DNA methylation-specific multiplex assays for body fluid identification

Differential Methylation Profile of Ovarian Cancer in Tissues and Plasma

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood

Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework.

Microbial communities present in body fluids can assist in distinguishing between types of body fluids. Metagenomic studies have reported bacterial genera which are core to specific body fluids and are greatly influenced by geographical location and ethnicity. Bacteria in body fluids could also be due to bacterial infection; hence, it would be worthwhile taking into consideration bacterial species associated with diseases. The present review reports bacterial species characteristic of diseased and healthy body fluids across geographical locations, and bacteria described in forensic studies, with the aim of collating a set of bacteria to serve as the core species-specific markers for forensic body fluid identification. The most widely reported saliva-specific bacterial species are Streptococcus salivarius, Prevotella melaninogenica, Neisseria flavescens, with Fusobacterium nucleatum associated with increased diseased state. Lactobacillus crispatus and Lactobacillus iners are frequently dominant in the vaginal microbiome of healthy women. Atopobium vaginae, Prevotella bivia, and Gardnerella vaginalis are more prevalent in women with bacterial vaginosis. Semen and urine-specific bacteria at species level have not been reported, and menstrual blood bacteria are indistinguishable from vaginal fluid. Targeting more than one bacterial species is recommended for accurate body fluid identification. Although metagenomic sequencing provides information of a broad microbial profile, the specific bacterial species could be used to design biosensors for rapid body fluid identification. Validation of microbial typing methods and its application in identifying body fluids in a mixed sample would allow regular use of microbial profiling in a forensic workflow.

Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs

Recent evidence has indicated an important role for DNA methylation in the control of gene expression, silencing of transposons and maintenance of genome stability. The current study utilised the Amplified Methylation Polymorphism (AMP) technique to investigate the extent of DNA methylation polymorphisms and their biological importance in several plant species. DNA methylation patterns were examined in response to different environments and propagation through tissue culture, and in different tissue types of one plant species. The technique was also used to investigate the extent of DNA loss in transgenic sugarcane as a result of genetic engineering procedures. Widespread DNA methylation changes were detected in response to the environment in both species investigated, namely sugarcane (a complex monocot polyploid) and Arabidopsis thaliana (the simple dicot diploid). Sugarcane cultivar Q155 grown at three diverse locations in Queensland, Australia, showed location-specific DNA methylation patterns, and when clones were moved to common glasshouses they showed new glasshouse-specific DNA methylation patterns whilst generally retaining the older location-specific DNA methylation signatures. In addition, prolonged cold treatment (vernalisation) in Arabidopsis induced DNA methylation changes. These findings indicate that DNA methylation may be a general control mechanism for mediating phenotypic responses to environmental change. Plant genome instability with passage through tissue culture was shown to vary greatly between plant species, with wheat cultivar Hartog being particularly stable in comparison to sugarcane cultivars. It was also demonstrated that the process of producing double haploids of Hartog resulted in few, if any DNA methylation or sequence changes, thereby validating this approach for producing mapping populations for mapping important traits in wheat. In sugarcane, on the other hand, genome instability in conventionally tissue culture-propagated plants was demonstrated to be a major problem. Furthermore, gene transfer and/or selection processes increased the extent of genome instability in the form of DNA loss compared to that resulting from the conventional tissue culture-propagation process alone. While genome instability in conventionally tissue culture-propagated and genetically engineered sugarcane is currently a major problem, AMP analysis of non-transgenic sugarcane plants propagated by a new direct regeneration method indicated that the prospects of overcoming this problem of genome instability in sugarcane are encouraging. AMP profiles were shown to be distinguishable between leaf and pollen DNA in lily (Lilium longiforum), however, the extent of tissue-specific DNA methylation changes was shown to be less than that reported for mammals. Throughout the present study, the AMP technique has been proven to be a robust and efficient tool for analysing plant genome instability and assessing the DNA methylation status of specific loci throughout the genome. This study has also demonstrated the potential importance of DNA methylation in the plant response mechanism to changing environments, including responses to stresses imposed by the tissue culture process. However, more studies are necessary to elucidate the environmentally-induced DNA methylation changes. In particular, the priority would be the cloning, sequencing and mapping of the environmentally-induced DNA methylation changes. In view of the fact that the Arabidopsis genome is completely sequenced, mapping of the vernalisation-induced DNA methylation markers is currently achievable in this species and could greatly enhance our understanding of vernalisation and the control of flowering time in plants. Future applications of the AMP protocol would include efficient screening for genome integrity in tissue culture-derived and genetically engineered plants, assessing the effect of tissue culture conditions and gene transfer and selection procedures on genome instability, and further identification and characterisation of genomic regions displaying environment-specific DNA methylation.

Identification and the time since deposition (TsD) estimation of body fluid stains from a crime scene could provide valuable information for solving the cases and are always difficult for forensics. Microbial characteristics were considered as a promising biomarker to address the issues. However, changes in the microbiota may damage the specific characteristics of body fluids. Correspondingly, incorrect body fluid identification may result in inaccurate TsD estimation. The mutual influence is not well understood and limited the codetection. In the current study, saliva, semen, vaginal secretion, and menstrual blood samples were exposed to indoor conditions and collected at eight time points (from fresh to 30 days). High-throughput sequencing based on the 16S rRNA gene was performed to characterize the microbial communities. The results showed that a longer TsD could decrease the discrimination of different body fluid stains. However, the accuracies of identification still reached a quite high value even without knowing the TsD. Correspondingly, the mean absolute error (MAE) of TsD estimation significantly increased without distinguishing the types of body fluids. The predictive TsD of menstrual blood reached a quite low MAE (1.54 ± 0.39 d). In comparison, those of saliva (6.57 ± 1.17 d), semen (6.48 ± 1.33 d), and vaginal secretion (5.35 ± 1.11 d) needed to be further improved. The great effect of individual differences on these stains limited the TsD estimation accuracy. Overall, microbial characteristics allow for codetection of body fluid identification and TsD estimation, and body fluids should be identified before estimating TsD in microbiome-based stain analyses.IMPORTANCEEmerged evidences suggest microbial characteristics could be considered a promising tool for identification and time since deposition (TsD) estimation of body fluid stains. However, the two issues should be studied together due to a potential mutual influence. The current study provides the first evidence to understand the mutual influence and determines an optimal process for codetection of identification and TsD estimation for unknown stains for forensics. In addition, we involved aged stains into our study for identification of body fluid stains, rather than only using fresh stains like previous studies. This increased the predictive accuracy. We have preliminary verified that individual differences in microbiotas limited the predictive accuracy of TsD estimation for saliva, semen, and vaginal secretion. Microbial characteristics could provide an accurate TsD estimation for menstrual blood. Our study benefits the comprehensive understanding of microbiome-based stain analyses as an essential addition to previous studies.

<div>Abstract<p>Taxanes are standard therapy in clinical practice for metastatic breast cancer; however, primary or acquired chemoresistance are a common cause of mortality. Breast cancer patient-derived xenografts (PDX) are powerful tools for the study of cancer biology and drug treatment response. Specific DNA methylation patterns have been associated to different breast cancer subtypes but its association with chemoresistance remains unstudied. Aiming to elucidate docetaxel resistance mechanisms, we performed genome-wide DNA methylation in breast cancer PDX models, including luminal and triple-negative breast cancer (TNBC) models sensitive to docetaxel, their matched models after emergence of chemoresistance and residual disease after short-term docetaxel treatment. We found that DNA methylation profiles from breast cancer PDX models maintain the subtype-specific methylation patterns of clinical samples. Two main DNA methylation clusters were found in TNBC PDX and remain stable during the emergence of docetaxel resistance; however, some genes/pathways were differentially methylated according to docetaxel response. A DNA methylation signature of resistance able to segregate TNBC based on chemotherapy response was identified. Transcriptomic profiling of selected sensitive/resistant pairs and integrative analysis with methylation data demonstrated correlation between some differentially methylated and expressed genes in docetaxel-resistant TNBC PDX models. Multiple gene expression changes were found after the emergence of docetaxel resistance in TNBC. DNA methylation and transcriptional changes identified between docetaxel-sensitive and -resistant TNBC PDX models or residual disease may have predictive value for chemotherapy response in TNBC.</p>Implications:<p>Subtype-specific DNA methylation patterns are maintained in breast cancer PDX models. While no global methylation changes were found, we uncovered differentially DNA methylated and expressed genes/pathways associated with the emergence of docetaxel resistance in TNBC.</p></div>

Body fluid identification at crime scenes is the first step in the forensic biology workflow, leading to the identification of the perpetrator and/or, in some cases, the victim. Current methods that are regularly used in forensic criminal evidence analysis utilize well-studied properties of each fluid as the foundation of the protocol. Among these approaches, alternative light sources, chemical reactions, lateral flow immunochromatographic tests, and microscopic detection stand out to identify the main body fluids encountered at crime scenes: blood, semen, and saliva. However, these often come with limits for specificity and sensitivity. There is also difficulty with fluid mixtures, environmental degradation, and destruction of the sample by the method used. Other fluids, like vaginal fluid and fecal matter, lack standardized protocols and require innovative ideas for accurate analysis without compromising the sample. Emerging technologies based on molecular methods have been the focus of body fluid research, with emphasis on topics such as mRNA, microRNA, epigenetics, and microbial analysis. Additional information alongside the determination of fluid origin could be an advantage from new molecular techniques, such as the identification of donors from SNP analysis, if regular STR analysis is not possible. Validation studies and the integration of such research have the potential to expand and enhance the laboratory practices of forensic science. This article will provide an overview of the current methods applied in the crime lab for body fluid identification before exploring active research in this field, pointing out the potential of these techniques for application in forensic cases to overcome present issues and expand the variety of body fluids identified.

The nucleosome remodeling and deacetylation (NuRD) complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered a key player in the formation of a functional NuRD complex. The recruitment of MBD3 to methylated promoters may require other prerequisite factors. We show that cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), an essential gene for early embryonic development, plays a role in pluripotency of ESC by engaging MBD3 to the promoter region of Wnt signaling genes. The occupancy of MBD3 on several promoters of Wnt genes was significantly lost in the absence of CDK2AP1, resulting in hyperactivation of Wnt. We propose that the transcriptional modulation of the Wnt pathway mediated by NuRD requires the presence of essential auxiliary components such as CDK2AP1, which may aid the association of the complex with specific focal regions of the target promoters.

Forensic sciences are a multidisciplinary science that covers all branches of science for justice. Forensic genetics is one of the sub-branches of forensic sciences. It is a branch of science that contributes to establishing a connection between crime and criminal by using all kinds of biological evidence (blood, semen, saliva, urine, vaginal fluid, hair, hair, sweat, saliva, etc.) and reaching the perpetrator with the genetic information obtained. Serological and immunological tests are used in the identification of body fluids found as evidence at the crime scene. The number of serological tests used in the past and today is limited; not all body fluids can be analyzed routinely. Many of the existing tests are not directly specific to the body fluid but are intended to identify intermediate metabolites. One such example is the urine sample. In the identification of urine, the presence of creatinine and amines in the urine is often used. In addition, these tests require the expenditure of separate consumables to identify each body fluid. For this reason, with the development of technology, in recent years, the use of spectroscopic methods that provide cost-free and easy analysis in the identification of body fluids has increased. Fourier Transform Infrared (FTIR) spectroscopy is one of the spectroscopic methods used to identify body fluids from biological samples found at the crime scene without loss of quantity and without damaging the sample, quickly and without loss of consumables. This study investigated the identification of urine stains obtained from the crime scene with FTIR spectroscopy without using serological materials. Functional groups, which we can express as identification regions, were determined as 13 different distinguishing points in the relevant molecules and their usability in forensic serological analyses was determined.

In forensic sciences, the serological methods used to determine which body fluid was collected from the crime scene are merely presumptive or labor intensive since they rely on protein detection or on microscopic identification of cells. Given that certain forensic cases may need the precise identification of a body fluid to determine criminal contact, such is the example of a suspected sexual assault of a minor; certainty in the body fluid of origin may depict a precise picture of the events. The identification of loci that show differences in methylation according to the tissue of origin can aid forensic analysts in determining the origin of a DNA sample. The process of DNA methylation occurs naturally in the genome of living organisms and consists in the presence of a methyl group on the carbon 5 of a cytosine, which is typically followed by a guanine (CpG). Analyzing patterns of DNA methylation in body fluids collected from a crime scene is preferential to the analysis of proteins or mRNA since the same extracted DNA used for STR typing can be used for DNA methylation analysis. We have validated and identified loci able to discriminate blood, saliva, semen and vaginal epithelia. In the current study, we have also established the minimum amount of DNA able to provide reliable results using methodologies such as pyrosequencing and high-resolution melt (HRM) analysis for the different markers identified. Lastly, we performed an alternative bioinformatic analysis of data collected using an array that studied methylation in over 450,000 individual cytosines on the human genome. We were able to sort the locations that showed potentially higher methylation differences between body fluids and investigated over 100 of them using HRM analysis. The results of that study, allowed the identification of three new loci able to distinguish blood and two new loci able to distinguish saliva and vaginal epithelia, respectively. The use of DNA methylation patterns to aid forensic investigations started with a publication in 2010, therefore each small contribution such as this work may, similarly to what occured in the biochemistry field, result in the discovery of a method able to put the technology in the hands of forensic analysts.

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain