Dionex ICS 3000

In this study, a new strategy for the simultaneous quantization of five serotonin 5-hydroxytryptamine receptor antagonists—ondansetron, azasetron, ramosetron, granisetron, and tropisetron—either in infusion samples or in injection dosage form was first established based on high-performance liquid chromatography combined with a quantitative analysis of multiple components by a single marker. The quantitative analysis of multicomponents by a single marker method was conducted with ondansetron as an internal reference substance and performed using relative retention time and ultraviolet spectral similarity as the double indicator. The quantitative analysis of the 5-HT3 receptor antagonists was calculated and investigated based on the relative correction factors. Chromatographic separation was achieved using a C18 column (150 mm × 4.6 mm, 5.0 μm), and the mobile phase was composed of acetonitrile-0.05 mol·L−1 potassium dihydrogen phosphate (pH 4.0) (25 : 75) at a flow rate of 1.0 mL·min−1 and detection wavelengths of 307 nm (ondansetron, azasetron, ramosetron), 302 nm (granisetron), and 285 nm (tropisetron). In addition, the accuracy of the quantitative analysis of multicomponents by a single marker method was compared with an external standard method, and no significant difference was observed between the two methods. The established method is rapid, is easy, and does not require many reference substances, and it can been successfully applied as part of the quality control of the five 5-HT3 receptor antagonists in their injection dosage form and infusion sample drugs in hospitals.

Fuchao Chen, Baoxia Fang, Peng Li, Sicen Wang. International Journal of Analytical Chemistry, 2021, 1-9, 2021.

Background. Renal interstitial fibrosis is a pathological manifestation of progression of chronic kidney disease induced by various factors. Shen Shuai II Recipe (SSR) has been used in clinical practice for more than 20 years, and clinical studies have confirmed that SSR significantly improves the renal function of patients with chronic kidney disease. However, the specific mechanisms underlying its efficacy require further research. This study aims to explore the influencing factors of renal interstitial fibrosis in the context of hypoxia via the IL-1β/c-Myc pathway and the potential molecular mechanisms of SSR intervention in vivo and in vitro. Methods. A rat model of chronic renal failure was developed by performing 5/6 (ablation/infarction, A/I) surgery on randomly selected, male Sprague Dawley rats. Thirty-six successfully modeled rats were randomly divided into three groups: 5/6 (A/I), 5/6 (A/I) + SSR, and 5/6 (A/I) + losartan. Another 12 rats were used as the sham group. After 8 weeks of the corresponding intervention, renal function, liver function, and protein expression of renal-fibrosis-related factors, HIF-1α, IL-1β, and c-Myc, were detected. In vitro analysis was performed using hypoxia-induced rat renal tubular epithelial cells (NRK-52E) and IL-1β-stimulated rat renal interstitial fibroblasts (NRK-49F). IL-1β concentration in the culture medium and IL-1β protein expression in hypoxic NRK-52E treated with different concentrations of SSR were investigated. Furthermore, we also studied the changes in protein expression of c-Myc and fibrosis-related factors after c-Myc gene silencing in IL-1β-stimulated NRK-49F treated with SSR. Results. Shen Shuai II Recipe significantly reduced RIF and downregulated the expression of HIF-1α, c-Myc, and IL-1β proteins in 5/6 (A/I) rats with chronic renal failure. It also inhibited IL-1β secretion from NRK-52E induced by hypoxia, which in turn inhibited fibroblast activation mediated by the IL-1β/c-Myc pathway, and finally reduced the overproduction of the extracellular matrix. Conclusion. The renoprotective effects of SSR in rats with chronic renal failure may be related to its inhibition of hypoxia via the IL-1β/c-Myc pathway. Thus, SSR is a potentially effective drug for delaying the progression of renal interstitial fibrosis.

Liuyi Yang, Meng Wang, Yuan Zhou, Jing Yang, Chaoyang Ye, Chen Wang. Evidence-Based Complementary and Alternative Medicine, 2021, 1-13, 2021.

The coronavirus disease 2019 (COVID-19) has become a serious problem for public health since it was identified in the province of Wuhan (China) and spread around the world producing high mortality rates and economic losses. Nowadays, the WHO recognizes traditional, complementary, and alternative medicine for treating COVID-19 symptoms. Therefore, we investigated the antiviral potential of the hydroalcoholic extract of Uncaria tomentosa stem bark from Peru against SARS-CoV-2 in vitro. The antiviral activity of U. tomentosa against SARS-CoV-2 in vitro was assessed in Vero E6 cells using cytopathic effect (CPE) and plaque reduction assay. After 48 h of treatment, U. tomentosa showed an inhibition of 92.7% of SARS-CoV-2 at 25.0 μg/mL () by plaque reduction assay on Vero E6 cells. In addition, U. tomentosa induced a reduction of 98.6% () and 92.7% () in the CPE caused by SARS-CoV-2 on Vero E6 cells at 25 μg/mL and 12.5 μg/mL, respectively. The EC50 calculated for the U. tomentosa extract by plaque reduction assay was 6.6 μg/mL (4.89–8.85 μg/mL) for a selectivity index of 4.1. The EC50 calculated for the U. tomentosa extract by TCID50 assay was 2.57 μg/mL (1.05–3.75 μg/mL) for a selectivity index of 10.54. These results showed that U. tomentosa, known as cat's claw, has an antiviral effect against SARS-CoV-2, which was observed as a reduction in the viral titer and CPE after 48 h of treatment on Vero E6 cells. Therefore, we hypothesized that U. tomentosa stem bark could be promising in the development of new therapeutic strategies against SARS-CoV-2.

Andres F. Yepes-Perez, Oscar Herrera-Calderón, Cristian A. Oliveros, Lizdany Flórez-Álvarez, María I. Zapata-Cardona, Lina Yepes, Wbeimar Aguilar-Jimenez, María T. Rugeles, Wildeman Zapata. Evidence-Based Complementary and Alternative Medicine, 2021, 1-11, 2021.

Aflibercept is an inhibitor of vascular endothelial growth factor (VEGF) used to treat macular edema following branch retinal vein occlusion (BRVO). Despite well-documented efficacy, there is limited knowledge about proteome changes following aflibercept intervention in BRVO. Proteome changes may provide insights into mechanisms of action as well as aspects related to safety profile. In seven Danish Landrace pigs, BRVO was induced with a well-established experimental model of argon laser-induced BRVO. BRVO was induced in both eyes. Three days after the induced BRVO, aflibercept was injected intravitreally in the right eyes, while the left eyes received intravitreal isotonic saline water. Retinas were collected 15 days after the induced BRVO and analyzed with label-free quantification liquid chromatography tandem mass spectrometry (LFQ LC-MS/MS). Fourteen proteins were changed in expression following aflibercept intervention in the BRVO model. LFQ LC-MS/MS identified an upregulation of DnaJ homolog subfamily C member 17 (DNAJC17) (fold change = 6.19) and a modest downregulation of isoform 2 of the protein encoded by N-myc downstream regulated gene 2 (NDRG2) (fold change = 0.40). NDRG2 was unchanged by Western blotting. In the additional significantly regulated proteins, only discrete changes were observed (fold changes 0.52–1.59). Our study is the first to report an association between aflibercept intervention and the heat shock protein DNAJC17. Our results indicate that the role of heat shock proteins in the treatment of BRVO should be further explored.

Lasse Jørgensen Cehofski, Anders Kruse, Alexander Nørgård Alsing, Benn Falch Sejergaard, Jonas Ellegaard Nielsen, Shona Pedersen, Danson Vasanthan Muttuvelu, Svend Kirkeby, Bent Honoré, Henrik Vorum. Journal of Ophthalmology, 2021, 1-9, 2021.

The therapeutic arsenal for the treatment of leishmaniasis is limited and has serious obstacles, such as variable activity, high toxicity, and costs. To overcome such limitations, it becomes urgent to characterize new bioactive molecules. Plants produce and accumulate different classes of bioactive compounds, and these molecules can be studied as a strategy to combat leishmaniasis. The study presented herein evaluated the leishmanicidal effect of maslinic acid isolated from the leaves of Hyptidendron canum (Lamiaceae) and investigated the morphological that occurred on Leishmania (Leishmania) infantum upon treatment. Maslinic acid was active and selective against promastigote and amastigote forms in a dose-dependent manner. Additionally, it was not toxic to peritoneal macrophages isolated from golden hamsters, while miltefosine and amphotericin B showed mild toxicity for macrophages. Morphological changes in promastigotes of L. (L.) infantum treated with maslinic acid were related to cytoplasmic degeneration, intense exocytic activity, and blebbing in the kDNA; disruption of mitochondrial cristae was observed in some parasites. The nucleus of promastigote forms seems to be degraded and the chromatin fragmented, suggesting that maslinic acid triggers programmed cell death. These results indicate that maslinic acid may be an interesting molecule to develop new classes of drugs against leishmaniasis.

Jéssica Adriana Jesus, Márcia Dalastra Laurenti, Matheus Lopes Silva, João Henrique Ghilardi Lago, Luiz Felipe Domingues Passero. Evidence-Based Complementary and Alternative Medicine, 2021, 1-10, 2021.

β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with K_m and V_max values of 1.0 mM and 250 μmol min⁻¹ mg⁻¹ respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a K_i of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (K_m) and conversion efficiency (V_max). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X₂, X₃ and X₄). Catalytic conversion of X₂, X₃ and X₄ decreased (V_max and k_cat) with increasing chain length.

Narin Kirikyali, Jonathan Wood, Ian F Connerton. AMB Express, 4 (1), 2014.

Background: Exploring a simple and versatile technique for direct immobilization of target enzymes from cell lysate without prior purification is urgently needed. Thus, a novel all-in-one strategy for purification and immobilization of β-1, 3-xylanase was proposed, the target enzymes were covalently immobilized on silica nanoparticles via ELP-based biomimetic silicification and SpyTag/SpyCatcher spontaneous reaction. Thus, the functional carriers that did not require the time-consuming surface modification step were quickly and efficiently prepared. These carriers could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification. Results: The ELPs-SpyCatcher hardly leaked from the carriers (0.5%), and the immobilization yield of enzyme was up to 96%. Immobilized enzyme retained 85.6% of the initial activity and showed 88.6% of the activity recovery. Compared with free ones, the immobilized β-1, 3-xylanase showed improved thermal stability, elevated storage stability and good pH tolerance. It also retained more than 70.6% of initial activity after12 reaction cycles, demonstrating its excellent reusability. Conclusions: The results clearly highlighted the effectiveness of the novel enzyme immobilization method proposed here due to the improvement of overall performance of immobilized enzyme in respect to free form for the hydrolysis of macromolecular substrates. Thus, it may have great potential in the conversion of algae biomass as well as other related fields.

Lixi Cai, Yunmeng Chu, Xin Liu, Yue Qiu, Zhongqi Ge, Guangya Zhang. 2019.

Background: Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight, a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. While previous studies have shown F. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that there remain many secondary metabolites awaiting description.Methods: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions.Results: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed.Conclusions: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosomeevolution and the biosynthetic potential of F. poae isolates.

Thomas Witte, Linda Harris, Hai Nguyen, Anne Hermans, Anne Johnston, Amanda Sproule, Jeremy Dettman, Christopher Boddy, David Overy. 2020.

Background. Kappaphycus alvarezii (Doty) Doty ex P.C.Silva is a widely used seaweed that has antioxidant and antiglycation activities. The purpose of this study was to examine the ability of active fraction from Kappaphycus alvarezii to decrease glucose level and inhibit glycation process. Methods. This study used bioassay-guided fractionation through three stages of the extraction, partition, and fractionation processes that were monitored using Thin Layer Chromatography and BSA-Glucose test. Inhibition of glycation was known by calculating percentage of inhibition and IC₅₀. Selected active fraction was used for in vivo tests using 24 Wistar male rats. Measurement of glucose levels used GOD-PAP method, while levels of glycated albumin (GA) and Nε- (carboxymethyl) lysine (CML) were measured using ELISA. Analysis of RAGE gene expression used qPCR. Results Glycation test showed a significant difference (p < 0.05) between all treatments. Chloroform extract showed higher percentage of inhibition (62.4 ± 3.45%) with lower IC₅₀ (0.33 ± 0.01 mg/ml) compared to methanol extract (0.52 ± 0.03 mg/ml). Methanol-soluble extracts had a higher percentage of inhibition (51.10 ± 1.64%) with IC₅₀ 0.45 ± 0.05 mg/ml compared to methanol-insoluble extract (1.25 ± 0.05 mg/ml). Fraction II had a higher percentage of inhibition (53.37 ± 1.92%) with IC₅₀ 0.12 ± 0.01 mg/ml compared to other fractions. Selected active fraction reduced blood glucose by 1.3% and 5.2% and CML levels by 50.6% and 42.4% at concentrations of 0.17 and 0.255 mg/ml in diabetic rats. RAGE gene expression was lower in the diabetic rat groups treated with active fraction compared to untreated diabetic group. Conclusions The active fraction has ability for reducing blood glucose, antiglycation, or reducing CML levels, and RAGE gene expression.

Mae Sri Hartati Wahyuning, Evy - Yulianti, Sunarti - Sunarti. 2020.

Backgrounds: Cordyceps militaris is a well-known medicinal fungus. Cordycepin, a metabolite of this fungus, has strong biological activities against leukemia, oxidative stress, aging, tumors, and inflammation. Methods: HPLC analysis was conducted to measure the content of corydycepin in the extract. Real time PCR was performed to evaluate the cytokines. Immunoactivity including the polarization, phagocytic activity and cellular differentiations were evaluated by flow cytometry. Results: The yields of cordycepin and adenosine in the extract were 11.75 µg and 1.25 µg (per gram fresh mycelium), respectively. From measurements of the bioactivity in the extract, the levels of TNF-α and IL-1β in macrophages treated with lipopolysaccharides (LPS) were found to be approximately 4 and 48 times higher than those in the control, as shown by qRT-PCR. However, cells treated with 1 µg/mL of the extract showed 13 and 10-fold lower TNF-α and IL-1β levels when compared to LPS-treated cells. This was corroborated by flow-cytometry, where their levels were 20 and 14 times lower, respectively. Addition of the extract to LPS-treated cells enhanced M2 polarization and attenuated M1 polarization. In addition, the extract also dose-dependently activated macrophage phagocytosis. Under treatment with the extract conditioned medium, DCs were strongly differentiated toward CD11b⁺ and Xcr1⁺ cells as their density were 13.6 and 6.26 times higher than those in the LPS conditioned medium, respectively. Moreover, the number of T_reg and NKT cells differentiated in the extract conditioned medium were increased about 1.67 and 6.73 times than those in the LPS conditioned medium, respectively. Conclusions: These results suggest that the C. militaris hydrolytic extract has strong effects on the modulation of immune actors, such as macrophages and dendritic cells, under inflammatory stress.

Seoyoon Choi, Ahran Song, Yoonjin Park, Jinkwan Kim, Boyong Kim, Seunggwan Lee. 2020.

Cellular RNA polymerases can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, or enter dormancy. How RNA polymerase recycling into active states is achieved and balanced with quiescence remains elusive. We structurally analyzed Bacillus subtilis RNA polymerase bound to the NTPase HelD. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNA polymerase secondary channel, dismantling the active site and displacing RNA; a unique helical protrusion inserts into the main channel, prying β and β’ subunits apart and dislodging DNA, aided by the δ subunit. HelD release depends on ATP, and a dimeric structure resembling hibernating RNA polymerase I suggests that HelD can induce dormancy at low energy levels. Our results reveal an ingenious mechanism by which active RNA polymerase pools are adjusted in response to the nutritional state.

Markus Wahl, Hao-Hong Pei, Tarek Hilal, Zhuo Chen, Yong-Heng Huang, Yuan Gao, Nelly Said, Bernhard Loll, Juri Rappsilber, Georgiy Belogurov, Irina Artsimovitch. 2020.

Advances in genomics have revealed many of the genetic underpinnings of human disease, but exposomics methods are currently inadequate to obtain a similar level of understanding of environmental contributions to human disease. Exposomics methods are limited by low abundance of xenobiotic metabolites and lack of authentic standards, which precludes identification using solely mass spectrometry-based criteria. Here, we develop and validate a method for enzymatic generation of xenobiotic metabolites for use with high-resolution mass spectrometry (HRMS) for chemical identification. Generated xenobiotic metabolites were used to confirm identities of respective metabolites in mice and human samples based upon accurate mass, retention time and co-occurrence with related xenobiotic metabolites. The results establish a generally applicable enzyme-based identification (EBI) for mass spectrometry identification of xenobiotic metabolites.

Ken Liu, Choon Lee, Grant Singer, Michael Woodworth, Thomas Ziegler, Colleen Kraft, Gary Miller, Shuzhao Li, Young-Mi Go, Edward Morgan, Dean Jones. 2020.

Hypoxia selectively enhances mRNA translation despite suppressed mammalian target of rapamycin complex 1 activity, contributing to gene expression reprogramming that promotes metastasis and survival of cancer cells. Little is known about how this paradoxical control of translation occurs. Here, we report a new pathway that links hypoxia to selective mRNA translation. Transglutaminase 2 (TG2) is a hypoxia-inducible factor 1–inducible enzyme that alters the activity of substrate proteins by polyamination or crosslinking. Under hypoxic conditions, TG2 polyaminated eukaryotic translation initiation factor 4E (eIF4E)-bound eukaryotic translation initiation factor 4E-binding proteins (4EBPs) at conserved glutamine residues. 4EBP1 polyamination enhances binding affinity for Raptor, thereby increasing phosphorylation of 4EBP1 and cap-dependent translation. Proteomic analyses of newly synthesized proteins in hypoxic cells revealed that TG2 activity preferentially enhanced the translation of a subset of mRNA containing G/C-rich 5′UTRs but not upstream ORF or terminal oligopyrimidine motifs. These results indicate that TG2 is a critical regulator in hypoxia-induced selective mRNA translation and provide a promising molecular target for the treatment of cancers.

Sung-Yup Cho, Seungun Lee, Jeonghun Yeom, Hyo-Jun Kim, Jin-Haeng Lee, Ji-Woong Shin, Mee-ae Kwon, Ki Baek Lee, Eui Man Jeong, Hee Sung Ahn, Dong-Myung Shin, Kyunggon Kim, In-Gyu Kim. Life Science Alliance, 3 (3), e201900565, 2020.