Mass Spectrometry Probe Combined with Machine Learning to Capture the Relationship between Metabolites and Mitochondrial Complex Activity at the Whole-Cell Level.

Mitochondrial complex activity controls a multitude of physiological processes by regulating the cellular metabolism. Current methods for evaluating mitochondrial complex activity mainly focus on single metabolic reactions within mitochondria. These methods often require fresh samples in large quantities for mitochondria purification or intact mitochondrial membranes for real-time monitoring. Confronting these limitations, we shifted the analytical perspective toward interactive metabolic networks at the whole-cell level to reflect mitochondrial complex activity. To this end, we compiled a panel of mitochondrial respiratory chain-mapped metabolites (MRCMs), whose perturbations theoretically provide an overall reflection on mitochondrial complex activity. By introducing N-dimethyl-p-phenylenediamine and N-methyl-p-phenylenediamine as a pair of mass spectrometry probes, an ultraperformance liquid chromatography-tandem mass spectrometry method with high sensitivity (LLOQ as low as 0.2 fmol) was developed to obtain accurate quantitative data of MRCMs. Machine learning was then combined to capture the relationship between MRCMs and mitochondrial complex activity. Using Complex I as a proof-of-concept, we identified NADH, alanine, and phosphoenolpyruvate as metabolites associated with Complex I activity based on the whole-cell level. The effectiveness of using their concentrations to reflect Complex I activity was further validated in external data sets. Hence, by capturing the relationship between metabolites and mitochondrial complex activity at the whole-cell level, this study explores a novel analytical paradigm for the interrogation of mitochondrial complex activity, offering a favorable complement to existing methods particularly when sample quantities, type, and treatment timeliness pose challenges. More importantly, it shifts the focus from individual metabolic reactions within mitochondria to a more comprehensive view of an interactive metabolic network, which should serve as a promising direction for future research into the functional architecture between mitochondrial complexes and metabolites.

Innovation for continuous aerobic granular sludge process in actual municipal sewage treatment: Self-circulating up-flow fluidized bed process.

Aerobic granular sludge (AGS) capable of nitrogen and phosphorus removal is mainly limited to the applications in sequencing batch reactors. This study introduced an innovative continuous self-circulating up-flow fluidized bed process (Zier process) using separate aeration. The process was composed of an anoxic column (Zier-A), aeration column (Zier-OO) and aerobic column (Zier-O), and was used to treat actual municipal sewage continuously for 170 days. The process achieved self-circulation of 20-32 times and an up-flow velocity within the reactor of 7-16 m/h which were accurately controlled with only separate aeration. The larger proportion of self-circulating multiple times contributed to particle formation and stability, providing hydraulic shear conditions, and screened the precipitation performance of the granular sludge (GS). Meanwhile, the dissolved oxygen (DO) of Zier-O was controlled at 0.1-0.3 mg/L, and the DO of Zier-A input water was zero. The accurate oxygen supply enhanced simultaneous nitrification and denitrification (SND) as well as short-cut nitrification and denitrification in Zier-O and improved the COD utilization rate and the nitrogen removal rate in Zier-A. The COD treatment capacity reached 2.46 kg-COD/(m³·d). With a hydraulic retention time of 10 h, the process consistently ensured that the average concentrations of ammonia nitrogen and total nitrogen in the effluent were maintained below 5 and 15 mg/L, respectively. Moreover, the process maintained the shape and stability of GS, the median diameter of GS ranged between 300-1210 µm, the percentage of mass with particle size distribution < 200 µm at a height of 150 cm within Zier-A and Zier-O accounted for as low as 0.04%-0.05%, and showed good settling performance. The suspended solids in effluent can be maintained at 50-80 mg/L. Overall, the unique structural setting and control method of the Zier process provide another approach for the application of continuous AGS treatment for municipal sewage.

Targeted Quantification of Glutathione/Arginine Redox Metabolism Based on a Novel Paired Mass Spectrometry Probe Approach for the Functional Assessment of Redox Status.

Glutathione (GSH) redox control and arginine metabolism are critical in regulating the physiological response to injury and oxidative stress. Quantification assessment of the GSH/arginine redox metabolism supports monitoring metabolic pathway shifts during pathological processes and their linkages to redox regulation. However, assessing the redox status of organisms with complex matrices is challenging, and single redox molecule analysis may not be accurate for interrogating the redox status in cells and in vivo. Herein, guided by a paired derivatization strategy, we present a new ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based approach for the functional assessment of biological redox status. Two structurally analogous probes, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and newly synthesized 2-methyl-6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (MeAQC), were set for paired derivatization. The developed approach was successfully applied to LPS-stimulated RAW 264.7 cells and HDM-induced asthma mice to obtain quantitative information on GSH/arginine redox metabolism. The results suggest that the redox status was remarkably altered upon LPS and HDM stimulation. We expect that this approach will be of good use in a clinical biomarker assay and potential drug screening associated with redox metabolism, oxidative damage, and redox signaling.

The regulatory mechanism of rapid lignification for timely anther dehiscence.

Anther dehiscence is a crucial event in plant reproduction, tightly regulated and dependent on the lignification of the anther endothecium. In this study, we investigated the rapid lignification process that ensures timely anther dehiscence in Arabidopsis. Our findings reveal that endothecium lignification can be divided into two distinct phases. During Phase I, lignin precursors are synthesized without polymerization, while Phase II involves simultaneous synthesis of lignin precursors and polymerization. The transcription factors MYB26, NST1/2, and ARF17 specifically regulate the pathway responsible for the synthesis and polymerization of lignin monomers in Phase II. MYB26-NST1/2 is the key regulatory pathway responsible for endothecium lignification, while ARF17 facilitates this process by interacting with MYB26. Interestingly, our results demonstrate that the lignification of the endothecium, which occurs within approximately 26 h, is much faster than that of the vascular tissue. These findings provide valuable insights into the regulation mechanism of rapid lignification in the endothecium, which enables timely anther dehiscence and successful pollen release during plant reproduction.

Enzyme-Driven LC-HRMS Approach for Specific Recognition of 12α-Hydroxy Bile Acids.

The synthesis of 12α-hydroxylated bile acids (12HBAs) and non-12α-hydroxylated bile acids (non-12HBAs) occurs via classical and alternative pathways, respectively. The composition of these BAs is a crucial index for pathophysiologic assessment. However, accurately differentiating 12HBAs and non-12HBAs is highly challenging due to the limited standard substances. Here, we innovatively introduce 12α-hydroxysteroid dehydrogenase (12α-HSDH) as an enzymatic probe synthesized by heterologous expression in Escherichia coli, which can specifically and efficiently convert 12HBAs in vitro under mild conditions. Coupled to the conversion rate determined by liquid chromatography-high resolution mass spectrometry (LC-HRMS), this enzymatic probe allows for the straightforward distinguishing of 210 12HBAs and 312 non-12HBAs from complex biological matrices, resulting in a BAs profile with a well-defined hydroxyl feature at the C12 site. Notably, this enzyme-driven LC-HRMS approach can be extended to any molecule with explicit knowledge of enzymatic transformation. We demonstrate the practicality of this BAs profile in terms of both revealing cross-species BAs heterogeneity and monitoring the alterations of 12HBAs and non-12HBAs under asthma disease. We envisage that this work will provide a novel pattern to recognize the shift of BA metabolism from classical to alternative synthesis pathways in different pathophysiological states, thereby offering valuable insights into the management of related diseases.

FNICM: A New Methodology To Identify Core Metabolites Based on Significantly Perturbed Metabolic Subnetworks.

Metabolomics has emerged as a powerful tool in biomedical research to understand the pathophysiological processes and metabolic biomarkers of diseases. Nevertheless, it is a significant challenge in metabolomics to identify the reliable core metabolites that are closely associated with the occurrence or progression of diseases. Here, we proposed a new research framework by integrating detection-based metabolomics with computational network biology for function-guided and network-based identification of core metabolites, namely, FNICM. The proposed FNICM methodology is successfully utilized to uncover ulcerative colitis (UC)-related core metabolites based on the significantly perturbed metabolic subnetwork. First, seed metabolites were screened out using prior biological knowledge and targeted metabolomics. Second, by leveraging network topology, the perturbations of the detected seed metabolites were propagated to other undetected ones. Ultimately, 35 core metabolites were identified by controllability analysis and were further hierarchized into six levels based on confidence level and their potential significance. The specificity and generalizability of the discovered core metabolites, used as UC's diagnostic markers, were further validated using published data sets of UC patients. More importantly, we demonstrated the broad applicability and practicality of the FNICM framework in different contexts by applying it to multiple clinical data sets, including inflammatory bowel disease, colorectal cancer, and acute coronary syndrome. In addition, FNICM was also demonstrated as a practicality methodology to identify core metabolites correlated with the therapeutic effects of Clematis saponins. Overall, the FNICM methodology is a new framework for identifying reliable core metabolites for disease diagnosis and drug treatment from a systemic and a holistic perspective.

UPLC-Q-TOF-MS/MS-based urine metabolomics studies on the toxicity and detoxication of Tripterygium wilfordii Hook. f. after roasting.

Tripterygium wilfordii (TW), a well-known traditional Chinese medicine, was widely used in the treatment of autoimmune disorders and inflammatory diseases. However, the clinical use of TW was limited by severe toxicities, such as hepatotoxicity and nephrotoxicity. Our previous studies indicated that roasting was an effective approach for reducing TW-induced toxicity. After roasting, celastrol was completely decomposed, partially converted into 1-hydroxy-2,5,8-trimethyl-9-fluorenone and the total alkaloids content were significantly reduced. However, the detoxication mechanisms of roasting on TW were poorly unknown. This study aimed to explore the toxicity and detoxification mechanisms of TW after roasting based on urine metabolomics. Promising biomarkers were evaluated by multiple comparison analyses. Sixteen toxicity biomarkers were identified between control group and total extract group. Twelve toxicity biomarkers were identified between control group and total alkaloids group. Eight toxicity biomarkers were identified between control group and celastrol group. These metabolites were mainly involved in seven metabolic pathways, summarized as pentose and glucuronate interconversions, lipid metabolism (sphingolipid metabolism, glycerophospholipid metabolisms, fatty acid biosynthesis and steroid hormone biosynthesis) and amino acid metabolism (taurine and hypotaurine metabolism, tryptophan metabolism). After roasting, the toxicities of total extract, total alkaloids and celastrol were relieved by ameliorative serum parameters and pathological changes in hepatic and renal tissues which revealed that the reduction of celastrol and total alkaloids played important roles in the detoxification of roasting on TW. Furthermore, roasting regulated the levels of fourteen potential biomarkers in the total extract group, ten potential biomarkers in the total alkaloids group and seven candidate biomarkers in the celastrol group to normal levels. Biological pathway analysis revealed that roasting may ameliorate TW-induced metabolic disorders in pentose and glucuronate interconversions, lipid metabolism and amino acid metabolism. This study provided evidence for the application of roasting in TW.

Hyposterolactone A, a 3α-Hydroxy Steroidal Lactone from the Deep-Sea-Derived Fungus Hypocrea sp. ZEN14.

Chemical investigation of the deep-sea-derived fungus Hypocrea sp. ZEN14 afforded a new 3α-hydroxy steroidal lactone, hyposterolactone A (1) and 25 known secondary metabolites (2-26). The structure of the new compound was established by detailed spectroscopic analysis, electronic circular dichroism (ECD) calculation as well as a J-based configuration analysis. Compound 10 showed potent cytotoxicity against Huh7 and Jurkat cells with IC50 values of 1.4 µM and 6.7 µM, respectively.

Pseudotargeted metabolomics-based random forest model for tracking plant species from herbal products.

BACKGROUND: The "one-to-multiple" phenomenon is prevalent in medicinal herbs. Accurate species identification is critical to ensure the safety and efficacy of herbal products but is extremely challenging due to their complex matrices and diverse compositions. PURPOSE: This study aimed to identify the determinable chemicalome of herbs and develop a reasonable strategy to track their relevant species from herbal products. METHODS: Take Astragali Radix-the typical "one to multiple" herb, as a case. An in-house database-driven identification of the potentially bioactive chemicalome (saponins and flavonoids) in AR was performed. Furthermore, a pseudotargeted metabolomics method was first developed and validated to obtain high-quality semi-quantitative data. Then based on the data matrix, the random forest algorithm was trained to predict Astragali Radix species from commercial products. RESULTS: The pseudotargeted metabolomics method was first developed and validated to obtain high-quality semi-quantitative data (including 56 saponins and 49 flavonoids) from 26 batches of AR. Then the random forest algorithm was well-trained by importing the valid data matrix and showed high performance in predicting Astragalus species from ten commercial products. CONCLUSION: This strategy could learn species-special combination features for accurate herbal species tracing and could be expected to promote the traceability of herbal materials in herbal products, contributing to manufacturing standardization.

One-stage partial nitrification/anammox system with short sludge retention time and low mixed liquor suspended solids long-term control: Establishment, performance and sludge characteristics.

Anammox is a green, economical and efficient nitrogen removal process. Most successful anammox studies are based on biofilm- or granule-based systems, but pure floc sludge partial nitrification (PN) and anammox (A) systems that are not inoculated with anaerobic ammonia oxidizing bacteria (AnAOB) are rarely reported. If the anammox process occurs in floc-based systems, the large specific surface areas provide more efficient nitrogen removal, and are much more economical in terms of construction and investment. This study investigated the establishment, performance and sludge characteristics of a one-stage PN/A system with pure floc sludge and exhibited a short sludge retention time (SRT) and low mixed liquor suspended solids (SS) content. The experiment was run for approximately 1260 days and divided into five stages based on the SRTs and influent ammonia concentrations treating synthetic wastewater with no organic matter. The results showed that the AnAOB were successfully cultivated and enriched with ordinary nitrification and denitrification sludge, which formed a pure floc-based anammox system with a short SRT (at least 14 days) and a low SS control. The maximum nitrogen removal efficiency and sludge removal loading rate reached 87.1 % and 3.16 kg N/(kg VSS·d) with ammonia loading rates = 0.55 and 0.56 kg-N/(m3·d), dissolved oxygen = 0.2 and 0 mg/L, temperature = 30 and 28 °C, mixed liquor volatile suspended solid (VSS) = 800 and 130 mg/L, free ammonia (FA)/VSS = 3.5 and 47.5 mg NH3-N/g VSS and SRT = 30 and 15 days, respectively. Moreover, the FA/VSS ratio was used to determine the operating performance of the PN/A system, and the thresholds for inhibiting nitrite-oxidizing bacteria and ammonia-oxidizing bacteria, including AnAOB, were 0.5-50 and above 50 mg NH3-N/g VSS, respectively. The floc-based one-stage PN/A systems proposed in this study provide reductions in the volumes, and floor areas for the reactor tanks, and in the cost of the carrier.

Comparison of Semen Aesculi species from different geographical origins and horse chestnut based on metabolomics and chemometrics analysis.

BACKGROUND: Semen Aesculi, a traditional Chinese herbal medicine, has a long history of use for treating chest and abdominal pain with distension. In addition, the horse chestnut (Aesculus hippocastanum L.) is another species of Aesculus in Europe and has notable clinical significance in alleviating chronic venous insufficiency, hemorrhoids, and postoperative edema. Thus, highlighting the comparative study of Semen Aesculi and horse chestnut may broaden clinical applications. OBJECTIVES: To conduct a comprehensive comparative analysis on the chemical profiling of these two varieties and determine whether they have equivalent clinical efficacy by integrating plant metabolomics and multivariate statistical methods. METHODS: Initially, a comprehensive characterisation was performed using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) platform, and in total 44 active ingredients were identified. Then, untargeted metabolomics combined with principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) was applied for the discrimination of a German species and three official Chinese species. Next, 24 marker compounds responsible for the discrimination of different species were screened out and used to predict the species of unknown samples by genetic algorithm-optimised support vector machine (GA-SVM) with a high prediction accuracy. Finally, a heatmap visualisation was employed for clarifying the distribution of the identified active ingredients. RESULTS: The three species of Chinese Semen Aesculi showed distinct separation from each other, while European horse chestnut and Aesculus chinensis Bunge were similar in chemical composition. CONCLUSIONS: This work provided experimental evidence for further expanding the clinical application of Chinese Semen Aesculi and promoted the species identification and quality control of Semen Aesculi.

Reactive oxygen species contribute to delirium-like behavior by activating CypA/MMP9 signaling and inducing blood-brain barrier impairment in aged mice following anesthesia and surgery.

Postoperative delirium (POD) is common in the elderly and is associated with poor clinical outcomes. Reactive oxygen species (ROS) and blood-brain barrier (BBB) damage have been implicated in the development of POD, but the association between these two factors and the potential mechanism is not clear. Cyclophilin A (CypA) is a specifically chemotactic leukocyte factor that can be secreted in response to ROS, which activates matrix metalloproteinase 9 (MMP9) and mediates BBB breakdown. We, therefore, hypothesized that ROS may contribute to anesthesia/surgery-induced BBB damage and delirium-like behavior via the CypA/MMP9 pathway. To test these hypotheses, 16-month-old mice were subjected to laparotomy under 3% sevoflurane anesthesia (anesthesia/surgery) for 3 h. ROS scavenger (N-acetyl-cysteine) and CypA inhibitor (Cyclosporin A) were used 0.5 h before anesthesia/surgery. A battery of behavior tests (buried food test, open field test, and Y maze test) was employed to evaluate behavioral changes at 24 h before and after surgery in the mice. Levels of tight junction proteins, CypA, MMP9, postsynaptic density protein (PSD)-95, and synaptophysin in the prefrontal cortex were assessed by western blotting. The amounts of ROS and IgG in the cortex of mice were observed by fluorescent staining. The concentration of S100ß in the serum was detected by ELISA. ROS scavenger prevented the reduction in TJ proteins and restored the permeability of BBB as well as reduced the levels of CypA/MMP9, and further alleviated delirium-like behavior induced by anesthesia/surgery. Furthermore, the CypA inhibitor abolished the increased levels of CypA/MMP, which reversed BBB damage and ameliorated delirium-like behavior caused by ROS accumulation. Our findings demonstrated that ROS may participate in regulating BBB permeability in aged mice with POD via the CypA/MMP9 pathway, suggesting that CypA may be a potential molecular target for preventing POD.

Least absolute shrinkage and selection operator-based prediction of collision cross section values for ion mobility mass spectrometric analysis of lipids.

Collision cross section (CCS) values generated from ion mobility mass spectrometry (IM-MS) have commonly been employed to facilitate lipid identification. However, this is hindered by the limited available lipid standards. Recently, CCS values were predicted by means of computational calculations, though the prediction precision was generally not good and the predicted CCS values of the lipid isomers were almost identical. To address this challenge, a least absolute shrinkage and selection operator (LASSO)-based prediction method was developed for the prediction of lipids' CCS values in this study. In this method, an array of molecular descriptors were screened and optimized to reflect the subtle differences in structures among the different lipid isomers. The use of molecular descriptors together with a wealth of standard CCS values for the lipids (365 in total) significantly improved the accuracy and precision of the LASSO model. Its accuracy was externally validated with median relative errors (MREs) of <1.1% using an independent data set. This approach was demonstrated to allow differentiation of cis/trans and sn-positional isomers. The results also indicated that the LASSO-based prediction method could practically reduce false-positive identifications in IM-MS-based lipidomics.

An in-house database-driven untargeted identification strategy for deep profiling of chemicalome in Chinese medicinal formula.

Deep profiling of chemicalome in Chinese medicinal formulas is vital for disclosing the secret underlying their effectiveness. To address this issue, an in-house database-driven untargeted identification strategy was proposed with the use of ultra-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry. Firstly, an in-house mass spectral database for the analyzed herbs was constructed, and database querying was performed for rapid recognition of known compounds. Secondly, a chemical diagnostic characteristics algorithm was originally developed for deep mining unrecorded ions, and thus expanding coverage of components beyond the database. Additionally, we proposed evaluation criteria for the untargeted identification of compounds with different confidence levels. As a case study, the integrated strategy was applied to comprehensively characterize complex multi-type components in Gegen-Qinlian Decoction. A total of 381 compounds were characterized and annotated with four different confidence levels, and 88.40% of these annotated compounds were successfully re-identified in triplicate analyses with a different instrument. The integrated strategy was demonstrated powerful in deep profiling of chemicalome in Chinese medicinal formulas with higher throughput, analytical sharpness, and lower omission ratios.

Dexmedetomidine Combined with Femoral Nerve Block Provides Effective Analgesia Similar to Femoral Nerve Combined with Sciatic Nerve Block in Patients Undergoing Total Knee Arthroplasty: A Randomized Controlled Study.

BACKGROUND: Total knee arthroplasty (TKA) is a severe traumatic procedure, and femoral nerve block (FNB) combined with a sciatic nerve block (SNB) is widely used in TKA. However, injury of the sciatic nerve is clinically reported. Dexmedetomidine (DEX) could reduce stress and inflammation, as well as improve pain in TKA. This study aims to observe the analgesic impact of DEX combined with FNB in TKA. METHODS: Eighty-eight patients undergoing TKA were included and randomly divided into two groups: DF group (FNB combined with DEX 0.6µg/kg before surgery, followed by DEX 0.2-0.4µg/kg/h until articular closure) and SF group (FNB combined with SNB). Each nerve was blocked with 0.375% ropivacaine 20mL, and all patients received general anesthesia routinely. The primary endpoint was the pain visual analog scale (VAS) score during activities at postoperative 24 hours. RESULTS: There was no statistical difference in the pain VAS scores at any time point. The mean duration of analgesia for patients with rescue analgesic requests was comparable between the two groups: 25.4 ± 6.3 hours in the DF group vs 24.8 ± 6.4 hours in the SF group (two-sample t-test, p=0.738). The total dose of sufentanil was similar between groups (P=0.355). The maintenance dose of propofol and dose of rescue analgesics were comparable (all P>0.05). There were no statistical differences in the incidence of adverse events. However, the time to extubate in the DF group was significantly longer than those in the SF group (P<0.001). CONCLUSION: DEX combined with FNB could provide effective analgesia similar to SNB combined with FNB in TKA. CLINICAL TRIAL REGISTRATION: The trial was registered at the Chinese Clinical Trial Registry on November 17, 2019 (identifier: ChiCTR1900027552).

Targeting tryptophan metabolism reveals Clematichinenoside AR alleviates triptolide-induced hepatotoxicity.

Liver toxicity induced by Triptolide (TP) has limited its clinical application on rheumatoid arthritis (RA). Saponins have been proved as an efficacious remedy to mitigate hepatotoxicity. However, the mechanism of reducing hepatotoxicity by saponins intervention remains incompletely characterized. Tryptophan (Trp) metabolites activate transcriptional regulators to mediate host detoxification responses. Our study aimed to investigate whether Clematichinenoside AR (C-AR) could attenuate TP-induced liver damage by regulating Trp metabolism. We used targeted metabolomics to quantify Trp metabolites in the serum and liver samples of collagen-induced arthritis rats treated by TP. Multiple comparison analyses helped the evaluation of promising biomarkers. The pronounced changed levels of Trp, indole acetic acid, and indole-3-carboxaldehyde in the serum and indole acetic acid, indole, and tryptamine in the liver are relevant to TP-induced liver injury. Intervention with C-AR could relieve TP-induced hepatotoxicity evidenced by ameliorative serum parameters and hepatic histology. In addition, C-AR regulated the levels of these indoles biomarker candidates to normal. Therapeutic modulation with natural compounds might be a useful clinical strategy to ameliorate toxicity induced by TP. Deciphering Trp metabolism will facilitate a better understanding of the pathogenesis of diseases and drug responding.

Metabolomics Tools Assisting Classic Screening Methods in Discovering New Antibiotics from Mangrove Actinomycetia in Leizhou Peninsula.

Mangrove actinomycetia are considered one of the promising sources for discovering novel biologically active compounds. Traditional bioactivity- and/or taxonomy-based methods are inefficient and usually result in the re-discovery of known metabolites. Thus, improving selection efficiency among strain candidates is of interest especially in the early stage of the antibiotic discovery program. In this study, an integrated strategy of combining phylogenetic data and bioactivity tests with a metabolomics-based dereplication approach was applied to fast track the selection process. A total of 521 actinomycetial strains affiliated to 40 genera in 23 families were isolated from 13 different mangrove soil samples by the culture-dependent method. A total of 179 strains affiliated to 40 different genera with a unique colony morphology were selected to evaluate antibacterial activity against 12 indicator bacteria. Of the 179 tested isolates, 47 showed activities against at least one of the tested pathogens. Analysis of 23 out of 47 active isolates using UPLC-HRMS-PCA revealed six outliers. Further analysis using the OPLS-DA model identified five compounds from two outliers contributing to the bioactivity against drug-sensitive A. baumannii. Molecular networking was used to determine the relationship of significant metabolites in six outliers and to find their potentially new congeners. Finally, two Streptomyces strains (M22, H37) producing potentially new compounds were rapidly prioritized on the basis of their distinct chemistry profiles, dereplication results, and antibacterial activities, as well as taxonomical information. Two new trioxacarcins with keto-reduced trioxacarcinose B, gutingimycin B (16) and trioxacarcin G (20), together with known gutingimycin (12), were isolated from the scale-up fermentation broth of Streptomyces sp. M22. Our study demonstrated that metabolomics tools could greatly assist classic antibiotic discovery methods in strain prioritization to improve efficiency in discovering novel antibiotics from those highly productive and rich diversity ecosystems.

Blood Levels of Glycated Hemoglobin, D-Dimer, and Fibrinogen in Diabetic Retinopathy.

PURPOSE: This study aimed to describe the levels of glycated hemoglobin (HbA1c), D-dimer (D-D), and fibrinogen (FIB) in different types of diabetic retinopathy (DR). METHODS: A total of 61 patients with diabetes, who were treated in our department between November 2017 and May 2019, were selected. According to their non-mydriatic fundus photography and fundus angiography results, patients were divided into three groups, ie, the non-DR (NDR) group (n=23), the non-proliferative DR (NPDR) group (n=17), and the proliferative DR (PDR) group (n=21). A control group of 20 people who had tested negative for diabetes was also included. The levels of HbA1c, D-D, and FIB were measured and compared, respectively. RESULTS: The mean values of HbA1c were 6.8% (5.2%, 7.7%), 7.4% (5.8%, 9.0%), and 8.5% (6.3%, 9.7%) in the NDR, NPDR, and PDR groups, respectively. The control group values were 4.9% (4.1%, 5.8%). These results indicated a significant statistical difference between groups. The mean values of D-D were 0.39 ± 0.21 mg/L, 1.06 ± 0.54 mg/L, and 1.39 ± 0.59 mg/L in the NDR, NPDR, and PDR groups, respectively. The control group result was 0.36 ± 0.17 mg/L. The values of the NPDR and PDR groups were significantly higher than those of the NDR and control groups, and the value of the PDR group was significantly higher than that of the NPDR group, indicating a significant difference between the groups (P < 0.001). The mean values of FIB were 3.07 ± 0.42 g/L, 4.38 ± 0.54 g/L, and 4.46 ± 1.09 g/L in the NDR, NPDR, and PDR groups, respectively. The control group result was 2.97 ± 0.67 g/L. The difference between the groups was statistically significant (P < 0.05). CONCLUSION: Blood levels of HbA1c, D-D, and FIB in the PDR group were significantly higher than in the NPDR group.

Measurement of Intracellular Nitric Oxide with a Quantitative Mass Spectrometry Probe Approach.

Nitric oxide (NO) is a molecule of physiological importance, and the function of NO depends on its concentration in biological systems, particularly in cells. Concentration-based analysis of intracellular NO can provide insight into its precise role in health and disease. However, current methods for detecting intracellular NO are still inadequate for quantitative analysis. In this study, we report a quantitative mass spectrometry probe approach to measure NO levels in cells. The probe, Amlodipine (AML), comprises a Hantzsch ester group that reacts with NO to form a pyridine, Dehydro Amlodipine (DAM). Quantification of DAM by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) allows specific measurement of intracellular NO levels. Notably, the AML/NO reaction proceeds rapidly (within 1 s), which is favorable for NO detection considering its large diffusivity and short half-life. Meanwhile, studies under simulated physiological conditions revealed that the AML response to NO is proportional and selective. The presented UPLC-MS/MS method showed high sensitivity (LLOQ = 0.24 nM) and low matrix interference (less than 15%) in DAM quantification. Furthermore, the mass spectrometry probe approach was demonstrated by enabling the measurement of endogenous and exogenous NO in cells. Hence, the quantitative UPLC-MS/MS method developed using AML as a probe is expected to be a new method for intracellular NO analysis.