SPERM HY-LITER™ for the identification of spermatozoa from sexual assault evidence.

Accurate microscopic identification of human spermatozoa is important in sexual assault cases. We have compared the results of examinations with (1) a fluorescent microscopy method, SPERM HY-LITER™, and (2) Baecchi's method for identification of human spermatozoa. In 35 artificial, forensic type samples, spermatozoa were identified in 45.7% with SPERM HY-LITER™ in Copenhagen, in 54.3% in the laboratory of the manufacturer of SPERM HY-LITER™, and 40.0% of the samples with Baecchi's staining method. When differences occurred between the two methods, it was significantly more often that SPERM HY-LITER™ detected spermatozoa when Baecchi's method did not (ts=6.567, df=1, P=0.048). This trend was also seen in selected compromised or degraded samples and in selected adjudicative samples. The reactions with spermatozoa from dog, horse, pig and bull were negative with SPERM HY-LITER™, whereas Baecchi's method was non-selective. Data from forensic casework samples in Copenhagen from two years (2008 and 2009) are presented. The samples from 2008 were investigated using Baecchi's method, while those from 2009 were investigated using SPERM HY-LITER™. The frequencies of positive results were similar between the two methods for the two years (27.9% and 32.1% respectively). Analysis of acid phosphatase (ACP) activity for the positive results obtained for these two years does not support the use of a negative ACP result as a prescreen for microscopic analysis for spermatozoa.

Developmental validation of RSID™-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen.

Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate-specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID™-Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID™-Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross-react with other human or nonhuman tissues tested. RSID™-Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA-based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID™-Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.

Developmental validation of the SPERM HY-LITER™ kit for the identification of human spermatozoa in forensic samples.

With sexual assault evidence, the visualization of spermatozoa confirms that ejaculation has occurred. However, microscopic examination of spermatozoa is a laborious process and can sometimes result in sperm cells being overlooked. Here, we present the developmental validation of the SPERM HY-LITER™ kit, which contains a human sperm-specific mouse monoclonal antibody coupled to a fluorescent Alexa 488 dye. The kit was tested using samples of human semen, saliva, blood, and urine, various animal semen extracts, sexual lubricants, and a commercially available spermicidal film. Postcoital vaginal swabs, degraded semen samples, and samples prepared with sample fixation techniques that deviated from the kit-provided protocol were also tested. In each case, the SPERM HY-LITER™ kit was demonstrated to bind only to human sperm cell heads. Limitations to this fluorescent staining procedure include nonspecific staining and increased background fluorescence with extreme heat fixation in some samples.

Skp2 directs Myc-mediated suppression of p27Kip1 yet has modest effects on Myc-driven lymphomagenesis.

The universal cyclin-dependent kinase inhibitor p27(Kip1) functions as a tumor suppressor, and reduced levels of p27(Kip1) connote poor prognosis in several human malignancies. p27(Kip1) levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCF(Skp2) complex following its phosphorylation by the cyclin E-cyclin-dependent kinase 2 complex. Binding of phospho-p27(Kip1) is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Emu-Myc transgenic mice triggers p27(Kip1) destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Emu-Myc lymphomas and of human Burkitt lymphoma that bear MYC/Immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27(Kip1) was abolished in Skp2-null Emu-Myc B cells. However, the effect of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared with the effects of Cks1 loss. Collectively, these findings suggest that Cks1 targets, in addition to p27(Kip1), are critical for Myc-driven proliferation and tumorigenesis.

Developmental validation of RSID-saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva.

Current methods for forensic identification of saliva generally assay for the enzymatic activity of alpha-amylase, an enzyme long associated with human saliva. Here, we describe the Rapid Stain IDentification (RSID-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID-Saliva detects less than 1 microL of saliva. The sensitivity of RSID-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.

Developmental validation of a novel lateral flow strip test for rapid identification of human blood (Rapid Stain Identification--Blood).

Human blood is the body fluid most commonly encountered at crime scenes, and blood detection may aid investigators in reconstructing what occurred during a crime. In addition, blood detection can help determine which items of evidence should be processed for DNA-STR testing. Unfortunately, many common substances can cause red-brown stains that resemble blood. Furthermore, many current human blood detection methods are presumptive and prone to false positive results. Here, the developmental validation of a new blood identification test, Rapid Stain Identification--Blood (RSID--Blood), is described. RSID--Blood utilizes two anti-glycophorin A (red blood cell membrane specific protein) monoclonal antibodies in a lateral flow strip test format to detect human blood. We present evidence demonstrating that this test is accurate, reproducible, easy to use, and highly specific for human blood. Importantly, RSID--Blood does not cross-react with ferret, skunk, or primate blood and exhibits no high-dose hook effect. Also, we describe studies on the sensitivity, body fluid specificity, and species specificity of RSID--Blood. In addition, we show that the test can detect blood from a variety of forensic exhibits prior to processing for DNA-STR analysis. In conclusion, we suggest that RSID--Blood is effective and useful for the detection of human blood on forensic exhibits, and offers improved blood detection when compared to other currently used methods.

Myc targets Cks1 to provoke the suppression of p27Kip1, proliferation and lymphomagenesis.

Reduced levels of the cyclin-dependent kinase inhibitor p27(Kip1) connote poor prognosis in cancer. In human Burkitt lymphoma and in precancerous B cells and lymphomas arising in Emu-Myc transgenic mice, p27(Kip1) expression is markedly reduced. We show that the transcription of the Cks1 component of the SCF(Skp2) complex that is necessary for p27(Kip1) ubiquitylation and degradation is induced by Myc. Further, Cks1 expression is elevated in precancerous Emu-Myc B cells, and high levels of Cks1 are also a hallmark of Emu-Myc lymphoma and of human Burkitt lymphoma. Finally, loss of Cks1 in Emu-Myc B cells elevates p27(Kip1) levels, reduces proliferation and markedly delays lymphoma development and dissemination of disease. Therefore, Myc suppresses p27(Kip1) expression, accelerates cell proliferation and promotes tumorigenesis at least in part through its ability to selectively induce Cks1.

Nfkb 1 is dispensable for Myc-induced lymphomagenesis.

Rel/NF-kappaB transcription factors are critical arbiters of immune responses, cell survival, and transformation, and are frequently deregulated in cancer. The p50 NF-kappaB 1 component of Rel/NF-kappaB DNA-binding dimers regulates genes involved in both cell cycle traverse and apoptosis. Nfkb 1 loss accelerates B cell growth and leads to increased B cell turnover in vivo, phenotypes akin to those manifested in B cells of Emu-Myc transgenic mice, a model of human Burkitt lymphoma. Interestingly, Emu-Myc B cells express reduced levels of cytoplasmic and nuclear NF-kappaB 1 and have reduced Rel/NF-kappaB DNA-binding activity, suggesting that Myc-mediated repression of NF-kappaB 1 might mediate its proliferative and apoptotic effects on B cells. Furthermore, Nfkb 1 expression was reduced in the majority of Emu-Myc lymphomas and was also suppressed in human Burkitt lymphoma. Nonetheless, loss of Nfkb 1 did not appreciably affect Myc's proliferative or apoptotic responses in B cells and had no effect on lymphoma development in Emu-Myc mice. Therefore, Nfkb 1 is dispensable for Myc-induced lymphomagenesis..

Targeting ornithine decarboxylase in Myc-induced lymphomagenesis prevents tumor formation.

Checkpoints that control Myc-mediated proliferation and apoptosis are bypassed during tumorigenesis. Genes encoding polyamine biosynthetic enzymes are overexpressed in B cells from E mu-Myc transgenic mice. Here, we report that disabling one of these Myc targets, Ornithine decarboxylase (Odc), abolishes Myc-induced suppression of the Cdk inhibitors p21(Cip1) and p27(Kip1), thereby impairing Myc's proliferative, but not apoptotic, response. Moreover, lymphoma development was markedly delayed in E mu-Myc;Odc(+/-) transgenic mice and in E mu-Myc mice treated with the Odc inhibitor difluoromethylornithine (DFMO). Strikingly, tumors ultimately arising in E mu-Myc;Odc(+/-) transgenics lacked deletions of Arf, suggesting that targeting Odc forces other routes of transformation. Therefore, Odc is a critical Myc transcription target that regulates checkpoints that guard against tumorigenesis and is an effective target for cancer chemoprevention.