Metabolic pathway prediction of core microbiome based on enterotype and orotype.

Introduction: Identification of key microbiome components has been suggested to help address the maintenance of oral and intestinal health in humans. The core microbiome is similar in all individuals, whereas the diverse microbiome varies across individuals, based on their unique lifestyles and phenotypic and genotypic determinants. In this study, we aimed to predict the metabolism of core microorganisms in the gut and oral environment based on enterotyping and orotyping. Materials and methods: Gut and oral samples were collected from 83 Korean women aged 50 years or older. The extracted DNA was subjected to next-generation sequencing analysis of 16S rRNA hypervariable regions V3-V4. Results: Gut bacteria were clustered into three enterotypes, while oral bacteria were clustered into three orotypes. Sixty-three of the core microbiome between the gut and oral population were correlated, and different metabolic pathways were predicted for each type. Eubacterium_g11, Actinomyces, Atopobium, and Enterococcus were significantly positively correlated between the gut and oral abundance. The four bacteria were classified as type 3 in orotype and type 2 in enterotype. Conclusion: Overall, the study suggested that collapsing the human body's multidimensional microbiome into a few categories may help characterize the microbiomes better and address health issues more deeply.

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.

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.

Microbial analyses of blood spot surfaces collected from a laboratory and the bathroom of a female single-person household under different environmental conditions.

Many people spend most of their time indoors, thereby exposing themselves to indoor environmental microbial communities that might interact with the human microbiota. These potential interactions have only been considered for personal identification; however, accumulating evidence indicates that these microbial interactions are potentially implicated with the identification of human interactions and location-specific factors including time and seasonal variations in the microbial community. To augment the potential of metagenomics-based forensic tools, we compared the composition of microbial communities in blood spot surfaces from healthy adults placed in different environments, such as in the bathroom of a female single-person household and on a laboratory, which were sampled across seasons and time points. The laboratory samples showed more changes in the bacterial community over time owing to the higher number of individuals using the laboratory, whereas the microbial communities in the bathroom samples remained relatively stable over time. Moreover, the two locations could be distinguished according to their specific bacterial community compositions. Variations were also observed related to changes in temperature and humidity, allowing for prediction of season-based microbial community. These findings offer a new perspective regarding the use of microbial community analysis in forensic science.

Individual Identification with Short Tandem Repeat Analysis and Collection of Secondary Information Using Microbiome Analysis.

Forensic investigation is important to analyze evidence and facilitate the search for key individuals, such as suspects and victims in a criminal case. The forensic use of genomic DNA has increased with the development of DNA sequencing technology, thereby enabling additional analysis during criminal investigations when additional legal evidence is required. In this study, we used next-generation sequencing to facilitate the generation of complementary data in order to analyze human evidence obtained through short tandem repeat (STR) analysis. We examined the applicability and potential of analyzing microbial genome communities. Microbiological supplementation information was confirmed for two of four failed STR samples. Additionally, the accuracy of the gargle sample was confirmed to be as high as 100% and was highly likely to be classified as a body fluid sample. Our experimental method confirmed that anthropological and microbiological evidence can be obtained by performing two experiments with one extraction. We discuss the advantages and disadvantages of using these techniques, explore prospects in the forensic field, and highlight suggestions for future research.

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.

Comparison of Swabbing Solution Volume and gDNA Extraction Kits on DNA Recovery from Rigid Surface.

For bacteria sampling studies, various collection methods have been used to identify bacteria. To obtain accurate information about bacteria, high quality samples should be obtained. In order to obtain a high quality sample, a stable and large number of DNA copies must be collected. This study compared the efficiency of different methods of bacterial gDNA extraction and bacteria collection according to swabbing solution volumes and types. The efficiency of bacterial genomic DNA extraction was compared using a AccuPrep® Genomic DNA Extraction kit, a QIAamp® DNA Mini kit, and a MOBIO® DNeasy PowerSoil kit. The DNA Mini kit was shown to extract the highest amount of gDNA, and sub-experiments were conducted using this kit. Phosphate-buffered saline and phosphate-buffered saline with 0.1% Tween 20 were used as collection solutions of various volumes (0, 40, 50, 60, 70, 80, 90, 100, 110, and 120 µL) using cotton swabs. Bacteria collection efficiency was highest when 70 µL PBS was used. The target strains collected in this experiment were Staphylococcus aureus and Escherichia coli, and these were quantified using the number of colony-forming units, DNA concentrations, and the number of DNA copies. These results can be used to efficiently bacterial collection for experiments in various fields.

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.

Internal standard metabolites for obtaining absolute quantitative information on the components of bloodstains by standardization of samples.

Analysis of the components of bloodstains found at crime scenes can provide important information for solving the crime. However, components of blood and bloodstains vary with volume and various other unpredictable factors. Therefore, it is necessary to specify the volume of the initial liquid blood droplet and standardize the analysis. In this study, internal standard metabolites that remained constant in a certain amount of bloodstain, long after deposition of the stain, were identified. Liquid chromatography-electrospray ionization-tandem mass spectrometry of the metabolites extracted from the bloodstain samples at various time points (0, 7, 14, 21, and 28 days) was performed. The coefficient of variation (CV) of the obtained molecular features was calculated for each criterion: time point, subject, and all data (time and subject, triplicate of each). Five molecular features with average CVs of less than or equal to 5% were selected as candidates. Partial least squares discriminant analysis and principal component analysis showed that the effect on the candidates was very low over time. The fold-change value of abundances was confirmed according to time. Stigmasterol exhibited the most stable pattern; l-methionine remained stable until day 14 and after day 21. This study was the first attempt to identify internal standard metabolites that were maintained at a constant level in a bloodstain for a sufficiently long time. Analysis of internal standard metabolites in bloodstains will facilitate determination of the initial blood volume from which the bloodstain was made. Moreover, this method will provide an approach for standardization of bloodstains to obtain absolute quantitative information of bloodstain components at crime scenes.

Estimation of Age of Bloodstains by Mass-Spectrometry: A Metabolomic Approach.

Bloodstains are common evidence in crime scenes, containing significant information, including genetic information. Although efforts have been made to reliably determine the time of incident by analyzing the elapsed time of the bloodstain, there has been limited success. To identify candidate metabolites in bloodstains over time, we prepared bloodstain samples using filter paper and analyzed the metabolites by high-performance liquid chromatography-mass spectrometry (HPLC-MS)/MS over a 21-day period. Using Venn diagrams and by multivariate analysis, we selected 62 candidate molecular features. We found by partial least-squares discriminant analysis (PLS-DA) that the group can be classified with an accuracy of 75.0%, and the R2 and Q2 values were 0.7513 and 0.6998, respectively. Five metabolites were successfully identified based on candidate molecular features. The level of two metabolites, l-tryptophan and ergothioneine, decreased with time. The concentration of candidate metabolites that we propose reliably increased or decreased with time, thus, enabling the measurement of elapsed time of the bloodstain. This study is the first to identify markers used to analyze the elapsed time of bloodstains through metabolomics analysis.

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.

Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction.

BACKGROUND: Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR). METHODS: The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI) genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing. RESULTS: Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously. CONCLUSION: This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection.

Melatonin modulates adiponectin expression on murine colitis with sleep deprivation.

AIM: To determine adiponectin expression in colonic tissue of murine colitis and systemic cytokine expression after melatonin treatments and sleep deprivation. METHODS: The following five groups of C57BL/6 mice were used in this study: (1) group I, control; (2) group II, 2% DSS induced colitis for 7 d; (3) group III, 2% DSS induced colitis and melatonin treatment; (4) group IV, 2% DSS induced colitis with sleep deprivation (SD) using specially designed and modified multiple platform water baths; and (5) group V, 2% DSS induced colitis with SD and melatonin treatment. Melatonin (10 mg/kg) or saline was intraperitoneally injected daily to mice for 4 d. The body weight was monitored daily. The degree of colitis was evaluated histologically after sacrificing the mice. Immunohistochemical staining and Western blot analysis was performed using anti-adiponectin antibody. After sampling by intracardiac punctures, levels of serum cytokines were measured by ELISA. RESULTS: Sleep deprivation in water bath exacerbated DSS induced colitis and worsened weight loss. Melatonin injection not only alleviated the severity of mucosal injury, but also helped survival during stressful condition. The expression level of adiponectin in mucosa was decreased in colitis, with the lowest level observed in colitis combined with sleep deprivation. Melatonin injection significantly (P < 0.05) recovered the expression of adiponectin. The expression levels of IL-6 and IL-17 were increased in the serum of mice with DSS colitis but decreased after melatonin injection. CONCLUSION: This study suggested that melatonin modulated adiponectin expression in colonic tissue and melatonin and adiponectin synergistically potentiated anti-inflammatory effects on colitis with sleep deprivation.

Low ambient oxygen prevents atherosclerosis.

Large population studies have shown that living at higher altitudes, which lowers ambient oxygen exposure, is associated with reduced cardiovascular disease mortality. However, hypoxia has also been reported to promote atherosclerosis by worsening lipid metabolism and inflammation. We sought to address these disparate reports by reducing the ambient oxygen exposure of ApoE-/- mice. We observed that long-term adaptation to 10% O2 (equivalent to oxygen content at ∼5000 m), compared to 21% O2 (room air at sea level), resulted in a marked decrease in aortic atherosclerosis in ApoE-/- mice. This effect was associated with increased expression of the anti-inflammatory cytokine interleukin-10 (IL-10), known to be anti-atherogenic and regulated by hypoxia-inducible transcription factor-1α (HIF-1α). Supporting these observations, ApoE-/- mice that were deficient in IL-10 (IL10-/- ApoE-/- double knockout) failed to show reduced atherosclerosis in 10% oxygen. Our study reveals a specific mechanism that can help explain the decreased prevalence of ischemic heart disease in populations living at high altitudes and identifies ambient oxygen exposure as a potential factor that could be modulated to alter pathogenesis. Key messages: Chronic low ambient oxygen exposure decreases atherosclerosis in mice. Anti-inflammatory cytokine IL-10 levels are increased by low ambient O2. This is consistent with the established role of HIF-1α in IL10 transactivation. Absence of IL-10 results in the loss of the anti-atherosclerosis effect of low O2. This mechanism may contribute to decreased atherosclerosis at high altitudes.

Synergistic effect of interleukin-6 and hyaluronic acid on cell migration and ERK activation in human keratinocytes.

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-κB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-κB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.

Increased expression of interleukin-1ß in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1ß (IL-1ß) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1ß, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1ß, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1ß expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1ß expression by TG-treated macrophages may play a role during atherogenesis.

Suppressing effect of resveratrol on the migration and invasion of human metastatic lung and cervical cancer cells.

The antioxidant 3,4',5 tri-hydroxystilbene (resveratrol), a phytoalexin found in grapes, shows cancer preventive activities, including inhibition of migration and invasion of metastatic tumors. However, the molecular mechanism underlying the effect of resveratrol on tumor metastasis, especially in human metastatic lung and cervical cancers is not clear. A non-cytotoxic dosage of resveratrol causes a reduction in the generation of reactive oxygen species, and suppresses phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration in both A549 and HeLa cells. Resveratrol also decreases both the expression and the enzymatic activity of matrix metalloproteinase-9 (MMP-9), and the promoter activity of PMA-stimulated MMP-9 is also inhibited. However, resveratrol does not affect either the expression or the proteolytic activity of MMP-2. Our results also show that resveratrol suppresses the transcription of MMP-9 by the inhibition of both NF-κB and AP-1 transactivation. These results indicate that resveratrol inhibits both NF-κB and AP-1 mediated MMP-9 expression, leading to suppression of migration and invasion of human metastatic lung and cervical cancer cells. Resveratrol has potential for clinical use in preventing invasion by human metastatic lung and cervical cancers.

N-acetyl cysteine suppresses the foam cell formation that is induced by oxidized low density lipoprotein via regulation of gene expression.

Foam cells derived from macrophages have been implicated as markers of early stage atherosclerosis development. In this study, we found that N-acetyl cysteine (NAC), a well-known inhibitor of reactive oxygen species (ROS), decreased the generation of ROS and suppressed foam cell formation in the presence of oxidized low density lipoprotein through down-regulation of cluster of differentiation 36 expression. We investigated gene expression profiles in order to determine the effects of NAC on foam cell formation using a microarray analysis. The level of apolipoprotein E, which is involved in lipid efflux, was increased and the levels of the antioxidant genes glutathione peroxidase 1 and 3 were also increased. The expression levels of the oxidative stress response and the DNA repair genes were decreased. These results were confirmed using quantitative real-time PCR. Our results indicate that oxidative stress plays an important role in foam cell formation, and that regulation of oxidation using antioxidants is a potential therapeutic method for blocking atherosclerosis development.

Inhibitory effect of Trolox on the migration and invasion of human lung and cervical cancer cells.

The antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) is implicated in migration and invasion of metastatic tumors. However, the molecular mechanism underlying the effect of Trolox on metastatic cancer cells is not known. We found that a non-cytotoxic dose of Trolox decreased phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration of both A549 and HeLa cancer cells. We also found that Trolox suppressed both the expression and the proteolytic activity of matrix metalloproteinase-9 (MMP-9), and that the promoter activity of PMA-induced MMP-9 was inhibited by Trolox. Our results show that Trolox inhibits the transcriptional activity of MMP-9 by suppression of NF-κB transactivation. These results indicate that Trolox inhibits NF-κB-mediated MMP-9 expression, leading to the suppression of migration and invasion in lung and cervical cancer cells. Trolox is a potential agent for clinical use in preventing the invasion and metastasis of human malignant lung and cervical cancers.