Focus-independent particle size measurement from streak images: a comparison of multivariate methods.

Our laboratories have recently developed a flow-through imaging photometer to characterize and classify fluorescent particles between 3 and 47 µm in size. The wide aperture of the objective lens (0.7 NA) required for measuring spectral fluorescence of single particles restricts the depth of field, such that a large sample volume results in many particles that are out of focus. Here, we describe numerical methods for determining the size of these objects, regardless of their distance from the focal plane, using image processing and multivariate calibration. An intensity profile is extracted from the images and is used as the input for a variety of calibration methods, including partial least squares, neural networks, and support vector machines. The capabilities of these methods are examined to establish the best method for particle sizing that is independent of focus. We found that support vector machines provided the best results, with size estimation error of ±3.1 µm.

A quantitative method for determining a representative detection limit of the forensic luminol test for latent bloodstains.

The luminol test has been used for over 60 years by forensic investigators for presumptive identification of blood and visualization of blood splatter patterns. Multiple studies have estimated the limit of detection (LD) for bloodstains when luminol is employed, with results ranging from 100× to 5,000,000× dilute. However, these studies typically have not identified and controlled important experimental variables which may affect the luminol LD for bloodstains. Without control of experimental parameters in the laboratory, variables which affect the potential of presumptive bloodstain test methods remain largely unknown, and comparisons required to establish new, more powerful detection methods are simply impossible. We have developed a quantitative method to determine the relationship between the amount of blood present and its reaction with luminol by measuring, under controlled conditions, the resulting chemiluminescent intensity with a video camera, combined with processing of the digital intensity data. The method resulted in an estimated LD for bloodstains on cotton fabric at ∼200,000× diluted blood with a specific luminol formulation. Although luminol is the focus of this study, the experimental protocol used could be modified to study effects of variables using other blood detection reagents.

The New Inhibitor of Monoamine Oxidase, M30, has a Neuroprotective Effect Against Dexamethasone-Induced Brain Cell Apoptosis.

Stress detrimentally affects the brain and body and can lead to or be accompanied by depression. Although stress and depression may contribute to each other, the exact molecular mechanism underlying the effects is unclear. However, there is a correlation between stress and an increase in glucocorticoid secretion which causes a subsequent increase in monoamine oxidase (MAO) activity during stress. Consequently, MAO inhibitors have been used as traditional antidepressant drugs. Cellular treatment with the synthetic glucocorticoid, dexamethasone (a cellular stressor), has been reported to markedly increase both MAO A and MAO B catalytic activities, as well as apoptosis. This study compares the neuroprotective abilities of M30 (a new generation inhibitor of both MAO A and MAO B) with rasagiline (Azilect(®), another new MAO B inhibitor) and selegiline (Deprenyl(®), a traditional MAO B inhibitor) in the prevention of dexamethasone-induced brain cell death and MAO activity in human neuroblastoma cells, SH-SY5Y. M30 demonstrated the highest inhibitory effect on MAO A; however, M30 showed the lowest inhibitory effect on MAO B enzymatic activity in comparison to rasagiline and selegiline. Although, M30 exhibited the greatest neuroprotective effect by decreasing cell death rates and apoptotic DNA damage compared to rasagiline and selegiline, these neuroprotective effects of M30 were, overall, similar to rasagiline. Summarily, M30 has a generally greater impact on neuroprotection than the MAO B inhibitors, selegiline and rasagiline. Our results suggest that M30 may have great potential in alleviating disorders involving increases in both MAO A and MAO B, such as stress-induced disorders.

Comparative neuroprotective effects of rasagiline and aminoindan with selegiline on dexamethasone-induced brain cell apoptosis.

Stress can affect the brain and lead to depression; however, the molecular pathogenesis is unclear. An association between stress and stress-induced hypersecretion of glucocorticoids occurs during stress. Dexamethasone (a synthetic glucocorticoid steroid) has been reported to induce apoptosis and increase the activity of monoamine oxidase (MAO) (Youdim et al. 1989). MAO is an enzyme for the degradation of aminergic neurotransmitters; dopamine, noradrenaline and serotonin and dietary amines and MAO inhibitors are classical antidepressant drugs. In this study, we have compared the ability of rasagiline (Azilect) and its main metabolite, R-aminoindan with selegiline (Deprenyl) in prevention of dexamethasone-induced brain cell death employing human neuroblastoma SH-SY5Y cells and glioblastoma 1242-MG cells. Dexamethasone reduced cell viability as measured by MTT test, but rasagiline, selegiline, and 1-R-aminoindan could significantly prevent dexamethasone-induced brain cell death. Among three drugs, rasagiline had the highest neuroprotective effect. Furthermore, the inhibitory effects of these drugs on MAO B catalytic activity and on apoptotic DNA damage (TUNEL staining) were examined. Rasagiline exhibited highest inhibition on MAO B enzymatic activity and prevention on DNA damage as compared to selegiline and 1-R-aminoindan. In summary, the greater neuroprotective effect of rasagiline may be associated with the combination of the parent drug and its metabolite 1-R-aminoindan.

The Neuroprotective Effect of Antidepressant Drug via Inhibition of TIEG2-MAO B Mediated Cell Death.

Alcohol use disorders are common in the world. However, the development of novel drugs to prevent alcohol-induced brain damage is based upon an improved neurobiological understanding on the cellular changes that take place in the brain. We previously reported that ethanol exposure lowered cell proliferation and increased cell apoptosis in all cell types, but affects brain cell lines the most, while ethanol and the anti-depressant drug deprenyl, an monoamine oxidase B (MAO B) inhibitor, exposure in unison increases cell viability. Here we investigated the molecular mechanism of the neuroprotective effect of deprenyl (0.25 nM) on ethanol (75 mM)-induced harmful effect. Transforming growth factor-beta-inducible early gene 2 (TIEG2) is an activator for MAO B. MAO B levels increase has been shown to contribute to neuronal cell death. This study uses the neuronal cell line to address whether ethanol induced cell death is through the activation of TIEG2-MAO B apoptotic pathway, and whether deprenyl protects cells from the effects of alcohol through the inhibition of this pathway. We have found that ethanol exposure increases the levels of mRNA and protein/catalytic activity for both TIEG2 and MAO B, while ethanol and deprenyl exposure in unison reduce the expression of both TIEG2 and MAO B, however it increases cell viability. Additionally, TIEG2-overexpressed cells display more cellular death-induced by ethanol than control cells. In summary, this study demonstrates the role of TIEG2 in ethanol induced cell death. The inhibition of the TIEG2-MAO B pathway may be one of the mechanisms for the neuroprotective effect of deprenyl.