Potential Applications of MicroRNA Profiling to Forensic Investigations

Within the forensic science community, there is a continued push to develop novel tools to aid in criminal investigations. microRNA (miRNA) analysis has been the focus of many researcher’s attention in the biomedical field since its discovery in 1993; however, the forensic application of miRNA analysis has only been suggested within the last 10 years and has been gaining considerable traction recently. The primary focus of the forensic application of miRNA analysis has been on body fluid identification to provide confirmatory universal analysis of unknown biological stains obtained from crime scenes or evidence items. There are, however, other forensic applications of miRNA profiling that have shown potential, yet are largely understudied, and warrant further investigation such as organ tissue identification, donor age estimation, and more. This review paper aims to evaluate the current literature and future potential of miRNA analysis within the forensic science field

A Comparative Analysis of Protein and Peroxidase Blood Enhancement Reagents Following Laundering and their Impact on DNA Recovery

Blood is a commonly encountered biological fluid in criminal investigations concerning a violent incident, and visual traces of the fluid on a suspect’s clothing can be diminished through laundering. This study aims to analyze the effects of laundering and the application of commercially available blood enhancement reagents commonly used to improve visualization of dilute bloodstains and their impact on DNA recovery. Enhancement reagents Hungarian Red, Coomassie Blue, Amido Black, luminol, Bluestar® Forensic Magnum, and aqueous Leuco Crystal Violet (LCV) were used to enhance human blood on cotton, polyester, denim, and wool following laundering. DNA was extracted from these samples using a QIAamp® DNA Investigator Mini Kit and quantified using a NanoDrop™ OneC UV-Vis spectrophotometer. This study revealed the peroxidase based reagents to produce the greatest sensitivity on the natural fabrics, reacting positively down to a blood dilution of 1:1000. The protein reagents produced greater sensitivity on the synthetic fabrics, reacting positively down to a blood dilution of 1:10. Peroxidase stains relying on chemiluminescent properties rather than colorimetric results produced positive results on the dark colored fabrics as sufficient color contrast was not achieved with the protein stains. The resulting yields of extracted DNA suggest that quantifiable amounts of DNA originating from bloodstains persist despite laundering and enhancement. Additionally, measurements indicated that the application of some blood enhancement reagents, particularly Amido Black, may affect DNA recovery

The Effect of Fabric Type and Laundering Conditions on the Detection of Semen Stains

There has been little research into the effect of fabric type and different laundering conditions on the ability to detect semen stains on washed fabrics. This study aimed to investigate three potential factors affecting semen identification on laundered clothing: fabric type, water temperature during washing, and whether the stain was dry at the time of washing. Following laundering, semen stains on four fabric types (cotton, polyester, denim, and wool) were examined and tested with three common methods used to detect semen; screening with an alternate light source, acid phosphatase press test, and histological staining of spermatazoa. It was determined that semen was difficult to detect if it was still wet when the semen-stained article was washed. There did not appear to be any difference based on the temperature of the wash cycle. It was also determined that synthetic fabrics such as polyester may not effectively retain the components of semen during laundering, making detection more difficult

Investigating the isolation and amplification of MicroRNAs for Forensic Body Fluid Identification

Background: The discovery of forensic DNA typing evolved molecular biology far beyond what could have been expected in terms of its forensic application, and now there exists other developments in molecular biology which are ready for application to forensic challenges. One such challenge is the identification of the body fluid source of stains recovered from evidence items and crime scenes. Currently there are significant efforts in the research field to develop novel methods for the molecular identification of body fluids, with microRNAs (miRNAs) revealing great potential. MiRNAs have been shown to have high tissue specificity and are less susceptible to degradation as a result of their small size, which infers great advantages to their potential role for identifying forensically relevant body fluids. Objective: This study investigated the isolation and amplification of miRNAs from forensically relevant body fluids. Method: Venous blood, menstrual blood, semen, saliva, and vaginal material samples were extracted using; miRNeasy® mini kit (Qiagen), mirVana™ miRNA isolation kit (Ambion), and a modified mirVana™ method, and the quality/quantity of isolated miRNA was determined. miRNAs previously identified to show specificity for particular forensically relevant body fluids were examined. Real Time-Quantitative PCR (RT-qPCR) was performed targeting 5 miRNAs of interest, miR-451, miR-412, miR-891a, miR-205 and miR-124a. Results: This study identified the miRNeasy® mini kit as the optimal method of the three methods investigated for the extraction of miRNAs from body fluids and further validates a selection of miRNAs previously suggested as potential biomarkers. Conclusion: This research highlights the potential of miRNAs as novel markers for the identification of forensically relevant body fluids

The Use of Raman Spectroscopy for the Identification of Forensically Relevant Body Fluid Stains

Raman spectroscopy is fast becoming a popular technique in the forensic science discipline, and more recently its focus has turned to biological samples. This study reveals the ability of Raman spectroscopy to identify some forensically relevant body fluids, both individually and within mixed samples, that can be crucial in some forensic investigations. This study also further demonstrates the capabilities of Raman as a means for human blood identification in simulated crime scene samples to include bloodstains on a variety of fabrics, at varying dilutions, following laundering, and with the use of various blood-enhancement reagents. The impact of washing and blood-enhancement reagents reveals the importance of the choice of method and its bearing on subsequent Raman analysis

Screening of Exosomal MicroRNAs From Colorectal Cancer Cells

BACKGROUND: Cells release extracellular membrane vesicles including microvesicles known as exosomes. Exosomes contain microRNAs (miRNAs) however the full range within colorectal cancer cell secreted exosomes is unknown. OBJECTIVE: To identify the full range of exosome encapsulated miRNAs secreted from 2 colorectal cancer cell lines and to investigate engineering of exosomes over-expressing miRNAs. METHODS: Exosomes were isolated from HCT-116 and HT-29 cell lines. RNA was extracted from exosomes and microRNA array performed. Cells were engineered to express miR-379 (HCT-116-379) or a non-targeting control (HCT-116-NTC) and functional effects were determined. Exosomes secreted by engineered cells were transferred to recipient cells and the impact examined. RESULTS: Microvesicles 40-100 nm in size secreted by cell lines were visualised and confirmed to express exosomal protein CD63. HT-29 exosomes contained 409 miRNAs, HCT-116 exosomes contained 393, and 338 were common to exosomes from both cell lines. Selected targets were validated. HCT-116-379 cells showed decreased proliferation (12-15% decrease, p \u3c 0.001) and decreased migration (32-86% decrease, p \u3c 0.001) compared to controls. HCT-116-379 exosomes were enriched for miR-379. Confocal microscopy visualised transfer of HCT-116-379 exosomes to recipient cells. CONCLUSIONS: Colorectal cancer cells secrete a large number of miRNAs within exosomes. miR-379 decreases cell proliferation and migration, and miR-379 enriched exosomes can be engineered

Impact of Tumour Epithelial Subtype on Circulating MicroRNAs in Breast Cancer Patients

While a range of miRNAs have been shown to be dysregulated in the circulation of patients with breast cancer, little is known about the relationship between circulating levels and tumour characteristics. The aim of this study was to analyse alterations in circulating miRNA expression during tumour progression in a murine model of breast cancer, and to detemine the clinical relevance of identified miRNAs at both tissue and circulating level in patient samples. Athymic nude mice received a subcutaneous or mammary fat pad injection of MDA-MB-231 cells. Blood sampling was performed at weeks 1, 3 and 6 following tumour induction, and microRNA extracted. MicroRNA microArray analysis was performed comparing samples harvested at week 1 to those collected at week 6 from the same animals. Significantly altered miRNAs were validated across all murine samples by RQ-PCR (n = 45). Three miRNAs of interest were then quantified in the circulation(n = 166) and tissue (n = 100) of breast cancer patients and healthy control individuals. MicroArray-based analysis of murine blood samples revealed levels of 77 circulating microRNAs to be changed during disease progression, with 44 demonstrating changes .2-fold. Validation across all samples revealed miR-138 to be significantly elevated in the circulation of animals during disease development, with miR-191 and miR-106a levels significantly decreased. Analysis of patient tissue and blood samples revealed miR-138 to be significantly up-regulated in the circulation of patients with breast cancer, with no change observed in the tissue setting. While not significantly changed overall in breast cancer patients compared to controls, circulating miR-106a and miR-191 were significantly decreased in patients with basal breast cancer. In tissue, both miRNAs were significantly elevated in breast cancer compared to normal breast tissue. The data demonstrates an impact of tumour epithelial subtype on circulating levels of miRNAs, and highlights divergent miRNA profiles between tissue and blood samples from breast cancer patients

Automatisierte Planung von digitalen Hochgeschwindigkeitsnetzen

Der Ausbau von digitalen Hochgeschwindigkeitsnetzen ist gekennzeichnet durch neuartige Anforderungen an den Planungsprozess. Diese Anforderungen erfordern wiederum den Einsatz von neuartigen Paradigmen, die eine effiziente und zugleich genaue Planung von flächendeckenden Glasfasernetzen ermöglichen. Hierbei können wiederkehrende Planungsaufgaben durch eine gezielte computergestützte Automatisierung effizienter und genauer ausgeführt, als es mit bisherigen Planungskonzepten möglich ist. Dieses Arbeitspapier beschreibt die computergestützte Ausführung eines Planungsprozesses auf Basis von fünf grundlegenden, iterativen Planungsschritten und gibt Empfehlungen für eine effiziente und genaue Planung von Glasfasernetzen. Der hier vorgestellte Ansatz ermöglicht es Netzbetreibern und Investoren, den Ausbau beliebiger Siedlungs- und Gewerbegebiete auf der zuverlässigen Basis von belastbarem Faktenwissen wirtschaftlich zu priorisieren

Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer