Gut metabolome and microbiota signatures predict response to treatment with exclusive enteral nutrition in a prospective study in children with active Crohn's disease.

BACKGROUND: Predicting response to exclusive enteral nutrition (EEN) in active Crohn's disease (CD) could lead to therapy personalization and pretreatment optimization. OBJECTIVES: This study aimed to explore the ability of pretreatment parameters to predict fecal calprotectin (FCal) levels at EEN completion in a prospective study in children with CD. METHODS: In children with active CD, clinical parameters, dietary intake, cytokines, inflammation-related blood proteomics, and diet-related metabolites, metabolomics and microbiota in feces, were measured before initiation of 8 wk of EEN. Prediction of FCal levels at EEN completion was performed using machine learning. Data are presented with medians (IQR). RESULTS: Of 37 patients recruited, 15 responded (FCal < 250 µg/g) to EEN (responders) and 22 did not (nonresponders). Clinical and immunological parameters were not associated with response to EEN. Responders had lesser (µmol/g) butyrate [responders: 13.2 (8.63-18.4) compared with nonresponders: 22.3 (12.0-32.0); P = 0.03], acetate [responders: 49.9 (46.4-68.4) compared with nonresponders: 70.4 (57.0-95.5); P = 0.027], phenylacetate [responders: 0.175 (0.013-0.611) compared with nonresponders: 0.943 (0.438-1.35); P = 0.021], and a higher microbiota richness [315 (269-347) compared with nonresponders: 243 (205-297); P = 0.015] in feces than nonresponders. Responders consumed (portions/1000 kcal/d) more confectionery products [responders: 0.55 (0.38-0.72) compared with nonresponders: 0.19 (0.01-0.38); P = 0.045]. A multicomponent model using fecal parameters, dietary data, and clinical and immunological parameters predicted response to EEN with 78% accuracy (sensitivity: 80%; specificity: 77%; positive predictive value: 71%; negative predictive value: 85%). Higher taxon abundance from Ruminococcaceae, Lachnospiraceae, and Bacteroides and phenylacetate, butyrate, and acetate were the most influential variables in predicting lack of response to EEN. CONCLUSIONS: We identify microbial signals and diet-related metabolites in feces, which could comprise targets for pretreatment optimization and personalized nutritional therapy in pediatric CD.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

Evaluation of the analgesic efficacy of grapiprant compared with robenacoxib in cats undergoing elective ovariohysterectomy in a prospective, randomized, masked, non-inferiority clinical trial.

OBJECTIVES: The main objective of this study was to compare the postoperative analgesic effects of grapiprant with those of robenacoxib in cats undergoing ovariohysterectomy (OVH). METHODS: In total, 37 female cats (age range 4 months-10 years, weighing ⩾2.5 kg) were enrolled in a prospective, randomized, masked, non-inferiority (NI) clinical trial. Cats received oral robenacoxib (1 mg/kg) or grapiprant (2 mg/kg) 2 h before OVH. Analgesia was assessed via the Feline Grimace Scale (FGS), the Glasgow Composite Measure Pain Scale-Feline (CMPS-F), von Frey monofilaments (vFFs) and pressure algometry (ALG) 2 h before treatment administration, at extubation, and 2, 4, 6, 8, 18 and 24 hours after extubation. Hydromorphone (<8 h postoperatively) or buprenorphine (>18 h postoperatively) were administered to cats with scores of ⩾5/20 on CMPS-F and/or ⩾4/10 on FGS. NI margins for CMPS-F and vFFs were set at 3 and -0.2, respectively. A mixed-effect ANOVA was used for FGS scores (P <0.05). Data are reported as mean ± SEM. RESULTS: The data from 33 cats were analyzed. The upper limit of the 95% confidence interval (CI) (0.35) was less than the NI margin of 3 for CMPS-F, and the lower limit of the 95% CI (0.055) was greater than the NI margin of -0.2 for vFFs, indicating NI of grapiprant. The FGS scores were greater than baseline at extubation for both treatments (1.65 ± 0.63; P = 0.001); however, there was no difference between treatments. There was no difference between treatments, nor treatment by time interaction, for vFFs (P <0.001). The CMPS-F scores for both treatments were higher at extubation but returned to baseline after 4 h (P <0.001). For ALG, there was no difference in treatment or treatment by time interaction. The robenacoxib group had lower pressure readings at extubation and 6 h compared with baseline. CONCLUSIONS AND RELEVANCE: These results indicate that grapiprant was non-inferior to robenacoxib for mitigating postsurgical pain in cats after OVH performed via ventral celiotomy. The impact of grapiprant for analgesia in OVH via the flank is unknown.

Anti-inflammatory potential of simvastatin and amfenac in ARPE-19 cells; insights in preventing re-detachment and proliferative vitreoretinopathy after rhegmatogenous retinal detachment surgery.

PURPOSE: Rhegmatogenous retinal detachment is a severe vision-threatening complication that can result into proliferative vitreoretinopathy (PVR) and re-detachment of the retina if recovery from surgery fails. Inflammation and changes in retinal pigment epithelial (RPE) cells are important contributors to the disease. Here, we studied the effects of simvastatin and amfenac on ARPE-19 cells under inflammatory conditions. METHODS: ARPE-19 cells were pre-treated with simvastatin and/or amfenac for 24 h after which interleukin (IL)-1α or IL-1ß was added for another 24 h. After treatments, lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) processing, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity, prostaglandin E2 (PGE2) level, and extracellular levels of IL-6, IL-8, monocytic chemoattractant protein (MCP-1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor, as well as the production of reactive oxygen species (ROS) were determined. RESULTS: Pre-treatment of human ARPE-19 cells with simvastatin reduced the production of IL-6, IL-8, and MCP-1 cytokines, PGE2 levels, as well as NF-κB activity upon inflammation, whereas amfenac reduced IL-8 and MCP-1 release but increased ROS production. Together, simvastatin and amfenac reduced the release of IL-6, IL-8, and MCP-1 cytokines as well as NF-κB activity but increased the VEGF release upon inflammation in ARPE-19 cells. CONCLUSION: Our present study supports the anti-inflammatory capacity of simvastatin as pre-treatment against inflammation in human RPE cells, and the addition of amfenac complements the effect. The early modulation of local conditions in the retina can prevent inflammation induced PVR formation and subsequent retinal re-detachment.

Engineering of 4-hydroxyphenylacetate 3-hydroxylase derived from Pseudomonas aeruginosa for the ortho-hydroxylation of ferulic acid.

Polyphenolic compounds have natural antioxidant properties, and their antioxidant activity is usually related to the number and position of hydroxyls. Here, we successfully applied the engineered 4-hydroxyphenylacetate 3-hydroxylases (4HPA3Hs) derived from Pseudomonas aeruginosa to catalyze ferulic acid (FA) synthesis of ortho-hydroxyferulic acid (5-hydroxyferulic acid, 5-OHFA). Through optimization of co-expression, the oxygenase component (PaHpaB) and the reductase component (PaHpaC) in E. coli, and optimization of whole-cell catalytic conditions, the engineered strain BC catalyzed ortho-hydroxylation of 2 g/L of FA with a yield of 75 % from 39 %. Through tunnel engineering of PaHpaB, the obtained mutants F301A and Q376A almost completely transformed 2 g/L of FA. Further, a multiple mutant L214A/F301A/Q376A converted 4 g/L FA into 5-OHFA within 12 h, and the yield reached 99.9 %, which was approximately 2.39-fold of the wild type. The kcat/Km value of L214A/F301A/Q376A was about 307 times greater than that of the wide type. Analysis of three-dimensional structural models showed that L214, F301, and Q376 mutated into Ala, which greatly shortened the side chain and broadened the tunnel size, thereby significantly improving the catalytic efficiency of L214A/F301A/Q376A. This biosynthesis of 5-OHFA is simple, efficient, and green, suggesting that it is useful for efficient biosynthesis of polyphenolic compounds.

Integrated gene co-expression network analysis and experimental validation revealed potential targets of human urine extract CDA-II in treating chronic myeloid leukemia.

BACKGROUND: Cell differentiation agent II (CDA-II) exhibits potent anti-proliferative and apoptosis-inducing properties against a variety of cancer cells. However, its mechanism of action in chronic myeloid leukemia (CML) remains unclear. METHODS: Cell counting Kit 8 (CCK-8) and flow cytometry were used to investigate the effects of CDA-II on the biological characteristics of K562 cells. Gene (mRNA and lncRNA) expression profiles were analyzed by bioinformatics to screen differentially expressed genes and to perform enrichment analysis. The Pearson correlation coefficients of lncRNAs and mRNAs were calculated using gene expression values, and a lncRNA/mRNA co-expression network was constructed. The MCODE and cytoHubba plugins were used to analyze the co-expression network. RESULTS: The Results, derived from CCK-8 and flow cytometry, indicated that CDA-II exerts dual effects on K562 cells: it inhibits their proliferation and induces apoptosis. From bioinformatics analysis, we identified 316 mRNAs and 32 lncRNAs. These mRNAs were predominantly related to the meiotic cell cycle, DNA methylation, transporter complex and peptidase regulator activity, complement and coagulation cascades, protein digestion and absorption, and cell adhesion molecule signaling pathways. The co-expression network comprised of 163 lncRNA/mRNA interaction pairs. Notably, our analysis results implicated clustered histone gene families and five lncRNAs in the biological effects of CDA-II on K562 cells. CONCLUSION: This study highlights the hub gene and lncRNA/mRNA co-expression network as crucial elements in the context of CDA-II treatment of CML. This insight not only enriches our understanding of CDA-II's mechanism of action but also might provide valuable clues for subsequent experimental studies of CDA-II, and potentially contribute to the discovery of new therapeutic targets for CML.

Modified Chinese quince oligomeric proanthocyanidin protects deep-frying oil quality by inhibiting oxidation.

Chinese quince (Chaenomeles sinensis) fruit is an underutilized resource, rich in proanthocyanidins with antioxidant ability but poor lipid solubility. In this study, a novel modified oligomeric proanthocyanidin (MOPA) was prepared, which exhibited favorable lipid solubility (354.52 mg/100 g). It showed higher radical scavenging abilities than commercial antioxidant-BHA (butylated hydroxyanisole), both at 0.4-0.5 mg/mL. The addition of MOPA (0.04 %wt.) significantly increased the oxidative stability index of the soybean oil from 5.52 to 8.03 h, which was slightly lower than that of BHA (8.35 h). Analysis of the physicochemical properties and composition of oil during deep-frying showed that MOPA demonstrated significant antioxidant effects and effectively restricted the oil oxidation. This inhibition also delays the formation of heterocyclic amines (HAs) in fried food, thereby reducing the migration of HAs from food to deep-frying oil. Therefore, MOPA is a promising novel liposoluble antioxidant for protecting the quality of deep-frying oil.

Intestinal Microbiome Metabolism of Cranberry (Vaccinium macrocarpon) Proanthocyanidin Dimers, but Not Trimers, Is Altered by Dysbiosis in Ulcerative Colitis Ex Vivo.

Cranberries contain proanthocyanidins with different interflavan bond types and degrees of polymerization. These chemical differences may impact the metabolism of proanthocyanidins by the intestinal microbiome. In our previous study, we found that healthy microbiomes produced higher concentrations of the phenolic acid metabolites 5-(3',4'-dihydroxyphenyl)-g-valerolactone and 3-hydroxyphenylacetic acid from the cranberry extract in comparison to ulcerative colitis (UC) microbiomes ex vivo. To understand this difference, LC-ESI-MS/MS was utilized to characterize the metabolism of the precursor proanthocyanidins. Healthy microbiomes metabolized procyanidin A2, procyanidin B2, and procyanidin dimeric intermediates but not A-type trimers, to a greater extent than UC microbiomes. The metabolism of procyanidin A2 and procyanidin B2 by fecal microorganisms was then compared to identify their derived phenolic acid metabolites. 5-(3',4'-Dihydroxyphenyl)-g-valerolactone and 3-hydroxyphenylacetic acid were identified as unique metabolites of procyanidin B2. Based on these results, the metabolism of procyanidin B2 contributed to the differential metabolism observed between healthy and UC microbiomes.

Effect of meloxicam or robenacoxib administration timing on renal function and postoperative analgesia in cats undergoing ovariohysterectomy: A randomized, blinded, controlled clinical trial.

We evaluated the effect of administration timing of meloxicam and robenacoxib on renal function, platelet cyclo-oxygenase and perioperative analgesia in 60 cats undergoing ovariohysterectomy, in a prospective randomized blinded controlled study. Twelve cats were randomly allocated to one subcutaneous treatment group: meloxicam (0.2 mg/kg) or robenacoxib (2 mg/kg) at admission (MA, RA), at induction (MI, RI) and robenacoxib at the end of surgery (RE). All cats received the same anaesthesia protocol. Plasma renin activity (PRA), plasma creatinine, drug concentrations and serum thromboxane (TxB2) were measured sequentially. Anaesthesia significantly increased PRA, as activity at end of the surgery was higher than 2 h later (mean ± SD: 26.6 ± 2.8 versus 10.0 ± 3.9 ng/mL/h). PRA remained higher at 2 h post-surgery in admission groups compared to induction groups (p = .01). Serum TxB2 was lower with meloxicam than robenacoxib (p = .001), and was lower in the MA than each robenacoxib group at catheter placement. Admission groups (16/24 from RA and MA groups) received earlier rescue analgesia than other groups (p = .033). In conclusion, the renin-angiotensin system was activated during anaesthesia despite cyclo-oxygenase inhibition, possibly due to hypotension or surgical stimulation. There was no effect of drug or timing on the markers of renal function but one cat receiving meloxicam at induction had suspected IRIS grade II acute kidney injury.

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

The Influence of Topical Perioperative Nepafenac on Retinal Vasculature Following Phacoemulsification Using Optical Coherence Tomography Angiography: A Randomized Clinical Trial.

PURPOSE: To investigate topical nepafenac drops (TND) effect on retinal vasculature following uneventful phacoemulsification. METHODS: 54 eyes were randomly assigned to G1: TND 3 days preoperatively and 1 month postoperatively; G2: TND for 1 month postoperatively; and G3 no TND (control). BCVA, macular OCTA were compared baseline, 1 week and 1 month. RESULTS: No statistically significant differences found between 3 groups in OCTA parameters during study visits. However, at 1 month (compared to baseline) there were a statistically significant increase in superficial capillary plexus (SCP) capillary vessel density (CVD) in G2 (p=0.036); increase in central foveal thickness (CFT) in G2 (p=0.011) and G3 (control) (p=0.001); and reduced number of patients with SCP disorganization in G2 (p=0.04). There were no significant correlation/association between most of OCTA parameters and final BCVA. CONCLUSION: TND use perioperative did not show any effect on retinal OCTA microvasculature and might affect macular edema by other mechanisms.

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins.

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

Antiproliferative, antimigratory, and apoptotic effects of diffractaic and vulpinic acids as thioredoxin reductase 1 inhibitors on cervical cancer.

Cervical cancer is among the most frequently observed cancer types in females. New therapeutic targets are needed because of the side impacts of existing cancer drugs and the inadequacy of treatment methods. Thioredoxin reductase 1 (TrxR1) is often overexpressed in many cancer cells, and targeting TrxR1 has become an attractive target for cancer therapy. This study investigated the anticancer impacts of diffractaic and vulpinic acids, lichen secondary metabolites, on the cervical cancer HeLa cell line. XTT findings demonstrated showed that diffractaic and vulpinic acids suppressed the proliferation of HeLa cells in a dose- and time-dependent manner and IC50 values were 22.52 µg/ml and 66.53 µg/ml at 48 h, respectively. Each of these lichen metabolites significantly suppressed migration. Diffractaic acid showed an increase in both the BAX/BCL2 ratio by qPCR analysis and the apoptotic cell population via flow cytometry analysis on HeLa cells. Concerning vulpinic acid, although it decreased the BAX/BCL2 ratio in this cells, it increased apoptotic cells according to the flow cytometry analysis results. Diffractaic and vulpinic acids significantly suppressed TrxR1 enzyme activity rather than the gene and protein expression levels in HeLa cells. This research demonstrated for the first time, that targeting TrxR1 with diffractaic and vulpinic acids was an effective therapeutic strategy for treating cervical cancer.

Modified Podophyllotoxin Phenoxyacetamide Phenylacetate Derivatives: Tubulin/AKT1 Dual-Targeting and Potential Anticancer Agents for Human NSCLC.

Cancer is a major disease threatening human health worldwide, among which non-small-cell lung cancer (NSCLC) is the most deadly. Clinically, almost all anticancer drugs eventually fail to consistently benefit patients due to serious drug resistance. AKT is a key effector of the PI3K/AKT/mTOR pathway, which is closely related to the occurrence, development, and drug resistance of tumors. Herein, we first designed and synthesized 20 kinds of novel hybrid molecules targeting both tubulin and AKT based on a podophyllotoxin (PPT) skeleton through computer-aided drug design. By CCK8 assay, we screened the compound D1-1 (IC50 = 0.10 µM) with the strongest inhibitory activity against H1975 cells, and its activity was 100 times higher than PPT (IC50 = 12.56 µM) and 300 times higher than gefitinib (IC50 = 32.15 µM). Affinity analysis results showed that D1-1 not only retained the tubulin targeting of PPT but also showed strong AKT targeting. Subsequent pharmacological experiments showed that D1-1 significantly inhibited the proliferation and metastasis of H1975 cells and slightly induced their apoptosis by inhibiting both tubulin polymerization and the AKT pathway activation. Collectively, these data demonstrate that the novel hybrid molecule D1-1 may be an excellent lead compound for the treatment of human NSCLC as a dual inhibitor of tubulin and AKT.

Cyclodextrin-enabled nepafenac eye drops with improved absorption open a new therapeutic window.

Nepafenac is a highly effective NSAID used for treating postoperative ocular inflammation and pain after cataract surgery and its advantage over conventional topical NSAIDs has been proved many times. However, Nevanac® is a suspension eye drop, which clearly lacks patient compliance causing irritation, blurred vision, foreign body sensation along with problematic dosage due to its sticky, inhomogeneous consistence. In this study, nepafenac containing eye drops were prepared using hydroxypropyl-ß-cyclodextrin to ensure complete dissolution of nepafenac, sodium hyaluronate to provide mucoadhesion and adequate viscosity and a preservative-free officinal formula, Oculogutta Carbomerae containing carbomer (just like Nevanac®), therefore providing a similar base for the new formulations. According to an experimental design, 11 formulations were tested in vitro including two reference formulations by measuring their viscosity, mucoadhesion, drug release and corneal permeability. Finally, two formulations were found promising and investigated further on porcine eyes ex vivo and corneal distribution of nepafenac was determined by RAMAN mapping. The results showed that one formulation possessed better bioavailability ex vivo than Nevanac® 0.1 % suspension, while the other formulation containing only 60 % of the original dose were ex vivo equivalent with Nevanac® opening the way to nepafenac-containing eye drops with better patient compliance in the future.

Metabolites from the plant endophytic fungus Penicillium sp. CPCC 401423 and their cytotoxic activity against MIA PaCa-2 cells.

Twenty-two metabolites were isolated from Penicillium sp. CPCC 401423 cultured on rice. The structures of all compounds were elucidated mainly by MS and NMR analysis as well as the necessary CD experimental evidence, of which penicillidione A (1), penicillidione B (2), (E)-4-[(4-acetoxy-3-methyl-2-butenyl)oxy]phenylacetic acid (3), (S)-2-hydroxy-2-{4-[(3-methyl-2-butenyl)oxy]phenyl} (4), (S)-4-(2,3-dihydroxy-3-methyl-butoxy)phenylacetic acid (5), (E)-4-[(3-carboxy-2-butenyl)oxy]benzoic acid (6), (Z)-4-[(4-hydroxy-3-methyl-2-butenyl)oxy]benzoic acid (7), open-cycled N-demethylmelearoride A (12), and penostatin M (16) were identified as new compounds. The cytotoxic activity against human pancreatic carcinoma cell line MIA PaCa-2a was detected. Among them, compounds 13-15 and 22 displayed significant cytotoxicity against MIA-PaCa-2 cells with IC50 values of 8.9, 36.5, 31.8, and 22.3 µM, respectively (positive control gemcitabine IC50 65.0 µM).

The many forms of alpha-methoxy phenylacetic acid in the gas phase: flexibility, internal dynamics, and their intramolecular interactions.

We present a rotational spectroscopy study of alpha-methoxy phenylacetic acid in the gas phase. This acid is a derivative of mandelic acid and is used in various organic reactions. The conformational landscape of alpha-methoxy phenylacetic acid was explored to gain insight into its intramolecular dynamics. A rich rotational spectrum was obtained using chirped-pulse Fourier transform microwave spectroscopy in the 2-8 GHz range. Five conformers out of six calculated low-energy forms were identified in the spectrum, and the assignment of the 13C singly substituted isotopologues for the lowest-energy conformer led to its accurate structure determination. Splitting patterns were analyzed and attributed to the internal rotation of a methyl top. The analysis of the non-covalent interactions within the molecule highlights the subtle balance in the stabilization of the different conformers. We thus provide high-level structural and intramolecular dynamics information that is also used to benchmark the performance of quantum-chemical calculations.

The antibacterial mechanism of phenylacetic acid isolated from Bacillus megaterium L2 against Agrobacterium tumefaciens.

Background: Agrobacterium tumefaciens T-37 can infect grapes and other fruit trees and cause root cancer. Given the pollution and damage of chemical agents to the environment, the use of biological control has become an important area of focus. Bacillus megaterium L2 is a beneficial biocontrol strain isolated and identified in the laboratory, which has a good antibacterial effect on a variety of plant pathogens. The antibacterial metabolites of L2 were separated and purified to obtain a bioactive compound phenylacetic acid (PAA). Methods: The potential antibacterial mechanism of PAA against A. tumefaciens T-37 strain was determined by relative conductivity, leakage of nucleic acids, proteins, and soluble total sugars, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reactive oxygen species (ROS). Results: PAA showed good antibacterial activity against strain A. tumefaciens T-37 with IC50 of 0.8038 mg/mL. Our data suggested that after treatment with PAA, the relative conductivity, nucleic acid, protein, and total soluble sugar of T-37 were increased significantly compared with the chloramphenicol treatment group and the negative treatment group. The total protein synthesis of T-37 cells was inhibited, the consumption of phosphorus decreased with the increase of incubation time, and the content of ROS was significantly higher than that in the negative treatment group. Meanwhile, the activity of two key enzymes (MDH and SDH) involved in the tricarboxylic acid cycle (TCA cycle) decreased. In addition, T-37 cells were found to be damaged by scanning electron microscopy observation. Our results showed that PAA can destroy cell membrane integrity, damage cell structures, affect cell metabolism, and inhibit protein synthesis to exert an antibacterial effect. Conclusions: We concluded that the mechanism of action of the PAA against strain T-37 might be described as PAA exerting antibacterial activity by affecting cell metabolism, inhibiting protein synthesis, and destroying cell membrane integrity and cell ultrastructure. Therefore, PAA has a promising application prospect in the prevention and treatment of root cancer disease caused by A. tumefaciens.

The Nurr7 agonist Cytosporone B differentially regulates inflammatory responses in human polarized macrophages.

The orphan nuclear receptor Nur77 is involved in diverse cellular processes such as inflammation, proliferation, differentiation and survival. Stimuli like lipopolysaccharide (LPS) and tumor necrosis factor (TNF) increase Nur77 expression in human and murine macrophages, and it has been proposed that Nur77 plays a major role in dampening the inflammatory response. Here, we evaluated the expression and function of Nur77 in human anti-inflammatory and pro-inflammatory macrophages derived from blood monocytes cultured with macrophage colony-stimulating factor (M-MDMs) or granulocyte/macrophage colony-stimulating factor (GM-MDMs), respectively. Nur77 mRNA expression was significantly enhanced in M-MDMs compared with GM-MDMs, both constitutively and upon exposure to Toll-like receptor (TLR)2, 3, and 4 ligands. Nur77 activation with the agonist Cytosporone B (CsnB) significantly suppressed the production of TNF, interleukin (IL)-1ß, IL-6, and IL-8 in GM-MDMs stimulated with LPS. In contrast, it tended to enhance the production of the anti-inflammatory cytokine IL-10. This effect was associated with reduced NF-κB p65 nuclear translocation. Similarly, Nur77 knockdown enhanced TNF production in GM-MDMs. CsnB effectively stimulated the transactivation activity of Nur77 in M-MDMs, but it did not alter cytokine synthesis or p65 nuclear translocation. However, Nur77 seemed to have a role in maintaining the anti-inflammatory profile of M-MDMs, since Nur77-deficient M-MDMs constitutively produced higher levels of TNF transcripts. Thus, in the absence of exogenous agonists, Nur77 activity favors the anti-inflammatory function of M-MDMs, whereas agonistic activation of this receptor preferentially drives attenuation of inflammation in inflammatory macrophages.

Non-target metabolomics reveals the changes of small molecular substances in duck breast meat under different preservation time.

Poultry products are an essential animal source of protein for humans. Many factors could destroy the balance of the poultry production chain and cause an overstock of products, which need to be stored in the frozen storage warehouse for a long time. The long-term frozen storage may affect the quality of meat products. In this study, the changes of small molecular substances were revealed in duck meat during long-term storage using non-targeted metabolomics. The results showed that compared with fresh meat, even if the meat is stored under frozen storage conditions, the number of differential metabolites of frozen storage meat continues to increase with the prolongation of storage time, indicating that the meat composition has changed significantly with the storage time increased. With the increase in storage time, the nitrogen-containing small molecular compounds in duck meat increased (carnosine and anserine, aspartic acid, and tyrosine, 1H-indole-3-acetamide, 2-Hydroxyphenethylamine, 2-Naphylamine, allocystathionine, and O-phosphoethanolamine), the nucleotides decomposition process strengthened (IMP and AMP, GMP and UMP), and the content of organic acid increased (5-hydroxy indole acetic acid, 5-hydroxypentanoic acid and phenylacetate, taurine) and carbohydrate (1-O-sinapoyl-beta-d-glucose, 4-O-beta-d-glucopyranosyl-d-mannose, and alpha-d-glucose). These small molecular substances can be used as biomarkers to detect long-term stored duck meat deterioration. KEGG enrichment analysis showed that protein catabolism, nucleotide catabolism, fat decomposition and oxidation, and carbohydrate decomposition were the main metabolic processes of meat deterioration during the long-term storage of duck meat. In addition, Non-target metabolome technology is a powerful tool to reveal the meat deterioration process during long-term storage systematically. This study provided a reference for optimizing domestic poultry meat storage methods and ensuring food safety.