Uncertainty in estimating the number of contributors from simulated DNA mixture profiles, with and without allele dropout, from Chinese, Malay, Indian, and Caucasian ethnic populations.

Determining the number of contributors (NOC) accurately in a forensic DNA mixture profile can be challenging. To address this issue, there have been various studies that examined the uncertainty in estimating the NOC in a DNA mixture profile. However, the focus of these studies lies primarily on dominant populations residing within Europe and North America. Thus, there is limited representation of Asian populations in these studies. Further, the effects of allele dropout on the NOC estimation has not been explored. As such, this study assesses the uncertainty of NOC in simulated DNA mixture profiles of Chinese, Malay, and Indian populations, which are the predominant ethnic populations in Asia. The Caucasian ethnic population was also included to provide a basis of comparison with other similar studies. Our results showed that without considering allele dropout, the NOC from DNA mixture profiles derived from up to four contributors of the same ethnic population could be estimated with confidence in the Chinese, Malay, Indian and Caucasian populations. The same results can be observed on DNA mixture profiles originating from a combination of differing ethnic populations. The inclusion of an overall 30% allele dropout rate increased the probability (risk) of underestimating the NOC in a DNA mixture profile; even a 3-person DNA mixture profile has a > 99% risk of underestimating the NOC as two or fewer contributors. However, such risks could be mitigated when the highly polymorphic SE33 locus was included in the dataset. Lastly there was a negligible level of risk in misinterpreting the NOC in a mixture profile as deriving from a single source profile. In summary, our studies showcased novel results representative of the Chinese, Malay, and Indian ethnic populations when examining the uncertainty in NOC estimation in a DNA mixture profile. Our results would be useful in the estimation of NOC in a DNA mixture profile in the Asian context.

A practical study on direct PCR amplification using the GlobalFiler™ PCR Amplification Kit on human bloodstains collected with microFLOQ™ Direct swabs.

Rapid DNA profiling of casework samples is a powerful tool that can support law enforcement agencies in the quick apprehension of perpetrators before they re-offend or escape the jurisdiction. This present study evaluated the feasibility of direct PCR amplification, using the microFLOQ™ Direct swab, for generating DNA profiles (from bloodstains) within 3 h. The swab tip is coated with nylon fibers pre-treated with cell lysing agent, which allows for the direct PCR amplification of collected samples without DNA extraction and quantification, thereby shortening the time required to obtain a DNA profile. Samples collected were directly amplified using GlobalFiler™ PCR Amplification Kit with and without the presence of a PCR additive. Addition of the PCR additive enhanced the peak heights of DNA profiles by approximately 2 fold. Hence, an additive could improve results obtained in the absence of a DNA purification step, especially since casework samples may contain PCR inhibitors. Subsequently, these swabs, amplified using the GlobalFiler™ PCR Amplification Kit with PCR additive, were evaluated on common substrates encountered in routine casework samples submitted with bloodstains, such as denim jeans, knife blade, tissue paper, leather belt, shirt, and blood swabs. The minimum peak heights observed were generally above the analytical and stochastic thresholds established by the laboratory. Finally, the microFLOQ™ Direct swab workflow was compared to the laboratory's standard workflow of DNA profiling comprising of conventional processing steps such as extraction using the DNA-IQ™ chemistry on Maxwell® 16, followed by quantification, amplification and capillary electrophoresis. The average peak heights of the DNA profiles generated by direct PCR amplification were similar or exceeded those generated using the standard workflow. This study clearly demonstrates that direct PCR amplification using microFLOQ™ Direct swab can be used in a rapid workflow to obtain DNA profiles from casework samples.

Identification of p10 as a neurotoxic product generated from the proteolytic cleavage of the neuronal Cdk5 activator.

The involvement of cyclin-dependent kinase-5 (Cdk5) and p25, the proteolytic fragment of activator p35, has long been implicated in the development of neuron-fibrillary tangles (NFTs), a hallmark of Alzheimer's disease (AD). Findings in this area over the past decade have been highly controversial and inconclusive. Here we report unprecedented detection of endogenous p10, the smaller proteolytic fragment of the Cdk5 activator p35 in treated primary cortical neurons that underwent significant apoptosis, triggered by proteasome inhibitors MG132 and lactacystin, and protein kinase inhibitor staurosporine (STS). p10 appeared exclusively in the detergent-resistant fraction made up of nuclear matrix, membrane-bound organelles, insoluble membrane proteins, and cytoskeletal components. Intriguingly, transient overexpression of p10 in neural cells induced apoptotic morphologies, suggesting that p10 may play an important role in mediating neuronal cell death in neurodegenerative diseases. We demonstrated for the first time that p10-mediated apoptosis occurred via a caspases-independent pathway. Furthermore, as p10 may contain the myristoylation signal for p35 which is responsible for binding p35 to several intracellular components and the membrane, all in all these novel results present that the accumulation of p10 to the detergent-insoluble fraction may be a crucial pathological event to triggering neuronal cell death.

Annexin A3 is associated with cell death in lactacystin-mediated neuronal injury.

Massive neuronal apoptosis and accumulation of protein aggregates in the cortex and hippocampus of the brain are hallmarks of several neurodegenerative disorders, indicating ubiquitin proteasome system (UPS) dysfunction. Lactacystin, a classical proteasome inhibitor, is used to simulate ubiquitin proteasome system dysfunction in neurons to mimic pathological features of neurodegenerative disorders. Based on Western blot analyses, we reported for the first time that annexin A3 (AnxA3) is not only endogenously expressed in mouse cortical neurons but also more importantly, by gene expression microarray and real-time RT-PCR that it is greatly transcriptional up-regulated to approximately 11- and 15-fold, respectively in murine primary cortical neurons with 1µM lactacystin for 24h. Up-regulation of AnxA3 expression occurred after 12-15h post-lactacystin treatment, which corresponded with the onset of neuronal injury, with approximately 25% of the neurons being non-viable by that time interval. Western blot analysis with anti-AnxA3 antibodies further validated that up-regulation of AnxA3 only occurs with onset of neuronal death, and not with the onset of proteasome inhibition, which occurs at 4.5h post-lactacystin treatment. Over-expression studies suggested AnxA3 might be involved in death promotion during lactacystin-mediated neuronal death, since caspase-3 activation was significantly stronger upon neuronal AnxA3 over-expression. We propose AnxA3 up-regulation may have significant relevance in the elucidation of neurodegenerative pathophysiology.

pH dependent high transfection efficiency of mouse neuroblastomas using TransFectin.

Mouse neuroblastoma cell lines are often used in lieu of mouse primary neurons in ex vivo experiments, as they provide an easier platform for transfection, compared to the latter. A well-known inherent problem with this strategy is the relatively low transfection efficiency (15-30%) of mouse neuroblastoma cell lines such as neuro-2A and N1E-115. We were able to improve the transfection efficiency of these cell lines by using the cationic lipid reagent, TransFectin (Bio-Rad, Hercules, CA, USA) to optimise the transfection conditions. Our results, based on fluorescence intensity determinations and Western blotting for enhanced green fluorescence protein (EGFP) over-expression in neuro-2A, demonstrated that pH is a crucial factor in determining the transfection efficiency. Under pH-optimised transfection conditions, flow cytometric analysis revealed high EGFP transfection efficiencies of 76.4 +/- 0.5 and 60.9 +/- 0.6% for neuro-2A and N1E-115, respectively. Notably, the optimised TransFectin-based transfection system did not result in any detectable cytotoxicity to the mouse neuroblastomas. The resultant optimised system is economical, easy to use and does not require any specialised equipment.