Robert Ta

A paramagnetic chemical exchange-based MRI probe metabolized by cathepsin D: design, synthesis and cellular uptake studies.

Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimers disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE).

Mg2+- and MgATP-inhibited and Ca2+/calmodulin-sensitive TRPM7-like current in hepatoma and hepatocytes

Although understood to be ubiquitously expressed, the functional identification and significance of Mg(2+)-inhibited, nonspecific cation currents has been established in only a few cell types. Here we identified an outwardly rectifying nonspecific cation current in quiescent rat hepatocytes and the proliferating and polarized rat hepatoma, WIF-B. Under whole cell recording conditions in which cells were bathed and dialyzed with Na-gluconate solutions, the latter Ca(2+) and Mg(2+) free, current reversed close to 0 mV, was time independent, and was greater than 10 times higher at +120 mV compared with -120 mV. Outward current at -120 mV developed slowly, from 17.7 +/- 10.3 pA/pF at patch rupture to 106.6 +/- 15.6 pA/pF at 12 min in WIF-B cells, and 4.9 +/- 2.7 to 20.6 +/- 5.6 pA/pF in rat hepatocytes. The nonspecific TRP channel inhibitor, 2-aminoethoxyphenylborate (2-APB), inhibited current (IC(50) = 72 +/- 13 microM) and caused apoptotic cell death in WIF-B cells. Rat hepatocyte survival was more resistant to 2-APB. Dialysis of WIF-B cells with physiological concentrations of Mg(2+) and Mg-ATP, but not ATP alone, inhibited current development, suggesting that Trpm7 rather than Trpm6 underlies this current. RT-PCR demonstrated that both Trpm6 and Trpm7 are expressed at similar levels in both cell types, suggesting that the functional differences noted are not transcript dependent. Intracellular Ca(2+) (IC(50) = 125 +/- 35 nM) also inhibited current development, and this could be partially relieved by the calmodulin and Ca(2+)/calmodulin-dependent kinase inhibitors W-7, staurosporine, KN-93, or calmodulin kinase II (CaMKII) inhibitory peptide. To summarize, our results show that in addition to their established Mg(2+) sensitivity, Trpm7-like channels are inhibited by cytosolic Ca(2+) in a CaMKII-dependent manner and may support hepatocellular survival during proliferation.

A novel MRI-compatible brain ventricle phantom for validation of segmentation and volumetry methods†

To create a standardized, MRI‐compatible, life‐sized phantom of the brain ventricles to evaluate ventricle segmentation methods using T1‐weighted MRI. An objective phantom is needed to test the many different segmentation programs currently used to measure ventricle volumes in patients with Alzheimers disease.

A dual magnetic resonance imaging/fluorescent contrast agent for Cathepsin-D detection.

Currently there are no approved biomarkers for the pre-symptomatic diagnosis of Alzheimers disease (AD). Cathepsin-D (Cat-D) is a lysosomal protease that is present at elevated levels in amyloid plaques and neurons in patients with AD and is also elevated in some cancers. We have developed a magnetic resonance imaging (MRI)/fluorescent contrast agent to detect Cat-D enzymatic activity. The purpose of this study was to investigate the cellular and tissue uptake of this MRI/fluorescent contrast agent. The agent consists of an MRI probe [DOTA-caged metal ion (Gd³⁺ or Tm³⁺)] and a fluorescent probe coupled to a cell-penetrating-peptide sequence by a Cat-D recognition site. The relaxivity of Gd³⁺-DOTA-CAT(cleaved) was measured in 10% heat-treated bovine serum albumin (BSA) phantoms to assess contrast efficacy at magnetic fields ranging from 0.24 mT to 9.4 T. In vitro, Tm³⁺-DOTA-CAT was added to neuronal SN56 cells over-expressing Cat-D and live-cell confocal microscropy was performed at 30 min. Tm³⁺-DOTA-CAT was also intravenously injected into APP/PS1-dE9 Alzheimers disease mice (n = 9) and controls (n = 8). Cortical and hippocampal uptake was quantified at 30, 60 and 120 min post-injection using confocal microscopy. The liver and kidneys were also evaluated for contrast agent uptake. The relaxivity of Gd³⁺-DOTA-CAT(cleaved) was 3.3 (mM s)⁻¹ in 10% BSA at 9.4 T. In vitro, cells over-expressing Cat-D preferentially took up the contrast agent in a concentration-dependent manner. In vivo, the contrast agent effectively crossed the blood-brain barrier and exhibited a distinct time course of uptake and retention in APP/PS1-dE9 transgenic mice compared with age-matched controls. At clinical and high magnetic field strengths, Gd³⁺-DOTA-CAT produced greater T₁ relaxivity than Gd³⁺-DTPA. Tm³⁺-DOTA-CAT was taken up in a dose-dependent manner in cells over-expressing Cathepsin-D and was shown to transit the blood-brain barrier in vivo. This strategy may be useful for the in vivo detection of enzyme activity and for the diagnosis of Alzheimers disease.

Diffuse Alveolar Hemorrhage in Systemic Lupus Erythematosus: Histopathologic Features and Clinical Correlations

The case of a 16-year-old African-American girl with systemic lupus erythematosus, who developed diffuse alveolar hemorrhage with fatal consequences, is described. Diffuse alveolar hemorrhage is a rare but serious complication of systemic lupus. It occurs in three distinct but overlapping phenotypes, acute capillaritis, bland pulmonary hemorrhage, and diffuse alveolar damage, each of which is associated with a different group of underlying conditions. Diffuse alveolar hemorrhage is a medical emergency: choice of treatment depends on early diagnosis and determination of the underlying etiology. Acute infection, superimposed on diffuse alveolar hemorrhage in the setting of immune compromise, is often a terminal event, as in this case.

miR-223 potentially targets SWI/SNF complex protein SMARCD1 in atypical proliferative serous tumor and high-grade ovarian serous carcinoma

Ovarian cancer is the fifth most common cancer in women worldwide and has the highest mortality amongst gynecological cancers. miRNAs are a class of non-coding RNAs, approximately 22 nt long, that negatively regulate gene expression and have roles in cell growth, differentiation, metabolism, apoptosis and tumorigenesis. Dysregulated miRNA-223 expression has been implicated in a wide range of cancer subtypes. SMARCD1 is an integral protein component of the SWI/SNF complex, which remodels chromatin, and which has important roles in transcriptional control, DNA replication, recombination and repair. In this study, we examined whether the expression levels of miR-223 and SMARCD1 are altered in ovarian serous neoplasia and whether miR-223 functionally regulates the gene and protein expression of SMARCD1 in vivo, as has been predicted by in silico methods. Benign, atypical proliferative serous tumors (borderline) and malignant serous tumors (n = 144) were laser-capture microdissected, and relative expression levels of miR-223 and SMARCD1 were quantified by RT-PCR. Ovarian cancer cell line OC316 was reverse transfected with a miR-223 mimic, and relative expression levels of miR-223 and SMARCD1 were quantified by reverse-transcription polymerase chain reaction; protein expression of SMARCD1 was evaluated by Western blot. miR-223 expression was up-regulated in high-grade ovarian serous carcinoma samples (median RQ = 4.8881, P = .0045), whilst SMARCD1 was down-regulated (median RQ = 0.5107, P = .0492). In OC316 cells transfected with a miR-223 mimic, SMARCD1 gene expression was down-regulated 3-fold (P = .001), and SMARCD1 protein expression was down-regulated 2-fold (P = .002). These results suggest a regulatory role for miR-223 in ovarian serous neoplasia, linking it with SMARCD1.

Helmet-Wearing Practices and Barriers in Toronto Bike-Share Users: a Case-Control Study.

BACKGROUND Helmet use among bike-share users is low. We sought to characterize helmet-use patterns, barriers to helmet use, and cycling safety practices among bike-share users in Toronto. METHODS A standardized survey of public bike-share program (PBSP) users at semi-random distribution of PBSP stations was undertaken. By maintaining a ratio of one helmet-wearer (HW): two non-helmet-wearers (NHW) per survey period, we controlled for location, day, time, and weather. RESULTS Surveys were completed on 545 (180 HW, 365 NHW) unique users at 48/80 PBSP locations, from November 2012 to August 2013. More females wore helmets (F: 41.1%, M: 30.9%, p=0.0423). NHWs were slightly younger than HWs (NHW mean age 34.4 years vs HW 37.3, p=0.0018). The groups did not differ by employment status, education, or income. Helmet ownership was lower among NHWs (NHW: 62.4% vs HW: 99.4%, p<0.0001), as was personal bike ownership (NHW: 65.8%, vs HW: 78.3%, p=0.0026). NHWs were less likely to always wear a helmet on personal bikes (NHW: 22.2% vs HW: 66.7%, p<0.0001), and less likely to wear a helmet always or most of the time on PBSP (NHW: 5.8% vs HW: 92.3%, p<0.0001). Both groups, but more HWs, had planned to use PBSP when leaving their houses (HW: 97.2% vs NHW: 85.2%, p<0.0001), primarily to get to work (HW: 88.3% vs NHW: 84.1%, p=0.19). NHWs were more likely to report that they would wear a helmet more (NHW: 61.4% vs HW: 13.9%, p<0.0001), and/or cycle less (NHW: 22.5% vs HW: 4.4%) if helmet use was mandatory. CONCLUSIONS PBSP users surveyed appear to make deliberate decisions regarding helmet use. NHWs tended to be male, slightly younger, and less likely to use helmets on their personal bikes. As Toronto cyclists who do not wear helmets on PBSP generally do not wear helmets on their personal bikes, interventions to increase helmet use should target both personal and bike-share users. Legislating helmet use and provision of rental helmets could improve helmet use among bike-share users, but our results suggest some risk of reduced cycling with legislation.

In-Vitro and In-Vivo Study of a Magnetic Resonance Imaging (MRI)/Fluorescent Contrast Agent for Detection in Alzheimer's Disease

uptake of this MRI/fluorescent contrast agent targeting CatD. Methods: SN56 cells, a neuronal cell line, were cultured and differentiated. To simulate conditions of increased CatD, cells were transfected with a plasmid which stimulates high level expression of human CatD tagged with red fluorescent protein. Varying concentrations (5, 10, 50, 100 mM) of DOTA-CAT were added to the media of SN56 cells and confocal microscopy was performed at 30 minutes. Uptake was quantified by counting the proportion of cells which were labeled with DOTA-CAT expressed as Mean 6 SEM. Lastly, cleavage experiments on DOTA-CAT were performed using purified CatD and analyzed using electrospray-ionization mass spectrometry (ESI-MS). Results: In uptake experiments, 19 6 2% of cells exposed to the agent at 5 mM took up DOTA-CAT, and this number increased with concentration such that at 100 mM, 78 6 7% of cells took up DOTA-CAT. In contrast, a maximum of 5 6 1% of cells not over-expressing CatD took up DOTA-CAT over the entire concentration range. DOTA-CAT was observed intracellularly and in intracellular vesicles frequently co-localized with red fluorescent protein tagged-CatD within lysosomes. In cleavage experiments, incubation of DOTA-CAT with CatD produced a 1906.6 Da fragment confirming the cleavage of DOTA-CAT at the predicted CatD recognition sequence. Conclusions: These experiments demonstrate that DOTA-CAT interacts with CatD and is cleaved as predicted. Furthermore, DOTA-CAT is preferentially taken up by SN56 cells over-expressing CatD in a concentration dependant manner. Therefore, DOTA-CAT shows significant potential as a molecular imaging probe that will be further evaluated in future experiments using an in-vivo model of AD.

A novel MRI-compatible brain ventricle phantom for validation of lateral ventricle segmentation programs

three cohorts (equal sample size) according to their rates of change in ADAS-Cog total scores. Patients with most positive rates of change in ADAS-cog total scores (i.e., worsening) showed significantly more rapid hippocampal atrophy as compared with to patients that showed the most negative rates of change in ADAS-Cog total scores (i.e., improving), and patients with intermediate, near-zero, rates of change in ADAS-cog total scores (i.e., stable) (trend). Conclusions: The rate of hippocampal degeneration may be altered in DAT patients who respond to treatment with memantine or donepezil as compared to patients who are treated with these drugs but who do not respond. However, we were not able to detect drug-specific changes in hippocampal degeneration. Cognitive improvement and slowing of disease progression in AD as a result of drug treatment may be dependent upon neuroanatomical factors that influence who will respond to treatment. Improving our understanding of these factors way lead to the development of more effective therapies.

In-Vitro Cellular Uptake of a Magnetic Resonance Imaging (MRI)/Fluorescent Contrast Agent for Detection of Cathepsin-D Activity in Alzheimer's Disease

correlated moderately with change in MMSE score. Conclusions: We conclude that WBA, VE and HV rates of change from our institute are not significantly different than those from the DRC and CIND, respectively. We also observed that the measured WBA, VE and HV rate changes were significantly higher in AD subjects relative to the normal control group, showing that our methods were sensitive enough to detect changes in subjects with AD.