Contrasting roles for dopamine D1 and D2 receptor subtypes in the dorsomedial striatum but not the nucleus accumbens core during behavioral inhibition in the stop-signal task in rats.

Dopamine and dopamine-receptor function are often implicated in behavioral inhibition, and deficiencies within behavioral inhibition processes linked to attention deficit/hyperactivity disorder (ADHD), schizophrenia, obsessive-compulsive disorder, and drug addiction. In the stop-signal task, which measures the speed of the process of inhibition [stop-signal reaction time (SSRT)], psychostimulant-related improvement of SSRT in ADHD is linked with dopamine function. However, the precise nature of dopaminergic control over SSRT remains unclear. This study examined region- and receptor-specific modulation of SSRT in the rat using direct infusions of the dopamine D1 receptor (DRD1) antagonist SCH 23390 or dopamine D2 receptor (DRD2) antagonist sulpiride into the dorsomedial striatum (DMStr) or nucleus accumbens core (NAcbC). DRD1 and DRD2 antagonists had contrasting effects on SSRT that were specific to the DMStr. SCH 23390 decreased SSRT with little effect on the go response. Conversely, sulpiride increased SSRT but also increased go-trial reaction time and reduced trial completion at the highest doses. These results suggest that DRD1 and DRD2 function within the DMStr, but not the NAcbC, may act to balance behavioral inhibition in a manner that is independent of behavioral activation.

Silica-modified oligonucleotide-gold nanoparticle conjugate enables closed-tube colorimetric polymerase chain reaction.

A facile silica coating significantly enhances the thermal stability and polymerase chain reaction (PCR) compatibility of oligonucleotide-gold nanoparticle conjugates, thus enabling colorimetric detection of PCR results in a closed-tube format. This method is specific, sensitive, and generally applicable. Its simplicity, visual readout, and carryover contamination-free features hold promise for point-of-care or on-site DNA testing.

Characterization of plasma ß-secretase (BACE1) activity and soluble amyloid precursor proteins as potential biomarkers for Alzheimer's disease.

Reduction in cerebrospinal fluid (CSF) amyloid ß42 (Aß42) and elevation in total tau and phospho-thr181 tau consistently differentiate between Alzheimer's disease (AD) and age-matched control subjects. In contrast, CSF ß-site APP-cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and ß (sAPPα and sAPPß) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPß, and sAPPα assays and investigated their ability to distinguish AD from age-matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1-specific inhibitor. Plasma sAPPß and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ-secretase inhibitor led to significant lowering of plasma sAPPß with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPß, sAPPα, and Aß42 (P < 0.001) compared with age-matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies.

Development of a microplate fluorescence assay for kynurenine aminotransferase.

Inhibition of kynurenine aminotransferases (KATs) is a strategy to therapeutically reduce levels of kynurenic acid (KYNA), an endogenous antagonist of glutamatergic N-methyl-D-aspartate (NMDA) and cholinergic α7 nicotinic receptors. Several methods of measuring KAT activity in vitro have been developed, but none is well-suited to high throughput and automation. In this article, we describe a modification of existing high-performance liquid chromatography (HPLC)-based methods that enables the development of a 96-well microplate assay in both enzyme- and cell-based formats using human KAT I as an example. KYNA enzymatically produced from L-kynurenine is measured directly in a reaction mixture fluorimetrically.

First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Chelators at the cancer coalface: desferrioxamine to Triapine and beyond.

The importance of iron and copper in cancer biology has been well established. Iron plays a fundamental role in cellular proliferation and copper has been shown to be a significant cofactor for angiogenesis. Early observations with the chelator used for the treatment of iron overload, desferrioxamine, showed that it had promise as an anticancer agent. These results sparked great interest in the possibility of developing more effective iron chelators for cancer therapy. The recent entry into clinical trials of the iron-binding drug, Triapine, provides evidence of the potential of this antitumor strategy. Likewise, chelators originally designed to treat disorders of copper overload, such as penicillamine, trientine, and tetrathiomolybdate, have also emerged as potential anticancer drugs, as they are able to target the key angiogenic cofactor, copper. In this review, we will discuss the development of these and other chelators that show potential as anticancer agents.

Precipitation of PEG/Carboxyl-Modified Gold Nanoparticles with Magnesium Pyrophosphate: A New Platform for Real-Time Monitoring of Loop-Mediated Isothermal Amplification.

Gold nanoparticles have proven to be promising for decentralized nucleic acid testing by virtue of their simple visual readout and absorbance-based quantification. A major challenge toward their practical application is to achieve ultrasensitive detection without compromising simplicity. The conventional strategy of thermocycling amplification is unfavorable (because of both instrumentation and preparation of thermostable oligonucleotide-modified gold nanoparticle probes). Herein, on the basis of a previously unreported co-precipitation phenomenon between thiolated poly(ethylene glycol)/11-mercaptoundecanoic acid co-modified gold nanoparticles and magnesium pyrophosphate crystals (an isothermal DNA amplification reaction byproduct), a new ultrasensitive and simple DNA assay platform is developed. The binding mechanism underlying the co-precipitation phenomenon is found to be caused by the complexation of carboxyl and pyrophosphate with free magnesium ions. Remarkably, poly(ethylene glycol) does not hinder the binding and effectively stabilizes gold nanoparticles against magnesium ion-induced aggregation (without pyrophosphate). In fact, a similar phenomenon is observed in other poly(ethylene glycol)- and carboxyl-containing nanomaterials. When the gold nanoparticle probe is incorporated into a loop-mediated isothermal amplification reaction, it remains as a red dispersion for a negative sample (in the absence of a target DNA sequence) but appears as a red precipitate for a positive sample (in the presence of a target). This results in a first-of-its-kind gold nanoparticle-based DNA assay platform with isothermal amplification and real-time monitoring capabilities.

Assessing the Efficacy of Restricting Access to Barbecue Charcoal for Suicide Prevention in Taiwan: A Community-Based Intervention Trial.

OBJECTIVE: Charcoal-burning suicide has recently been spreading to many Asian countries. There have also been several cases involving this new method of suicide in Western countries. Restricting access to suicide means is one of the few suicide-prevention measures that have been supported by empirical evidence. The current study aims to assess the effectiveness of a community intervention program that restricts access to charcoal to prevent suicide in Taiwan. METHODS AND FINDINGS: A quasi-experimental design is used to compare method-specific (charcoal-burning suicide, non-charcoal-burning suicide) and overall suicide rates in New Taipei City (the intervention site, with a population of 3.9 million) with two other cities (Taipei City and Kaohsiung City, the control sites, each with 2.7 million residents) before (Jan 1st 2009- April 30th 2012) and after (May 1st 2012-Dec. 31st 2013) the initiation of a charcoal-restriction program on May 1st 2012. The program mandates the removal of barbecue charcoal from open shelves to locked storage in major retail stores in New Taipei City. No such restriction measure was implemented in the two control sites. Generalized linear regression models incorporating secular trends were used to compare the changes in method-specific and overall suicide rates before and after the initiation of the restriction measure. A simulation approach was used to estimate the number of lives saved by the intervention. Compared with the pre-intervention period, the estimated rate reduction of charcoal-burning suicide in New Taipei City was 37% (95% CI: 17%, 50%) after the intervention. Taking secular trends into account, the reduction was 30% (95% CI: 14%, 44%). No compensatory rise in non-charcoal-burning suicide was observed in New Taipei City. No significant reduction in charcoal-burning suicide was observed in the other two control sites. The simulation approach estimated that 91 (95%CI [55, 128]) lives in New Taipei City were saved during the 20 months of the intervention. CONCLUSION: Our results demonstrate that the charcoal-restriction program reduced method-specific and overall suicides. This study provides strong empirical evidence that restricting the accessibility of common lethal methods of suicide can effectively reduce suicide rates.

Efficacy of dual PI-3K and mTOR inhibitors in vitro and in vivo in acute lymphoblastic leukemia.

The major regulators of human acute lymphoblastic leukemia (ALL) cell growth and survival mediate their effects through the phosphoinositide 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. We have shown that the mTOR inhibitor everolimus extended survival in a non-obese diabetic/severe combined immune-deficient (NOD/SCID) mouse xenograft model of human ALL. Since PI-3K has mTOR dependent and independent functions we examined the effect of the dual PI-3K/mTOR inhibitors BEZ235 and BGT226. These agents inhibited the proliferation of ALL cell lines with a three log greater potency than everolimus. However, the induction of cell death differed, with BGT226 being cytotoxic in the low micromolar range while a two log higher concentration of BEZ235 was required to produce the same effect. While all three agents extended the survival of NOD/SCID mice engrafted with human ALL, the responses of individual xenografts varied. Although differential phosphorylation of AKT on Ser(473) and Thr(308) in response to everolimus exposure was observed, this did not entirely explain the different in vivo responses to the drugs. Our data suggests that while dual PI-3K/mTOR inhibitors may improve therapeutic outcomes for a subset of ALL patients, patient selection will be important, with some patients likely to respond better to single mTOR inhibition.

Intracellular clusterin interacts with brain isoforms of the bridging integrator 1 and with the microtubule-associated protein Tau in Alzheimer's disease.

Sporadic or late-onset Alzheimer's disease (AD) is expected to affect 50% of individuals reaching 85 years of age. The most significant genetic risk factor for late-onset AD is the e4 allele of APOE gene encoding apolipoprotein E, a lipid carrier shown to modulate brain amyloid burden. Recent genome-wide association studies have uncovered additional single nucleotide polymorphisms (SNPs) linked to AD susceptibility, including those in the CLU and BIN1 genes encoding for clusterin (CLU) and the bridging integrator 1 (BIN1) proteins, respectively. Because CLU has been implicated in brain amyloid-ß (Aß) clearance in mouse models of amyloid deposition, we sought to investigate whether an AD-linked SNP in the CLU gene altered Aß42 biomarker levels in the cerebrospinal fluid (CSF). Instead, we found that the CLU rs11136000 SNP modified CSF levels of the microtubule-associated protein Tau in AD patients. We also found that an intracellular form of CLU (iCLU) was upregulated in the brain of Tau overexpressing Tg4510 mice, but not in Tg2576 amyloid mouse model. By overexpressing iCLU and Tau in cell culture systems we discovered that iCLU was a Tau-interacting protein and that iCLU associated with brain-specific isoforms of BIN1, also recently identified as a Tau-binding protein. Through expression analysis of CLU and BIN1 variants, we found that CLU and BIN1 interacted via their coiled-coil motifs. In co-immunoprecipitation studies using human brain tissue, we showed that iCLU and the major BIN1 isoform expressed in neurons were associated with modified Tau species found in AD. Finally, we showed that expression of certain coding CLU variants linked to AD risk led to increased levels of iCLU. Together, our findings suggest that iCLU and BIN1 interaction might impact Tau function in neurons and uncover potential new mechanisms underlying the etiology of Tau pathology in AD.

Antitumor activity of metal-chelating compound Dp44mT is mediated by formation of a redox-active copper complex that accumulates in lysosomes.

The metal-chelating compound Dp44mT is a di-2-pyridylketone thiosemicarbazone (DpT) which displays potent and selective antitumor activity. This compound is receiving translational attention, but its mechanism is poorly understood. Here, we report that Dp44mT targets lysosome integrity through copper binding. Studies using the lysosomotropic fluorochrome acridine orange established that the copper-Dp44mT complex (Cu[Dp44mT]) disrupted lysosomes. This targeting was confirmed with pepstatin A-BODIPY FL, which showed redistribution of cathepsin D to the cytosol with ensuing cleavage of the proapoptotic BH3 protein Bid. Redox activity of Cu[Dp44mT] caused cellular depletion of glutathione, and lysosomal damage was prevented by cotreatment with the glutathione precursor N-acetylcysteine. Copper binding was essential for the potent antitumor activity of Dp44mT, as coincubation with nontoxic copper chelators markedly attenuated its cytotoxicity. Taken together, our studies show how the lysosomal apoptotic pathway can be selectively activated in cancer cells by sequestration of redox-active copper. Our findings define a novel generalized strategy to selectively target lysosome function for chemotherapeutic intervention against cancer.

Online adaptive radiotherapy for muscle-invasive bladder cancer: results of a pilot study.

PURPOSE: To determine the advantages and disadvantages of daily online adaptive image-guided radiotherapy (RT) compared with conventional RT for muscle-invasive bladder cancer. METHODS AND MATERIALS: Twenty-seven patients with T2-T4 transitional cell carcinoma of the bladder were treated with daily online adaptive image-guided RT using cone-beam computed tomography (CBCT). From day 1 daily soft tissue-based isocenter positioning was performed using CBCT images acquired before treatment. Using a composite of the initial planning CT and the first five daily CBCT scans, small, medium, and large adaptive plans were created. Each of these adaptive plans used a 0.5-cm clinical target volume (CTV) to planning target volume expansion. For Fractions 8-32, treatment involved daily soft tissue-based isocenter positioning and selection of suitable adaptive plan of the day. Treating radiation therapists completed a credentialing program, and one radiation oncologist performed all the contouring. Comparisons were made between adaptive and conventional treatment on the basis of CTV coverage and normal tissue sparing. RESULTS: All 27 patients completed treatment per protocol. Bladder volume decreased with time or fraction number (p < 0.0001). For the adaptive component (Fractions 8-32) the small, medium, large, and conventional plans were used in 9.8%, 49.2%, 39.5%, and 1.5% of fractions, respectively. For the adaptive strategy, 2.7% of occasions resulted in a CTV V95 <99%, compared with 4.8% of occasions for the conventional approach (p = 0.42). Mean volume of normal tissue receiving a dose >45 Gy was 29% (95% confidence interval, 24-35%) less with adaptive RT compared with conventional RT. The mean volume of normal tissue receiving >5 Gy was 15% (95% confidence interval, 11-18%) less with adaptive RT compared with conventional RT. CONCLUSIONS: Online adaptive radiotherapy is feasible in an academic radiotherapy center. The volume of normal tissue irradiated can be significantly smaller without reducing CTV coverage.

Adaptive radiotherapy for bladder cancer reduces integral dose despite daily volumetric imaging.

We studied the integral radiation dose in 27 patients who had adaptive radiotherapy for bladder cancer using kilo voltage cone beam CT imaging. Compared to conventional radiotherapy the reduction in margin and choice of best plan of three for the day resulted in a lower total dose in most patients despite daily volumetric imaging.