Analysis of biological mixtures is a significant problem for forensic laboratories, particularly ... more Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.
Raman spectroscopy for forensic body fluid analysis has received some attention due to the nondes... more Raman spectroscopy for forensic body fluid analysis has received some attention due to the nondestructive nature and potential application for identification at the crime scene; however, its usage has been limited by low detection sensitivity. Surface enhanced Raman scattering (SERS) was evaluated for blood identification for forensic applications. Specifically, a SERS-active substrate was fabricated, composed of nickel nanotips coated with Ag nanoparticles. Compared with a conventional substrate, the SERS substrate enhanced Raman scattering by more than two orders of magnitude and allowed blood to be identified to a dilution of 1:100,000. Blood was also successfully detected by swabbing the SERS substrate directly on mock evidence. Most importantly, Raman spectra obtained by swabbing the SERS substrate on blood stains were free of luminescence even when blood was deposited on luminescent fabrics. The nondestructive character, simplicity of sample preparation, and high sensitivity make SERS a prime candidate for field and laboratory-based blood identification.
Background: Despite the lack of agreement on their exact roles, it is known that miRNAs contribut... more Background: Despite the lack of agreement on their exact roles, it is known that miRNAs contribute to cancer progression. Many studies utilize methods to detect differential regulation of miRNA expression. It is prohibitively expensive to examine all potentially dysregulated miRNAs and traditionally, researchers have focused their efforts on the most extremely dysregulated miRNAs. These methods may overlook the contribution of less differentially expressed but more functionally relevant miRNAs. The purpose of this study was to outline a method that not only utilizes differential expression but ranks miRNAs based on the functional relevance of their targets. This work uses a networks based approach to determine the sum node degree for all experimentally verified miRNA targets to identify potential regulators of prostate cancer initiation, progression and metastasis. Results: Here, we present a method for identifying functionally relevant miRNAs that contribute to prostate cancer development. This paper shows that miRNAs preferentially regulate highly connected, central proteins within a protein-protein interaction network. Known targets of miRNAs differentially regulated during prostate cancer progression are enriched in pathways with known involvement in tumorigenesis. To demonstrate the applicability of our method, we utilized a unique model of prostate cancer progression to identify five miRNAs that may contribute to the oncogenic state of the cell. Three of these miRNAs have been shown by other studies to have a role in cancer but their exact role in prostate cancer remains undefined. Conclusion: Developing methods to determine which miRNAs to carry forward into biological and biochemical analyses is important as traditional approaches often overlook miRNAs that contribute to oncogenesis. Our method applied to a model of prostate cancer progression was able to identify miRNAs with roles in prostate cancer development.
Luminol, Bluestar ® , and Hemascein ® were tested to compare detection sensitivities to latent bl... more Luminol, Bluestar ® , and Hemascein ® were tested to compare detection sensitivities to latent blood. Untreated, EDTA-treated human blood, and a catalytically similar blood substitute were diluted (neat to 1:1,000,000) and pipetted onto a variety of substrates. Luminol and Bluestar ® performed similarly on all surfaces and fabrics. Hemascein ® yielded poor results on wood surfaces, but performed well in the detection of latent blood on fabrics. Results from untreated, EDTA-treated, and synthetic blood results indicate that EDTA-treated blood is similar or slightly less sensitive than untreated blood at all dilutions and on all substrates, and the synthetic blood is less sensitive than real blood, but consistent in detection threshold and thus is useful as a training aid. Additionally, some foods and household chemicals that have previously been shown to cross-react were tested with Bluestar ® , Hemascein ® , and luminol. Hemascein ® cross-reacted with many substances, while both luminol reagents were more discriminating.
We investigated Raman scattering from human blood as a function of parameters that are relevant f... more We investigated Raman scattering from human blood as a function of parameters that are relevant for forensic field analysis, such as substrate, sample dilution, individual from which the sample originates, and age of the sample. Peaks characteristic of blood components and in particular the hemoglobin peaks were routinely detected when blood was deposited on substrates that are not strongly luminescent, such as plastic, metal utensils and dry wall. Raman scattering from blood proved quite sensitive and blood samples with a dilution up to 1:250 could be measured for an excitation power of ∼2 mW measured at the sample plane. The sensitivity of Raman scattering to diluted blood allowed measurement using blood reconstituted from fabric substrates, thereby alleviating issues related to luminescence and scattering from the substrate. The dependence of Raman scattering on sample age and individual was also investigated. We found that the relative intensities of scattering peaks depended on sample age and history. For example, the relative intensity of oxyhemoglobin peaks decreases after blood has dried. Fresh blood drawn directly from a donor without intermediate storage exhibits also scattering peaks at 1155 and 1511 cm(-1) which disappear after drying. The origin of these peaks is under investigation. We noticed, however, that they were not found in blood that had been stored for longer than one week in EDTA containers before analysis, thus requiring the use of fresh blood for future studies and validation purposes. The relative intensity of scattering peaks was also found to be somewhat dependent on the donor and, for a same donor, on the day on which blood was drawn.
We hypothesize that black women experience accelerated biological aging in response to repeated o... more We hypothesize that black women experience accelerated biological aging in response to repeated or prolonged adaptation to subjective and objective stressors. Drawing on stress physiology and ethnographic, social science, and public health literature, we lay out the rationale for this hypothesis. We also perform a first population-based test of its plausibility, focusing on telomere length, a biomeasure of aging that may be shortened by stressors. Analyzing data from the Study of Women's Health Across the Nation (SWAN), we estimate that at ages 49-55, black women are 7.5 years biologically "older" than white women. Indicators of perceived stress and poverty account for 27% of this difference. Data limitations preclude assessing objective stressors and also result in imprecise estimates, limiting our ability to draw firm inferences. Further investigation of black-white differences in telomere length using large-population-based samples of broad age range and with detailed measures of environmental stressors is merited.
Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordin... more Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordinate regulation of CTP:phosphocholine cytidylyltransferase-a (CTa) and the group VIA calcium-independent phospholipase A 2 (iPLA 2 ). CTa expression is modulated by sterol and this is mediated in part through sterol regulatory element binding proteins (SREBP). In this report, we investigate the possibility that iPLA 2 expression is controlled in a similar manner. When Chinese hamster ovary (CHO) cells were cultured under sterol-depleted conditions, iPLA 2 catalytic activity, mRNA, and protein were induced by between two-and threefold. These inductions were suppressed when the cells were supplemented with exogenous sterols. Luciferase reporter assays indicated that sterol depletion induced transcription of iPLA 2 , an analysis of the 5Vflanking region suggested that the iPLA 2 gene contained a putative sterol regulatory element (SRE), and electrophoretic mobility shift assay (EMSA) analysis indicated that this element can bind SREBP-2. Notably, a mutant CHO cell line (SRD4) that constitutively generates mature SREBP proteins exhibited increased iPLA 2 activity and expression compared to wild-type cells. These data suggest that iPLA 2 expression is regulated in a manner consistent with other important genes in sterol and glycerophospholipid metabolism. Such coordinate regulation may be essential for maintaining the lipid composition of cell membranes. D
Analysis of biological mixtures is a significant problem for forensic laboratories, particularly ... more Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.
Raman spectroscopy for forensic body fluid analysis has received some attention due to the nondes... more Raman spectroscopy for forensic body fluid analysis has received some attention due to the nondestructive nature and potential application for identification at the crime scene; however, its usage has been limited by low detection sensitivity. Surface enhanced Raman scattering (SERS) was evaluated for blood identification for forensic applications. Specifically, a SERS-active substrate was fabricated, composed of nickel nanotips coated with Ag nanoparticles. Compared with a conventional substrate, the SERS substrate enhanced Raman scattering by more than two orders of magnitude and allowed blood to be identified to a dilution of 1:100,000. Blood was also successfully detected by swabbing the SERS substrate directly on mock evidence. Most importantly, Raman spectra obtained by swabbing the SERS substrate on blood stains were free of luminescence even when blood was deposited on luminescent fabrics. The nondestructive character, simplicity of sample preparation, and high sensitivity make SERS a prime candidate for field and laboratory-based blood identification.
Background: Despite the lack of agreement on their exact roles, it is known that miRNAs contribut... more Background: Despite the lack of agreement on their exact roles, it is known that miRNAs contribute to cancer progression. Many studies utilize methods to detect differential regulation of miRNA expression. It is prohibitively expensive to examine all potentially dysregulated miRNAs and traditionally, researchers have focused their efforts on the most extremely dysregulated miRNAs. These methods may overlook the contribution of less differentially expressed but more functionally relevant miRNAs. The purpose of this study was to outline a method that not only utilizes differential expression but ranks miRNAs based on the functional relevance of their targets. This work uses a networks based approach to determine the sum node degree for all experimentally verified miRNA targets to identify potential regulators of prostate cancer initiation, progression and metastasis. Results: Here, we present a method for identifying functionally relevant miRNAs that contribute to prostate cancer development. This paper shows that miRNAs preferentially regulate highly connected, central proteins within a protein-protein interaction network. Known targets of miRNAs differentially regulated during prostate cancer progression are enriched in pathways with known involvement in tumorigenesis. To demonstrate the applicability of our method, we utilized a unique model of prostate cancer progression to identify five miRNAs that may contribute to the oncogenic state of the cell. Three of these miRNAs have been shown by other studies to have a role in cancer but their exact role in prostate cancer remains undefined. Conclusion: Developing methods to determine which miRNAs to carry forward into biological and biochemical analyses is important as traditional approaches often overlook miRNAs that contribute to oncogenesis. Our method applied to a model of prostate cancer progression was able to identify miRNAs with roles in prostate cancer development.
Luminol, Bluestar ® , and Hemascein ® were tested to compare detection sensitivities to latent bl... more Luminol, Bluestar ® , and Hemascein ® were tested to compare detection sensitivities to latent blood. Untreated, EDTA-treated human blood, and a catalytically similar blood substitute were diluted (neat to 1:1,000,000) and pipetted onto a variety of substrates. Luminol and Bluestar ® performed similarly on all surfaces and fabrics. Hemascein ® yielded poor results on wood surfaces, but performed well in the detection of latent blood on fabrics. Results from untreated, EDTA-treated, and synthetic blood results indicate that EDTA-treated blood is similar or slightly less sensitive than untreated blood at all dilutions and on all substrates, and the synthetic blood is less sensitive than real blood, but consistent in detection threshold and thus is useful as a training aid. Additionally, some foods and household chemicals that have previously been shown to cross-react were tested with Bluestar ® , Hemascein ® , and luminol. Hemascein ® cross-reacted with many substances, while both luminol reagents were more discriminating.
We investigated Raman scattering from human blood as a function of parameters that are relevant f... more We investigated Raman scattering from human blood as a function of parameters that are relevant for forensic field analysis, such as substrate, sample dilution, individual from which the sample originates, and age of the sample. Peaks characteristic of blood components and in particular the hemoglobin peaks were routinely detected when blood was deposited on substrates that are not strongly luminescent, such as plastic, metal utensils and dry wall. Raman scattering from blood proved quite sensitive and blood samples with a dilution up to 1:250 could be measured for an excitation power of ∼2 mW measured at the sample plane. The sensitivity of Raman scattering to diluted blood allowed measurement using blood reconstituted from fabric substrates, thereby alleviating issues related to luminescence and scattering from the substrate. The dependence of Raman scattering on sample age and individual was also investigated. We found that the relative intensities of scattering peaks depended on sample age and history. For example, the relative intensity of oxyhemoglobin peaks decreases after blood has dried. Fresh blood drawn directly from a donor without intermediate storage exhibits also scattering peaks at 1155 and 1511 cm(-1) which disappear after drying. The origin of these peaks is under investigation. We noticed, however, that they were not found in blood that had been stored for longer than one week in EDTA containers before analysis, thus requiring the use of fresh blood for future studies and validation purposes. The relative intensity of scattering peaks was also found to be somewhat dependent on the donor and, for a same donor, on the day on which blood was drawn.
We hypothesize that black women experience accelerated biological aging in response to repeated o... more We hypothesize that black women experience accelerated biological aging in response to repeated or prolonged adaptation to subjective and objective stressors. Drawing on stress physiology and ethnographic, social science, and public health literature, we lay out the rationale for this hypothesis. We also perform a first population-based test of its plausibility, focusing on telomere length, a biomeasure of aging that may be shortened by stressors. Analyzing data from the Study of Women's Health Across the Nation (SWAN), we estimate that at ages 49-55, black women are 7.5 years biologically "older" than white women. Indicators of perceived stress and poverty account for 27% of this difference. Data limitations preclude assessing objective stressors and also result in imprecise estimates, limiting our ability to draw firm inferences. Further investigation of black-white differences in telomere length using large-population-based samples of broad age range and with detailed measures of environmental stressors is merited.
Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordin... more Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordinate regulation of CTP:phosphocholine cytidylyltransferase-a (CTa) and the group VIA calcium-independent phospholipase A 2 (iPLA 2 ). CTa expression is modulated by sterol and this is mediated in part through sterol regulatory element binding proteins (SREBP). In this report, we investigate the possibility that iPLA 2 expression is controlled in a similar manner. When Chinese hamster ovary (CHO) cells were cultured under sterol-depleted conditions, iPLA 2 catalytic activity, mRNA, and protein were induced by between two-and threefold. These inductions were suppressed when the cells were supplemented with exogenous sterols. Luciferase reporter assays indicated that sterol depletion induced transcription of iPLA 2 , an analysis of the 5Vflanking region suggested that the iPLA 2 gene contained a putative sterol regulatory element (SRE), and electrophoretic mobility shift assay (EMSA) analysis indicated that this element can bind SREBP-2. Notably, a mutant CHO cell line (SRD4) that constitutively generates mature SREBP proteins exhibited increased iPLA 2 activity and expression compared to wild-type cells. These data suggest that iPLA 2 expression is regulated in a manner consistent with other important genes in sterol and glycerophospholipid metabolism. Such coordinate regulation may be essential for maintaining the lipid composition of cell membranes. D
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Papers by Sarah Seashols