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The N -methyl- d -aspartate (NMDA) receptor is a glutamate-activated cation channel that is critical to many processes in the brain. Genome-wide association studies suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity are important for body weight homeostasis 1 . Here we report the engineering and preclinical development of a bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease. GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects neuroplasticity in the hypothalamus and brainstem. Importantly, targeting of MK-801 to GLP-1 receptor-expressing brain regions circumvents adverse physiological and behavioural effects associated with MK-801 monotherapy. In summary, our approach demonstrates the feasibility of using peptide-mediated targeting to achieve cell-specific ionotropic receptor modulation and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for safe and effective obesity treatment. Unimolecular integration of NMDA receptor antagonism with GLP-1 receptor agonism effectively reverses obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease.

Previously, we prepared a pirarubicin (THP)‐encapsulated micellar drug using styrene–maleic acid copolymer (SMA) as the drug carrier, in which active THP was non‐covalently encapsulated. We have now developed covalently conjugated SMA‐THP (SMA‐THP conjugate) for further investigation toward clinical development, because covalently linked polymer–drug conjugates are known to be more stable in circulation than drug‐encapsulated micelles. The SMA‐THP conjugate also formed micelles and showed albumin binding capacity in aqueous solution, which suggested that this conjugate behaved as a macromolecule during blood circulation. Consequently, SMA‐THP conjugate showed significantly prolonged circulation time compared to free THP and high tumor‐targeting efficiency by the enhanced permeability and retention (EPR) effect. As a result, remarkable antitumor effect was achieved against two types of tumors in mice without apparent adverse effects. Significantly, metastatic lung tumor also showed the EPR effect, and this conjugate reduced metastatic tumor in the lung almost completely at 30 mg/kg once i.v. (less than one‐fifth of the maximum tolerable dose). Although SMA‐THP conjugate per se has little cytotoxicity in vitro (1/100 of free drug THP), tumor‐targeted accumulation by the EPR effect ensures sufficient drug concentrations in tumor to produce an antitumor effect, whereas toxicity to normal tissues is much less. These findings suggest the potential of SMA‐THP conjugate as a highly favorable candidate for anticancer nanomedicine with good stability and tumor‐targeting properties in vivo. Covalently amide linked SMA‐THP conjugate showed high stability in circulation and thus prolonged circulation time and tumor targeting property based on the EPR effect. Consequently marked in vivo antitumor effects were achieved, including metastatic lung cancer, with less adverse effects, suggesting the potential of SMA‐THP conjugate as a promising candidate for cancer treatment.

This study developed a high-performance liquid chromatography-tandem mass spectrometry method to simultaneously determine the concentrations of flupirtine and its major active metabolite D-13223 in human plasma in order to assess the bioequivalence (BE) of two flupirtine maleate capsules among healthy male Chinese volunteers under fasting and fed conditions. There were two single-center, randomized, single-dose, open-label, laboratory-blinded, two-period, cross-over studies which included 24 healthy male Chinese volunteers under fasting and fed conditions, respectively. Plasma samples were collected prior to and up to 48 h after dosing. The concentrations of flupirtine and its major active metabolite D-13223 in plasma samples were determined by a validated method, that is, high-performance liquid chromatography coupled with a tandem mass spectrometry detector. Pharmacokinetic metrics of area from time zero to the last measurable concentration (AUC ), area under the plasma concentration-time curve from administration to infinite time (AUC ), and C were used for BE assessment. Forty-eight healthy volunteers who met the criteria were enrolled and completed the study. According to the observation of vital signs and laboratory measurement, no volunteers had any adverse reactions. Under fasting condition, the geometric mean ratios (90% CI) of the test/reference drug for flupirtine were 103.0% (98.1%-108.2%) for AUC , 102.9% (98.2%-107.9%) for AUC , and 97.0% (85.9%-109.5%) for C . Under fed condition, the geometric mean ratios (90% CI) of the test/reference drug for flupirtine were 101.7% (98.4%-105.1%) for AUC , 101.6% (98.5%-104.8%) for AUC , and 103.5% (94.7%-113.0%) for C . The difference between test and reference formulations, T , was not statistically significant. The 90% CIs of the test/reference AUC ratio and C ratio of D-13223 were also within the acceptance range for BE both under fasting and fed conditions. The two formulations of flupirtine maleate capsule were bioequivalent (the test and the reference products) under fasting and fed conditions, and thus both can be used interchangeably in the clinical setting.

This study aimed to develop a multienzymatic system for synthesis of l -malate. First, recombinant Escherichia coli strains were constructed expressing maleic acid cis – trans isomerase (MaiA) or fumarase C (FumC) from different sources. Serratia marcescens MaiA (SMaiA) and E. coli FumC (ECFumC) showed good catalytic performance. Next, six co-expression systems for SMaiA and ECFumC were constructed. E. coli BL21 (DE3)-pRSF Duet-1 - ecfumC-smaiA (named strain pFM2) had the highest l -malate catalytic activity. In 7-L fed-batch fermentation, the SMaiA and ECFumC activities of strain pFM2 wet cells were 43.4 and 154.5 U/g, respectively, 2.4- and 10.7-fold the values that were obtained in shaken flasks. Finally, a whole-cell catalytic process was established for the production of l -malate by strain pFM2 with maleate as the substrate. When the dose of pFM2 wet cells was 0.5 g/100 mL and 1 mol/L maleate was the substrate, the catalytic process was completed within 4 h. Notably, the intermediate fumarate was almost absent during the conversion process. The concentration of l -malate reached 143.8 g/L with a yield of 0.60 g/(L·min). The molar conversion rate of the substrate was 98.4%. These findings lay a foundation for the industrial application of multienzymatic synthesis of l -malate. Graphical Abstract

The potential of fumaric acid as a raw material in the polymer industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w/w from glucose, the latter raw material is three times cheaper. Besides, the fermentation fixes CO₂. Production of fumaric acid by Rhizopus species and the involved metabolic pathways are reviewed. Submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production include metabolic engineering approaches to achieve low pH fermentations.

The saffron bulb mite, Rhizoglyphus robini Claparede ( Acari: Acaridae ), is the most important pest of the saffron crop in Iran. This pest attacks and feeds on saffron corms. For this reason, the corms are treated with acaricides before planting. The high activity of detoxification enzymes in arthropods may reduce their pesticide sensitivity. Diethyl maleate (DEM) is an inhibitor of glutathione S-transferases (GSTs), piperonyl butoxide (PBO) is an inhibitor of cytochrome P450 monooxygenases, and triphenyl phosphate (TPP) is an inhibitor of esterase activity. A filter paper method was used to determine the efficiency of these synergists in inhibiting the activity of detoxifying enzymes of R. robini . Adult mites were treated with these three synergists for 6, 12, 24, and 48 h, respectively. The activity of each detoxifying enzyme was measured and compared to the control treatment, and the inhibition percentage was calculated each time. The results showed that DEM reduced GST activity by 59.9% after 48 h, PBO inhibited cytochrome P450 activity by 30%, and TPP suppressed esterase activity by 38.5%. The most statistically significant inhibition occurred 24 h after pretreatment with each synergist. Bioassays with 24 h pretreatment showed that the sensitivity of R. robini to propargite increased by 1.6 times with PBO, 1.7 times with TPP, and 2.5 times with DEM. In conclusion, synergists and efficient inhibition of detoxifying enzymes can play a significant role in increasing the sensitivity of agricultural pests to pesticides and can be considered in managing pesticide resistance.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62–1000 nM) or diethyl maleate (5.62–1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Acid-sensing ion channels (ASICs) are proton-gated members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout the central and peripheral nervous systems. The homotrimeric splice variant ASIC1a has been implicated in nociception, fear memory, mood disorders and ischemia. Here, we extract full-length chicken ASIC1 (cASIC1) from cell membranes using styrene maleic acid (SMA) copolymer, elucidating structures of ASIC1 channels in both high pH resting and low pH desensitized conformations by single-particle cryo-electron microscopy (cryo-EM). The structures of resting and desensitized channels reveal a reentrant loop at the amino terminus of ASIC1 that includes the highly conserved ‘His-Gly’ (HG) motif. The reentrant loop lines the lower ion permeation pathway and buttresses the ‘Gly-Ala-Ser’ (GAS) constriction, thus providing a structural explanation for the role of the His-Gly dipeptide in the structure and function of ASICs.

The NMDA ( N -methyl- d -aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1–GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer’s disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation. A high-resolution X-ray structure and molecular dynamics simulations of the N -methyl- d -aspartate receptor in complexes with channel-blocking ligands reveals the molecular basis of the ligand binding and channel block.

Endocrine disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling and thereby adversely affecting human health. Recent reports provide evidence for the presence of EDCs in commercially available bottled water, including steroid receptor agonists and antagonists. However, since these findings are based on biological data the causative chemicals remain unidentified and, therefore, inaccessible for toxicological evaluation. Thus, the aim of this study is to assess the antiestrogenic and antiandrogenic activity of bottled water and to identify the causative steroid receptor antagonists. We evaluated the antiestrogenic and antiandrogenic activity of 18 bottled water products in reporter gene assays for human estrogen receptor alpha and androgen receptor. Using nontarget high-resolution mass spectrometry (LTQ-Orbitrap Velos), we acquired corresponding analytical data. We combined the biological and chemical information to determine the exact mass of the tentative steroid receptor antagonist. Further MS(n) experiments elucidated the molecule's structure and enabled its identification. We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%. Nontarget chemical analysis revealed that out of 24520 candidates present in bottled water one was consistently correlated with the antagonistic activity. By combining experimental and in silico MS(n) data we identified this compound as di(2-ethylhexyl) fumarate (DEHF). We confirmed the identity and biological activity of DEHF and additional isomers of dioctyl fumarate and maleate using authentic standards. Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water. Applying a novel approach to combine biological and chemical analysis this is the first study to identify so far unknown EDCs in bottled water. Notably, dioctyl fumarates and maleates have been overlooked by science and regulation to date. This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.