Enhanced Antibacterial Activity against Escherichia coli Based on Cationic Carbon Dots Assembling with 5-Aminolevulinic Acid.

Carbon dots (CDs) with positive surface charges are considered one of the encouraging nanomedications for antibacterial applications. However, due to the distinctive membrane structure of Gram-negative bacteria, cationic CDs with relatively high concentrations are usually required for effective treatment, which might bring out serious safety issues at high doses. Therefore, it is of substantial significance to improve the killing efficiency of cationic CDs on Gram-negative bacteria at appropriately low concentrations. In this work, optimized cationic CDs (bPEI25 000-CDs) were prepared via a hydrothermal method with citric acid and branched PEI25000, which offered a positive surface potential, elimination abilities against Escherichia coli, and relatively high biosafety. The optimized bPEI25 000-CDs can further assemble with the clinical photodynamic therapy (PDT) drug 5-aminolevulinic acid (5-ALA) through electrostatic interaction. Moreover, compared with bPEI25 000-CDs and 5-ALA, the bacterial survival rate was significantly reduced by the ALA-bPEI25 000-CD-induced PDT effect. Even when the dose of bPEI25 000-CD carrier was halved, the bacterial survival could be reduced by 44.4% after light exposure compared to those incubated in the dark. The investigation of the bacterial morphology, membrane potential, and intracellular ROS production suggested that the enhanced antibacterial activity may be due to the membrane dysfunction and cell damage resulting from the high interaction between positively charged ALA-bPEI25 000-CDs and the bacterial cell membrane. Meanwhile, the cationic ALA-bPEI25 000-CDs may facilitate the cellular uptake of 5-ALA, resulting in a more efficient PDT effect. In summary, the antibacterial strategy proposed in this study will provide a novel approach for expanding the application of CD-based nanomedications.

Aptamer-functionalized two-photon SiO2@GQDs hybrid-based signal amplification strategy for targeted cancer imaging.

Targeted imaging is playing an increasingly important role in the early detection and precise diagnosis of cancer. This need has motivated research into sensory nanomaterials that can be constructed into imaging agents to serve as biosensors. Graphene quantum dots (GQDs) as a valuable nanoprobe show great potential for use in two-photon biological imaging. However, most as-prepared GQDs exhibit a low two-photon absorption cross-section, narrow spectral coverage, and "one-to-one" signal conversion mode, which greatly hamper their wide application in sensitive early-stage cancer detection. Herein, a versatile strategy has been employed to fabricate an aptamer Sgc8c-functionalized hybrid as a proof-of-concept of the signal amplification strategy for targeted cancer imaging. In this study, GQDs with two-photon imaging performance, and silica nanoparticles (SiO2 NPs) as nanocarriers to provide amplified recognition events by high loading of GQD signal tags, were adopted to construct a two-photon hybrid-based signal amplification strategy. Thus, the obtained hybrid (denoted SiO2@GQDs) enabled extremely strong fluorescence with a quantum yield up to 0.49, excellent photostability and biocompatibility, and enhanced bright two-photon fluorescence up to 2.7 times that of bare GQDs (excitation at 760 nm; emission at 512 nm). Moreover, further modification with aptamer Sgc8c showed little disruption to the structure of the SiO2@GQDs-hybrid and the corresponding two-photon emission. Hence, SiO2@GQDs-Sgc8c showed specific responses to target cells. Moreover, it could be used as a signal-amplifying two-photon nanoprobe for targeted cancer imaging with high specificity and great efficiency, which exhibits a distinct green fluorescence compared to that of GQDs-Sgc8c or SiO2@GQDs. This signal amplification strategy holds great potential for the accurate early diagnosis of tumors and offers new tools for the detection a wide variety of analytes in clinical application.

Danggui Buxue decoction alleviates cyclophosphamide-induced myelosuppression by regulating ß-hydroxybutyric acid metabolism and suppressing oxidative stress.

CONTEXT: Danggui Buxue Decoction (DBD) is an effective complementary medicine in alleviating myelosuppression after chemotherapy (MAC). However, its mechanism of action is elusive. OBJECTIVE: To illustrate that regulating ß-hydroxybutyric acid (ß-OHB) metabolism and suppressing oxidative stress could be a potential mechanism of action for DBD in alleviating MAC. MATERIALS AND METHODS: After HPLC quantification and dose testing (3, 6 and 10 g/kg, gavage) of DBD, Sprague-Dawley rats were divided into control, cyclophosphamide (CTX) (30 mg/kg CTX for 5 days, intraperitoneal administration) and CTX + DBD groups (6 g/kg DBD for 14 days, gavage). Blood cell counts, thigh bone histological examination, ß-OHB levels, oxidative stress indices and HDAC1 activity were tested. The biological function of ß-OHB was verified in vitro (hBMSC cells were incubated in culture mediums that contained 40 µM CTX and ß-OHB in 0, 1, 2.5, 5, 10 mM) and in vivo (MAC rat model, 3 g/kg ß-OHB for 14 days, gavage). RESULTS: Rats in the CTX + DBD group showed upregulated blood cell counts (118-243%), ß-OHB levels (495 nmol/mL in blood, 122 nmol/mg in marrow supernatant) and downregulated HDAC1 activity (59%), and oxidative stress indices (60-85%). In vitro, 5 mM ß-OHB improved hBMSC cell migration (123%) and proliferation (131%). In vivo, rats treated with 3 g/kg ß-OHB showed upregulated blood cell counts (121-182%) and downregulated HDAC1 activity (64%) and oxidative stress indices (65-83%). DISCUSSION AND CONCLUSIONS: DBD, a traditional Chinese medicine, alleviates MAC by intervening in ß-OHB metabolism and oxidative stress.

Deaminative Cyclization of Tertiary Amines for the Synthesis of 2-Arylquinoline Derivatives with a Nonsubstituted Vinylene Fragment.

With triethylamine as a vinylene source, a convenient protocol for the regioselective synthesis of ß,γ-nonsubstituted 2-arylquinolines from aldehydes and arylamines has been accomplished. The deaminative cyclization is also extended to long-chain tertiary alkylamines, enabling diverse alkyl groups to be concurrently installed into the pyridine rings. This process demonstrates a new conversion pathway for the simultaneous dual C(sp3)-H bond functionalization of tertiary amines, wherein the transient acyclic enamines generated in situ undergo the Povarov reaction.

Association between valproate treatment for acute phase schizophrenia and risk of new onset hypothyroidism.

AIM: To assess the association between acute phase treatment by valproate as an adjunctive drug and risk of new onset hypothyroidism in a large cohort of patients affected by schizophrenia. METHODS: We conducted a retrospective cohort study in a psychiatric hospital in China between January 2016 and December 2018. We obtained approval from the Ethics Committee of the study hospital prior to the commencement of the study. Patients who were diagnosed with schizophrenia and admitted to the study hospital during the study period with thyroid function tests at admission and during hospitalization were included. Patients with abnormal thyroid function at admission were excluded. Hypothyroidism, defined as TSH>4.2 mU/L or on L-thyroxine treatment, was the primary outcome. The primary exposure was adjunctive valproate plus atypical antipsychotics (AAPD), the secondary exposure was lithium plus AAPD and the comparison group was AAPD only. Adjusted relative risk (RR) and 95% confidence interval (CI) were estimated by log-binomial model to assess the independent association between valproate treatment and risk of hypothyroidism. RESULTS: A total of 1622 eligible patients were included the final analysis. Rate of new onset hypothyroidism was 10.7% and 20.9% in AAPD only and valproate plus AAPD groups, respectively. Adjusted RR (95% CI) for valproate plus AAPD was 1.85 (1.44-2.38), with AAPD only group as reference. Similarly, adjusted RR (95% CI) for lithium plus AAPD was 1.93 (1.32-2.69). CONCLUSION: Similar with lithium, valproate as adjunctive drug is associated with increased risk of new onset hypothyroidism during acute phase treatment for schizophrenia.

Integrated Metabolomics and Network Pharmacology Strategy-Driven Active Traditional Chinese Medicine Ingredients Discovery for the Alleviation of Cisplatin Nephrotoxicity.

Renal injury is the main adverse reaction of cisplatin, and many traditional Chinese medicines (TCMs) were proven active against renal toxicity. Here, an integrated metabolomics and network pharmacology strategy was proposed to discover active TCM ingredients for the alleviation of cisplatin nephrotoxicity. First, by interrogating the Human Metabolome Database (HMDB) we collected targets connected to 149 cisplatin nephrotoxicity-related metabolites. Second, targets of kidney damage were obtained from the Therapeutic Target Database (TTD), PharmGKB, Online Mendelian Inheritance in Man (OMIM), and Genetic Association Database (GAD). Common targets of both dysregulated metabolites and kidney damage were then used for TCM active ingredient screening by applying the network pharmacology approach. Eventually, 22 ingredients passed screening criteria, and their antinephrotoxicity activity was assessed in human kidney tubular epithelial (HK2) cells. As a result, 14 ingredients were found to be effective, in which kaempferol showed relatively better activity. Further metabolomics analysis revealed that kaempferol exerted an antinephrotoxicity effect in rats by regulating amino acid, pyrimidine, and purine metabolism as well as lipid metabolism. Collectively, this proposed integrated strategy would promote the transformation of metabolomics research in the field of drug pair discovery for the purpose of reduced toxicity and increased efficiency.

An extendable all-in-one injection twin derivatization LC-MS/MS strategy for the absolute quantification of multiple chemical-group-based submetabolomes.

The ability of LC-MS/MS for high coverage metabolite analysis lags behind the requirements of global metabolomics. The introduction of chemical derivatizations could significantly extend the ability of LC-MS/MS with enhanced MS response and improved LC separation, which has been serving as a promising quantitative tool for metabolomic analysis. However, as one specific derivatization reagent usually targets to a certain moiety, only a single chemical-group-based submetabolome could be analyzed in one injection. Therefore, the coverage of detected metabolites by derivatization-based LC-MS/MS is largely limited. To overcome this technical obstacle of derivatization-based LC-MS and increase submetabolome coverage, we proposed an extendable all-in-one injection LC-MS/MS strategy. 5-dimethylamino-naphthalene-1-sulfonyl chloride (Dns-Cl)/5-diethylamino-naphthalene-1-sulfonyl chloride (Dens-Cl) and 5-dimethylamino-naphthalene-1-sulfonyl piperazine (Dns-PP)/5-diethylamino-naphthalene-1-sulfonyl piperazine (Dens-PP) were used as twins labeling reagents for amino/phenol and carboxyl submetabolomes, respectively. "Series Mode" and "Parallel Mode" were proposed and investigated using eight representative standards with the consideration of interaction between different derivatization systems, time-consumption, and extendability. As a result, we found that "Series Mode" led to yield reduction, while "Parallel Mode" gave identical results with those of individual derivatization. Finally, a "Parallel Mode" was chosen to develop an extendable all-in-one injection twin derivatization LC-MS/MS strategy to quantify eighty metabolites assigned to five classes of microbial metabolites, including polyamines, amino acids, indole derivatives, bile acids, and free fatty acids. This well-validated method quantified 67 metabolites absolutely and discovered additional 40 differential metabolites compared with the untargeted method in rat serum from irinotecan (CPT-11)-induced gastrointestinal toxicity model.

Biomarker Discovery for Cytochrome P450 1A2 Activity Assessment in Rats, Based on Metabolomics.

Cytochrome P450 1A2 (CYP1A2) is one of the major CYP450 enzymes (CYPs) in the liver, and participates in the biotransformation of various xenobiotics and endogenous signaling molecules. The expression and activity of CYP1A2 show large individual differences, due to genetic and environmental factors. In order to discover non-invasive serum biomarkers associated with hepatic CYP1A2, mass spectrometry-based, untargeted metabolomics were first conducted, in order to dissect the metabolic differences in the serum and liver between control rats and ß-naphthoflavone (an inducer of CYP1A2)-treated rats. Real-time reverse transcription polymerase chain reaction and pharmacokinetic analysis of phenacetin and paracetamol were performed, in order to determine the changes of mRNA levels and activity of CYP1A2 in these two groups, respectively. Branched-chain amino acids phenylalanine and tyrosine were ultimately focalized, as they were detected in both the serum and liver with the same trends. These findings were further confirmed by absolute quantification via a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics approach. Furthermore, the ratio of phenylalanine to tyrosine concentration was also found to be highly correlated with CYP1A2 activity and gene expression. This study demonstrates that metabolomics can be a potentially useful tool for biomarker discovery associated with CYPs. Our findings contribute to explaining interindividual variations in CYP1A2-mediated drug metabolism.

Pharmacometabolomic prediction of individual differences of gastrointestinal toxicity complicating myelosuppression in rats induced by irinotecan.

Pharmacometabolomics has been already successfully used in toxicity prediction for one specific adverse effect. However in clinical practice, two or more different toxicities are always accompanied with each other, which puts forward new challenges for pharmacometabolomics. Gastrointestinal toxicity and myelosuppression are two major adverse effects induced by Irinotecan (CPT-11), and often show large individual differences. In the current study, a pharmacometabolomic study was performed to screen the exclusive biomarkers in predose serums which could predict late-onset diarrhea and myelosuppression of CPT-11 simultaneously. The severity and sensitivity differences in gastrointestinal toxicity and myelosuppression were judged by delayed-onset diarrhea symptoms, histopathology examination, relative cytokines and blood cell counts. Mass spectrometry-based non-targeted and targeted metabolomics were conducted in sequence to dissect metabolite signatures in predose serums. Eventually, two groups of metabolites were screened out as predictors for individual differences in late-onset diarrhea and myelosuppression using binary logistic regression, respectively. This result was compared with existing predictors and validated by another independent external validation set. Our study indicates the prediction of toxicity could be possible upon predose metabolic profile. Pharmacometabolomics can be a potentially useful tool for complicating toxicity prediction. Our findings also provide a new insight into CPT-11 precision medicine.

Metabolomics Reveals the Efficacy of Caspase Inhibition for Saikosaponin D-Induced Hepatotoxicity.

Saikosaponin d (SSd) is a major hepatoprotective component of saikosaponins derived from Radix Bupleuri, which was also linked to hepatotoxicity. Previous studies have demonstrated that caspases play a key role in SSd-induced liver cell death. Our in vitro and in vivo studies also showed that treatment with caspase inhibitor z-VAD-fmk could significantly reduce the L02 hepatocyte cells death and lessen the degree of liver damage in mice caused by SSd. In order to further reveal the underlying mechanisms of caspase inhibition in SSd-induced hepatotoxicity, mass spectrometry based untargeted metabolomics was conducted. Significant alterations in metabolic profiling were observed in SSd-treated group, which could be restored by caspase inhibition. Bile acids and phospholipids were screened out to be most significant by spearman correlation analysis, heatmap analysis and S-Plot analysis. These findings were further confirmed by absolute quantitation of bile acids via targeted metabolomics approach. Furthermore, cytokine profiles were analyzed to identify potential associations between inflammation and metabolites. The study could provide deeper insight into the hepatotoxicity of SSd and the efficacy of caspase inhibition.

Combination of LC/MS and GC/MS based metabolomics to study the hepatotoxic effect of realgar nanoparticles in rats.

Realgar nanoparticles (NPs) are increasingly used as therapeutic agents for their enhanced anti-proliferation effect and cytotoxicity on cancer cells. However, the alteration of particle size may enhance biological reactivity as well as toxicity. A LC/MS and GC/MS based metabolomics approach was employed to explore the mechanism of realgar NPs-induced hepatotoxicity and identify potential biomarkers. Male Sprague-Dawley rats were administrated intragastrically with realgar or realgar NPs at a dose of 1.0 g·kg-1·d-1 for 28 days and toxic effects of realgar NPs on liver tissues were examined by biochemical indicator analysis and histopathologic examination. Increased levels of serum enzymes and high hepatic steatosis were discovered in the realgar NPs treated group. Multivariate data analysis revealed that rats with realgar NPs-induced hepatotoxicity could be distinctively differentiated from the animals in the control and realgar treated groups. In addition, 21 and 32 endogenous metabolites were apparently changed in the serum and live extracts, respectively. Realgar NPs might induce free fatty acid and triglyceride accumulation, resulting in hepatotoxicity. In conclusion, the present study represents the first comprehensive LC/MS- and GC/MS-based metabolomics analysis of realgar NPs-induced hepatotoxicity, which may help further research of nanotoxicity.

Time-resolved metabolomics analysis of individual differences during the early stage of lipopolysaccharide-treated rats.

Lipopolysaccharide (LPS) can lead to uncontrollable cytokine production and eventually cause fatal sepsis syndrome. Individual toxicity difference of LPS has been widely reported. In our study we observed that two thirds of the rats (24/36) died at a given dose of LPS, while the rest (12/36) survived. Tracking the dynamic metabolic change in survival and non-survival rats in the early stage may reveal new system information to understand the inter-individual variation in response to LPS. As the time-resolved datasets are very complex and no single method can elucidate the problem clearly and comprehensively, the static and dynamic metabolomics methods were employed in combination as cross-validation. Intriguingly, some common results have been observed. Lipids were the main different metabolites between survival and non-survival rats in pre-dose serum and in the early stage of infection with LPS. The LPS treatment led to S-adenosly-methionine and total cysteine individual difference in early stage, and subsequent significant perturbations in energy metabolism and oxidative stress. Furthermore, cytokine profiles were analyzed to identify potential biological associations between cytokines and specific metabolites. Our collective findings may provide some heuristic guidance for elucidating the underlying mechanism of individual difference in LPS-mediated disease.

A novel liquid chromatography tandem mass spectrometry method for simultaneous determination of branched-chain amino acids and branched-chain α-keto acids in human plasma.

Branched-chain amino acids (BCAAs) and branched-chain α-keto acids (BCKAs) play significant biological roles as they are involved in protein and neurotransmitter synthesis as well as energy metabolism pathways. To routinely and accurately study the dynamics of BCAAs and BCKAs in human diseases, e.g. cerebral infarction, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated. The plasma samples were deproteinized with acetonitrile, and then separated on a reversed phase C18 column with a mobile phase of 0.1 % formic acid (solvent A)-methanol (solvent B) using gradient elution. The detection of BCAAs and BCKAs was conducted in multiple reaction monitoring with positive/negative electrospray ionization switching mode. Biologically relevant isomers such as leucine and isoleucine were individually quantified by combining chromatographic separation and fragmentation. Good linearity (R (2) > 0.99) was obtained for all six analytes with the limits of detection from 0.1 to 0.2 µg/mL. The intra-day and inter-day accuracy ranged from 93.7 to 108.4 % and the relative standard deviation (RSD) did not exceed 15.0 %. The recovery was more than 80 % with RSD less than 14.0 %. The main improvements compared to related, state-of-the-art methods included enhanced sensitivity, enhanced separation of isomers, and reduced complexity of sample processing. Finally, the validated method was applied to analyze the plasma samples of healthy volunteers and patients suffering cerebral infarction, and significant differences in the concentration levels of BCAAs and BCKAs were observed.

Metabolomics based on liquid chromatography with mass spectrometry reveals the chemical difference in the stems and roots derived from Ephedra sinica.

To better understand different traditional uses of the stems (known as Mahuang) and roots (known as Mahuanggen) of Ephedra sinica, their chemical difference should be investigated. In this study, an ultra-fast liquid chromatography coupled with ion trap time-of-flight mass spectrometry untargeted metabolomics approach was established to reveal global chemical difference between Mahuang and Mahuanggen. Clear separation was observed in scores plots of principal component analysis and orthogonal partial least squares-discriminant analysis. Twenty two chemical markers responsible for such separation were screened out and unambiguously/tentatively characterized. Then chemical markers of pharmacologically important ephedrine and pseudoephedrine were absolutely quantified using liquid chromatography coupled with tandem mass spectrometry under multiple reaction monitoring mode. The results showed that Mahuang was rich in ephedrine-type alkaloids, while Mahuanggen was rich in macrocyclic spermine alkaloids. Additionally, different types of flavan-3-ols and flavones exist in Mahuang and Mahuanggen extracts. This research facilitates a better understanding of different traditional uses of Mahuang and Mahuanggen and provides references for chemical analysis of other medicinal plants.

Medicinal uses, phytochemistry and pharmacology of the genus Dictamnus (Rutaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Seven species from the genus Dictamnus are distributed throughout Europe and North Asia and only two species grow in China. One is Dictamnus dasycarpus Turcz., which could be found in many areas of China and has been recorded in Chinese Pharmacopoeia. The other is Dictamnus angustifolius G. Don ex Sweet, which is only present in Xinjiang province and has been used as an alternative for Dictamnus dasycarpus in the local for the treatment of rheumatism, bleeding, itching, jaundice, chronic hepatitis and skin diseases. The present paper reviewed the traditional uses, phytochemistry, pharmacology and toxicology of the genus Dictamnus. MATERIALS AND METHODS: Information on the Dictamnus species was collected from classic books about Chinese herbal medicine and globally accepted scientific databases including PubMed, Elsevier, ASC, Scopus, Google Scholar, Web of Science, CNKI and others. RESULTS: About 170 chemical compounds, which include quinoline alkaloids, limonoids, sesquiterpenes, coumarins, flavonoids and steroids, have been isolated from the genus Dictamnus. The characteristic and active constituents of Dictamnus species are considered to be quinoline alkaloids and limonoids, which exhibited a broad spectrum of biological activities such as anti-cancer, anti-inflammation, anti-microbe, anti-platelet-aggregation, vascular-relaxation, anti-insect, anti-HIV, anti-allergy and neuroprotection. Moreover, quinoline alkaloids and limonoids could be used as quality control markers to distinguish different species from the genus Dictamnus. However, there were also some reports on the toxic hepatitis and phototoxic effect of Dictamnus species, and the related research needs to be further studied. CONCLUSION: In this review, we summarized the chemical constituents, pharmacology, quality control and toxicology of the species from genus Dictamnus. Phytochemical investigations indicated that quinoline alkaloids and limonoids were the major bioactive components with potential cytotoxic, neuroprotective, anti-inflammatory, antimicrobial, anti-platelet-aggregation and vascular relaxing activities. These two kinds of compounds have attracted great interests in the past few years and may have great potential to be new drug lead compounds.