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.

Identification and Validation of SAA4 as a Rheumatoid Arthritis Prescreening Marker by Liquid Chromatography Tandem-mass Spectrometry.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that progresses into systemic inflammation and joint deformity. RA diagnosis is a complicated procedure, and early diagnostic methods are insufficient. Therefore, in this study, we attempted to identify new markers to improve the accuracy of RA prescreening. e identified differentially expressed proteins (DEPs) by using liquid chromatography tandem-mass spectrometry in health-prescreening sera with high rheumatoid factor (RF) values, and compared the findings with those from sera with normal RF values. We identified 93 DEPs; of these, 36 were upregulated, and 57 were downregulated in high-RF sera. Pathway analysis revealed that these DEPs were related to immune responses. Additionally, four DEPs were statistically analyzed by proteomic analysis; of these, SAA4 was significantly validated in individual enzyme-linked immunosorbent assays. Moreover, SAA4 was significantly upregulated in RA patients (n = 40, 66.43 ± 12.97 ng/mL) compared with normal controls (n = 40, 4.79 ± 0.95 ng/mL) and had a higher area under the curve than C-reactive protein. Thus, we identified SAA4 as a protein that was positively correlated with RF and RA. SAA4 may represent a novel prescreening marker for the diagnosis of RA.

Proteomics Reveals Plasma Biomarkers for Ischemic Stroke Related to the Coagulation Cascade.

Stroke has a high incidence rate and often leads to permanent disability, particularly if it is not treated promptly. However, no blood biomarkers for early diagnosis are available to date. Therefore, we sought to detect stroke-specific blood biomarkers by identifying proteins associated with the underlying coagulation mechanism, which accounts for more than 80% of all stroke cases. Protein profiling was performed using blood samples from 16 healthy controls and 18 patients who suffered a stroke as the discovery set. We identified upregulated proteins (> 1.5-fold change and p value < 0.05) in patients who suffered a stroke relative to the corresponding levels in healthy controls by nano-liquid chromatography-tandem mass spectrometry using data-independent acquisition based on sequential window acquisition of all theoretical mass spectra, which was developed to improve the consistency and accuracy of candidate proteins. Pathway analysis confirmed that the upregulated proteins were mainly involved in blood coagulation. Among these, we selected prothrombin, plasminogen, fibrinogen alpha-chain, and histidine-rich glycoprotein as candidate biomarkers. Multiple reaction monitoring analysis was performed on a validation set of 61 serum samples (31 healthy controls and 30 stroke patients) to assess the diagnostic value of the candidate biomarkers. All four proteins showed higher expression levels in patients with stroke than in healthy controls. The areas under the receiver operating characteristic curve were greater than 0.9, confirming their clinical value. These four blood coagulation proteins may help in diagnosing stroke more accurately and quickly.

Influence of Tacrolimus on Depressive-Like Behavior in Diabetic Rats Through Brain-Derived Neurotrophic Factor Regulation in the Hippocampus.

The neurotoxicity of immunosuppressive agents and diabetes mellitus are known risk factors of neurological complications in kidney transplant recipients. The aim of the present study was to investigate the influence of tacrolimus on brain-derived neurotrophic factor (BDNF), the critical protein for maintenance of neuronal functions, in the hippocampus in a diabetic condition. A diabetic rat model was established by a single streptozotocin injection (60 mg/kg). Control and diabetic rats then received daily tacrolimus (1.5 mg/kg per day) injections for 6 weeks. BDNF expression in the hippocampus was examined in the dentate gyrus (DG) and CA3 region using immunohistochemistry. There was a significant decrease of BDNF expression in the DG and CA3 region in tacrolimus-treated and diabetic rats compared with that of the control group injected with vehicle only. However, there was no difference in BDNF expression between the two experimental groups. Tacrolimus treatment in diabetic rats further decreased the BDNF expression level in the DG and CA3 region. Interestingly, mossy fiber sprouting, demonstrated by prominent punctate immunolabeling of BDNF with synaptoporin, was observed in the diabetic group treated with tacrolimus, which localized at the stratum oriens of the CA3 region. These data suggest that tacrolimus treatment or a diabetic condition decreases BDNF expression in the hippocampus, and that tacrolimus treatment in the diabetic condition further injures the CA3 region of the hippocampus. In addition to BDNF expression, decreased locomotor activity and evident depressive behavior were observed in tacrolimus-treated diabetic rats. Moreover, there were significant decreases of the mRNA levels of γ-aminobutyric acid and serotonin receptors in the diabetic hippocampus with tacrolimus treatment. This finding suggests that tacrolimus treatment may cause further psychiatric and neurological complications for patients with diabetes, and should thus be used with caution.

Proteomics analysis reveals differential pattern of widespread protein expression and novel role of histidine-rich glycoprotein and lipopolysaccharide-binding protein in rheumatoid arthritis.

Rheumatoid factor (RF) is an auto-antibody against antigen-antibody immune complexes. RF is valuable as a biomarker for the screening of autoimmune and infectious diseases. However, it is suggested that RF would be a more powerful biomarker when used complementarily with RF-correlated proteins. In this study, we utilized a proteomic approach to analyze global protein expression in RF-low and RF-high subjects using high-performance liquid chromatography tandem mass spectrometry. Histidine-rich glycoprotein (HRG) and lipopolysaccharide-binding protein (LBP) were found to be differentially expressed between RF-low and RF-high subjects (cut-off > 2-fold, p < 0.05), which was validated by enzyme-linked immunosorbent assay. To evaluate whether both proteins allow discriminating rheumatoid arthritis patients from healthy controls, receiver-operating characteristic (ROC) curves were analyzed. Areas under the ROC curves of HRG and LBP were 0.861 and 0.888, respectively. The correlation between RF and HRG was statistically significant (p = 0.003), and LBP was also correlated with RF (p = 0.044), as indicated by correlation analysis. HRG and LBP are reportedly involved in RF-producing and RF-correlated diseases. Thus, we propose that HRG and LBP could be useful screening markers for RF-correlated diseases.