Discovery of donor age markers from bloodstain by LC-MS/MS using a metabolic approach.

Bloodstains are frequently encountered at crime scenes and they provide important evidence about the incident, such as information about the victim or suspect and the time of death or other events. Efforts have been made to identify the age of the bloodstain's donor through genomic approaches, but there are some limitations, such as the availability of databases and the quality dependence of DNA. There is a need for the development of a tool that can obtain information at once from a small blood sample. The aim of this study is to identify bloodstain metabolite candidates that can be used to determine donor age. We prepared bloodstain samples and analyzed metabolites using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eighteen molecular features (MFs) were selected as candidates using volcano plots and multivariate analysis. Based on the MS/MS spectrum of the MFs, the following nine metabolites were identified from the METaboliteLINk database: Δ2-cis eicosenoic acid, ergothioneine, adenosine 5'-monophosphate, benzaldehyde, phenacylamine, myristic acid ethyl ester, p-coumaric acid, niacinamide, and N-arachidonoyl-L-alanine. These nine age markers at high or low abundances could be used to estimate the age of a bloodstain's donor. This study was the first to develop metabolite age markers that can be used to analyze crime scene bloodstains.

Enhanced detection of cell-free DNA (cfDNA) enables its use as a reliable biomarker for diagnosis and prognosis of gastric cancer.

Although circulating cell-free DNA (cfDNA) is a promising biomarker for the diagnosis and prognosis of various tumors, clinical correlation of cfDNA with gastric cancer has not been fully understood. To address this, we developed a highly sensitive cfDNA capture system by integrating polydopamine (PDA) and silica. PDA-silica hybrids incorporated different molecular interactions to a single system, enhancing cfDNA capture by 1.34-fold compared to the conventional silica-based approach (p = 0.001), which was confirmed using cell culture supernatants. A clinical study using human plasma samples revealed that the diagnostic accuracy of the new system to be superior than the commercially available cfDNA kit, as well as other serum antigen tests. Among the cancer patients, plasma cfDNA levels exhibited a good correlation with the size of a tumor. cfDNA was also predicative of distant metastasis, as the median cfDNA levels of metastatic cancer patients were ~60-fold higher than those without metastasis (p = 0.008). Furthermore, high concordance between tissue biopsy and cfDNA genomic analysis was found, as HER2 expression in cfDNA demonstrated an area under ROC curve (AUC) of 0.976 (p <0.001) for detecting patients with HER2-positive tumors. The new system also revealed high prognostic capability of cfDNA, as the concentration of cfDNA was highly associated with the survival outcomes. Our novel technology demonstrates the potential to achieve efficient detection of cfDNA that may serve as a reliable biomarker for gastric tumor.

OmpA of Klebsiella pneumoniae ATCC 13883 induces pyroptosis in HEp-2 cells, leading to cell-cycle arrest and apoptosis.

Klebsiella pneumoniae is an opportunistic pathogenic bacterium that commonly causes pneumonia in elderly people. OmpA, a toxin that is highly expressed in the outer membrane of the bacterium, is one of the primary factors implicated in the pulmonary pathogenesis of K. pneumoniae. To evaluate the associated pyroptosis mechanism of infection, the ompA gene was cloned, and the protein was expressed, extracted, and used to treat human larynx epithelial cells. We observed that OmpA induces reactive oxygen species production and cell-cycle arrest in the G2/M phase in host cells, leading to subsequent apoptosis. Moreover, OmpA was found to induce IL-1ß and IL-18 production in host cells, resulting in caspase-1 activation, which simultaneously stimulated pyroptosis, thus leading to the death of the host cells. We next sought to examine differential gene expression via RNA sequencing to better elucidate the mechanisms associated with these cellular changes, and found that genes associated with these pathways were more highly expressed in OmpA-treated cells than in K. pneumoniae-infected cells. Thus, cell-cycle arrest, apoptosis, and pyroptosis may serve as the primary defenses employed by host cells against OmpA. These results provide novel insights into the host defense against K. pneumoniae infection.

Sub-lethal hyperthermia promotes epithelial-to-mesenchymal-like transition of breast cancer cells: implication of the synergy between hyperthermia and chemotherapy.

Thermotherapy has demonstrated a potential to be an effective non-surgical technique to treat breast cancer. Despite its advantages, including low toxicity and high repeatability, thermotherapy is typically required to be applied in combination with other treatments since the residual tumor cells that survive after hyperthermal treatment often cause recurrence. In this study, we confirmed that breast cancer cells tolerate temperature of up to 47 °C by synthesizing a large amount of heat shock proteins. Further changes in the molecular properties of the heat-exposed cells were investigated using western blotting, quantitative reverse transcription polymerase chain reaction, and immunocytochemistry. We found that low-temperature hyperthermia promoted epithelial-to-mesenchymal-like transition (EMT), as observed by the increased mesenchymal marker expression levels while decreasing epithelial markers. Moreover, cell morphology changed from cobblestone-like to a more spindle-like appearance, in addition to significantly enhanced cell motility upon heat treatment. These results all support that sub-lethal hyperthermal stress induces EMT. In addition, we examined changes in the chemo-sensitivity of the heat-treated cells. Addition of a chemo-drugs caused increased cytotoxicity of the heat-treated cells compared to the cells that were not co-treated with heat. Our study demonstrates that thermotherapy alone may cause undesirable EMT, which could be well overcome through a synergistic effect when applied with chemotherapy.

Global gene expression patterns and induction of innate immune response in human laryngeal epithelial cells in response to Acinetobacter baumannii outer membrane protein A.

The outer membrane protein A of Acinetobacter baumannii (AbOmpA) is an important pathogen-associated molecular pattern that induces host cell death. We determined the gene expression profiles of human laryngeal epithelial HEp-2 cells in response to the sublethal concentration of recombinant AbOmpA (rAbOmpA) and investigated the molecular mechanisms by which rAbOmpA induces an innate immune response. The microarray analysis showed that rAbOmpA sequentially regulated a relatively small set of genes, including those associated with signal transductions and molecules involved in immune response. Among the differentially expressed genes involved in innate immune responses, the surface expression of Toll-like receptor 2 and the production of inducible nitric oxide synthase (iNOS) were prominently observed. However, rAbOmpA did not induce the production of proinflammatory cytokines and chemokines. rAbOmpA activated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs). Inhibition of JNK MAPK suppressed iNOS production in the rAbOmpA-treated HEp-2 cells. These results suggest that interaction of laryngeal epithelial cells with AbOmpA has a significant impact on the induction of innate immunity during the early stages of A. baumannii infection.

Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells.

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial infection. Despite considerable clinical and epidemiological data regarding the role of A. baumannii in nosocomial infection, the specific virulence factor or pathogenic mechanism of this organism has yet to be elucidated. This study investigated the molecular mechanism of apoptosis on the infection of human laryngeal epithelial HEp-2 cells with A. baumannii and examined the contribution of outer membrane protein 38 (Omp38) on the ability of A. baumannii to induce apoptosis of epithelial cells. A. baumannii induced apoptosis of HEp-2 cells through cell surface death receptors and mitochondrial disintegration. The Omp38-deficient mutant was not as able to induce apoptosis as the wild-type A. baumannii strain. Purified Omp38 entered the cells and was localized to the mitochondria, which led to a release of proapoptotic molecules such as cytochrome c and apoptosis-inducing factor (AIF). The activation of caspase-3, which is activated by caspase-9, degraded DNA approximately 180 bp in size, which resulted in the appearance of a characteristic DNA ladder. AIF degraded chromosomal DNA approximately 50 kb in size, which resulted in large-scale DNA fragmentation. These results demonstrate that Omp38 may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stage of A. baumannii infection.