Characteristics, predictors and outcomes of new-onset QT prolongation in sepsis: a multicenter retrospective study.

BACKGROUND: Sepsis-induced myocardial injury is a serious complication of sepsis. QT prolongation is a proarrhythmic state which reflects myocardial injury in a group of heterogeneous disorders. However, the study on the clinical value of QT prolongation in sepsis is limited. METHODS: We aimed to investigate the clinical characteristics and predictors of new-onset QT prolongation in sepsis and its impact on the outcome in a multicenter retrospective cohort study. Electrocardiographic and clinical data were collected from patients with sepsis from the wards and intensive care units of four centers after exclusion of QT-influencing medications and electrolyte abnormalities. Clinical outcomes were compared between patients with and without QT prolongation (QTc > 450 ms). Multivariate analysis was performed to ascertain whether QT prolongation was an independent predictor for 30-day mortality. The factors predicting QT prolongation in sepsis were also analyzed. RESULTS: New-onset QT prolongation occurred in 235/1024 (22.9%) patients. The majority demonstrated similar pattern as type 1 long QT syndrome. Patients with QT prolongation had a higher 30-day in-hospital mortality (P < 0.001), which was also associated with increased tachyarrhythmias including paroxysmal atrial fibrillation or tachycardia (P < 0.001) and ventricular arrhythmia (P < 0.001) during hospitalization. QT prolongation independently predicted 30-day mortality (P = 0.044) after multivariate analysis. History of coronary artery disease (P = 0.001), septic shock (P = 0.008), acute respiratory (P < 0.001), heart (P = 0.021) and renal dysfunction (P = 0.013) were independent predictors of QT prolongation in sepsis. CONCLUSIONS: New-onset QT prolongation in sepsis was associated with increased mortality as well as atrial and ventricular arrhythmias, which was predicted by disease severity and organ dysfunction.

Serum lipidomics reveals distinct metabolic profiles for asymptomatic hyperuricemic and gout patients.

OBJECTIVES: This study aimed to characterize the systemic lipid profile of patients with asymptomatic hyperuricemia (HUA) and gout using lipidomics, and to find potential underlying pathological mechanisms therefrom. METHODS: Sera were collected from Affiliated Hospital of Nanjing University of Chinese Medicine as centre 1 (discovery and internal validation sets) and Suzhou Hospital of Traditional Chinese Medicine as centre 2 (external validation set), including 88 normal subjects, 157 HUA and 183 gout patients. Lipidomics was performed by ultra high performance liquid chromatography plus Q-Exactive mass spectrometry (UHPLC-Q Exactive MS). Differential metabolites were identifed by both variable importance in the projection ≥1 in orthogonal partial least-squares discriminant analysis mode and false discovery rate adjusted P ≤ 0.05. Biomarkers were found by logistic regression and receiver operating characteristic (ROC) analysis. RESULTS: In the discovery set, a total of 245 and 150 metabolites, respectively, were found for normal subjects vs HUA and normal subjects vs gout. The disturbed metabolites included diacylglycerol, triacylglycerol (TAG), phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, etc. We also found 116 differential metabolites for HUA vs gout. Among them, the biomarker panel of TAG 18:1-20:0-22:1 and TAG 14:0-16:0-16:1 could differentiate well between HUA and gout. The area under the receiver operating characteristic ROC curve was 0.8288, the sensitivity was 82% and the specificity was 78%, at a 95% CI 0.747, 0.9106. In the internal validation set, the predictive accuracy of TAG 18:1-20:0-22:1 and TAG 14:0-16:0-16:1 panel for differentiation of HUA and gout reached 74.38%, while it was 84.03% in external validation set. CONCLUSION: We identified serum biomarkers panel that have the potential to predict and diagnose HUA and gout patients.

Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production.

L-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to L-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and L-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance L-leucine production and cell growth. Subsequently, the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters PleuA and PilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter Ptuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36 g/L L-leucine in shake flask fermentation.

Lensless Computational Imaging Technology Using Deep Convolutional Network.

Within the framework of Internet of Things or when constrained in limited space, lensless imaging technology provides effective imaging solutions with low cost and reduced size prototypes. In this paper, we proposed a method combining deep learning with lensless coded mask imaging technology. After replacing lenses with the coded mask and using the inverse matrix optimization method to reconstruct the original scene images, we applied FCN-8s, U-Net, and our modified version of U-Net, which is called Dense-U-Net, for post-processing of reconstructed images. The proposed approach showed supreme performance compared to the classical method, where a deep convolutional network leads to critical improvements of the quality of reconstruction.

Toxicity Reduction of Euphorbia kansui Stir-Fried with Vinegar Based on Conversion of 3-O-(2'E,4'Z-Decadi-enoyl)-20-O-acetylingenol.

The dried roots of Euphorbia kansui S.L.Liou ex S.B.Ho have long been used to treat edema in China. However, the severe toxicity caused by Euphorbia kansui (EK) has seriously restricted its clinical application. Although EK was processed with vinegar to reduce its toxicity, the detailed mechanisms of attenuation in toxicity of EK stir-fried with vinegar (VEK) have not been well delineated. Diterpenoids are the main toxic ingredients of EK, and changes in these after processing may be the underlying mechanism of toxicity attenuation of VEK. 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3-O-EZ) is one of the diterpenoids derived from EK, and the content of 3-O-EZ was significantly reduced after processing. This study aims to explore the underlying mechanisms of toxicity reduction of VEK based on the change of 3-O-EZ after processing with vinegar. Based on the chemical structure of 3-O-EZ and the method of processing with vinegar, simulation experiments were carried out to confirm the presence of the product both in EK and VEK and to enrich the product. Then, the difference of peak area of 3-O-EZ and its hydrolysate in EK and VEK were detected by ultra-high-performance liquid chromatography (UPLC). Furthermore, the toxicity effect of 3-O-EZ and its hydrolysate, as well as the underlying mechanism, on zebrafish embryos were investigated. The findings showed that the diterpenoids (3-O-EZ) in EK can convert into less toxic ingenol in VEK after processing with vinegar; meanwhile, the content of ingenol in VEK was higher than that of EK. More interestingly, the ingenol exhibited less toxicity (acute toxicity, developmental toxicity and organic toxicity) than that of 3-O-EZ, and 3-O-EZ could increase malondialdehyde (MDA) content and reduce glutathione (GSH) content; cause embryo oxidative damage by inhibition of the succinate dehydrogenase (SDH) and superoxide dismutase (SOD) activity; and induce inflammation and apoptosis by elevation of IL-2 and IL-8 contents and activation of the caspase-3 and caspase-9 activity. Thus, this study contributes to our understanding of the mechanism of attenuation in toxicity of VEK, and provides the possibility of safe and rational use of EK in clinics.

Chemical Property Changes and Thermal Analysis during the Carbonizing Process of the Pollen Grains of Typha.

Carbonized pollen grains of Typha (CPT) were widely used in clinical for antithrombosis, wound and bleeding in China. In order to ensure the role of drugs, it is very important to control the quality of drugs. However, there is a lack of monitoring methods in the process of charcoal preparation. To characterize the process of CPT, we used thermal analysis, scanning electron microscope (SEM), color measurement, Fourier transform infrared spectrometry (FTIR) and HPLC. In this study, 7 min was the optimal processing time and the heating process condition should be controlled under 272.35 ± 7.23 °C. This comprehensive strategy to depict the whole carbonizing process would provide new ideas for researches on quality control of Traditional Chinese Medicine (TCM) and processing theory of charcoal medicine.

A Review on the Phytochemistry, Pharmacology, and Pharmacokinetics of Amentoflavone, a Naturally-Occurring Biflavonoid.

Amentoflavone (C30H18O10) is a well-known biflavonoid occurring in many natural plants. This polyphenolic compound has been discovered to have some important bioactivities, including anti-inflammation, anti-oxidation, anti-diabetes, and anti-senescence effects on many important reactions in the cardiovascular and central nervous system, etc. Over 120 plants have been found to contain this bioactive component, such as Selaginellaceae, Cupressaceae, Euphorbiaceae, Podocarpaceae, and Calophyllaceae plant families. This review paper aims to profile amentoflavone on its plant sources, natural derivatives, pharmacology, and pharmacokinetics, and to highlight some existing issues and perspectives in the future.

Chemical Constituents from Euphorbia kansui.

In this research, a new triterpenoid, tirucalla-8,24-diene-3ß,11ß-diol-7-one (1), and eupha-8,24-diene-3ß,11ß-diol-7-one (2), which was isolated from Euphorbia kansui for the first time, together with twelve other known compounds (3-14), were isolated from the ethyl acetate extract of Euphorbia kansui. Their structures were elucidated based on High resolution electrospray ionization mass spectrometry (HR-ESI-MS), Infrared Spectroscopy (IR), 1D and 2D Nuclear Magnetic Resonance (NMR) data. Both constituents 1 and 2 exhibited moderate cytotoxicity against colon cancer HCT-116, gastric cancer MKN-45 and breast cancer MCF-7.

Antioxidant capacity of Typha angustifolia extracts and two active flavonoids.

CONTEXT: The pollen of Typha angustifolia L. (Typhaceae) has been used as a traditional Chinese medicine for improving the microcirculation and promoting wound healing. Flavonoids are the main constituent in the plant, but little is known about the antioxidant activity of the principal constituent of the pollen in detail. OBJECTIVES: To assess the antioxidant activities of ethanol and water extracts and two constituents of the pollen. MATERIALS AND METHODS: Plant material (1 g) was extracted by 95% ethanol and water (10 mL × 2, 1 h each), respectively. The extracted activities (0.8-2.6 mg/mL) were measured by DPPH and the reducing activity of ferric chloride (1.7-2.6 mg/mL). Typhaneoside and isorhamnetin-3-O-neohesperidoside (I3ON) (2.8-70 µmol/L) were investigated on the relationship between NO, MDA and SOD in HUVECs treated with 100 µg/mL of LPS for 24 h. RESULTS: Nine compounds were identified by UPLC-MS. Ethanol extract showed IC50 values in DPPH (39.51 ± 0.72) and Fe3+ reducing activity (82.76 ± 13.38), higher than the water extract (50.85 ± 0.74) and (106.33 ± 6.35), respectively. Typhaneoside and I3ON promoted cell proliferation at the respective concentration range of 2.8 to 70 µmol/L (p < 0.01). This two compounds decreased MDA (1.91 ± 0.10, 1.80 ± 0.34, p < 0.05) and NO levels (14.64 ± 0.08, 13.10 ± 0.88, p < 0.01), respectively, and increased SOD level (22.94 ± 2.48, 23.57 ± 2.38, p < 0.01) at the concentration of 70 µmol/L compared with LPS group. CONCLUSIONS: The constituents from Typha angustifolia could be a novel therapeutic strategy for LPS-induced inflammation.

A Review of the Botany, Phytochemistry, Pharmacology and Toxicology of Rubiae Radix et Rhizoma.

Rubia cordifolia Linn (Rubiaceae) is a climbing perennial herbal plant, which is widely distributed in China and India. Its root and rhizome, Rubiae Radix et Rhizoma (called Qiancao in China and Indian madder in India), is a well known phytomedicine used for hematemesis, epistaxis, flooding, spotting, traumatic bleeding, amenorrhea caused by obstruction, joint impediment pain, swelling and pain caused by injuries from falls. In addition, it is a kind of pigment utilized as a food additive and a dye for wool or fiber. This review mainly concentrates on studies of the botany, phytochemistry, pharmacology and toxicology of this Traditional Chinese Medicine. The phytochemical evidences indicated that over a hundred chemical components have been found and isolated from the medicine, such as anthraquinones, naphthoquinones, triterpenoids, cyclic hexapeptides and others. These components are considered responsible for the various bioactivities of the herbal drug, including anti-oxidation, anti-inflammation, immunomodulation, antitumor, effects on coagulation-fibrinolysis system, neuroprotection and other effects. Additionally, based on these existing results, we also propose some interesting future research directions. Consequently, this review should help us to more comprehensively understand and to more fully utilize the herbal medicine Rubiae Radix et Rhizoma.

Recent Advances in Topical Hemostatic Materials.

Untimely or improper treatment of traumatic bleeding may cause secondary injuries and even death. The traditional hemostatic modes can no longer meet requirements of coping with complicated bleeding emergencies. With scientific and technological advancements, a variety of topical hemostatic materials have been investigated involving inorganic, biological, polysaccharide, and carbon-based hemostatic materials. These materials have their respective merits and defects. In this work, the application and mechanism of the major hemostatic materials, especially some hemostatic nanomaterials with excellent adhesion, good biocompatibility, low toxicity, and high adsorption capacity, are summarized. In the future, it is the prospect to develop multifunctional hemostatic materials with hemostasis and antibacterial and anti-inflammatory properties for promoting wound healing.

Mechanism investigation of Shi-Xiao-San in treating blood stasis syndrome based on network pharmacology, molecular docking and in vitro/vivo pharmacological validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Shixiao San (SXS) is a traditional Chinese formula that has been widely used in clinical practice to treat blood stasis syndromes, such as hyperlipidemia, atherosclerotic, thrombosis and coronary heart disease. However, the effectiveness and mechanism of SXS have not been studied in detail yet. AIM OF THE STUDY: Current study aimed to identify the compounds in SXS, evaluate the formula efficacies using network pharmacology, molecular docking, and verify the pharmacological effects by in vivo and in vitro experiments. MATERIALS AND METHODS: The compounds in SXS were analyzed using UPLC-QTOF-MS. Potential target genes for identified compounds were obtained from three databases. DAVID database was used to perform GO and KEGG pathway enrichment analyses. PPI network was constructed to screen core targets. Molecular docking was used to examine interactions between active compounds and potential targets. The mechanism was also verified by model of acute blood stasis rats and human umbilical vein cells. RESULTS: In total, 45 compounds were identified from SXS. Among the detected phytochemicals, quercetin, isorhamnetin, kaempferol, D-catechin, naringenin and amentoflavone were identified as the active constituents. SXS is primarily involved in the modulation of hypoxic state, vascular regulation, and inflammation response, according to GO and KGG pathway enrichment analysis. A network of protein-protein interactions (PPIs) was constructed and five core targets were identified as VEGFA, AKT1, EGFR, PTGS2, and MMP9. Molecular docking simulation revealed good binding affinity of the five putative targets with the corresponding compounds. SXS reduced HIF-1α and COX-2 levels and increased the eNOS expression levels in hypoxic HUVECs. SXS can reduce the whole blood viscosity in adrenaline induced acute blood stasis rats and relieve blood stasis. CONCLUSIONS: SXS removes blood stasis might through VEGFA/AKT/eNOS/COX-2 pathway and flavonoids are the main active components in the formula.

[Spectrum-active relation research on Typha angustifolia before and after carbonized].

OBJECTIVE: To study the components of Typha angustifolia change before and after carbonized and the correlation with its effective. METHODS: The chemical constitutions between the pollen and its carbonized product were compared by UPLC-MASS and their thrombin activity was tested. RESULTS: The change of components was significant especially the flavonoids. The content of flavonoid glycoside was reduced obviously and the thrombin activity showed the main difference was quercetin and isorhamnetin. CONCLUSION: The flavonoids in pollen typha may be the main factors in their thrombin activity.

The complete plastome of Garcinia subelliptica, Merr. 1909 (Clusiaceae).

The complete plastome of G. subelliptica, Merr. 1909. The complete length is 158,356 bp, with the typical structure and gene content of angiosperm plastomes, including a large single-copy region (LSC) of 86,220 bp, a repeat region (IRB), and a reverse repeat region (IRA) of 27,399 bp, respectively, and a small single-copy region (SSC) of 17,338 bp. The plastome contains 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The total G/C content of the plastome is 36.1%.

Discovery of processing-associated Q-marker of carbonized traditional Chinese medicine: An integrated strategy of metabolomics, systems pharmacology and in vivo high-throughput screening model.

BACKGROUND: Carbonized traditional Chinese medicine (TCM) is a kind of distinctive traditional medicine, which has been widely used to cure various bleeding syndromes in clinic for over 2000 years. However, there are no effective quality control methods developed on carbonized TCM so far. PURPOSE: This study aimed at developing a processing-associated quality marker (Q-marker) discovery strategy, which would enable to promote the quality control study of carbonized TCM. METHODS: Carbonized Typhae Pollen (CTP), a typical carbonized TCM with fantastic efficacy of stanching bleeding and removing blood stasis, was used as an example. First, a ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) method was established to characterize four types of CTP in different processing degrees. Second, chemometric method was applied to screen candidate Q-markers. Third, peak area changes and Aratio changes of each candidate markers in 57 batches samples were described (Traceability and Transitivity). Fourth, systems pharmacology and two high-throughput zebrafish models: cerebral hemorrhage model and thrombus model were used to furtherly screen Q-markers (Effectiveness). Finally, a ultraperformance liquid chromatographic coupled with triple quadrupole tandem mass spectrometry (UPLC-TQ-MS) method was established and applied to quantify Q-markers in additional 10 batches of CTP samples (Measurability). RESULTS: The chemical profiles of Typhae Pollen during the carbonized process were investigated. Then, 12 candidate compounds were screened in chemometric part. Six Q-markers (isorhamnetin-3-O-neohesperidoside, isorhamnetin-3-O-rutinoside, kaempferol-3-O-neohesperidoside, naringenin, quercetin and isorhamnetin) were subsequently screened out using three principles of Q-markers combined with content changes and two in vivo zebrafish models. Their average contents in additional 10 batches of CTP were 316.8 µg/g, 13.7 µg/g, 6.1 µg/g, 197.8 µg/g, 12.9 µg/g and 199.3 µg/g, respectively. Their content proportion was about 25: 1: 0.5: 15: 1: 15. CONCLUSION: A processing-associated Q-marker discovery strategy was developed for carbonized TCM. It might provide a novel insight to solve the problem of 'Chao Tan Cun Xing' in carbonized process.

Defensing against oxidative stress in Caenorhabditis elegans of a polysaccharide LFP-05S from Lycii fructus.

Oxidative stress is closely associated with the initiation and progression of aging. Considerable interest centers in the potential application of natural polysaccharides in oxidative stress alleviation and senescence delay. Herein, LFP-05S, an acidic heteropolysaccharide from Lycii fructus, was purified and structurally characterized based on a combination strategy of molecular weight (MW) distribution, monosaccharide composition, methylation and NMR spectroscopy analysis. The dominant population of LFP-05S was composed of long homogalacturonan (HG) backbone interspersed with alternating sequences of intra-rhamnogalacturonans-I (RG-I) domains and branched arabinogalactan and arabinan. Orally supplied LFP-05S exhibited defensive modulation in paraquat (PQ)-damaged oxidative stress Caenorhabditis elegans by strengthening the internal defense systems. Under normal conditions, LFP-05S extended the lifespan without significant impairment of propagation. Overall, these results suggested LFP-05S and L. fructus are worth further exploration as promising redox-based candidates for the prevention and management of aging and related disorders.

Structural determination and pro-angiogenic effect of polysaccharide from the pollen of Typha angustifolia L.

Four fractions of polysaccharides (TPP-1, TPP-2, TPP-3, and TPP-4) were isolated and purified from the pollen of Typha angustifolia L., and the structure of TPP-3 was furtherly determined by HPGPC (High Performance Gel Permeation Chromatography), monosaccharide composition analysis, methylation analysis and NMR (Nuclear Magnetic Resonance). TPP-3 was found to be a homogeneous heteropolysaccharide with an average molecular weight of 5.5 × 104 Da and composed of eight types of monosaccharides. The pro-angiogenic activities of TPP-3 were verified on HUVECs and VEGFR tyrosine kinase inhibitor II (VRI)-induced vascular defect zebrafish model. Furthermore, the underlying mechanism investigation showed that its pro-angiogenic activities were closely related with the activation of VEGF/PI3K/Akt signaling pathway.

[Study on the chemical constituents of vinegar Schisandra chinensis].

OBJECTIVE: To study the chemical constituents of vinegar Schisandra chinensis. METHODS: Column chromatography was used in the isolation procedure. The structures of isolated compounds were elucidated by spectral data. RESULTS: Five compounds were isolated and their structures were identified as kadsuranin (I), beta-sitosterol (II), schizandrin (III), 5,5'-[oxybis (methylene)] bis-2-furancarboxaldehyde (IV) and (NH)-form-2-Hydroxyquinoline4-carboxylic acid (V). CONCLUSION: Compound 5 is isolated from this genus for the first time.

Effects of carbonized process on quality control, chemical composition and pharmacology of Typhae Pollen: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Carbonized Typhae Pollen (CTP), a processed product of Typhae Pollen after stir-fried, is a well-known Traditional Chinese Medicine (TCM) with functions of removing blood stasis and hemostasis. AIM OF REVIEW: The aim of this study is to summarize and discuss up-to-date information on quality control of CTP, and effects of carbonized process on phytochemistry and biological activities. We hope this review could provide feasible insights for further studies of CTP on its material basis and pharmacological effect mechanism. MATERIAL AND METHODS: The information of TP before and after carbonized process was collected from online databases (PubMed, CNKI, Google Scholar, Baidu Xueshu, Web of Science, SpringerLink, Wiley Online Library, SciFinder and Chemical book). Meanwhile local books, published and unpublished Ph.D., MSc. dissertations were also taken into consideration. RESULTS: A total of 27 Ph.D., MSc. dissertations and 208 articles were collected from online database, from which 122 compounds of TP were collected, but only two researches focused on the chemical compositions of CTP. Introductions of new technologies and intelligent processing equipment developments are considered as two main solutions to the quality control of CTP. CTP is a well-known ethnic medicine in China with a fantastic efficacy in curing bleeding caused by blood stasis. Flavonoids were reported as the main active compounds for removing blood stasis while the enhanced hemostatic activity were consistent with flavonoid aglycones. Modern pharmacological researches showed that CTP has wound healing activity, effects on blood vessels, antithrombotic activity, hemostatic activity, antioxidant activity and immunomodulatory activity. CONCLUSIONS: Although CTP has been widely used in clinic, there are some problems blocking its further development. Unknown mechanism and uncertain active compounds might be the main reasons for few rules on controlling the quality of CTP. It is necessary to investigate the mechanisms and the relationship between carbonized process and the changes in constituents as well as pharmacological effects. This is essential to promote the safe clinical use of CTP.

The toxicity mechanism of toxic compounds from Euphorbiae pekinensis Radix on zebrafish embryos.

Euphorbiae pekinensis Radix (EP) is effective in treating various diseases, but it's toxicity is a major obstacle in use in clinical. Although EP was processed with vinegar to reduce it's toxicity, the detailed mechanism of toxicity in EP have not been clearly delineated. This study investigate the toxicity attenuation-mechanism of Euphorbiae pekinensis after being processed with vinegar (VEP) and the toxic mechanism of four compounds from EP on zebrafish embryos. The contents of four compounds decreased obviously in VEP. Correspondingly, slower development on embryos can be seen as some symptoms like reduction of heart rate, liver area and gastrointestinal peristalsis after exposed to the compounds. Some obvious pathological signals such as pericardial edema and yolk sac edema were observed. Furthermore, the compounds could increase the contents of MDA and GSH-PX and induce oxidative damage by inhibiting the activity of SOD. Also, four compounds could provoke apoptosis by up-regulating the expression level of p53, MDM2, Bax, Bcl-2 and activating the activity of caspase-3, caspase-9. In conclusion, the four compounds play an important role in the toxicity attenuation effects of VEP, which may be related to the apoptosis induction and oxidative damage. This would contribute to the clinical application and further toxicity-reduction mechanism research.