Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site.

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

Molecular mechanisms underlying the vulnerability of Pacific abalone (Haliotis discus hannai) to Vibrio harveyi infection at higher water temperature.

Climate change is one of the most important threats to farmed abalone worldwide. Although abalone is more susceptible to vibriosis at higher water temperatures, the molecular mode of action underlying this has not been fully elucidated. Therefore, this study aimed to address the high susceptibility of Halitotis discus hannai to V. harveyi infection using abalone hemocytes exposed to low and high temperatures. Abalone hemocytes were divided into four groups, 20C, 20 V, 25C, and 25 V, depending on co-culture with (V)/without (C) V. harveyi (MOI = 12.8) and incubation temperature (20 °C or 25 °C). After 3 h of incubation, hemocyte viability and phagocytic activity were measured, and RNA sequencing was performed using Illumina Novaseq. The expression of several virulence-related genes in V. harveyi was analyzed using real-time PCR. The viability of hemocytes was significantly decreased in the 25 V group compared to cells in the other groups, whereas phagocytic activity at 25 °C was significantly higher than at 20 °C. Although a number of immune-associated genes were commonly upregulated in abalone hemocyte exposed to V. harveyi, regardless of temperature, pathways and genes regarding pro-inflammatory responses (interleukin-17 and tumor necrosis factor) and apoptosis were significantly overexpressed in the 25 V group compared to the 25C group. Notably, in the apoptosis pathway, genes encoding executor caspases (casp3 and casp7) and pro-apoptotic factor, bax were significantly up-regulated only in the 25 V group, while the apoptosis inhibitor, bcl2L1 was significantly up-regulated only in the 20 V group compared to the control group at the respective temperatures. The co-culture of V. harveyi with abalone hemocytes at 25 °C up-regulated several virulence-related genes involved in quorum sensing (luxS), antioxidant activity (katA, katB, and sodC), motility (flgI), and adherence/invasion (ompU) compared to those at 20 °C. Therefore, our results showed that H. discus hannai hemocytes exposed to V. harveyi at 25 °C were highly stressed by vigorously activated inflammatory responses and that the bacterial pathogen overexpressed several virulence-related genes at the high temperature tested. The transcriptomic profile of both abalone hemocytes and V. harveyi in the present study provide insight into differential host-pathogen interactions depending on the temperature conditions and the molecular backgrounds related to increased abalone vulnerability upon global warming.

Impact of intratumoral heterogeneity on the metabolic profiling of breast cancer tissue using high-resolution magic angle spinning magnetic resonance spectroscopy.

High-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) is a useful metabolic profiling technique for human tissue. However, the impact of intratumoral heterogeneity on the metabolite levels of breast cancers is not yet established. The purpose of this prospective study was to investigate whether the tumor cell fraction of core needle biopsy (CNB) specimens of breast cancers affect metabolic profiles assessed with HR-MAS MRS. From June 2015 to December 2016, 46 patients with 47 breast cancers were enrolled. HR-MAS MRS was used for the metabolic profiling of 285 CNB specimens from the 47 cancers. Multiple CNB samples (range 2-8) for the HR-MAS MRS experiment were obtained from surgical specimens under ultrasound guidance following surgical removal of the tumor. Tumor cell fraction was expressed as a percentage of the tumor cell volume relative to the total tumor volume contained in each CNB sample. Metabolite quantification levels were compared according to primary tumor characteristics using the t-test. Multivariate analyses were performed including primary tumor characteristics and tumor cell percentages as variables. Correlations between tumor cell percentage and metabolite levels in the CNB specimens were assessed according to the immunohistochemical status of the primary tumor. In univariate analysis, levels of choline-containing compounds, glutamate, glutamine, glycine, serine, and taurine were correlated with primary tumor characteristics. In multivariate analysis, most metabolite levels were not affected by tumor cell percentage. Tumor cell percentage showed poor correlation with metabolite levels in hormone receptor-positive cancer and triple-negative cancer, and poor to fair correlation with metabolite levels in HER2-positive cancer. This study showed that differences in the tumor cell fraction of CNB samples do not affect predictions on the primary cancer from which the samples are obtained.

Downregulated pol-miR-140-3p induces the expression of the kinesin family member 5A against Streptococcus parauberis infection in olive flounder.

MicroRNAs (miRNAs) are small non-coding RNAs that participate in various biological and cellular processes by regulating target gene expression. miRNAs are also known to play vital roles in the pathogenesis of various diseases, including infections, as well as the disease progression and defense responses. In this study, we examined the expression levels of pol-miR-140-3p and its target gene, kinesin family member 5A (KIF5A), in association with the Streptococcus parauberis (S. parauberis) infection, a major bacterial pathogen that causes streptococcosis in olive flounder (Paralichthys olivaceus). KIF5A is a heavy chain isoform of kinesin-1, which is known to be brain-specific, and this study is the first examination of KIF5A expression related to the regulation of miRNA in olive flounder (named PoKIF5A). There were significant differences in expression levels between infected and healthy olive flounder as the expression of pol-miR-140-3p in the infected fish was lower than that in the control, while the expression of PoKIF5A was higher in the infected fish than in the healthy controls. These contradictory results suggest that downregulated pol-miR-140-3p induces the expression of PoKIF5A against S. parauberis infection in olive flounder.

Using intracellular metabolic profiling to identify novel biomarkers of cisplatin-induced acute kidney injury in NRK-52E cells.

The aim of this study was to investigate changes in the intracellular metabolism resulting from cisplatin (CDDP)-induced nephrotoxicity in normal kidney tubular epithelial NRK-52E cells. Cytotoxicity, cell cycle analysis, and apoptotic cell death were all evaluated in NRK-52E cells treated with CDDP. Subsequently, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to investigate cellular metabolic profiles. CDDP-induced nephrotoxicity was determined in vivo model. Cytotoxicity in the NRK-52E cells significantly rose following treatment with CDDP and these increases were found to be concentration-dependent. Both p53 and Bax protein expression was increased in CDDP-treated NRK-52E cells, correlating with enhanced cellular apoptosis. In addition, a number of metabolites were altered in both media and cell lysates in these cells. In cell lysates, citrate, creatinine, and acetate levels were dramatically reduced following treatment with 20 µM CDDP concentrations, while glutamate level was elevated. Lactate and acetate levels were significantly increased in culture media but citrate concentrations were reduced following high 20 µM CDDP concentrations incubation. In addition, excretion of clusterin, calbindin, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), selenium binding protein 1 (SBP1), and pyruvate kinase M2 (PKM2) into the culture media was significantly increased in CDDP-treated cells while expression of acetyl CoA synthetase 1 (AceCS1) was markedly reduced in these cells. These findings suggest that acetate-dependent metabolic pathway may be a reliable and useful biomarker for detecting CDDP-induced nephrotoxicity. Taken together, data demonstrate that the discovery of novel biomarkers by metabolite profiling in target cells may contribute to the detection of nephrotoxicity and new drug development.

Metabolomics profiling of valproic acid-induced symptoms resembling autism spectrum disorders using 1H NMR spectral analysis in rat model.

Prenatal exposure to valproic acid (VPA) has been implicated in the manifestation of autism spectrum disorder (ASD)-like behavioral and functional changes both in human and rodents including mice and rats. The objective of this study was to determine metabolomics profiling and biomarkers related to VPA-induced symptoms resembling ASD using proton nuclear magnetic resonance (1H-NMR) spectral data. VPA was administered to pregnant rats at gestation day 12.5 and effects measured subsequently in male 4-week-old offspring pups. The sociability of VPA-treated animals was significantly diminished and exhibited ASD-like behavior as evidenced by reduction of social adaptation disorder and lack of social interactions. To find biomarkers related to ASD, the following were collected prefrontal brain cortices, urine bladder and blood samples directly from heart puncture. In all samples, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) displayed significant clustering pattern differences between control and treated groups. Valine, taurine, myo-inositol, 3-hydroxybutyrate and 1,3-dihydroxyacetone were significantly decreased in brain cortices in treated rats. Serum metabolites of glucose, creatine phosphate, lactate, glutamine and threonine were significantly increased in VPA-administered animals. Urinary metabolites of pimelate, 3-hydroxyisovalerate and valerate were significantly reduced in VPA-treated rat, whereas galactose and galactonate levels were elevated. Various metabolites were associated with mitochondrial dysfunction metabolism and central nervous system disorders. Data demonstrated that VPA-induced alterations in endogenous metabolites of serum, urine, and brain cortex which might prove useful as biomarkers for symptoms resembling ASD as a model of this disorder.

Regulation of the icl1 Gene Encoding the Major Isocitrate Lyase in Mycobacterium smegmatis.

Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl coenzyme A (succinyl-CoA) was shown to increase the affinity of binding of RamB to its binding sites and enable RamB to bind to RamBS2, which is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1; MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis. Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis, this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis- and trans-acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that a similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis.

Metabolic phenotyping of saliva to identify possible biomarkers of periodontitis using proton nuclear magnetic resonance.

AIM: The aim of this study was to propose biomarker candidates for periodontitis via untargeted metabolomics analysis. MATERIALS AND METHODS: Metabolic profiling was performed using saliva samples from 92 healthy controls (H) and 129 periodontitis patients (P) in the discovery cohort using proton nuclear magnetic resonance spectroscopy. Random forest was applied to identify metabolites that significantly differentiated the control group from the periodontitis group. Candidate metabolites were then validated in an independent validation cohort. RESULTS: In the discovery set, the metabolic profiles of the P group were clearly separated from those of the H group. A total of 31 metabolites were identified in saliva, and 7 metabolites were selected as candidate biomarkers. These metabolites were further confirmed in the validation set. Ethanol, taurine, isovalerate, butyrate, and glucose were finally confirmed as biomarkers. Furthermore, the biomarker panel showed more than 0.9 of the area under curve value in both discovery and validation sets, indicating that panels were more effective than individual metabolites for diagnosing periodontitis. CONCLUSIONS: We identified five metabolite biomarkers that discriminated patients with periodontitis from healthy controls in two independent cohorts. These biomarkers have the potential for periodontal screening, detection of periodontitis, and monitoring of the outcome of periodontal therapy.

Metabolomics approach to biomarkers of dry eye disease using 1H-NMR in rats.

Dry eye disease (DED) is a chronic and progressive lesion on the ocular surface and induces symptoms, such as burning sensation, itchy eyes, heavy eyes, tired eyes, dry feeling, facial flushing, and blurred vision. The present study was performed to develop DED biomarkers using metabolomics in a rat model. DED was induced by injecting scopolamine and exposing rats to a dry condition. Scopolamine (12 mg/kg/day for 7 days) was subcutaneously injected to male Sprague-Dawley rats. The rats were placed in dry condition with air-flow and dehumidifier. Tear volume and tear breakup time (TBUT) were measured, and eyes were examined through fluorescein staining to assess DED. Mucosal damage and immune reactions were also determined. Plasma and urinary endogenous metabolites were determined using 1H-NMR analysis. Compared with control tear and TBUT levels were significantly decreased in the DED group whereas corneal damage was significantly increased. The levels of interleukins (IL-6) and IL-1ß significantly elevated in the cornea and lacrimal glands in the DED group. TNF-α was numerically increased but not significantly different between groups. Pattern recognition using principal component analysis (PCA) and orthogonal projections to latent structure-discriminant analysis (OPLS-DA) of the NMR spectra in global profiling revealed different clusters between DED and control groups. Target profiling demonstrated that PCA and OPLS-DA score plots were separated between DED and controls in plasma and urine. Subsequently, 9 plasma metabolites were selected to examine different clustering between groups, and 26 urinary metabolites were also selected. Plasma metabolites showed a non-significant rising tendency in the DED group. Urinary phenylalanine, phenylacetate, pantothenate, glycine, succinate, methanol, valine, propylene glycol, histidine, threonine, lactate, and acetate were significantly different between control and DED rats. These results may contribute to understanding the metabolic regulation that is involved in DED and might be useful for potential biomarkers related to DED in rats.

Anti-Inflammatory Effect of Auranofin on Palmitic Acid and LPS-Induced Inflammatory Response by Modulating TLR4 and NOX4-Mediated NF-κB Signaling Pathway in RAW264.7 Macrophages.

Chronic inflammation, which is promoted by the production and secretion of inflammatory mediators and cytokines in activated macrophages, is responsible for the development of many diseases. Auranofin is a Food and Drug Administration-approved gold-based compound for the treatment of rheumatoid arthritis, and evidence suggests that auranofin could be a potential therapeutic agent for inflammation. In this study, to demonstrate the inhibitory effect of auranofin on chronic inflammation, a saturated fatty acid, palmitic acid (PA), and a low concentration of lipopolysaccharide (LPS) were used to activate RAW264.7 macrophages. The results show that PA amplified LPS signals to produce nitric oxide (NO) and various cytokines. However, auranofin significantly inhibited the levels of NO, monocyte chemoattractant protein-1, and pro-inflammatory cytokines, such as interleukin (IL)-1ß, tumor necrosis factor-α, and IL-6, which had been increased by co-treatment with PA and LPS. Moreover, the expression of inducible NO synthase, IL-1ß, and IL-6 mRNA and protein levels increased by PA and LPS were reduced by auranofin. In particular, the upregulation of NADPH oxidase (NOX) 4 and the translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by PA and LPS were suppressed by auranofin. The binding between the toll-like receptor (TLR) 4 and auranofin was also predicted, and the release of NO and cytokines was reduced more by simultaneous treatment with auranofin and TLR4 inhibitor than by auranofin alone. In conclusion, all these findings suggested that auranofin had anti-inflammatory effects in PA and LPS-induced macrophages by interacting with TLR4 and downregulating the NOX4-mediated NF-κB signaling pathway.

Integration of transcriptomics, proteomics and metabolomics identifies biomarkers for pulmonary injury by polyhexamethylene guanidine phosphate (PHMG-p), a humidifier disinfectant, in rats.

Polyhexamethylene guanidine phosphate (PHMG-p) was used as a humidifier disinfectant in Korea. PHMG induced severe pulmonary fibrosis in Koreans. The objective of this study was to elucidate mechanism of pulmonary toxicity caused by PHMG-p in rats using multi-omics analysis. Wistar rats were intratracheally instilled with PHMG-p by single (1.5 mg/kg) administration or 4-week (0.1 mg/kg, 2 times/week) repeated administration. Histopathologic examination was performed with hematoxylin and eosin staining. Alveolar macrophage aggregation and granulomatous inflammation were observed in rats treated with single dose of PHMG-p. Pulmonary fibrosis, chronic inflammation, bronchiol-alveolar fibrosis, and metaplasia of squamous cell were observed in repeated dose group. Next generation sequencing (NGS) was performed for transcriptome profiling after mRNA isolation from bronchiol-alveoli. Bronchiol-alveoli proteomic profiling was performed using an Orbitrap Q-exactive mass spectrometer. Serum and urinary metabolites were determined using 1H-NMR. Among 418 differentially expressed genes (DEGs) and 67 differentially expressed proteins (DEPs), changes of 16 mRNA levels were significantly correlated with changes of their protein levels in both single and repeated dose groups. Remarkable biological processes represented by both DEGs and DEPs were defense response, inflammatory response, response to stress, and immune response. Arginase 1 (Arg1) and lipocalin 2 (Lcn2) were identified to be major regulators for PHMG-p-induced pulmonary toxicity based on merged analysis using DEGs and DEPs. In metabolomics study, 52 metabolites (VIP > 0.5) were determined in serum and urine of single and repeated-dose groups. Glutamate and choline were selected as major metabolites. They were found to be major factors affecting inflammatory response in association with DEGs and DEPs. Arg1 and Lcn2 were suggested to be major gene and protein related to pulmonary damage by PHMG-p while serum or urinary glutamate and choline were endogenous metabolites related to pulmonary damage by PHMG-p.

Cost-Effective Electrochemical Activation of Graphitic Carbon Nitride on the Glassy Carbon Electrode Surface for Selective Determination of Serotonin.

A simple one-step electrochemical deposition/activation of graphitic carbon nitride (g-C3N4) is highly desired for sensor configurations and remains a great challenge. Herein, we attempt an electrochemical route to exfoliate the g-C3N4 nanosheets in an aqueous solution of pH 7.0 for constructing a sensor, which is highly sensitive for the detection of serotonin (5-HT). The significance of our design is to exfoliate the g-C3N4 nanosheets, a strong electrocatalyst for 5-HT detection. Investigations regarding the effect of neutral pH (pH 7.0) on the bulk g-C3N4 and g-C3N4 nanosheets, physical characterization, and electrochemical studies were extensively carried out. We demonstrate that the g-C3N4 nanosheets have a significant electrocatalytic effect for the 5-HT detection in a dynamic linear range from 500 pM to 1000 nM (R2 = 0.999). The limit of detection and sensitivity of the designed 5-HT sensor was calculated to be 150 pM and 1.03 µA µM-1 cm-2, respectively. The proposed sensor has great advantages such as high sensitivity, good selectivity, reproducibility, and stability. The constructed g-C3N4 nanosheets-based sensor platform opens new feasibilities for the determination of 5-HT even at the picomolar/nanomolar concentration range.

Co-Expression Network Analysis of Spleen Transcriptome in Rock Bream (Oplegnathus fasciatus) Naturally Infected with Rock Bream Iridovirus (RBIV).

Rock bream iridovirus (RBIV) is a notorious agent that causes high mortality in aquaculture of rock bream (Oplegnathus fasciatus). Despite severity of this virus, no transcriptomic studies on RBIV-infected rock bream that can provide fundamental information on protective mechanism against the virus have been reported so far. This study aimed to investigate physiological mechanisms between host and RBIV through transcriptomic changes in the spleen based on RNA-seq. Depending on infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as control (0C), heavy infected fish at week 0 (0H), heavy mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and light infected fish at week 3 (3L). We explored clusters from 35,861 genes with Fragments Per Kilo-base of exon per Million mapped fragments (FPKM) values of 0.01 or more through signed co-expression network analysis using WGCNA package. Nine of 22 modules were highly correlated with viral infection (|gene significance (GS) vs. module membership (MM) |> 0.5, p-value < 0.05). Expression patterns in selected modules were divided into two: heavy infected (0H and 0MH) and control and light-infected groups (0C, 3C, and 3L). In functional analysis, genes in two positive modules (5448 unigenes) were enriched in cell cycle, DNA replication, transcription, and translation, and increased glycolysis activity. Seven negative modules (3517 unigenes) built in this study showed significant decreases in the expression of genes in lymphocyte-mediated immune system, antigen presentation, and platelet activation, whereas there was significant increased expression of endogenous apoptosis-related genes. These changes lead to RBIV proliferation and failure of host defense, and suggests the importance of blood cells such as thrombocytes and B cells in rock bream in RBIV infection. Interestingly, a hub gene, pre-mRNA processing factor 19 (PRPF19) showing high connectivity (kME), and expression of this gene using qRT-PCR was increased in rock bream blood cells shortly after RBIV was added. It might be a potential biomarker for diagnosis and vaccine studies in rock bream against RBIV. This transcriptome approach and our findings provide new insight into the understanding of global rock bream-RBIV interactions including immune and pathogenesis mechanisms.

A Comparative Study on Processed Panax ginseng Products Using HR-MAS NMR-Based Metabolomics.

Panax ginseng is processed to diversify efficacy. Four processed ginsengs containing white ginseng (WG), tae-geuk ginseng (TG), red ginseng (RG), and black ginseng (BG) were analyzed using nuclear magnetic resonance (NMR) spectroscopy for screening overall primary metabolites. There were significant differences in the sugar content among these four processed ginseng products. WG had a high sucrose content, TG had a high maltose content, and BG had high fructose and glucose content. In the multivariate analyses of NMR spectra, the PCA score plot showed significant discrimination between the four processed ginsengs. For effective clustering, orthogonal partial least squares discriminant analyses (OPLS-DA) with a 1:1 comparison were conducted and all OPLS models were validated using the permutation test, the root mean square error of estimation (RMSEE), and the root mean square error of prediction (RMSEP). All OPLS-DA score plots showed clear separations of processed ginseng products, and sugars such as sucrose and fructose mainly contributed to these separations.

Metal Nanozyme with Ester Hydrolysis Activity in the Presence of Ammonia-Borane and Its Use in a Sensitive Immunosensor.

Metal nanoparticle surfaces are used for peroxidase- and oxidase-like nanozymes but not for esterase-like nanozymes. It is challenging to obtain rapid catalytic hydrolysis on a metal surface and even more so without a catalytically labile substrate. Here, we report that metal nanoparticle surfaces rapidly catalyze non-redox ester hydrolysis in the presence of redox H3 N-BH3 (AB). Metal hydrides are readily generated on a Pt nanoparticle (PtNP) from AB, and as a result the PtNP becomes electron-rich, which might assist nucleophilic attack of H2 O on the carbonyl group of an ester. The nanozyme system based on PtNP, AB, and 4-aminonaphthalene-1-yl acetate provides an electrochemical signal-to-background ratio much higher than natural enzymes, due to the rapid ester hydrolysis and redox cycling involving the hydrolysis product. The nanozyme system is applied in a sensitive electrochemical immunosensor for thyroid-stimulating hormone detection. The calculated detection limit is approximately 0.3 pg mL-1 , which indicates the high sensitivity of the immunosensor using the PtNP nanozyme.

An Algal Metabolite-Based PPAR-γ Agonist Displayed Anti-Inflammatory Effect via Inhibition of the NF-κB Pathway.

In our previous study, a synthetic compound, (+)-(R,E)-6a1, that incorporated the key structures of anti-inflammatory algal metabolites and the endogenous peroxisome proliferator-activated receptor γ (PPAR-γ) ligand 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), exerted significant PPAR-γ transcriptional activity. Because PPAR-γ expressed in macrophages has been postulated as a negative regulator of inflammation, this study was designed to investigate the anti-inflammatory effect of the PPAR-γ agonist, (+)-(R,E)-6a1. Compound (+)-(R,E)-6a1 displayed in vitro anti-inflammatory activity in lipopolysaccharides (LPS)-stimulated murine RAW264.7 macrophages. Compound (+)-(R,E)-6a1 suppressed the expression of proinflammatory factors, such as nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), possibly by the inhibition of the nuclear factor-κB (NF-κB) pathway. In macrophages, (+)-(R,E)-6a1 suppressed LPS-induced phosphorylation of NF-κB, inhibitor of NF-κB α (IκBα), and IκB kinase (IKK). These results indicated that PPAR-γ agonist, (+)-(R,E)-6a1, exerts anti-inflammatory activity via inhibition of the NF-κB pathway.

Protective Effect of Phloroglucinol on Oxidative Stress-Induced DNA Damage and Apoptosis through Activation of the Nrf2/HO-1 Signaling Pathway in HaCaT Human Keratinocytes.

Phloroglucinol (PG) is a component of phlorotannins, which are abundant in marine brown alga species. Recent studies have shown that PG is beneficial in protecting cells from oxidative stress. In this study, we evaluated the protective efficacy of PG in HaCaT human skin keratinocytes stimulated with oxidative stress (hydrogen peroxide, H2O2). The results showed that PG significantly inhibited the H2O2-induced growth inhibition in HaCaT cells, which was associated with increased expression of heme oxygenase-1 (HO-1) by the activation of nuclear factor erythroid 2-related factor-2 (Nrf2). PG remarkably reversed H2O2-induced excessive ROS production, DNA damage, and apoptosis. Additionally, H2O2-induced mitochondrial dysfunction was related to a decrease in ATP levels, and in the presence of PG, these changes were significantly impaired. Furthermore, the increases of cytosolic release of cytochrome c and ratio of Bax to Bcl-2, and the activation of caspase-9 and caspase-3 by the H2O2 were markedly abolished under the condition of PG pretreatment. However, the inhibition of HO-1 function using zinc protoporphyrin, a HO-1 inhibitor, markedly attenuated these protective effects of PG against H2O2. Overall, our results suggest that PG is able to protect HaCaT keratinocytes against oxidative stress-induced DNA damage and apoptosis through activating the Nrf2/HO-1 signaling pathway.

Metabolomics for Age Discrimination of Ginseng Using a Multiplex Approach to HR-MAS NMR Spectroscopy, UPLC-QTOF/MS, and GC × GC-TOF/MS.

(1) Background: The ability to determine the age of ginseng is very important because the price of ginseng depends on the cultivation period. Since morphological observation is subjective, a new scientific and systematic method for determining the age of ginseng is required. (2) Methods: Three techniques were used for a metabolomics approach. High-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy was used to analyze powdered ginseng samples without extraction. Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and gas chromatography quadrupole time-of-fight mass spectrometry (GC-TOF/MS) were used to analyze the extracts of 4-, 5-, and 6-year-old ginseng. (3) Results: A metabolomics approach has the potential to discriminate the age of ginseng. Among the primary metabolites detected from NMR spectroscopy, the levels of fumarate and choline showed moderate prediction with an area under the curve (AUC) value of more than 0.7. As a result of UPLC-QTOF/MS-based profiling, 61 metabolites referring to the VIP (variable importance in the projection) score contributed to discriminating the age of ginseng. The results of GC×GC-TOF/MS showed clear discrimination of 4-, 5-, and 6-year-old ginseng using orthogonal partial least-squares discriminant analysis (OPLS-DA) to 100% of the discrimination rate. The results of receiver operating characteristic (ROC) analysis, 16 metabolites between 4- and 5-year-old ginseng, and 18 metabolites between 5- and 6-year-old ginseng contributed to age discrimination in all regions. (4) Conclusions: These results showed that metabolic profiling and multivariate statistical analyses can distinguish the age of ginseng. Especially, it is meaningful that ginseng samples from different areas had the same metabolites for age discrimination. In future studies, it will be necessary to identify the unknown variables and to collaboratively study with other fields the biochemistry of aging in ginseng.

Roles of Alanine Dehydrogenase and Induction of Its Gene in Mycobacterium smegmatis under Respiration-Inhibitory Conditions.

Here we demonstrated that the inhibition of electron flux through the respiratory electron transport chain (ETC) by either the disruption of the gene for the major terminal oxidase (aa3 cytochrome c oxidase) or treatment with KCN resulted in the induction of ald encoding alanine dehydrogenase in Mycobacterium smegmatis A decrease in functionality of the ETC shifts the redox state of the NADH/NAD+ pool toward a more reduced state, which in turn leads to an increase in cellular levels of alanine by Ald catalyzing the conversion of pyruvate to alanine with the concomitant oxidation of NADH to NAD+ The induction of ald expression under respiration-inhibitory conditions in M. smegmatis is mediated by the alanine-responsive AldR transcriptional regulator. The growth defect of M. smegmatis by respiration inhibition was exacerbated by inactivation of the ald gene, suggesting that Ald is beneficial to M. smegmatis in its adaptation and survival under respiration-inhibitory conditions by maintaining NADH/NAD+ homeostasis. The low susceptibility of M. smegmatis to bcc1 complex inhibitors appears to be, at least in part, attributable to the high expression level of the bd quinol oxidase in M. smegmatis when the bcc1-aa3 branch of the ETC is inactivated.IMPORTANCE We demonstrated that the functionality of the respiratory electron transport chain is inversely related to the expression level of the ald gene encoding alanine dehydrogenase in Mycobacterium smegmatis Furthermore, the importance of Ald in NADH/NAD+ homeostasis during the adaptation of M. smegmatis to severe respiration-inhibitory conditions was demonstrated in this study. On the basis of these results, we propose that combinatory regimens including both an Ald-specific inhibitor and respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline are likely to enable a more efficient therapy for tuberculosis.

Serum and urine toxicometabolomics following gentamicin-induced nephrotoxicity in male Sprague-Dawley rats.

Gentamicin (GM) is an aminoglycoside antibiotic used in treatment of various types of bacterial infections, but the major adverse effect is drug-induced nephrotoxicity. This study aimed to determine biomarkers that might predict nephrotoxicity initiated by GM using serum or urinary proton nuclear magnetic resonance (1H NMR) spectral data in male Sprague-Dawley rats. GM (0, 30, or 300 mg/kg/d) was intraperitoneally administered for 3 consecutive days. Animals were sacrificed 2 d (D2) or 8 d (D8) after last administration of GM in order to perform analysis of serum biochemistries and histopathologic examination. Urine samples were collected every 24 h from prior to treatment until sacrifice. Serum and urinary 1H NMR spectral data revealed apparent differential clustering between control and GM-treated groups as evidenced by principal component analysis (PCA) and orthogonal projections to latent structure-discriminant analysis (OPLS-DA) in global and targeted profiling. The concentrations of endogenous serum metabolites including 3-hydroxybutyrate, alanine, citrate, creatine, glucose, and glycine were increased significantly on D2 or D8. Urinary levels of glucose, glycine, and succinate were significantly elevated on D2 or D8, whereas the concentration of hippurate was significantly decreased on D2 and D8. Correlation of serum and urinary 1H NMR OPLS-DA with serum biochemistry and renal histopathologic changes suggests that 1H NMR urinalysis may be used to reliably predict or screen for GM-induced nephrotoxicity. In contrast, Western blot analysis of kidney injury molecule-1 (KIM-1) demonstrated that protein expression was not markedly altered indicating this biomarker was not sensitive to detect GM-mediated renal damage. Data suggest that these altered metabolites might serve as specific and sensitive biomarkers for GM-mediated renal damage.