The algorithm is based on the segmentation of images acquired with a fast dual-echo pulse sequence, which incorporates both fluid attenuation and magnetization transfer contrast for superior brain/extrameningeal tissue contrast compared to conventional MR sequences. Evaluation of this technique using MR images of six volunteers indicates rapid and consistent removal of extrameningeal tissues.

Three-dimensional MR image registration of the human brain.

An automatic three-dimensional technique for registration of MR images of human brain is described. The algorithm was tested, using MR images of human brain, and was found to estimate angular offsets to within 0.5 degrees and translational offsets to within about 1 pixel. The quality of final registration was evaluated by histogram analysis. The algorithm was found to be computationally efficient and robust.

Relative concentrations of proton MR visible neurochemicals in gray and white matter in human brain.

The relative distributions of N-acetylaspartate (NAA) + N-acetylaspartylglutamate (NAAG), creatine + phosphocreatine (Cr/PCr), and choline (Cho) in the gray and white matter of human brain were determined by utilizing proton magnetic resonance spectroscopic imaging (SI). The SI data was processed using an automated spectroscopic image processing algorithm, and image segmentation was performed using a supervised technique. Linear regression analysis indicated that the NAA + NAAG (2.01 ppm) and Cr/PCr (3.02 ppm) peaks are greater in gray matter compared with white matter. The large intersubject variation observed in the Cho (3.20 ppm) resonance prevented the assessment of its regional distribution with confidence.

Controlling enzyme-catalyzed regioselectivity in sugar ester synthesis.

The rational control over enzyme-catalyzed regioselectivity has been studied using sucrose acylation by vinyl esters in organic media as a model. Subtilisins BPN' and Carlsberg preferentially acylate at the 1'-hydroxyl of sucrose with some acylation observed at the 6-hydroxyl. The preference for the 1'-hydroxyl is strongly affected by the hydrophobicity of the organic solvent and the chain length of the vinyl ester. Increasingly hydrophobic solvents and longer chain lengths lower the favorable formation of the 1'-acylation and improve 6-acylation. Molecular modeling of sucrose in the binding pocket of subtilisin BPN' shows that the 1'-acylation is favored in solvents that can solvate sugars (such as pyridine) as the glucose moiety is exposed to the medium, whereas 6-acylation leaves the entire sucrose molecule buried within the enzyme's binding pocket. Thus, 1'-acylation is sterically more favorable than 6-acylation. Increasingly hydrophobic solvents affect regioselectivity by changing the degree of solvation of the glucose moiety in the medium and forcing the sucrose 1'-ester completely into the binding pocket. In a related modeling, the vinyl ester chain length was shown to modulate regioselectivity by controlling the bond angles between the resulting acylenzymes and the sucrose thereby affecting the positioning of the sucrose in the binding pocket of subtilisin BPN'. This study shows that control over enzymic regioselectivity can be achieved by rational choices of substrate and solvent.

The relationship between a nutrition education program and nutrition knowledge and eating behaviors of the elderly.

An experimental study was conducted to examine the relationship between a nutrition education program and nutrition knowledge and eating behaviors of older adults. Twenty-four subjects were randomly assigned to an experimental or a control group. Prior to any intervention, all subjects completed a demographic data sheet, a 25-item True/False questionnaire and a 24-Hour Dietary Recall interview. The experimental group attended four one-hour nutrition education classes over a two-week period. Following the educational intervention, both groups of subjects again completed the True/False questionnaire and the 24-Hour Dietary Recall interview. Controlling for pretest, posttest differences on the two dependent variables were tested using repeated measures analysis of variance. No significant differences were found between the two groups.