Hemoglobin subunit beta protein as a novel marker for time since deposition of bloodstains at crime scenes.

Dried bloodstains at crime scenes provide abundant information for analyzing criminal identity of victims or suspects, morphological characteristics, and biological and chemical compounds. Therefore, they are considered important evidence by investigators at crime scenes. Moreover, the age of bloodstains can be used to determine the timeline of incidents at crime scenes; Inappropriately handled bloodstains may cause degradation of blood components. In this study, we identified a novel marker, hemoglobin subunit beta protein, as an internal standard to determine the age of bloodstains at crime scenes. We found that the target spot between 20 and 30 kDa in two-dimensional electrophoresis gradually increased in size. The hemoglobin subunit beta protein was identified from this spot using liquid chromatography-tandem mass spectrometry and verified using western blotting. Sample bloodstains were exposed to various environmental conditions (humidity: 30%, 60%, 90% at room temperature [RT]). Furthermore, the hemoglobin subunit protein extracted from the sample bloodstains at various time points (0 h to 30 d) was dissolved in our newly developed buffer solution and in deionized or distilled water. We also analyzed the expression levels of the protein in the sample bloodstains, dried at RT and under various humidity over time, using western blotting. In addition, we evaluated the protein extraction capacity of deionized or distilled water and the newly developed buffer from the sample bloodstains over time. At RT and 60% humidity, using the newly developed buffer, the hemoglobin subunit beta protein levels showed a gradually increasing pattern. Finally, we quantitated human hemoglobin subunit beta protein using western blotting and enzyme-linked immunosorbent assay, which revealed significant differences among the samples. In particular, the time points from 36 h to 30 days were considered for analysis. Thus, the hemoglobin subunit beta protein dried at RT and 60% humidity and further dissolved in the newly developed buffer solution can be used to determine the age of bloodstains at crime scenes.

Bacteroides fragilis Toxin Induces Intestinal Epithelial Cell Secretion of Interleukin-8 by the E-Cadherin/ß-Catenin/NF-κB Dependent Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) has emerged as a gut microbiome pathogen that can promote colitis associated cancer in humans. ETBF secretes the metalloprotease, B. fragilis toxin (BFT), which can induce ectodomain cleavage of E-cadherin and IL-8 secretion through the ß-catenin, NF-κB, and MAPK pathways in intestinal epithelial cells. However, it is still unclear whether E-cadherin cleavage is required for BFT induced IL-8 secretion and the relative contribution of these signaling pathways to IL-8 secretion. Using siRNA knockdown and CRISPR knockout studies, we found that E-cadherin cleavage is required for BFT mediated IL-8 secretion. In addition, genetic ablation of ß-catenin indicates that ß-catenin is required for the BFT induced increase in transcriptional activity of NF-κB, p65 nuclear localization and early IL-8 secretion. These results suggest that BFT induced ß-catenin signaling is upstream of NF-κB activation. However, despite ß-catenin gene disruption, BFT still activated the MAPK pathway, suggesting that the BFT induced activation of the MAPK signaling pathway is independent from the E-cadherin/ß-catenin/NF-κB pathway. These findings show that E-cadherin and ß-catenin play a critical role in acute inflammation following ETBF infection through the inflammatory response to BFT in intestinal epithelial cells.

Development of novel extraction reagents for analyzing dried blood spots from crime scenes.

Evidence of dried blood is very valuable in forensic science. Since the discovery of luminescence with Luminol and dried blood spots (DBSs) in 1928, interest and research on blood have continued to date. One of the most important factor that DBSs have is genes. However, the current use of distilled water (DDW) to collect and extract blood samples has disadvantages related to DNA stability. Therefore, this study aimed to develop an extraction reagent that is most suitable for gene extraction from DBSs. Blood was collected from 45 healthy adult men and women in vacuum blood containers without coagulants or anticoagulants. The collected blood was dried in various settings to check the performance of the extraction reagent. Extraction with Tris-EDTA (TE) and phosphate-buffered saline (PBS) was found more suitable in terms of gene interference effects compared with DDW; their performance was also compared with those of the newly developed extraction reagents. Upon comparing the results of polymerase chain reaction for human genomic DNA samples using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as the target, the performance of the newly developed extraction reagents, modified TE and PBS, was found to be relatively good. To determine the optimal composition of the developed extraction reagents, 12 new extraction reagents were developed with different pH and sodium concentrations. Among them, the best results were found when the DNA was extracted using extraction reagent No. 3 with pH 8.0 and containing 1 M NaCl. Next, the four extraction reagents, DDW, TE, PBS, and No. 3 were compared under nine different temperature and humidity conditions. Similarly, under various environmental conditions, extraction reagent No. 3 performed better than other reagents. It is proposed that modified TE and PBS mixed extraction reagents are the most suitable for collecting and preserving crime site samples. The proposed composition for a DNA extraction reagent can contribute greatly to crime scene reconstruction.

Enterotoxigenic Bacteroides fragilis infection exacerbates tumorigenesis in AOM/DSS mouse model.

The azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model is commonly used to study colitis-associated cancer. The human commensal bacterium, enterotoxigenic Bacteroides fragilis (ETBF) secretes the Bacteroides fragilis toxin (BFT) which is necessary and sufficient to cause colitis. We report that BALB/c mice infected with WT-ETBF and administered three cycles of AOM/DSS developed numerous, large-sized polyps predominantly in the colorectal region. In addition, AOM/DSS-treated BALB/c mice orally inoculated with wild-type nontoxigenic Bacteroides fragilis (WT-NTBF) overexpressing bft (rETBF) developed numerous polyps whereas mice infected with WT-NTBF overexpressing a biologically inactive bft (rNTBF) did not promote polyp formation. Unexpectedly, the combination of AOM+ETBF did not induce polyp formation whereas ETBF+DSS did induce polyp development in a subset of BALB/c mice. In conclusion, WT-ETBF promoted polyp development in AOM/DSS murine model with increased colitis in BALB/c mice. The model described herein provides an experimental platform for understanding ETBF-induced colonic tumorigenesis and studying colorectal cancer in wild-type mice.

Microarray and proteome array in an atherosclerosis mouse model for identification of biomarkers in whole blood.

Cardiovascular disease (CVD) is highly fatal, and 80 percent of the mortality is attributed to heart attack and stroke. Atherosclerosis is a disease that increases a patient's risk to CVD and is characterized by atheroma formed by immune cells, lipids, and smooth muscle cells. When an atherosclerotic lesion grows and blocks blood vessels or when an atheroma ruptures and blocks blood vessels by embolism, sudden angina, or stroke can occur. It is therefore important to diagnose atherosclerosis early and prevent its progression to more severe disease. Although myeloperoxidase, plasma fibrinogen, cardiac troponin-I, and C-reactive protein have been considered as diagnostic markers for multiple cardiac risks, specific biomarkers for atherosclerosis have not been clearly determined yet. Particularly, reliable biomarkers for the diagnosis of atherosclerosis using whole blood are not yet available. In this study, we screened potential biomarker genes and proteins from whole blood of apolipoprotein E knockout (ApoE-/- ) mice maintained on a Western diet, by comparing them to ApoE+/+ mice. We used whole blood for microarray and proteome array. Candidate genes and proteins identified from each method were confirmed with quantitative real-time PCR and ELISA. Based on our data, we speculate that Lilrb4a, n-R5s136, and IL-5 are potential targets that can be developed into novel biomarkers of atherosclerosis. Our study contributes to the diagnosis of atherosclerosis using whole blood in clinical settings.

Adipose tissue-derived mesenchymal stem cells cultured at high density express IFN-ß and TRAIL and suppress the growth of H460 human lung cancer cells.

Although mesenchymal stem cells (MSCs) have been reported to inhibit tumor growth, the mechanism controlling this tumor suppression function is unclear. Here, we report that high-density (40,000 cells/cm2) cultured adipose tissue-derived MSCs (40K-ASCs) expressed interferon (IFN)-ß and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); we also found that serum deprivation during cell culture induced the expression of IFN-ß and TRAIL. In addition, the mRNA expression of IFN-ß, but not TRAIL, was increased during the washing step required for the transplantation of normal-density (5000 cells/cm2) cultured ASCs (5K-ASCs). When the human lung cancer cell line H460 was co-cultured with 40K-ASCs, necrotic cell death was dramatically increased in vitro. When ASCs were injected after four washes, both 5K-ASCs and 40K-ASCs substantially reduced tumor weight in H460-derived cancer animal models. These results suggest that serum deprivation during the culture of 40K-ASCs or during the washing step of 5K-ASCs can induce IFN-ß and/or TRAIL expression, ultimately leading to the tumor suppression capability of ASCs.

Gene and Protein Expression Profiles in a Mouse Model of Collagen-Induced Arthritis.

The risk of rheumatoid arthritis (RA), an autoimmune disease, in the elderly population increases along with that of atherosclerosis, cardiovascular disease, type 2 diabetes, and Alzheimer's disease. Identifying specific biomarkers for RA can clarify the underlying molecular mechanisms and can aid diagnosis and patient care. To this end, the present study investigated the genes and proteins that are differentially expressed in RA using a mouse collagen-induced arthritis (CIA) model. We performed gene microarray and proteome array analyses using blood samples from the mice and found that 50 genes and 24 proteins were upregulated and 48 genes were downregulated by more than 2-fold in the CIA model relative to the control. The gene microarray and proteome array results were validated by evaluating the expression levels of select genes and proteins by real-time PCR and western blotting, respectively. We found that the level of integrin α2, which has not been previously reported as a biomarker of RA, was significantly increased in CIA mice as compared to controls. These findings provide a set of novel biomarkers that can be useful for diagnosing and evaluating the progression of RA.

Bacteroides fragilis Toxin Induces IL-8 Secretion in HT29/C1 Cells through Disruption of E-cadherin Junctions.

Enterotoxigenic Bacteroides fragilis (ETBF) is a human gut commensal bacteria that causes inflammatory diarrhea and colitis. ETBF also promotes colorectal tumorigenesis in the Min mouse model. The key virulence factor is a secreted metalloprotease called B. fragilis toxin (BFT). BFT induces E-cadherin cleavage, cell rounding, activation of the ß-catenin pathway and secretion of IL-8 in colonic epithelial cells. However, the precise mechanism by which these processes occur and how these processes are interrelated is still unclear. E-cadherin form homophilic interactions which tethers adjacent cells. Loss of E-cadherin results in detachment of adjacent cells. Prior studies have suggested that BFT induces IL-8 expression by inducing E-cadherin cleavage; cells that do not express E-cadherin do not secrete IL-8 in response to BFT. In the current study, we found that HT29/C1cells treated with dilute trypsin solution induced E-cadherin degradation and IL-8 secretion, consistent with the hypothesis that E-cadherin cleavage causes IL-8 secretion. However, physical damage to the cell monolayer did not induce IL-8 secretion. We also show that EDTA-mediated disruption of E-cadherin interactions without E-cadherin degradation was sufficient to induce IL-8 secretion. Finally, we determined that HT29/C1 cells treated with LiCl (ß-catenin activator) induced IL-8 secretion in a dose-dependent and time-dependent manner. Taken together, our results suggest that BFT induced IL-8 secretion may occur by the following process: E-cadherin cleavage, disruption of cellular interactions, activation of the ß-catenin pathway and IL-8 expression. However, we further propose that E-cadherin cleavage per se may not be required for BFT induced IL-8 secretion.

Enterotoxigenic Bacteroides fragilis causes lethal colitis in Mongolian gerbils.

Enterotoxigenic Bacteroides fragilis (ETBF) causes inflammatory diarrhea in humans and livestock and is implicated in colorectal cancer. In this report, we show for the first time that ETBF can induce profuse diarrhea and an acute, lethal colitis in Mongolian gerbils.