Waters Xevo QTof

Pyroptosis is a proinflammatory form of regulated cell death that plays an important role in ischemic stroke. Gualou Guizhi granule (GLGZG) is a classic prescription that has been shown to exert neuroprotective effects against cerebral ischemia reperfusion injury. In the present study, we examined the involvement of pyroptosis and its associated mechanism in protecting nerve function. Methods. Primary neurons were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) conditions in the presence or absence of GLGZG. Cellular viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay. The number of apoptoic cells was detected by NeuN and NSE protein expression. The expression levels of the pyroptosis markers, namely, NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, interleukin-18 (IL-18), and IL-1β were determined by quantitative real-time PCR analysis, western blot, and ELISA analyses as appropriate. Moreover, the expression levels of the PI3K/Akt pathway key proteins were determined by quantitative real-time PCR analysis and western blot assays. To determine the PI3K/Akt pathway involvement in GLGZG-mediated neuroprotection, the PI3K inhibitor LY294002 (LY, 10 μM) was added. The expression levels of NeuN, Akt, and p-Akt were evaluated. Results. It was found that GLGZG could inhibit OGD/R-induced cell apoptosis, increase neuronal cell viability, decrease the production of IL-18 and IL-1β, and downregulate the expression levels of pyroptosis markers (NLRP3, ASC, and caspase-1). Furthermore, GLGZG could modulate the PI3K/Akt signaling pathway. Pharmacological inhibition of the PI3K pathway not only abrogated the effects of GLGZG on Akt but also neutralized its prosurvival and antipyroptotic actions. Conclusions. The findings indicated that GLGZG pretreatment effectively reduced OGD/R-induced injury by inhibiting cell pyroptosis and activating the PI3K/Akt pathway. These data provide important evidence for the therapeutic applications of this regimen in ischemic stroke.

Yuqin Zhang, Hongyun Wang, Huang Li, Lihong Nan, Wei Xu, Yu Lin, Kedan Chu. Evidence-Based Complementary and Alternative Medicine, 2021, 1-14, 2021.

Triumfetta welwitschii has been used as a traditional medicine in Africa. It is documented as a rich source of phytochemicals with antibacterial activities. To further explore the antibacterial potential of these phytochemical components, the phytochemical profile of the dichloromethane: methanol leaf extract from T. welwitschii was investigated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Compounds were isolated from the extract using column chromatography and thin-layer chromatography. Compound B1 was isolated from the fraction eluted by 90 hexane:10 ethyl acetate using column chromatography. The antibacterial activity of B1 against Pseudomonas aeruginosa was evaluated in vitro using the broth microdilution method and the iodonitrotetrazolium (INT) colorimetric assay. The antibiofilm activities of the extract and B1 against P. aeruginosa were determined by quantifying the biofilms using crystal violet. The effect of the extract and B1 on capsular polysaccharide and extracellular DNA content of biofilm formed by P. aeruginosa was determined using phenol-sulphuric acid and propidium iodide, respectively. A total of 28 peaks were detected and identified using UPLC-MS/MS. The three most abundant phytochemicals identified were catechin, umbelliferone, and a luteolin derivative. B1 showed antibacterial activity against P. aeruginosa with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of 25 μg/ml. Only 38% and 6% of the biofilms were formed in the presence of the extract and B1, respectively. The extract and B1 reduced the capsular polysaccharide content in biofilms formed in P. aeruginosa by 40% and 65%, respectively. The extract and B1 significantly reduced the extracellular DNA content of biofilms by 29% and 72%, respectively. The results of this study provide evidence of the antibacterial and antibiofilm activities of B1 and leaf extracts from T. welwitschii. Future work should identify the chemical structure of B1 using nuclear magnetic resonance and mass spectrometry.

Molly Mombeshora, Godloves Fru Chi, Stanley Mukanganyama. Biochemistry Research International, 2021, 1-13, 2021.

The kynurenine pathway of tryptophan degradation produces several neuroactive metabolites suggested to be involved in a wide variety of diseases and disorders, however, technical challenges in reliably detecting these metabolites hampers cross-comparisons. The main objective of this study was to develop an accurate, robust and precise bioanalytical method for simultaneous quantification of ten plasma kynurenine metabolites. As a secondary aim, we applied this method on blood samples taken from healthy subjects conducting 1 session of sprint interval exercise (SIE). It is well accepted that physical exercise is associated with health benefits and reduces risks of psychiatric illness, diabetes, cancer and cardiovascular disease, but also influences the peripheral and central concentrations of kynurenines. In line with this, we found that in healthy old adults ( n = 10; mean age 64 years), levels of kynurenine increased 1 hour ( P = .03) after SIE, while kynurenic acid (KYNA) concentrations were elevated after 24 hours ( P = .02). In contrast, no significant changes after exercise were seen in young adults ( n = 10; mean age 24 years). In conclusion, the described method performs well in reliably detecting all the analyzed metabolites in plasma samples. Furthermore, we also detected an age-dependent effect on the degree by which a single intense training session affects kynurenine metabolite levels.

Ada Trepci, Sophie Imbeault, Victoria L Wyckelsma, Håkan Westerblad, Sigurd Hermansson, Daniel C Andersson, Fredrik Piehl, Tomas Venckunas, Marius Brazaitis, Sigitas Kamandulis, Lena Brundin, Sophie Erhardt, Lilly Schwieler. International Journal of Tryptophan Research, 13, 117864692097824, 2020.

Background: The purpose of the study was to establish reference values of amino acids and acylcarnitines in newborns of the Tibet Autonomous Region for the first time and to provide an experimental basis for the diagnosis of genetic metabolic diseases.Methods: We detected concentrations of 43 kinds of amino acids, acylcarnitines and succinylacetone in the dried blood spots of 15029 newborns using liquid chromatography tandem mass spectrometry. We compared the indexes between Tibet and our lab, where most data come from an inland area and Han Chinese people. Then we compared amino acid and acylcarnitine levels of seven regions in Tibet and explored their impact factors. The distribution of amino acid and acylcarnitines were different in Tibet.Results: Reference intervals of amino acids and acyl carnitines in neonates from the Tibet Autonomous Region were defined according to the (P 0.5% ~ P 99.5%) of the values. Given the third reference range, the recall rate of statistical screening was significantly reduced to 2.16%.Conclusions: This study has contributed to the field by determining the actual values of amino acids and acylcarnitines in newborns from the Tibet Autonomous Region, which could be used as reference for a newborn metabolic screening project in this area.

Chunyan Zhang, Drun Dha, Yuxuan Cheng, Ya Ma, Yan Meng, Drun Tse, Dolma Ngawang, Pedrun Dekyi, Tao Jiang, Yang Shu, Jiayi Cui, Jing Li, Yaping Tian. 2019.

Background Autism spectrum disorder (ASD) is a developmental disorder with limited effective pharmacological treatments for the core autistic symptoms so far. Increasing evidences, especially the clinical studies in ASD patients, suggest a functional link between gut microbiota and development of ASD. However, the mechanisms linking gut microbiota and brain dysfunctions (gut-brain axis) in ASD are still not well-established. With genetic mutations and down-regulated expression in patients with ASD, EPHB6 , which is also important in homeostasis of gut, has been generally considered to be a candidate gene for ASD. Nonetheless, the role and mechanism of EPHB6 involved in regulating gut microbiota and development of ASD have been unclear. Results Here, we found deletion of EphB6 induced autism-like behavior and disturbed gut microbiota in mice. More importantly, transplanting fecal microbiota from EphB6-deficient mice resulted in autism-like behavior in antibiotics-treated C57BL/6J mice. Meanwhile, transplanting fecal microbiota from wild-type mice ameliorated autism-like behavior in EphB6-deficient mice. At the metabolic levels, disturbed gut microbiota led to vitamin B6 and dopamine defects in EphB6-deficient mice. At the cellular levels, excitation/inhibition (E/I) imbalance in medial prefrontal cortex was induced by gut microbiota-mediated defects of vitamin B6 metabolism in EphB6-deficient mice. Conclusions Our study uncovers a key role for gut microbiota in regulation of autism-like social behavior by mediating vitamin B6 metabolism, dopamine synthesis and E/I balance in EphB6-deficient mice, suggesting new strategies for understanding and treatment of ASD.

Jian-Ming Li, Ying Li, Zheng-Yi Luo, Yu-Ying Hu, Yue-Wei Bi, Jian-Ming Yang, Wen-Jun Zou, Yun-Long Song, Shi Li, Tong Shen, Shu-Ji Li, Lang Huang, Ai-Jun Zhou, Tian-Ming Gao. 2020.

Background Type 2 diabetes mellitus (T2DM) is the most common type of metabolic disorder involving multiple organ systems. Grape has been reported to improve the symptoms of T2DM, the precise mechanism of its action is unclear. Our study was aimed to determine the effect and mechanism of grape pomace extract in T2DM mice induced by high fat diet (HFD). Materials and methods Ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry were used to identify the main active compounds in grape pomace extract to improve T2DM. C57BLK/6J mice induced by HFD supplemented with or without quercetin were used to show the effects of quercetin improving T2DM. By online database research, bioinformatics analysis and molecular biology experiments, Estrogen receptor alpha (ERα)-lncSHGL (lncRNA suppressor of hepatic gluconeogenesis and lipogenesis) pathway was identify as the target for quercetin. Results Quercetin was identified as one of the most active compounds in grape pomace extract to improve T2DM. Quercetin could inhibit HFD-induced T2DM in mice by activing ERα. LncSHGL was identified as the downstream of ERα and inhibited HFD-induced T2DM. Conclusions Quercetin could be beneficial for T2DM by promoting lncSHGL transcription and activating the lncSHGL pathway, and may be used as a drug component to treat T2DM.

Hanlu Fan, Haiwen Li, Huijiao Liu, Peng Li, Xiaomeng Jia, Peilan Guo, Qingquan Yu, Xiru Li, Yangdong Guo, Xiangdong Li. 2020.

Background: Cholangiocarcioma (CCA) is a major health problem for people in Thailand. The treatment is challenging because most of patients are diagnosed when the disease is advanced, and cancer recurrence is the main problem after treatment, leading to low survival rates. Therefore, our understanding of the mechanism underlying CCA recurrence is essential in order to prevent CCA recurrence and improve patient outcome. Methods: We performed ¹H-NMR and UPLC-MS based-metabolomics on CCA serum. The differential metabolites were further analyzed using pathway analysis and potential biomarkers identification.Results: At an early stage, CCA patients with recurrence have a different metabolic profile compared with non-recurrence patients. In recurrence patients, the metabolites involved in energy metabolism, such as pyruvate metabolism and the TCA cycle, are down-regulated, while most lipids, including TGs, PCs, PEs and Pas, are up-regulated. This metabolic feature has been described in cancer stem-like cell (CSC) metabolism. Based on this, the correlation of putative CSC markers and proteins involved in identified pathways, together with recurrence-free survival was explored. The results revealed that the expression levels of the CSC markers CD44v6 and CD44v8-10 are associated with CD36 (a protein involved in lipid uptake) as well as with recurrence-free survival. Potential metabolic biomarkers were identified using ROC and Kaplan-Meier analyses. We found that citrate, sarcosine, succinate, creatine, creatinine and pyruvate and TGs have good predictive values for CCA recurrence and are associated with recurrence-free survival. Conclusions: These findings reveal an alteration of the metabolic profile associated with recurrence. These metabolic changes may be associated with the existence of CSCs that lead to CCA recurrence. Moreover, the alteration of metabolites was shown to provide suitable biomarkers for CCA recurrence. Therefore, the differential metabolites between patients with and without recurrence can be used as biomarkers for CCA recurrence.

Sureerat Padthaisong, Jutarop Phetcharaburanin, Poramate Klanrit, Jia V. Li, Nisana Namwat, Narong Khuntikeo, Attapol Titapun, Apiwat Jarearnrat, Arporn Wangwiwatsin, Panupong Mahalapbutr, Watcharin Loilome. 2020.

BackgroundPlantaginis semen has been widely used as folk medicine and health care food against hyperuricemia (HUA) and gout, but little was known about its pharmacological mechanism. MethodsThe model was established by potassium oxonate intragastric administration. 42 Sprague-Dawley (SD) male rats were randomly divided into the control group, model group, benzbromarone group (10 mg/kg) and three Plantaginis semen groups (n = 7). The Plantaginis semen groups were treated orally with Plantaginis semen at 0.9375, 1.875 and 3.75 g/kg for 28 days. The levels of serum uric acid (UA), creatinine (Cr), triacylglycerol (TG) and tumor necrosis factor-α (TNF-α) were detected using enzyme-linked immunosorbent assay kits. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) was used as the basis for serum lipidomics analysis, and orthogonal partial least squares discriminant analysis (OPLS-DA) was carried out for the pattern recognition and characteristic metabolites identification. The relative levels of critical regulatory factors of urate anion transporter 1(URAT1) and phosphatidylinositol 3-kinase/ protein kinases B (PI3K/Akt) were determined by quantitative real-time polymerase chain reaction (RT-qPCR). ResultsCompared with the model group, the levels of serum UA, Cr, and TG were significantly (p<0.01) decreased in benzbromarone and three Plantaginis semen groups and the level of serum TNF-α was significantly (p<0.05) decreased in benzbromarone and low dose of Plantaginis semen group. With lipidomics analysis, significant lipid metabolic perturbations were observed in HUA rats, 13 metabolites were identified as potential biomarkers and glycerophospholipid metabolism pathway was mostly affected. These perturbations can be partially restored via treatment of benzbromarone and Plantaginis semen. Additionally, the URAT1 and PI3K/Akt mRNA expression levels were significantly decreased (p<0.05) after treatment with benzbromarone and high dose of Plantaginis semen. ConclusionsPlantaginis semen had significant anti-HUA, anti-inflammatory and renal protection effects and could attenuate potassium oxonate-induced HUA through regulation of lipid metabolism disorder. Trial registrationNot applicable

Wenjun Shi, Fei Yang, Liting Wang, Nankun Qin, Chengxiang Wang, Guang Xu, Yuying Guo, Xue Yu, Qun Ma. 2020.

Background: Banana, an important cash and staple crop worldwide, suffers from various biotrophic and hemi-biotrophic pathogens. In plants’ defense against these pathogens, the phytohormone salicylic acid (SA) plays a key role in the regulation of immune response. Using a specific set of SA-responsive genes as markers is frequently adopted to monitor the onset of SA-mediated immune response. However, reliable SA-responsive genes marker genes have not been well established in bananas. Results: From the transcriptome analysis of SA-treated ‘Pei-Chiao’ banana roots, we identified 19 up-regulated and 3 down-regulated genes. Four of the up-regulated genes previously reported to play crucial roles in SA-mediated immunity in other species were further analyzed for their applicability in different tissues and cultivars of bananas using real-time quantitative reverse-transcription PCR. The analysis showed that WRKY40, WRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (DLO1) were significantly induced upon SA treatment in both the leaves and roots of ‘Pei-Chiao’ (AAA genome), ‘Pisang Awak’ (ABB genome), and ‘Lady Finger’ (AA genome) bananas.Conclusions: The uncovering of common marker genes WRKY40, WRKY70, and DLO1 for SA response in different banana genome types provides the stepping stone for studies towards understanding of SA-mediated immune response in bananas.

Yuh Tzean, Bo-Han Hou, Shu-Ming Tsao, Ho-Ming Chen, An-Po Cheng, Elena Gamboa Chen, Wei-Yi Chou, Chih-Ping Chao, Wei-Chiang Shen, Hsin-Hung Yeh. 2020.

Microbial surfactants (biosurfactants) have gained interest as promising substitutes of synthetic surface-active compounds. However, their production and purification are still challenging, with significant room for efficiency and costs optimization. In this work, we introduce a method for the enhanced production and purification of cyclic lipopeptides pseudofactins (PFs) from Pseudomonas fluorescens BD5 cultures. The method is directly applicable in a technical scale with the possibility of further upscaling. Comparing to the original protocol for production of PFs (cultures in mineral salt medium in shaken flasks followed by solvent-solvent extraction of PFs), our process offers not only ∼24-fold increased productivity, but also easier and more efficient purification. The new process combines high yield of PFs (∼7.2 grams of PFs per 30 L of working volume), with recovery levels of 80–90% and purity of raw PFs up to 60–70%. These were achieved with an innovative, single-step thermal co-precipitation and extraction of PFs directly from collected foam, as a large amount of PF-enriched foam was produced during the bioprocess. Besides we present a protocol for the selective production of PF structural analogs and their separation with high-performance liquid chromatography. Our approach can be potentially utilized in the efficient production and purification of other lipopeptides of Pseudomonas and Bacillus origin.

Piotr Biniarz, Marius Henkel, Rudolf Hausmann, Marcin Łukaszewicz. Frontiers in Bioengineering and Biotechnology, 8, 2020.

The glucagon receptor (GCGR) is activated by glucagon and is essential for glucose, amino acid, and lipid metabolism of animals. GCGR blockade has been demonstrated to induce hypoglycemia, hyperaminoacidemia, hyperglucagonemia, decreased adiposity, hepatosteatosis, and pancreatic α cells hyperplasia in organisms. However, the mechanism of how GCGR regulates these physiological functions is not yet very clear. In our previous study, we revealed that GCGR regulated metabolic network at transcriptional level by RNA-seq using GCGR mutant zebrafish (gcgr^−/−). Here, we further performed whole-organism metabolomics and lipidomics profiling on wild-type and gcgr^−/− zebrafish to study the changes of metabolites. We found 107 significantly different metabolites from metabolomics analysis and 87 significantly different lipids from lipidomics analysis. Chemical substance classification and pathway analysis integrated with transcriptomics data both revealed that amino acid metabolism and lipid metabolism were remodeled in gcgr-deficient zebrafish. Similar to other studies, our study showed that gcgr^−/− zebrafish exhibited decreased ureagenesis and impaired cholesterol metabolism. More interestingly, we found that the glycerophospholipid metabolism was disrupted, the arachidonic acid metabolism was up-regulated, and the tryptophan metabolism pathway was down-regulated in gcgr^−/− zebrafish. Based on the omics data, we further validated our findings by revealing that gcgr^−/− zebrafish exhibited dampened melatonin diel rhythmicity and increased locomotor activity. These global omics data provide us a better understanding about the role of GCGR in regulating metabolic network and new insight into GCGR physiological functions.

Xuanxuan Bai, Jianxin Jia, Qi Kang, Yadong Fu, You Zhou, Yingbin Zhong, Chao Zhang, Mingyu Li. Frontiers in Cell and Developmental Biology, 8, 2021.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus (Flaviviridae). In spite of the availability of vaccines, the virus is still causing substantial financial losses to the livestock industry. In this context, the use of antiviral agents could be an alternative strategy to control and reduce viral infections. The viral RNA-dependent RNA polymerase (RdRp) is essential for the replication of the viral genome and constitutes an attractive target for the identification of antiviral compounds. In a previous work, we have identified potential molecules that dock into an allosteric binding pocket of BVDV RdRp via a structure-based virtual screening approach. One of them, N-(2-morpholinoethyl)-2-phenylquinazolin-4-amine [1, 50% effective concentration (EC₅₀) = 9.7 ± 0.5 μM], was selected to perform different chemical modifications. Among 24 derivatives synthesized, eight of them showed considerable antiviral activity. Molecular modeling of the most active compounds showed that they bind to a pocket located in the fingers and thumb domains in BVDV RdRp, which is different from that identified for other non-nucleoside inhibitors (NNIs) such as thiosemicarbazone (TSC). We selected compound 2-[4-(2-phenylquinazolin-4-yl)piperazin-1-yl]ethanol (1.9; EC₅₀ = 1.7 ± 0.4 μM) for further analysis. Compound 1.9 was found to inhibit the in vitro replication of TSC-resistant BVDV variants, which carry the N264D mutation in the RdRp. In addition, 1.9 presented adequate solubility in different media and a high-stability profile in murine and bovine plasma.

Gabriela A. Fernández, Eliana F. Castro, Rocío A. Rosas, Daniela M. Fidalgo, Natalia S. Adler, Leandro Battini, Maria J. España de Marco, Matias Fabiani, Ana M. Bruno, Mariela Bollini, Lucia V. Cavallaro. Frontiers in Chemistry, 8, 2020.

This work presents the first transition metal-free synthesis of oxygen-linked aromatic polymers by integrating iterative exponential polymer growth (IEG) with nucleophilic aromatic substitution (S_NAr) reactions. Our approach applies methyl sulfones as the leaving groups, which eliminate the need for a transition metal catalyst, while also providing flexibility in functionality and configuration of the building blocks used. As indicated by 1) ¹H-¹H NOESY NMR spectroscopy, 2) single-crystal X-ray crystallography, and 3) density functional theory (DFT) calculations, the unimolecular polymers obtained are folded by nonclassical hydrogen bonds formed between the oxygens of the electron-rich aromatic rings and the positively polarized C–H bonds of the electron-poor pyrimidine functions. Our results not only introduce a transition metal-free synthetic methodology to access precision polymers but also demonstrate how interactions between relatively small, neutral aromatic units in the polymers can be utilized as new supramolecular interaction pairs to control the folding of precision macromolecules.

Tyler J. Jaynes, Mona Sharafi, Joseph P. Campbell, Jessica Bocanegra, Kyle T. McKay, Kassondra Little, Reilly Osadchey Brown, Danielle L. Gray, Toby J. Woods, Jianing Li, Severin T. Schneebeli. Frontiers in Chemistry, 9, 2021.