Modulation of Nogo receptor 1 expression orchestrates myelin-associated infiltration of glioblastoma.

As the clinical failure of glioblastoma treatment is attributed by multiple components, including myelin-associated infiltration, assessment of the molecular mechanisms underlying such process and identification of the infiltrating cells have been the primary objectives in glioblastoma research. Here, we adopted radiogenomic analysis to screen for functionally relevant genes that orchestrate the process of glioma cell infiltration through myelin and promote glioblastoma aggressiveness. The receptor of the Nogo ligand (NgR1) was selected as the top candidate through Differentially Expressed Genes (DEG) and Gene Ontology (GO) enrichment analysis. Gain and loss of function studies on NgR1 elucidated its underlying molecular importance in suppressing myelin-associated infiltration in vitro and in vivo. The migratory ability of glioblastoma cells on myelin is reversibly modulated by NgR1 during differentiation and dedifferentiation process through deubiquitinating activity of USP1, which inhibits the degradation of ID1 to downregulate NgR1 expression. Furthermore, pimozide, a well-known antipsychotic drug, upregulates NgR1 by post-translational targeting of USP1, which sensitizes glioma stem cells to myelin inhibition and suppresses myelin-associated infiltration in vivo. In primary human glioblastoma, downregulation of NgR1 expression is associated with highly infiltrative characteristics and poor survival. Together, our findings reveal that loss of NgR1 drives myelin-associated infiltration of glioblastoma and suggest that novel therapeutic strategies aimed at reactivating expression of NgR1 will improve the clinical outcome of glioblastoma patients.

Self-Powered Chemical Sensing Driven by Graphene-Based Photovoltaic Heterojunctions with Chemically Tunable Built-In Potentials.

Ultralow power chemical sensing is essential toward realizing the Internet of Things. However, electrically driven sensors must consume power to generate an electrical readout. Here, a different class of self-powered chemical sensing platform based on unconventional photovoltaic heterojunctions consisting of a top graphene (Gr) layer in contact with underlying photoactive semiconductors including bulk silicon and layered transition metal dichalcogenides is proposed. Owing to the chemically tunable electrochemical potential of Gr, the built-in potential at the junction is effectively modulated by absorbed gas molecules in a predictable manner depending on their redox characteristics. Such ability distinctive from bulk photovoltaic counterparts enables photovoltaic-driven chemical sensing without electric power consumption. Furthermore, it is demonstrated that the hydrogen (H2 ) sensing properties are independent of the light intensity, but sensitive to the gas concentration down to the 1 ppm level at room temperature. These results present an innovative strategy to realize extremely energy-efficient sensors, providing an important advancement for future ubiquitous sensing.

Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

Correction: Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells.

[This corrects the article DOI: 10.1371/journal.pbio.1002152.].

Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells.

Epidermal growth factor receptor variant III (EGFRvIII) has been associated with glioma stemness, but the direct molecular mechanism linking the two is largely unknown. Here, we show that EGFRvIII induces the expression and secretion of pigment epithelium-derived factor (PEDF) via activation of signal transducer and activator of transcription 3 (STAT3), thereby promoting self-renewal and tumor progression of glioma stem cells (GSCs). Mechanistically, PEDF sustained GSC self-renewal by Notch1 cleavage, and the generated intracellular domain of Notch1 (NICD) induced the expression of Sox2 through interaction with its promoter region. Furthermore, a subpopulation with high levels of PEDF was capable of infiltration along corpus callosum. Inhibition of PEDF diminished GSC self-renewal and increased survival of orthotopic tumor-bearing mice. Together, these data indicate the novel role of PEDF as a key regulator of GSC and suggest clinical implications.

miR-181b-3p promotes epithelial-mesenchymal transition in breast cancer cells through Snail stabilization by directly targeting YWHAG.

Epithelial-mesenchymal transition (EMT) is essential for increased invasion and metastasis during cancer progression. Among the candidate EMT-regulating microRNAs that we previously identified, miR-181b-3p was found to induce EMT in MCF7 breast cancer cells, as indicated by an EMT-characteristic morphological change, increased invasiveness, and altered expression of an EMT marker. Transfection with a miR-181b-3p inhibitor reduced the expression of mesenchymal markers and the migration and invasion of highly invasive breast cancer cells. miR-181b-3p induced the upregulation of Snail, a master EMT inducer and transcriptional repressor of E-cadherin, through protein stabilization. YWHAG was identified as a direct target of miR-181b-3p, downregulation of which induced Snail stabilization and EMT phenotypes. Ectopic expression of YWHAG abrogated the effect of miR-181b-3p, including Snail stabilization and the promotion of invasion. In situ hybridization and immunohistochemical analyses indicated that YWHAG expression was inversely correlated with the expression of miR-181b-3p and Snail in human breast cancer tissues. Furthermore, transfection with miR-181b-3p increased the frequency of metastatic nodule formation in the lungs of mice in experimental metastasis assays using MDA-MB-231 cells. Taken together, our data suggest that miR-181b-3p functions as a metastasis activator by promoting Snail-induced EMT, and may therefore be a therapeutic target in metastatic cancers.

Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

MicroRNAs (miRNAs) play pivotal roles in tumorigenesis as either tumor suppressors or oncogenes. In the present study, we discovered and demonstrated the tumor suppressive function of a novel miRNA miR-5582-5p. miR-5582-5p induced apoptosis and cell cycle arrest in cancer cells, but not in normal cells. GAB1, SHC1, and CDK2 were identified as direct targets of miR-5582-5p. Knockdown of GAB1/SHC1 or CDK2 phenocopied the apoptotic or cell cycle arrest-inducing function of miR-5582-5p, respectively. The expression of miR-5582-5p was lower in tumor tissues than in adjacent normal tissues of colorectal cancer patients, while the expression of the target proteins exhibited patterns opposite to that of miR-5582-5p. Intratumoral injection of a miR-5582-5p mimic or induced expression of miR-5582-5p in tumor cells suppressed tumor growth in HCT116 xenografts. Collectively, our results suggest a novel tumor suppressive function for miR-5582-5p and its potential applicability for tumor control.

Continuous Films of Self-Assembled Graphene Quantum Dots for n-Type Doping of Graphene by UV-Triggered Charge Transfer.

The demands to examine components serving as one of the active layers in heterostructures of 2D materials have been recently increasing. Nanomaterials synthesized from a solution process and their self-assembly can provide a promising route to build a new type of mixed dimensional heterostructures, and several methodologies have been reported previously to construct 2D assemblies from colloidal nanostructures in solution. Graphene quantum dots (GQDs), receiving much interest due to the tunable optical band gap and the capability of chemical functionalization, are considered as emerging nanomaterials for various optoelectronic and biological applications. This study fabricates a closely packed GQDs film (GQDF) from colloidal solutions using a solvent-assisted Langmuir Blodgett method, and investigates the optical and electrical characteristics of the heterostacked graphene/GQD film (G/GQDF) structures. It is observed that the GQDF plays a role not only as a buffer layer that isolates Chemical Vapor Deposited graphene (CVD graphene) from undesired p-doping but also as a photoactive layer that triggers n-doping of the heterostacked CVD graphene film. The n-doping density of the G/GQDF device is proportional to UV irradiation time, but its carrier mobility remains constant regardless of doping densities, which are unique characteristics that have not been observed in other doping methods.

Silencing of ST6Gal I enhances colorectal cancer metastasis by down-regulating KAI1 via exosome-mediated exportation and thereby rescues integrin signaling.

Aberrant sialylation has long been correlated with human cancer. Increased ST6 Gal I (ß-galactoside α 2, 6 sialyltransferase) and consequently higher levels of cell-surface α 2, 6 sialylation has been associated with human colorectal cancer (CRC) metastasis. We have extensive circumstantial data that sialylation is connected to cancer metastasis, but we do not understand in detail how sialylation can switch on/off multiple steps in cancer metastasis. To investigate the molecular mechanism underlying the ST6Gal I-mediated metastasis of CRC, we silenced the ST6Gal I gene in a metastatic SW620 CRC cell line (SW620-shST6Gal I) and examined the metastatic behavior of the cells. We found that various hallmarks of metastatic ability were considerably enhanced in ST6Gal 1-depleted SW620 clones, as assessed both in vitro and in vivo . In particular, the metastasis suppressor, KAI1, was down-regulated in ST6Gal I-deficient SW620 clones. This reflected the increased exosome-mediated exportation of KAI1, and was associated with a decrease in the KAI1-mediated inhibition of integrin. These findings indicate that gene silencing of ST6Gal I could enhance metastasis of CRC by down-regulating KAI1 activity and rescuing its negative effects on integrin signaling.

Ocular findings in patients with spastic type cerebral palsy.

BACKGROUND: Refractive errors, strabismus, nystagmus, amblyopia, and cortical visual impairment are observed in 50 to 90 % of patients with cerebral palsy. Ocular abnormalities are known to differ according to cerebral palsy type, and spastic type has been reported to be more likely to be associated with ocular defects than the athetoid and ataxic types. METHODS: A retrospective review of medical records was performed on 105 consecutive children with spastic type of cerebral palsy who underwent ophthalmologic examination between July 2003 and March 2006. The complete ophthalmological examination included measurement of visual acuity, ocular motility, stereoacuity, binocular vision, cycloplegic refraction along with the evaluation of the anterior segment and the posterior segment. RESULTS: The most common ocular abnormality was strabismus (70.5 %) followed by refractive errors (53.3 %). Exodeviation was more commonly found than esodeviation (46 vs 27 patients), and hyperopia was much more prevalent than myopia. A considerable number of patients with strabismus had abnormal ocular motility wherein 16 patients showed inferior oblique overaction and ten superior oblique overaction. Whereas inferior oblique overaction was accompanied similarly in exotropia and esotropia, superior oblique overaction was accompanied more by exotropia. CONCLUSIONS: Children with spastic type cerebral palsy have a high prevalence of strabismus and refractive errors. Exotropia and hyperopia are the most common ocular abnormalities. All children with spastic type of cerebral palsy may require a detailed ophthalmologic evaluation.

Aspirin Targets SIRT1 and AMPK to Induce Senescence of Colorectal Carcinoma Cells.

Cancer therapies attempt to destroy the entire tumor, but this tends to require toxic compounds and high doses of radiation. Recently, considerable attention has focused on therapy-induced senescence (TIS), which can be induced in cancer cells by low doses of therapeutic drugs or radiation and provides a barrier to tumor development. However, the molecular mechanisms governing TIS remain elusive. Special attention has been paid to the potential chemopreventive effect of aspirin against human colorectal cancer. In this study, we investigated the effects of aspirin on TIS of human colorectal carcinoma (CRC) cells and show that it occurs via sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK), two key regulators of cellular metabolism. Aspirin increased the senescence of CRC cells, increased the protein levels of SIRT1, phospho-AMPK (T172), and phospho-acetyl CoA carboxylase (S79), and reduced the cellular level of ATP. Small-interfering RNA-mediated downregulation or pharmacological inhibition of SIRT1 or AMPK significantly attenuated the aspirin-induced cellular senescence in CRC cells. In contrast, treatment with a SIRT1 agonist or an AMP analog induced cellular senescence. Remarkably, SIRT1 knockdown abrogated the aspirin-induced activation of AMPK, and vice versa. During the progression of aspirin-induced cellular senescence in CRC cells, SIRT1 showed increased deacetylase activity at a relatively early time point but was characterized by decreased activity with increased cytoplasmic localization at a later time point. Collectively, these novel findings suggest that aspirin could provide anticancer effects by inducing senescence in human CRC cells through the reciprocal regulation of SIRT1-AMPK pathways.

All-Solution-Processed High-Performance MoS2 Thin-Film Transistors with a Quasi-2D Perovskite Oxide Dielectric.

Assembling solution-processed van der Waals (vdW) materials into thin films holds great promise for constructing large-scale, high-performance thin-film electronics, especially at low temperatures. While transition metal dichalcogenide thin films assembled in solution have shown potential as channel materials, fully solution-processed vdW electronics have not been achieved due to the absence of suitable dielectric materials and high-temperature processing. In this work, we report on all-solution-processedvdW thin-film transistors (TFTs) comprising molybdenum disulfides (MoS2) as the channel and Dion-Jacobson-phase perovskite oxides as the high-permittivity dielectric. The constituent layers are prepared as colloidal solutions through electrochemical exfoliation of bulk crystals, followed by sequential assembly into a semiconductor/dielectric heterostructure for TFT construction. Notably, all fabrication processes are carried out at temperatures below 250 °C. The fabricated MoS2 TFTs exhibit excellent device characteristics, including high mobility (>10 cm2 V-1 s-1) and an on/off ratio exceeding 106. Additionally, the use of a high-k dielectric allows for operation at low voltage (∼5 V) and leakage current (∼10-11 A), enabling low power consumption. Our demonstration of the low-temperature fabrication of high-performance TFTs presents a cost-effective and scalable approach for heterointegrated thin-film electronics.

PTTG1 oncogene promotes tumor malignancy via epithelial to mesenchymal transition and expansion of cancer stem cell population.

The prognosis of breast cancer patients is related to the degree of metastasis. However, the mechanisms by which epithelial tumor cells escape from the primary tumor and colonize at a distant site are not entirely understood. Here, we analyzed expression levels of pituitary tumor-transforming gene-1 (PTTG1), a relatively uncharacterized oncoprotein, in patient-derived breast cancer tissues with corresponding normal breast tissues. We found that PTTG1 is highly expressed in breast cancer patients, compared with normal tissues. Also, PTTG1 expression levels were correlated with the degree of malignancy in breast cancer cell lines; the more migratory and invasive cancer cell lines MDA-MB-231 and BT549 displayed the higher expression levels of PTTG1 than the less migratory and invasive MCF7 and SK-BR3 and normal MCF10A cell lines. By modulating PTTG1 expression levels, we found that PTTG1 enhances the migratory and invasive properties of breast cancer cells by inducing epithelial to mesenchymal transition, as evidenced by altered morphology and epithelial/mesenchymal cell marker expression patterns and up-regulation of the transcription factor Snail. Notably, down-regulation of PTTG1 also suppressed cancer stem cell population in BT549 cells by decreasing self-renewing ability and tumorigenic capacity, accompanying decreasing CD44(high) CD24(low) cells and Sox2 expression. Up-regulation of PTTG1 had the opposite effects, increasing sphere-forming ability and Sox2 expression. Importantly, PTTG1-mediated malignant tumor properties were due, at least in part, to activation of AKT, known to be a key regulator of both EMT and stemness in cancer cells. Collectively, these results suggest that PTTG1 may represent a new therapeutic target for malignant breast cancer.

Importance of PKCδ signaling in fractionated-radiation-induced expansion of glioma-initiating cells and resistance to cancer treatment.

Brain tumors frequently recur or progress as focal masses after treatment with ionizing radiation. However, the mechanisms underlying the repopulation of tumor cells after radiation have remained unclear. In this study, we show that cellular signaling from Abelson murine leukemia viral oncogene homolog (Abl) to protein kinase Cδ (PKCδ) is crucial for fractionated-radiation-induced expansion of glioma-initiating cell populations and acquisition of resistance to anticancer treatments. Treatment of human glioma cells with fractionated radiation increased Abl and PKCδ activity, expanded the CD133-positive (CD133(+)) cell population that possesses tumor-initiating potential and induced expression of glioma stem cell markers and self-renewal-related proteins. Moreover, cells treated with fractionated radiation were resistant to anticancer treatments. Small interfering RNA (siRNA)-mediated knockdown of PKCδ expression blocked fractionated-radiation-induced CD133(+) cell expansion and suppressed expression of glioma stem cell markers and self-renewal-related proteins. It also suppressed resistance of glioma cells to anticancer treatments. Similarly, knockdown of Abl led to a decrease in CD133(+) cell populations and restored chemotherapeutic sensitivity. It also attenuated fractionated-radiation-induced PKCδ activation, suggesting that Abl acts upstream of PKCδ. Collectively, these data indicate that fractionated radiation induces an increase in the glioma-initiating cell population, decreases cellular sensitivity to cancer treatment and implicates activation of Abl-PKCδ signaling in both events. These findings provide insights that might prove pivotal in the context of ionising-radiation-based therapeutic interventions for brain tumors.

Tissue factor is a critical regulator of radiation therapy-induced glioblastoma remodeling.

Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated ß-galactosidase (SA-ßGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-ßGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse in vivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.

Dopamine receptor D2 regulates glioblastoma survival and death through MET and death receptor 4/5.

Recent studies indicate that signaling molecules traditionally associated with central nervous system function play critical roles in cancer. Dopamine receptor signaling is implicated in various cancers including glioblastoma (GBM) and it is a recognized therapeutic target, as evidenced by recent clinical trials with a selective dopamine receptor D2 (DRD2) inhibitor ONC201. Understanding the molecular mechanism(s) of the dopamine receptor signaling will be critical for development of potent therapeutic options. Using the human GBM patient-derived tumors treated with dopamine receptor agonists and antagonists, we identified the proteins that interact with DRD2. DRD2 signaling promotes glioblastoma (GBM) stem-like cells and GBM growth by activating MET. In contrast, pharmacological inhibition of DRD2 induces DRD2-TRAIL receptor interaction and subsequent cell death. Thus, our findings demonstrate a molecular circuitry of oncogenic DRD2 signaling in which MET and TRAIL receptors, critical factors for tumor cell survival and cell death, respectively, govern GBM survival and death. Finally, tumor-derived dopamine and expression of dopamine biosynthesis enzymes in a subset of GBM may guide patient stratification for DRD2 targeting therapy.

Secretome analysis of ionizing radiation-induced senescent cancer cells reveals that secreted RKIP plays a critical role in neighboring cell migration.

Cellular senescence is a physiological program of irreversible growth arrest that is considered to play an important role in tumor suppression. Recent studies demonstrated that senescent cells secrete multiple growth regulatory proteins that could alter the behavior of neighboring cells. In this study, we investigated the effect of secretory proteins from ionizing radiation (IR) induced senescent tumor cells on normal and tumor cells. Conditioned medium (CM) from IR-induced senescent MCF7 cells significantly increased cell proliferation, invasion, migration, and wound healing activity in MCF7 cells and HUVECs. Comparative proteomics analysis revealed 24 differentially secreted protein spots including Raf kinase inhibitor protein (RKIP), α-Enolase, AKAP9, and MARK4, and the findings were confirmed by Western blot analysis of IR-induced senescent cancer cells. We found that RKIP was secreted via the classical pathway, and the transfection of small interfering RNA against RKIP suppressed CM-induced migration in MCF7 cells. Treatment with recombinant human RKIP increased the migratory activity of MCF7 cells. Taken together, our results demonstrate that the senescence-associated secretory protein RKIP could be the principal target to prevent the potential effects of the secretome from IR-induced senescent tumor cells on neighboring cell migration.

Understanding the Y chromosome variation in Korea--relevance of combined haplogroup and haplotype analyses.

We performed a molecular characterization of Korean Y-chromosomal haplogroups using a combination of Y-chromosomal single nucleotide polymorphisms (Y-SNPs) and Y-chromosomal short tandem repeats (Y-STRs). In a test using DNA samples from 706 Korean males, a total of 19 different haplogroups were identified by 26 Y-SNPs including the newly redefined markers (PK4, KL2, and P164) in haplogroup O. When genotyping the SNPs, phylogenetic nonequivalence was found between SNPs M117 and M133, which define haplogroup O3a3c1 (O3a2c1a according to the updated tree of haplogroup O by Yan et al. (European Journal of Human Genetics 19:1013-1015, 2011)), suggesting that the position of the M133 marker should be corrected. We have shown that the haplotypes consisted of DYS392, DYS393, DYS437, DYS438, DYS448, and DYS388 loci, which exhibit a relatively lower mutation rate, can preserve phylogenetic information and hence can be used to roughly distinguish Y-chromosome haplogroups, whereas more rapidly mutating Y-STRs such as DYS449 and DYS458 are useful for differentiating male lineages. However, at the relatively rapidly mutating DYS447, DYS449, DYS458, and DYS464 loci, unusually short alleles and intermediate alleles with common sequence structures are informative for elucidating the substructure within the context of a particular haplogroup. In addition, some deletion mutations in the DYS385 flanking region and the null allele at DYS448 were associated with a single haplogroup background. These high-resolution haplogroup and haplotype data will improve our understanding of regional Y-chromosome variation or recent migration routes and will also help to infer haplogroup background or common ancestry.

Potential forensic application of DNA methylation profiling to body fluid identification.

DNA analysis of various body fluid stains at crime scenes facilitates the identification of individuals but does not currently determine the type and origin of the biological material. Recent advances in whole genome epigenetic analysis indicate that chromosome pieces called tDMRs (tissue-specific differentially methylated regions) show different DNA methylation profiles according to the type of cell or tissue. We examined the potential of tissue-specific differential DNA methylation for body fluid identification. Five tDMRs for the genes DACT1, USP49, HOXA4, PFN3, and PRMT2 were selected, and DNA methylation profiles for these tDMRs were produced by bisulfite sequencing using pooled DNA from blood, saliva, semen, menstrual blood, and vaginal fluid. The tDMRs for DACT1 and USP49 showed semen-specific hypomethylation, and the tDMRs for HOXA4, PFN3, and PRMT2 displayed varying degrees of methylation according to the type of body fluid. Preliminary tests using methylation-specific PCR for the DACT1 and USP49 tDMRs showed that these two markers could be used successfully to identify semen samples including sperm cells. Body fluid-specific differential DNA methylation may be a promising indicator for body fluid identification. Because DNA methylation profiling uses the same biological source of DNA for individual identification profiling, the determination of more body fluid-specific tDMRs and the development of convenient tDMR analysis methods will facilitate the broad implementation of body fluid identification in forensic casework.