Rational design, synthesis, and biological evaluation of progesterone-modified MRI contrast agents.
A series of contrast agents for magnetic resonance imaging (MRI) aimed at noninvasively determining the hormone receptor status of cancer in vitro was developed. These MRI contrast agents were prepared by conjugating progesterone to clinically used Gd(III) chelates. These agents exhibited higher progesterone receptor binding affinities in the nanomolar range and intracellular accumulation. High logP values of the modified compounds suggested that the lipophilicity of the steroid conjugates may have contributed to membrane permeability. Synchrotron radiation X-ray fluorescence microscopy and magnetic resonance images revealed that the synthesized conjugates showed the greatest cellular accumulation and significant increase in relaxivity in vitro compared to the previously developed steroid-modified agent. Transcriptional assays using the progesterone response element linked to luciferase indicated that the contrast agents entered the cell, interacted with the biological target, and drove specific progesterone-mediated transcription.

title: Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain.
abstract: Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.

title: Development of near-infrared fluorophore (NIRF)-labeled activity-based probes for in vivo imaging of legumain.
abstract: Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease.

title: Target deconvolution techniques in modern phenotypic profiling.
abstract: The past decade has seen rapid growth in the use of diverse compound libraries in classical phenotypic screens to identify modulators of a given process. The subsequent process of identifying the molecular targets of active hits, also called 'target deconvolution', is an essential step for understanding compound mechanism of action and for using the identified hits as tools for further dissection of a given biological process. Recent advances in 'omics' technologies, coupled with in silico approaches and the reduced cost of whole genome sequencing, have greatly improved the workflow of target deconvolution and have contributed to a renaissance of 'modern' phenotypic profiling. In this review, we will outline how both new and old techniques are being used in the difficult process of target identification and validation as well as discuss some of the ongoing challenges remaining for phenotypic screening.

title: The contribution of myelin to magnetic susceptibility-weighted contrasts in high-field MRI of the brain
abstract: T(2)*-weighted gradient-echo MRI images at high field (≥ 7T) have shown rich image contrast within and between brain regions. The source for these contrast variations has been primarily attributed to tissue magnetic susceptibility differences. In this study, the contribution of myelin to both T(2)* and frequency contrasts is investigated using a mouse model of demyelination based on a cuprizone diet. The demyelinated brains showed significantly increased T(2)* in white matter and a substantial reduction in gray-white matter frequency contrast, suggesting that myelin is a primary source for these contrasts. Comparison of in-vivo and in-vitro data showed that, although tissue T(2)* values were reduced by formalin fixation, gray-white matter frequency contrast was relatively unaffected and fixation had a negligible effect on cuprizone-induced changes in T(2)* and frequency contrasts.

title: On the contribution of deoxy-hemoglobin to MRI gray–white matter phase contrast at high field
abstract: High field (> or =7 T) MRI studies based on signal phase have been used to improve visualization of the fine structure of the brain, most notably the major white matter fiber bundles, the gray-white matter subdivision, and the laminar cortical architecture. The observed contrast has been attributed in part to local variations in magnetic susceptibility arising from iron in storage proteins and tissue lipid. Another contribution could come from the paramagnetic blood constituent deoxy-hemoglobin, the tissue concentration of which may vary through local variations in vascular density. To investigate this possibility, we examined phase contrast between gray and white matter in rats after intravenous administration of a superparamagnetic contrast agent at various dosages. At the maximum dosage (3 mg Fe/kg), which resulted in an estimated paramagnetic susceptibility shift 4-8 times larger than deoxy-hemoglobin, we observed a negligible increase in phase contrast between gray and white matter. This result suggests that endogenous deoxy-hemoglobin has no significant contribution to phase contrast between gray and white matter.

title: Anthocyanins and other polyphenolics in American elderberry (Sambucus canadensis) and European elderberry (S. nigra) cultivars.
abstract: Ten genotypes representing two elderberry species, Sambucus canadensis L. (eight genotypes) and S. nigra L. (two genotypes), were examined for their anthocyanins (ACY), total phenolics (TP),°Brix, titratable acidity (TA), and pH over two growing seasons.



Overall, fruit generally had higher ACY, TP, ACY/TP,°Brix, and pH in 2005 than 2004. All samples of S. canadensis had similar anthocyanin profiles to one another, but were distinctly different from S. nigra. Both species had cyanidin-based anthocyanins as major pigments. Previously unreported anthocyanins were identified in some samples in this study. Trace levels of delphinidin 3-rutinoside were present in all elderberry samples except cv. 'Korsør'. Also, petunidin 3-rutinoside was detected in cvs 'Adams 2', 'Johns', 'Scotia', 'York', and 'Netzer' (S. canadensis). The identified polyphenolics of both species were mainly composed of cinnamic acids and flavonol glycosides. The major polyphenolic compounds present in S. canadensis were neochlorogenic acid, chlorogenic acid, rutin, and isorhamnetin 3-rutinoside, while chlorogenic acid and rutin were found to be major polyphenolic compounds in S. nigra.



Sufficient variability was seen among these genotypes to suggest that a successful breeding program could be carried out to improve levels of the various compounds evaluated in this study.

title: Combined effect of oleic acid and polyethylene glycol 200 on buccal permeation of [D-ala2, D-leu5]enkephalin from a cubic phase of glyceryl monooleate.
abstract: The combined use of the lipophilic permeation enhancer, oleic acid together with polyethylene glycol 200 (PEG 200) as a co-enhancer and incorporated into the cubic liquid crystalline phase of glyceryl monooleate was investigated in the ex vivo buccal permeation of [D-Ala2, D-Leu5]enkephalin (DADLE) through porcine buccal mucosa mounted in a Franz cell. The addition of oleic acid (1%) and PEG 200 (1-10%) did not change the intact appearance of the cubic phase. PEG 200 increased the aqueous solubility of oleic acid incorporated into the cubic phase and hence promoted the transport of oleic acid into the porcine buccal mucosa. The solubilising effect of PEG 200 on oleic acid incorporated into the cubic phase was dependent on the PEG 200 concentration but was non-linear. The buccal permeation flux of DADLE significantly increased when 5% (P<0.01) or 10% (P<0.001) of PEG 200 was co-administered with 1% oleic acid compared with the cubic phase containing 1% oleic acid alone. The present results suggest that PEG 200 enhances the action of the lipophilic permeation enhancer oleic acid and that the combination of oleic acid and PEG 200 as a co-enhancer can be a useful tool to improve the membrane permeability in the buccal delivery of peptide drugs using a cubic liquid crystalline phase of glyceryl monooleate and water.

title: Guns and states: pediatric firearm injury.
abstract: A recent report indicates that firearm-related injuries are responsible for 30% of pediatric trauma fatality. The literature is however limited in examining pediatric firearm injuries and variations in state gun control laws. Therefore, we sought to examine the association between pediatric firearm injuries and the Stand-Your-Ground (SYG) and Child Access Protection (CAP) laws.



All pediatric (age, 0-20 years) hospitalizations with firearm injuries were identified from the Kids' Inpatient Database from 2006 and 2009. States were compared for SYG and CAP laws.



A total of 19,233 firearm injury hospitalizations were identified, with 64.7% assault, 27.2% accidental, and 3.1% suicide injury. Demographics for assault injury were as follows: mean age of 17.6 years, 88.4% male, 44.4% black, 18.2% Hispanic, 70.5% from metropolitan areas, and 50.1% from the poorest median income neighborhoods. Suicide injury cases were more likely to be white (57.8% vs. 16.6%, p < 0.001) and female (15.1% vs. 9.8%, p < 0.001). States with the SYG law were associated with increased accidental injury (odds ratio [OR], 1.282; p < 0.001). There was no statistical association between CAP law and the incidence of accidental injury or suicide. Multivariate logistic regression analysis found other predictive demographic factors for firearm injury: black (OR, 6.164), urban areas (OR, 1.557), poorest median income neighborhoods (OR, 2.785), male (OR, 28.602), and 16 years or older (OR, 37.308). Total economic burden was estimated at more than $1 billion dollars, with a median length of stay of 3 days, 8.4% discharge to rehabilitation, and 6.2% in-hospital mortality.



Pediatric firearm injuries continue to be a significant source of morbidity, mortality, and economic burden. A significant increase in accidental firearm injuries in states with the SYG law may highlight inadvertent effects of the law. Race, sex, and median income are additional contributing factors. Advocacy and focused educational efforts for specific socioeconomic and racial groups may potentially reduce firearm injuries.



Prognostic study, level II.