Examination of Methylation Sites for Forensic Age Determination from Semen

Methylation Sensitive High-Resolution Melt (MS-HRM) is based on quantitating the melt curve from an experimental sample against a standard of known methylation levels. Whereas most applications of age prediction using methylation markers are based upon pyrosequencing or SNaPshot technologies, these analysis methods are both cost and instrumentation prohibitive. This study sought to use to the varied methylation status of the ELOVL2 and FHL2 alleles, both having known correlation with age (Hamano et. al.), in a labor and time efficient manner to develop an age prediction model. A non-linear regression and standard curve was compiled from the methylation status in a sample (n=7) of extracted semen samples and compared to chronological age. The methylation status of ELVOL2 and FHL2 from each sample was obtained, with the conclusion that no correlation in methylation percentage and biological age existed for this sample of individuals aged 20-33. The principal objective of this study, to expand the application of MS-HRM age prediction from blood to other body fluids, will need further testing using larger sample sizes and broader age ranges prior to application in forensic casework.https://scholarscompass.vcu.edu/uresposters/1281/thumbnail.jp

Optical Tweezers as an Effective Tool for Spermatozoa Isolation from Mixed Forensic Samples

A single focus optical tweezer is formed when a laser beam is launched through a high numerical aperture immersion objective. This objective focuses the beam down to a diffraction-limited spot, which creates an optical trap where cells suspended in aqueous solutions can be held fixed. Spermatozoa, an often probative cell type in forensic investigations, can be captured inside this optical trap and dragged one by one across millimeter-length distances in order to create a cluster of cells which can be subsequently drawn up into a capillary for collection. Sperm cells are then ejected onto a sterile cover slip, counted, and transferred to a tube for DNA analysis workflow. The objective of this research was to optimize sperm cell collection for maximum DNA yield, and to determine the number of trapped sperm cells necessary to produce a full STR profile. A varying number of sperm cells from both a single-source semen sample and a mock sexual assault sample were isolated utilizing optical tweezers and processed using conventional STR analysis methods. Results demonstrated that approximately 50 trapped spermatozoa were required to obtain a consistently full DNA profile. A complete, single-source DNA profile was also achieved by isolating sperm cells via optical trapping from a mixture of sperm and vaginal epithelial cells. Based on these results, optical tweezers are a viable option for forensic applications such as separation of mixed populations of cells in forensic evidence

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3’-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression

Infrared Laser Heating Applied to Nanopore Sensing for DNA Duplex Analysis

Temperature studies coupled with resistive-pulse nanopore sensing enable the quantification of a variety of important thermodynamic properties at the single-molecule limit. Previous demonstrations of nanopore sensing with temperature control have utilized bulk chamber heating methodologies. This approach makes it difficult to rapidly change temperatures and enable optical access for other analytical techniques (i.e., single-molecule fluorescence). To address these issues, researchers have explored laser-based methodologies through either direct infrared (IR) absorption or plasmonic assisted heating. In this paper, we demonstrate the use of IR-based direct absorption heating with the DNA sensing capabilities of a biological nanopore. The IR heating enables rapid changes of the temperature in and around an α-hemolysin pore, and we use this to explore melting properties for short (≤50 bp) double-stranded DNA homopolymers. We also demonstrate that the IR heating enables one to measure the percentage of different-sized DNA molecules in a binary mixture

Next generation sequencing results in prostate cancer cell line model.

<p>Next generation sequencing results in prostate cancer cell line model.</p

Dysregulation of miR-125b and miR-22 expression in RNA extracted from prostate tissue samples by LCM.

<p><b>(A)</b> Frozen prostate samples were dissected via LCM into benign glandular epithelial versus tumor. Total RNA was extracted using the PicoPure RNA extraction method, as described in Materials and Methods. qRT-PCR was performed with specific primers against miR-125b and miR-22 using the TaqMan^® Universal Master Mix II. C_T values were normalized to RNU48 and reported as fold differences compared to benign tissue using the ΔΔCT method of Livak et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.ref023" target="_blank">23</a>]. Results are the average from frozen tissue samples (n = 4) repeated in 3 technical replicates. <b>(B)</b> From 10 successive slides of a single FFPE prostate sample, miR-125b was quantitated by qRT-PCR as described in panel A for RNA extracted from stroma, benign glandular tissue, BPH, PIN and tumor of stage G4.</p

Model for dual regulation by miR-125b and miR-22.

<p>RPPA data shows increases in ErbB3, ErbB2, MET, PI3K, pBAD and pERK protein expression in M12 cells relative to the parental p69 cell lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.t003" target="_blank">Table 3</a>: green targets). Western blot (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.g004" target="_blank">Fig 4D</a>) confirms a decrease in PTEN expression (red). These proteins figure prominently in signal transduction pathways that are targeted by miR-125b and miR-22. Intermediates in yellow (AKT, RAS and pMEK) were not included in the proteome analysis. miR-125b directly targets ErbB2, ErbB3 and MET and miR-22 targets PTEN [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.ref030" target="_blank">30</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.ref031" target="_blank">31</a>]. We propose that the loss of tumor suppressor miR-125b in the M12 cell line would result in enhanced signaling via the PI3K/AKT pathway and the RAS/pMEK pathway as regulated by MET. Conversely, miR-22 targets PTEN, the loss of which by increased expression of miR-22 further exacerbates PI3K signaling. In this scenario the dysregulation of these two miRs could contribute to the highly tumorigenic/metastatic phenotype displayed by the M12 cells compared to the parental p69 cell line and subsequently contribute to prostate tumorigenesis.</p

Restoration of miR-125b or inhibition of miR-22 impairs <i>in vitro</i> migratory and invasive potential of M12 cells but initially has little effect on cell proliferation.

<p><b>(A)</b> A representative growth curve of M12 versus M12+miR-22inhibitor cells is shown. Cells (20,000) were plated in triplicate and viable cells counted at 0, 48, and 72 hours in a Beckman Coulter Vi-CELL automated cell viability analyzer using trypan blue exclusion (cell viability >94%). The average viable cell number was plotted on an exponential curve with the standard deviation noted. Analysis of cell growth at the 48 and 72 hour time points using a student t-test yielded a p-value of 0.11 and 0.009, respectively <b>(B)</b> Cells (50,000) were plated in triplicate in serum free media in the top chamber of a ThinCert^™ tissue culture insert and processed as described in Materials and Methods. Panel A confirmed no significant difference in growth potential at 20 hours at which time the number of migrated cells was counted in 10 random fields and the total number of migratory cells estimated by summing up each of the individual fields. The average mean sum of each well is presented, along with the standard error. The invasion assay used the same experimental set up with the additional step of coating the membrane with Culturex^® growth factor reduced basement membrane (60 μl) prior to cell addition. Results are shown for parental M12 cells or M12 cells stably transformed with a scrambled RNA sequence as a negative control (M12+scrambled RNA), a miR-22 inhibitor (M12+miR-22 inhibitor) or restored miR-125b (M12+miR-125b). Data is the mean of 3 independent experiments, each performed in triplicate. ANOVA test indicates a significant difference with a P-value < 0.05.</p

Single nucleotide variations in prostate cancer cell line model.

<p>*Not present in M12 cell line</p><p>Single nucleotide variations in prostate cancer cell line model.</p

Validation of miR-125b and miR-22 binding to the 3'-UTR of ErbB2 and PTEN respectively.

<p><b>(A)</b> Identification of two miR-125b binding sites within the 3’-UTR of ErbB2. Site 1 (position -19 to -44) downstream of the stop codon is proven by Scott et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.ref030" target="_blank">30</a>]. Site 2 (position -70 to -96) is proposed here by an analysis with RNAhybrid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.ref056" target="_blank">56</a>]. Both sites display notable binding between ErbB2’s 3’-UTR (red) and miR-125b (green) with favorable MFEs. <b>(B)</b> The M12 cell line was transfected with a dual luciferase reporter construct containing various portions of ErbB2’s 3’-UTR as noted. Firefly luciferase expression is normalized to renilla luciferase activity. Results are the mean of 3 independent experiments each performed in triplicate. ANOVA test indicates a significant difference with a P-value < 0.05. <b>(C)</b> The effect of miR-22 binding on the wild type and mutant PTEN 3’-UTR target binding site was assessed on firefly versus renilla luciferase activity as described in panel B via transfection into the M12 cell line. Mutation of a single base (C to A) within the target to miR-22’s seed region [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142373#pone.0142373.ref031" target="_blank">31</a>] resulted in increased firefly luciferase activity. Results are the average of 3 experiments repeated in triplicate. <b>(D)</b> A representative western blot of whole cell extracts (30 μg) from M12, M12+miR-22i and p69 cells. Proteins were separated on a 4–12% Tris-Bis gel and incubated with a PTEN antibody. β-actin was used as a loading control. The position of migration of relevant protein size markers are noted. <b>(E)</b> Quantitation of western blot shown in panel D with PTEN levels normalized to β-actin.</p