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Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins has been associated with neurodegenerative diseases, although direct mechanisms are poorly defined. Here, we report on a maturation process for the cellular prion protein (PrP C ) that involves a conformational change after LLPS and is regulated by mutations and poly(4-styrenesulfonic acid- co -maleic acid) (PSCMA), a molecule that has been reported to rescue Alzheimer's disease-related cognitive deficits by antagonizing the interaction between PrP C and amyloid-β oligomers (Aβo). We show that PSCMA can induce reentrant LLPS of PrP C and lower the saturation concentration ( C sat ) of PrP C by 100-fold. Regardless of the induction method, PrP C molecules subsequently undergo a maturation process to restrict molecular motion in a more solid-like state. The PSCMA-induced LLPS of PrP C stabilizes the intermediate LLPS conformational state detected by NMR, though the final matured β-sheet-rich state of PrP C is indistinguishable between induction conditions. The disease-associated E200 K mutation of PrP C also accelerates maturation. This post-LLPS shift in protein conformation and dynamics is a possible mechanism of LLPS-induced neurodegeneration.

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The K D of xanthotoxin, bergapten, and naringenin were 5.14 μM, 4.57 μM, and 3.72 μM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: The first dengue outbreak in Sao Tome and Principe was reported in 2022. Entomological investigations were undertaken to establish the typology of Aedes larval habitats, the distribution of Ae. aegypti and Ae. albopictus, the related entomological risk and the susceptibility profile of Ae. aegypti to insecticides, to provide evidence to inform the outbreak response., Methodology/principal Findings: Entomological surveys were performed in all seven health districts of Sao Tome and Principe during the dry and rainy seasons in 2022. WHO tube and synergist assays using piperonyl butoxide (PBO) and diethyl maleate (DEM) were carried out, together with genotyping of F1534C/V1016I/V410L mutations in Ae. aegypti. Aedes aegypti and Ae. albopictus were found in all seven health districts of the country with high abundance of Ae. aegypti in the most urbanised district, Agua Grande. Both Aedes species bred mainly in used tyres, discarded tanks and water storage containers. In both survey periods, the Breteau (BI > 50), house (HI > 35%) and container (CI > 20%) indices were higher than the thresholds established by WHO to indicate high potential risk of dengue transmission. The Ae. aegypti sampled were susceptible to all insecticides tested except dichlorodiphenyltrichloroethane (DDT) (9.2% mortality, resistant), bendiocarb (61.4% mortality, resistant) and alpha-cypermethrin (97% mortality, probable resistant). A full recovery was observed in Ae. aegypti resistant to bendiocarb after pre-exposure to synergist PBO. Only one Ae. aegypti specimen was found carrying F1534C mutation., Conclusions/significance: These findings revealed a high potential risk for dengue transmission throughout the year, with the bulk of larval breeding occurring in used tyres, water storage and discarded containers. Most of the insecticides tested remain effective to control Aedes vectors in Sao Tome, except DDT and bendiocarb. These data underline the importance of raising community awareness and implementing routine dengue vector control strategies to prevent further outbreaks in Sao Tome and Principe, and elsewhere in the subregion., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kamgang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Biodegradable plastics (BDP) are increasingly applied; however, there has been of concerns about their environmental safety, especially from nondegradable additive compositions. Until now, data of ecotoxicity of BDP additives is scarce. Here, nematode C. elegans was used to comparatively evaluate toxicity of an isocyanate additive, i.e., Hexamethylene diisocyanate (HDI), a maleic anhydride, i.e., Diallyl maleate (DIM), and other four BDP organic additives. These additives caused lethality of nematodes at µg L -1 level, of lowest LC 50 value of HDI/DIM. Uniform exposure to these additives resulted in various degrees of inhibitions in body volumes and longevity, indicating developmental toxicity. Moreover, BDP additives induced significant elevations of gst-4 expression, especially mean 123.54 %/234.29 % increase in HDI/DIM group, but reduced ges-1 expression, which indicates oxidative damages and mitochondrial dysfunction. BDP additives further caused inhibition in locomotor and food intake/excretion behavior, and related damages of glutamatergic neurons and GABAergic neurons, indicating their neurotoxicity. We found HDI and DIM presented relatively strong effects on susceptible endpoints including lethality, gst-4, mean lifespan, food intake and excretion behavior. Overall, this study suggests prominent ecotoxic risk of isocyanates and maleic anhydrides as BDP additives, which is significant for the selection of environmentally friendly BDP additives., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Solubility-driven optimization of the salts of nitro benzothiopyranone 1, which targets DprE1, led to an antimycobacterial preclinical candidate 2. Five pharmaceutically acceptable salts, including the maleate (2), fumarate (3), citrate (4, 5), and l-malate (6) of compound 1, were prepared via the salt formation reaction and evaluated for their physicochemical and pharmacokinetic properties. Compared with 1, all the target salts exhibited greatly increased aqueous solubility and improved oral bioavailability in mice. Maleate salt 2, which displayed higher chemical stability and lower log P, showed substantially improved bioavailability in rats and a much better in vivo effect compared with free base 1 at the same dose. The X-ray crystal structure of 2 revealed that the exposed hydrophilic piperazine-maleate moiety in the crystal structure cell may be critical in increasing the solubility of 2. Thus, this maleate salt 2 overcame the poor druggability of benzothiopyranone derivatives and was identified as a promising preclinical candidate for treating tuberculosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Purpose: Tyrosine kinase inhibitors inhibit VEGF receptors. If delivered to the retina, they might inhibit oedema and neovascularization such as in age-related macular degeneration and diabetic retinopathy. The aim of this study was to formulate cediranib maleate, a potent VEGF inhibitor, as γ-cyclodextrin nanoparticle eye drops and measure the retinal delivery and overall ocular pharmacokinetics after a single-dose administration in rabbits., Methods: A novel formulation technology with 3% cediranib maleate as γ-cyclodextrin micro-suspension was prepared by autoclaving method. Suitable stabilizers were tested for heat-stable eye drops. The ophthalmic formulation was topically applied to one eye in rabbits. The pharmacokinetics in ocular tissues, tear film and blood samples were studied at 1, 3 and 6 hr after administration., Results: γ-cyclodextrin formed complex with cediranib maleate. The formation of γ-cyclodextrin nanoparticles occurred in concentrated complexing media. Combined stabilizers prevented the degradation of drug during the autoclaving process. Three hours after administration of the eye drops, treated eyes showed cediranib levels of 737 ± 460 nM (mean ± SD) in the retina and 10 ± 6 nM in the vitreous humour., Conclusions: Cediranib maleate in γ-cyclodextrin nanoparticles were stable to heat in presence of stabilizers. The drug as eye drops reached the retina in concentrations that are more than 100 times higher than the 0.4 nM IC 50 value reported for the VEGF type-II receptor and thus, presumably, above therapeutic level. These results suggest that γ-cyclodextrin-based cediranib maleate eye drops deliver effective drug concentrations to the retina in rabbits after a single-dose administration., (© 2022 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

In the present study, four novel ginsenosides fatty acid and aromatic acid derivatives were designed and synthesized, and their cytotoxic effects on human ovarian carcinoma cells (SKOV3) were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated that all derivatives inhibited SKOV3 cell growth, and Compound 3 showed the most outstanding anti-proliferative effect on SKOV3 cells. The IC 50 value of Compound 3 was 33.8 ± 2.21 µM, less than half of that of cis-platinum (70.1 ± 7.64 µM). Subsequent analysis revealed that Compound 3 could promote SKOV3 cell apoptosis, and the percentage of apoptotic cell population increased with increasing Compound 3 concentrations. In addition, the expression ratios of Bax/Bcl-2, cleaved-Caspase-3/Caspase-3 and cleaved-Caspase-9/Caspase-9 were gradually elevated in Compound 3-treated SKOV3 cells compared with control cells. Furthermore, translocation of Bax to mitochondria was associated with the release of Cytochrome C. Molecular docking analysis revealed three hydrogen-bonds existed in Compound 3 with poly(ADP-ribose)polymerase (PARP) receptor (PDB code: 5DSY), which may be the target of the anti-ovarian cancer effect of Compound 3. Altogether, our study indicates that Compound 3 induces SKOV3 cell apoptosis via reactive oxygen species (ROS)-dependent mitochondrial pathway, and can serve as an anti-cancer agent for treating ovarian carcinoma.

Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activation depends on prior itaconate synthesis. When added to human cells in supraphysiological concentrations, all three isomers reduce lactate levels, whereas itaconate is the strongest succinate dehydrogenase (SDH) inhibitor. In cells infected with influenza A virus (IAV), all three isomers profoundly alter amino acid metabolism, modulate cytokine/chemokine release and reduce interferon signalling, oxidative stress and the release of viral particles. Of the three isomers, citraconate is the strongest electrophile and nuclear factor-erythroid 2-related factor 2 (NRF2) agonist. Only citraconate inhibits catalysis of itaconate by cis-aconitate decarboxylase (ACOD1), probably by competitive binding to the substrate-binding site. These results reveal mesaconate and citraconate as immunomodulatory, anti-oxidative and antiviral compounds, and citraconate as the first naturally occurring ACOD1 inhibitor., (© 2022. The Author(s).)

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction., (© 2022. The Author(s).)

Since the protective activity of the Bordeaux mixture against plant disease caused by oomycetes was discovered, copper compounds have been used for more than a century as an effective plant protection strategy. However, the application of excessive copper can cause adverse effects through long-term heavy metal accumulation in soils. Therefore, it is necessary to develop new strategies to reduce or replace copper in pesticides based on organic and low-input farming systems. Organic acids are eco-friendly. In this study, we tested the antifungal and anti-oomycete activity of maleic acid (MA) and copper sulfate (CS) against 13 plant pathogens. Treatment with a mixture of MA and CS showed strong anti-oomycetes activity against Phytophthora xcambivora , P. capsici , and P. cinnamomi . Moreover, the concentration of CS in the activated mixture of MA and CS was lower than that in the activated CS only, and the mixture showed synergy or partial synergy effects on the anti-oomycete activity. Application of a wettable powder formulation of MA and CS mixture (MCS 30WP; 26.67% MA and 3.33% CS) had excellent protective activity in pot experiments with control values of 73% Phytophthora blight on red pepper, 91% damping-off on cucumber, and 84% Pythium blight on creeping bentgrass, which are similar to those of the CS wettable powder formulation (6.67% CS) containing two times the CS content of MCS 30WP. These observations suggest that the synergistic effect of the MA and CS combination is a sustainable alternative for effective management of destructive oomycete diseases.

Brain injury causes temporary or permanent impairment of brain function due to an accident or circulation disorders. Even after rehabilitation training, there are often persistent functional impairments. Recent advances in our understanding of the repair mechanisms of neural circuits after brain injury have led to the possibility that these mechanisms may offer potential therapeutic targets for drugs that promote functional recovery after brain injury. Neuroplasticity is believed to be important for the recovery process after brain injury in the brain regions associated with injured region for compensation. The effectiveness of drugs for restoring brain function after stroke investigated in a variety of animal models and clinical trials has been focused on drugs that act on the monoamine system to modulate neuroplasticity, as well as other targets such as NMDA receptors and CCR5. Recently, we focused on novel small compound, edonerpic maleate, as a drug which facilitates experience-dependent synaptic delivery of AMPA receptor. We found that edonerpic maleate binds to Collapsin-response mediator protein 2, a downstream molecule of Semaphorin and enhance synaptic plasticity by facilitating synaptic delivery of AMPA receptors, thereby promoting functional recovery in a rehabilitation-dependent manner after brain injury in rodents and non-human primates. Further investigations is needed to seek more appropriate drug targets from both preclinical animal studies and clinical trials, and to translate preclinical results into successful clinical trials.

Inhibiting sprouting of potatoes is an interesting subject needed for potato storage and industry. Sprouting degrades the quality of tuber along with releasing α-solanine and α-chaconine, which are harmful for health. Sprout suppressants, available in the market, are either costly or toxic to both health and environment. So, there is a need for developing countries to explore new sprouting suppressant compound which is cheap, non-toxic and reasonably efficient in comparison to commercial ones. We have established that simple maleic acid and L-tartaric acid are effective sprout suppressing agents. Both can hinder sprouting up to 6 weeks and 4 weeks post treatment respectively at room temperature in dark. These do not affect the quality parameters, retain the moisture content and maintain the stout appearance of the tubers along the total storage period. Thus maleic acid and L-tartaric acid would qualify as alternative, cheap, efficient sprout suppressant for potato storage and processing., (© 2021. The Author(s).)

Objective Evidence concerning the safety and efficacy of indacaterol maleate in a real-life setting is limited. The objective of this post-marketing surveillance was to evaluate the real-life safety and efficacy of indacaterol maleate in Japanese patients with chronic obstructive pulmonary disease (COPD). Methods This was a 52-week post-marketing surveillance conducted between April 2012 and December 2018. The safety endpoints included the incidence of adverse events (AEs), serious adverse events (SAEs), and adverse drug reactions (ADRs). The efficacy endpoints included the physician-reported global evaluation of treatment effectiveness (GETE), change from baseline in the COPD assessment test (CAT) results, forced vital capacity (FVC), forced expiratory volume in 1 second (FEV 1 ), and %FEV 1 following 4, 12, 26, and 52 weeks of indacaterol administration. Results Of the 1,846 enrolled patients, 1,726 were included in the safety and efficacy analyses. The mean age of the patients was 72.5 years old. Cough, pneumonia and COPD worsening were the most common AEs reported, while pneumonia (1.04%) was the most common SAE, and cough (1.68%) was the most common ADR. GETE showed that 69.70% of patients achieved an excellent/good/moderate response following indacaterol treatment. The CAT score decreased, and lung function parameters (FVC, FEV 1 and %FEV 1 ) improved across all the COPD stages following treatment with indacaterol. Conclusion Indacaterol showed a favorable safety and tolerability profile in Japanese patients with COPD without new safety signals observed in real-life settings. These findings demonstrated that indacaterol is an effective maintenance treatment in real-life practice for Japanese patients with COPD.

We demonstrate an intrinsic antitumor effect of polymer nanoparticles (P-NPs), which could re-program tumor-associated macrophages to pro-inflammatory phenotype. The intrinsic effect of P-NPs on macrophage repolarization and its combination with other therapies provide new ideas for drug delivery, macrophage regulation and immunotherapy in cancer treatment.

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of tumors that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein. The mainstay of treatment remains chemotherapy, but the therapeutic outcome remains inadequate. This paper investigates the potential of a duocarmycin derivative, tafuramycin A (TFA), as a new and more effective chemotherapy agent in TNBC treatment. To this extent, we optimized the chemical synthesis of TFA, and we encapsulated TFA in a micellar system to reduce side effects and increase tumor accumulation. In vitro and in vivo studies suggest that both TFA and SMA-TFA possess high anticancer effects in TNBC models. Finally, the encapsulation of TFA offered a preferential avenue to tumor accumulation by increasing its concentration at the tumor tissues by around four times in comparison with the free drug. Overall, the results provide a new potential strategy useful for TNBC treatment.

Introduction: Autophagy is a cellular catabolic mechanism that helps clear damaged cellular components and is essential for normal cellular and tissue function. The sigma-1 receptor (σ-1R) is a chaperone protein involved in signal transduction, neurite outgrowth, and plasticity, improving memory, and neuroprotection. Recent evidence shows that σ-1R can promote autophagy. Autophagy activation by the σ-1Rs along with other neuroprotective effects makes it an interesting target for the treatment of Alzheimer's disease. AF710B, T-817 MA, and ANAVEX2-73 are some of the σ-1R agonists which have shown promising results and have entered clinical trials. These molecules have also been found to induce autophagy and show cytoprotective effects in cellular models., Areas Covered: This review provides insight into the current understanding of σ-1R functions related to autophagy and their role in alleviating AD., Expert Opinion: We propose a mechanism through which the activation of σ-1R and autophagy could alter amyloid precursor protein processing to inhibit amyloid-β production by reconstituting cholesterol and gangliosides in the lipid raft to offer neuroprotection against AD. Future AD treatment could involve the combined targeting of the σ-1R and autophagy activation. We suggest that future studies investigate the link between autophagy the σ-1R and AD.

Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at the transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. We first determined the first exon usage in KEAP1 and NFE2L2 (encoding NRF2) and found the presence of transcript diversity. We then examined changes in isoform usage of NRF2 target genes under stress conditions and identified a few isoforms dominantly expressed in the majority of NRF2 target genes. The expression levels of isoforms determined by Iso-Seq analyses showed striking differences from those determined by short-read RNA-Seq; the latter could be misleading concerning the abundance of transcripts. These results support that transcript usage is tightly regulated to produce functional proteins under electrophilic stress. Our present study strongly argues that there are important benefits that can be achieved by long-read transcriptome sequencing., (Copyright © 2021 American Society for Microbiology.)

Many restoring formulations for damaged hair keratin have been developed. Some patents claim that the hair repair occurs through the reconstruction of disulfide bridges of keratin, through α,β-unsaturated Michael acceptors, such as shikimic acid and bis-aminopropyl diglycol dimaleate. To gain more insights into the possible repairing mechanism, this study is aimed at assessing, by IR and Raman spectroscopies coupled to scanning electron microscopy (SEM), the structural changes induced in keratin from bleached hair by the treatment with commercial reconstructive agents as well as shikimic acid and dimethyl maleate, chosen as model compounds. Vibrational spectroscopy revealed that shikimic acid- and maleate-based restoring agents interacted with hair fibers modifying both their cortex and cuticle regions. None of the investigated treatments induced an increase in the SS disulfide bridges content of the hair cortex, although it cannot be excluded that this phenomenon could have occurred in the cuticle. SS rearrangements were found to occur. None of our results can be interpreted as direct evidence of the sulfa-Michael reaction/cross-linking. From a morphological point of view, beneficial effects of the restoring agents were observed by SEM analyses, in terms of a more regular hair surface and more imbricated scales., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A new sponge-type hydrogel was obtained by cross-linking hyaluronic acid (HA) and poly(methylvinylether-alt-maleic acid) P(MVE-alt-MA) through a solvent-free thermal method. The sponge-type hydrogel was characterized and checked as a support for cell growth. The influence of concentration and weight ratio of polymers on the morphology and hydrogel stability was investigated. The total polymers concentration of 3% (w/w) and the weight ratio of 1:1 were optimal for the synthesis of a stable hydrogel (HA 3 P50) and to promote cell proliferation. The swelling measurements revealed a high-water absorption capacity of the hydrogel in basic medium. Diphenhydramine (DPH), lidocaine (Lid) and propranolol (Prop) were loaded within the hydrogel as a model drugs to investigate the ability of drug transport and release. In vitro studies revealed that HA 3 P50 hydrogel promoted the adhesion and proliferation of human hepatocellular carcinoma cell line HepG2, providing a good support for 3D cell culture to obtain surrogate tumor scaffold suitable for preclinical anti-cancer drug screening., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aedes aegypti (L.) is the primary vector of Zika, dengue, yellow fever, and chikungunya viruses. Insecticides used in mosquito control can help prevent the spread of vector-borne diseases. However, it is essential to determine insecticide resistance (IR) status before control measures are undertaken. Only the most effective insecticides should be used to avoid ineffective control and/or promotion of IR. Pyrethroids and organophosphates are the most commonly used insecticides for mosquito control. Here, the efficacy of two active ingredients (AIs; permethrin [pyrethroid], chlorpyrifos [organophosphate]), two formulated products (FPs; Biomist [AI: permethrin]) and (Mosquitomist [AI: chlorpyrifos]), and three synergists (piperonyl butoxide, diethyl maleate, S-S-S-tributyl phosphorotrithioate) was evaluated in two Ae. aegypti colonies (pyrethroid resistant and susceptible). Mosquitomist was most effective against the pyrethroid-resistant colony (100% mortality at diagnostic time). Pre-exposure to synergists did not increase the efficacy of AIs against the pyrethroid-resistant colony. Further research is needed to discover how synergists may affect the efficacy of insecticides when used on pyrethroid-resistant mosquitoes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acute respiratory distress syndrome (ARDS), characterized by acute hypoxic respiratory dysfunction or failure, is a manifestation of multiple organ failure in the lung, and the most common risk factor is sepsis. We previously showed that blocking α 2 -adrenoceptor (α 2 -AR) could attenuate lung injury induced by endotoxin in rats. α 2A -adrenoceptor (α 2A -AR), a subtype of α 2 -AR plays a key role in inflammatory diseases, but the mechanism remains unknown. Here, we explored the effect of BRL-44408 maleate (BRL), a specific α 2A -AR antagonist, on cecal ligation puncture (CLP)-induced ARDS in rats and the underlying mechanism. Preadministration of BRL-44408 maleate significantly alleviated CLP-induced histological injury, macrophage infiltration, inflammatory response, and wet/dry ratio in lung tissue. However, there was no statistical difference in survival rate between the CLP and CLP+BRL groups. Extracellular regulated protein kinase (ERK1/2), p38MAPK, and p65 were activated in the CLP group, and BRL-44408 maleate inhibited the activation of these signal molecules, c-Jun N-terminal kinase (JNK) and protein kinase A (PKA) showed no changes in activation between these two groups. BRL-44408 maleate decreased lipopolysaccharide (LPS)-induced expression of cytokines in NR8383 rat alveolar macrophages and reduced phosphorylation of ERK1/2, p38MAPK, and p65. JNK and PKA were not influenced by LPS. Together, these findings suggest that antagonism of α 2A -AR improves CLP-induced acute lung injury and involves the downregulation of ERK1/2, p38MAPK, and p65 pathway independent of the activation of JNK and PKA., (© 2020 Wiley Periodicals, Inc.)

α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 °C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Organic acids such as fumarate are commonly used as antimicrobials in foods. Apart from the classical mechanism of intracellular dissociation, weak acids are active through important additional mechanisms which are not well-defined. Fumarate, based on its low dissociation constants is expected to have a low antimicrobial activity which is not the case, suggesting additional antimicrobial effects. Previously, fumarate has been shown to inhibit the GAD system of E. coli and therefore, we investigated for first time how it affects this system in Listeria monocytogenes. We found that fumarate is highly antimicrobial towards L. monocytogenes under acidic conditions. We also show that in cell lysates and similarly to E. coli, fumarate inhibits the GAD system of L. monocytogenes. However, despite the inhibition and in contrast to E. coli, L. monocytogenes is able to counteract this and achieve a higher extracellular GAD output (measured by GABA export) in the presence of fumarate compared to its absence. The latter is achieved by a dramatic 9.44-fold increase in the transcription of gadD2 which is the main component of the extracellular GAD system. Interestingly, although maleate, the cis-isomer of fumarate results in a more dramatic 48.5-fold gadD2 upregulation than that of fumarate, the final GAD e output is lower suggesting that maleate might be a stronger inhibitor of the GAD system. In contrast, the GAD e removes more protons in the presence of fumarate than in the presence of HCl at the same pH. All the above suggest that there are additional effects by fumarate which might be associated with the intracellular GAD system (GAD i ) or other acid resistance systems. We assessed the GAD i output by looking at the intracellular GABA pools which were not affected by fumarate. However, there are multiple pathways (e.g. GABA shunt) that can affect GABA i pools and we cannot conclusively suggest that GAD i is affected. Furthermore, similarly to maleate, fumarate is able to eliminate L. monocytogenes in biofilms under acidic conditions. Overall, fumarate is a good candidate for L. monocytogenes decontamination and biofilm removal which is not toxic compared to the toxic maleate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Affibody molecules are the most studied class of engineered scaffold proteins (ESPs) in radionuclide molecular imaging. Attempts to use affibody molecules directly labelled with radiometals for targeted radionuclide therapy were hampered by the high uptake and retention of radioactivity in kidneys. Several promising strategies have been implemented to circumvent this problem. Here, we investigated whether a pharmacological approach targeting different components of the reabsorption system could be used to lower the uptake of [ 99m Tc]Tc-Z HER:2395 affibody molecule in kidneys. Pre-injection of probenecid, furosemide, mannitol or colchicine had no influence on activity uptake in kidneys compared to the control group. Mice pre-injected with maleate and fructose had 33% and 51% reduction in the kidney-associated activity, respectively, compared to the control group. Autoradiography images showed that the accumulation of activity after [ 99m Tc]Tc-Z HER2:2395 injection was in the renal cortex and that both maleate and fructose could significantly reduce it. Results from this study demonstrate that pharmacological intervention with maleate and fructose was effective in reducing the kidney uptake of affibody molecules. A presumable mechanism is the disruption of ATP-mediated cellular uptake and endocytosis processes of affibody molecules by tubular cells.

Metabolites regulate protein function via covalent and noncovalent interactions. However, manipulating these interactions in living cells remains a major challenge. Here, we report a chemical strategy for inducing cysteine S-succination, a nonenzymatic post-translational modification derived from the oncometabolite fumarate. Using a combination of antibody-based detection and kinetic assays, we benchmark the in vitro and cellular reactivity of two novel S-succination "agonists," maleate and 2-bromosuccinate. Cellular assays reveal maleate to be a more potent and less toxic inducer of S-succination, which can activate KEAP1-NRF2 signaling in living cells. By enabling the cellular reconstitution of an oncometabolite-protein interaction with physiochemical accuracy and minimal toxicity, this study provides a methodological basis for better understanding the signaling role of metabolites in disease.

Rationale: Maleic acid is an industrial-grade chemical that is often used in adhesives, stabilizers, and preservatives. It is unknown whether long-term consumption of maleic acid modified starch is harmful to humans. However, many studies have indicated that maleic acid causes renal tubular damage in animal models, even as the associated pathways remain unclear. Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is the most innovative of the label-free quantitative technologies which have better quantification performance. Therefore, SWATH technology was used to investigate the effect of maleic acid on the rat kidney proteome in this study., Methods: Sprague-Dawley(SD) rats were treated with 0 mg/kg (control), 6 mg/kg (low-dose), 10 mg/kg (medium-dose), and 60 mg/kg (high-dose) of maleic acid. After kidney protein extraction, 28% SDS-PAGE was used, followed by in-gel digestion and desalting. Next, the samples were analyzed with ultra-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS), and data-dependent acquisition (DDA) and SWATH technology were also used. The gene ontology and pathway analysis were accomplished. Ultimately, these protein biomarkers were validated by using scheduled high-resolution multiple reaction monitoring (sMRM HR )., Results: Comparisons of the control group with the other three groups revealed that 95, 130, and 103 proteins were expressed at significantly different levels in the control group and in the low-dose, medium-dose, and high-dose groups, respectively. According to the gene ontology analysis, the major processes that these proteins were involved in were metabolic processes, biological regulation, cellular processes, and responses to stimuli; the major functions that these proteins were involved in were binding, hydrolase activity, catalytic activity, and oxidoreductase activity; and the major cellular components hat they were involved in were the cytoplasm, extracellular region, membrane, and mitochondria. According to the KEGG pathway analysis, these proteins were involved in 35 pathways, five of which, the carbohydrate metabolism, folate biosynthesis, renal tubular resorption, amino acid metabolism, and Ras signaling pathways, are discussed in this study. Ultimately, 19 proteins involved in 12 important pathways were validated by sMRM HR ., Conclusions: It was demonstrated that maleic acid caused insufficient energy production, which might lead to a decrease in the activity of the sodium-potassium ATP pump and hydrogen ion ATP pump, which could in turn have caused renal tubular resorption and hydrogen ion regulation to be blocked, thus leading to the accumulation of hydrogen ions in the renal tubules, which would then result in renal tubular acidification followed finally by Fanconi syndrome., (© 2019 John Wiley & Sons, Ltd.)

The co-chaperone HspBP1 interacts with members of the hsp70 family, but also provides chaperone-independent functions. We report here novel biological properties of HspBP1 that are relevant to the formation of cytoplasmic stress granules (SGs). SG assembly is a conserved reaction to environmental or pathological insults and part of the cellular stress response. Our study reveals that HspBP1 (1) is an integral SG constituent, and (2) a regulator of SG assembly. Oxidative stress relocates HspBP1 to SGs, where it co-localizes with granule marker proteins and polyA-RNA. Mass spectrometry and co-immunoprecipitation identified novel HspBP1-binding partners that are critical for SG biology. Specifically, HspBP1 associates with the SG proteins G3BP1, HuR and TIA-1/TIAR. HspBP1 also interacts with polyA-RNA in vivo and binds directly RNA homopolymers in vitro. Multiple lines of evidence and single-granule analyses demonstrate that HspBP1 is crucial for SG biogenesis. Thus, HspBP1 knockdown interferes with stress-induced SG assembly. By contrast, HspBP1 overexpression promotes SG formation in the absence of stress. Notably, the hsp70-binding domains of HspBP1 regulate SG production in unstressed cells. Taken together, we identified novel HspBP1 activities that control SG formation. These features expand HspBP1's role in the cellular stress response and provide new mechanistic insights into SG biogenesis.

Purpose: Activating the cell survival modulator sigma 1 receptor (Sig1R) delays cone photoreceptor cell loss in Pde6βrd10/J (rd10) mice, a model of retinitis pigmentosa. Beneficial effects are abrogated in rd10 mice lacking NRF2, implicating NRF2 as essential to Sig1R-mediated cone neuroprotection. Here we asked whether activation of NRF2 alone is sufficient to rescue cones in rd10 mice., Methods: Expression of antioxidant genes was evaluated in 661W cells and in mouse retinas after treatment with monomethylfumarate (MMF), a potent NRF2 activator. Rd10 mice were administered MMF (50 mg/kg) or the Sig1R ligand (+)-pentazocine (PTZ; 0.5 mg/kg) intraperitoneally (every other day, P14-42). Mice were evaluated for visual acuity (optokinetic tracking response), retinal function (electroretinography) and architecture (SD-OCT); histologic retinal sections were evaluated morphometrically., Results: MMF treatment increased Nrf2, Nqo1, Cat, Sod1, and Hmox1 expression in vitro and in vivo. Visual acuity of (+)-PTZ-treated rd10 mice was similar to wild-type mice; however, MMF treatment did not alter acuity compared with nontreated rd10 mice. Cone electroretinography b-wave amplitudes were greater in PTZ-treated than nontreated or MMF-treated rd10 mice. SD-OCT assessment of retinal thickness was greater in (+)-PTZ-treated mice versus nontreated or MMF-treated rd10 mice. Morphometric assessment of the outer nuclear layer revealed approximately 18 cells/100 µm retinal length in (+)-PTZ-treated rd10 mice, but only approximately 10 to 12 cells/100 µm in MMF-treated and nontreated rd10 retinas., Conclusions: Activation of NRF2 using MMF, at least at our dosing regimen, is insufficient to attenuate catastrophic photoreceptor damage characteristic of rd10 mice. The data prompt investigation of additional mechanisms involved in Sig1R-mediated retinal neuroprotection.

Dimethylfumarate (DMF) has long been used as part of a fixed combination of fumaric acid esters (FAE) in some European countries and is now available as an oral monotherapy for psoriasis. The present investigation determined whether DMF and its main metabolite monomethylfumarate (MMF) interact with hepatic cytochrome P450 (CYP) enzymes and the P-glycoprotein (P-gp) transporter, and was performed as part of DMF's regulatory commitments. Although referred to in the available product labels/summary of product characteristics, the actual data have not yet been made publicly available. In vitro inhibition experiments using CYP-selective substrates with human liver microsomes showed 50% inhibitory concentrations (IC 50 ) of >666 µmol/L for DMF and >750 µmol/L for MMF. MMF (≤250 μmol/L; 72 hours) was not cytotoxic in cultured human hepatocyte experiments and mRNA expression data indicated no CYP induction by MMF (1-250 µmol/L). DMF (≤6.66 mmol/L) showed moderate-to-high absorption (apparent permeability [P app ] ≥2.3-29.7 x 10 -6  cm/s) across a Caucasian colon adenocarcinoma (Caco-2) cell monolayer, while MMF (≤7.38 mmol/L) demonstrated low-to-moderate permeability (P app 1.2-8.9 × 10 -6  cm/s). DMF was not a substrate for P-gp (net efflux ratios ≤1.22) but was a weak inhibitor of P-gp at supratherapeutic concentrations (estimated IC 50 relative to solvent control of 1.5 mmol/L; [ 3 H]digoxin efflux in Caco-2 cells). This inhibition is unlikely to be clinically relevant. MMF was not a substrate or inhibitor of P-gp. Thus, DMF and MMF should not affect the absorption, distribution, metabolism or excretion of coadministered drugs that are CYP and P-gp substrates., Competing Interests: Jordi Aubets, Josep‐Maria Jansat and Miquel Salva are employees of Almirall S.A., Barcelona, Spain. Vicky M. Birks, Richard J. Cole, Michael Hall, Jenny Lewis and Annabell Pitcher declared no conflicts of interest., (© 2019 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Purpose: The system x C - transporter is upregulated in cancer cells in response to oxidative stress (OS). 5-[ 18 F]fluoroaminosuberic acid ([ 18 F]FASu) has been reported as a novel positron emission tomography (PET) imaging agent, targeting system x C - . The goal of this study was to evaluate the utility of [ 18 F]FASu in monitoring cellular response to diethyl maleate (DEM) and radiation-induced OS fluctuations., Procedures: [ 18 F]FASu uptake by breast cancer cells was studied in correlation to OS biomarkers: glutathione (GSH) and reactive oxygen species (ROS), as well as transcriptional and translational levels of xCT (the functional subunit of x C - ). System x C - inhibitor, sulfasalazine (SSZ), and small interfering RNA (siRNA) knockdown were used as negative controls. Radiotracer uptake was evaluated in three breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1, at two-time points (1 h and 16 h) following OS induction. In vivo [ 18 F]FASu imaging and biodistribution were performed using MDA-MB-231 xenograft-bearing mice at 16 and 24 h post-radiation treatment., Results: [ 18 F]FASu uptake was positively correlated to intracellular GSH and SLC7A11 expression levels, and radiotracer uptake was induced both by radiation treatment and by DEM at time points longer than 3 h. In an in vivo setting, there was no statistically significant uptake difference between irradiated and control tumors., Conclusion: [ 18 F]FASu is a specific system x C - PET radiotracer and as such it can be used to monitor system x C - activity due to OS. As such, [ 18 F]FASu has the potential to be used in therapy response monitoring by PET. Further optimization is required for in vivo application.

Purpose of Review: Stroke is a devastating illness which severely attenuates quality of life because of paralysis. Despite recent advances in therapies during acute phase such as thrombolytic therapy, clinical option to intervene the process of rehabilitation is limited. No pharmacological intervention that could enhance the effect of rehabilitation has not been established. Recent articles, which are summarized in the review article, reported novel small compound which accelerates training-dependent motor function recovery after brain damage., Recent Findings: A novel small compound, edonerpic maleate, binds to collapsin response mediator protein 2 (CRMP2) and enhance synaptic plasticity leading to the acceleration of rehabilitative training-dependent functional recovery after brain damage in rodent and nonhuman primate. The clinical trial to test this effect in human is now ongoing. Future preclinical and clinical studies will delineate the potentials of this compound., Summary: A novel CRMP2-binding small compound, edonerpic maleate, accelerates motor function recovery after brain damage in rodent and nonhuman primate.

Detection of protein biomarkers is an important tool for medical diagnostics, typically exploiting concentration of particular biomarkers or biomarker release from tissues. We sought to establish whether proteins not normally released by living cells can be extracted without harming cells, with a view to extending this into biomarker harvest for medical diagnosis and other applications. Styrene maleic acid (SMA) is a polymer that extracts nanodiscs of biological membranes (containing membrane proteins) from cells. Hitherto it has been used to harvest SMA-lipid-membrane protein particles (SMALP) for biochemical study, by destroying the living cellular specimen. In this study, we applied SMA at low concentration to human primary cardiovascular cells and rat vascular tissue, to 'biopsy' cell proteins while avoiding significant reductions in cell viability. SMA at 6.25 parts per million harvested proteins from cells and tissues without causing significant release of cytosolic dye (calcein) or reduction in cell viability at 24 and 72 hours post-SMA (MTT assay). A wide range of proteins were recovered (20-200 kDa) and a number identified by mass spectrometry: this confirmed protein recovery from plasma membrane, intracellular membranes and cell cytosol without associated cell death. These data demonstrate the feasibility of non-lethally sampling proteins from cells, greatly extending our sampling capability, which could yield new physiological and/or pathological biomarkers.

Oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of neurodegenerative diseases and favor lipid peroxidation, leading to increased levels of 7β-hydroxycholesterol (7β-OHC) which induces oxiapoptophagy (OXIdative stress, APOPTOsis, autoPHAGY). The cytoprotective effects of dimethylfumarate (DMF), used in the treatment of relapsing remitting multiple sclerosis and of monomethylfumarate (MMF), its main metabolite, were evaluated on murine oligodendrocytes 158 N exposed to 7β-OHC (50 μM, 24 h) with or without DMF or MMF (25 μM). The activity of 7β-OHC in the presence or absence DMF or MMF was evaluated on several parameters: cell adhesion; plasma membrane integrity measured with propidium iodide (PI), trypan blue and fluoresceine diacetate (FDA) assays; LDH activity; antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)); generation of lipid peroxidation products (malondialdehyde (MDA), conjugated dienes (CDs)) and protein oxidation products (carbonylated proteins (CPs)); reactive oxygen species (ROS) overproduction conducted with DHE and DHR123. The effect on mitochondria was determined with complementary criteria: measurement of succinate dehydrogenase activity, evaluation of mitochondrial potential (ΔΨm) and mitochondrial superoxide anions (O 2 ●- ) production using DiOC 6 (3) and MitoSOX, respectively; quantification of mitochondrial mass with Mitotracker Red, and of cardiolipins and organic acids. The effects on mitochondrial and peroxisomal ultrastructure were determined by transmission electron microscopy. Intracellular sterol and fatty acid profiles were determined. Apoptosis and autophagy were characterized by staining with Hoechst 33,342, Giemsa and acridine orange, and with antibodies raised against caspase-3 and LC3. DMF and MMF attenuate 7β-OHC-induced cytotoxicity: cell growth inhibition; decreased cell viability; mitochondrial dysfunction (decrease of succinate dehydrogenase activity, loss of ΔΨm, increase of mitochondrial O 2 ●- production, alteration of the tricarboxilic acid (TCA) cycle, and cardiolipins content); oxidative stress induction (ROS overproduction, alteration of GPx, CAT, and SOD activities, increased levels of MDA, CDs, and CPs); changes in fatty acid and cholesterol metabolism; and cell death induction (caspase-3 cleavage, activation of LC3-I in LC3-II). Ultrastructural alterations of mitochondria and peroxisomes were prevented. These results demonstrate that DMF and MMF prevent major dysfunctions associated with neurodegenerative diseases: oxidative stress, mitochondrial dysfunction, apoptosis and autophagy., (Copyright © 2019. Published by Elsevier Ltd.)

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans loss-of-function mutants for the glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human, as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH-depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that differs in progression, recurrence, and prognosis from other forms of breast cancer. The heterogeneity of TNBC has remained a challenge as no targeted therapy is currently available. Previously, we and others have demonstrated that raloxifene, a selective oestrogen receptor modulator, was also acting independently of the oestrogen receptor-α. However, raloxifene is characterised by a low bioavailability in vivo . Thus, we encapsulated raloxifene into a styrene-maleic acid (SMA) micelle to improve its pharmacokinetics. The micellar raloxifene had higher cytotoxicity when compared to the free formulation, promoted a higher cellular uptake and affected critical signalling pathways. Furthermore, SMA-raloxifene reduced TNBC tumour growth more efficiently than free raloxifene. Finally, we showed that this effect was partially mediated through oestrogen receptor-β. In conclusion, we have provided new insight into the role of raloxifene nanoformulation in improving the management of TNBC.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a core enzyme of the aerobic glycolytic pathway with versatile functions and is associated with cancer development. Recently, Kornberg et al . published the detailed correlation between GAPDH and di - or monomethyl fumarate (DMF or MMF), which are well-known GAPDH antagonists in the immune system. As an extension, herein, we report the crystal structure of MMF-bound human GAPDH at 2.29 Å. The MMF molecule is covalently linked to the catalytic Cys152 of human GAPDH, and inhibits the catalytic activity of the residue and dramatically reduces the enzymatic activity of GAPDH. Structural comparisons between NAD + bound GAPDH and MMF-bound GAPDH revealed that the covalently linked MMF can block the binding of the NAD + cosubstrate due to steric hindrance of the nicotinamide portion of the NAD + molecule, illuminating the specific mechanism by which MMF inhibits GAPDH. Our data provide insights into GAPDH antagonist development for GAPDH-mediated disease treatment.

Post stroke recanalization has been associated with increased risk of oxidative stress. Stimulating endogenous antioxidant pathway by activation of nuclear factor erythroid-2-related factor-2 (Nrf2) plays a key role in neuronal defense against inflammation and oxidative stress in penumbra. Here, we explored whether monomethyl fumarate (MMF) could produce neuro-protection after ischemia/reperfusion (I/R) injury via Nrf2/HO1 activation. In male SD rats, middle cerebral artery was occluded for 90 min and confirmed using Laser Doppler flowmeter. MMF (10, 20 and 40 mg/kg) was administered in two divided doses at 30 min post ischemia and 5-10 min after reperfusion. After 24 h, effect on neurobehavioral parameters, infarct damage by TTC staining and MRI, oxidative stress and inflammatory cytokines were assessed. Expression studies of nuclear Nrf2 and cytoplasmic HO1 were performed in peri-infarct cortex and striatum; followed by dual immunofluorescence study to check the specific cell type. I/R induced neurobehavioral deficits and infarct damage were significantly (p < 0.05) attenuated by MMF (20 and 40 mg/kg). MMF, 20 mg/kg, significantly normalized I/R induced altered redox status and increased levels of TNF-α, IL-1β in the ipsilateral cortex. MRI data showed significantly reduced infarct in cortex but not in striatum after MMF treatment. Expression of nuclear Nrf2 and cytoplasmic HO1 were significantly (p < 0.05) increased in peri-infarct cortex after treatment with MMF. Additionally, dual immunofluorescence showed increased Nrf2 expression in neurons and HO1 expression in neurons as well as astrocytes in peri-infarct cortex after MMF treatment. Our results show the neuro-protective potential of MMF probably by restricting the progression of damage from striatum to cortex through activation of Nrf2/HO1 pathway in peri-infarct cortex., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Dimethylfumarate (DMF) has been approved the for treatment of relapsing-remitting multiple sclerosis. The mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood, notably for brain resident cells. Therefore we investigated potential direct effects of DMF and MMF on microglia and indirect effects on oligodendrocytes. Primary rat microglia were differentiated into M1-like, M2-like and M0 phenotypes and treated in vitro with DMF or MMF. The gene expression of pro-inflammatory and anti-inflammatory factors such as growth factors (IGF-1), interleukins (IL-10, IL-1β), chemokines (CCl3, CXCL-10) as well as cytokines (TGF-1β, TNFα), iNOS, and the mannose receptor (MRC1) was examined by determining their transcription level with qPCR, and on the protein level by ELISA and FACS analysis. Furthermore, microglia function was determined by phagocytosis assays and indirect effects on oligodendroglial proliferation and differentiation. DMF treatment of M0 and M1-like polarized microglia demonstrated an upregulation of gene expression for IGF-1 and MRC1, but not on the protein level. While the phagocytic activity remained unchanged, DMF and MMF treated microglia supernatants led to an enhanced proliferation of oligodendrocyte precursor cells (OPC). These results suggest that DMF has anti-inflammatory effects on microglia which may result in enhanced proliferation of OPC.

Objectives: To evaluate SmearOFF, 7% maleic acid (MA) and two different preparations of ethylenediaminetetraacetic acid (EDTA) in smear layer removal. Materials and methods: Fifty single-rooted teeth were separated into five groups, instrumented and irrigated as follows: (1) SmearOFF, (2) 7% MA, (3) 18% EDTA (pH 11.4), (4) 17% EDTA (pH 8.5) and (5) 0.9% saline. Teeth samples were blinded and examined by scanning electron microscopy with Image J software. Results: Eighteen percent EDTA was less efficient when compared to SmearOFF and MA at all thirds of the root canal system. There was no difference between SmearOFF and MA in the coronal and middle thirds. In the apical third, MA removed more smear layer. Seventeen percent EDTA was as efficient as SmearOFF and MA in coronal and middle third but not in the apical third. Eighteen percent EDTA removed smear layer less efficiently in the coronal and middle thirds than 17% EDTA; in the apical third, there was no difference observed. In the saline group, all specimens were heavily smeared. There was no significant difference between 18% EDTA and saline at all canal thirds. Conclusions: SmearOFF and 17% EDTA (pH 8.5) had better smear layer removal capability in the coronal and middle thirds of the root canal system. In the apical third, 7% MA was superior. 18% EDTA (pH 11.4) and saline had poor smear layer removal ability.

Aims: Sepsis is a potentially fatal illness that can lead to impairment of multiple organs such as liver. The condition is deeply associated with oxidative stress and inflammation. Monomethyl fumarate (MMF) has manifested antioxidant and immunomodulatory properties. The aim of current study was to evaluate protective effects of MMF in sepsis-induced hepatic dysfunction., Main Methods: Sepsis was induced by cecal ligation and puncture (CLP). Wistar rats were assigned to one of sham, CLP, CLP + dexamethasone (as positive control of inflammation) and CLP + MMF groups. Levels of serum IL-1β, IL-6, IL-10, AST, ALT and γ‑GT were quantified. Furthermore, Hepatic levels of GSH and MDA and mRNA expression of TNF and NFKBIA along with hepatic protein level of TLR-4 were assessed. Also, histopathological study of liver was carried out to evaluate hepatic injuries., Key Findings: Septic rats demonstrated risen levels of IL-1β, IL-6, IL-10, AST, ALT and γ‑GT, while treatment with dexamethasone or MMF attenuated these levels. Moreover, enhancements in protein level of TLR-4 and mRNA levels of TNF and NFKBIA were observed in CLP rats. These elevations were mitigated in CLP-induced rats that were treated with either dexamethasone or MMF. Treatment with dexamethasone or MMF also shifted sepsis-induced disturbance in the levels of GSH and MDA towards sham levels. Hepato-protective effects of dexamethasone and MMF were further confirmed by histopathological observations., Significance: Our findings imply that MMF alleviates sepsis-induced hepatic dysfunction by mitigating the inflammatory and oxidative state and this effect is at least partly mediated by the inhibition of TLR-4/NF-κB signalling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The purpose of this study was to investigate if AZD5329, a dual neurokinin NK1/2 receptor antagonist, is a suitable candidate for further development as an oral immediate release (IR) solid dosage form as a final product. The neutral form of AZD5329 has only been isolated as amorphous material. In order to search for a solid material with improved physical and chemical stability and more suitable solid-state properties, a salt screen was performed. Crystalline material of a maleic acid salt and a fumaric acid salt of AZD5329 were obtained. X-ray powder diffractiometry, thermogravimetric analysis, differential scanning calorimetry and dynamic vapor sorption were used to investigate the physicochemical characteristics of the two salts. The fumarate salt of AZD5329 is anhydrous, the crystallization is reproducible and the hygroscopicity is acceptable. Early polymorphism assessment work using slurry technique did not reveal any better crystal modification or crystallinity for the fumarate salt. For the maleate salt, the form isolated originally was found to be a solvate, but an anhydrous form was found in later experiments; by suspension in water or acetone, by drying of the solvate to 100-120 °C or by subjecting the solvate form to conditions of 40 °C/75%RH for 3 months. The dissolution behavior and the chemical stability (in aqueous solutions, formulations and solid-state) of both salts were also studied and found to be satisfactory. The compound displays sensitivity to low pH, and the salt of the maleic acid, which is the stronger acid, shows more degradation during stability studies, in line with this observation. The presented data indicate that the substance fulfils basic requirements for further development of an IR dosage form, based on the characterization on crystalline salts of AZD5329., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Brain damage such as stroke is a devastating neurological condition that may severely compromise patient quality of life. No effective medication-mediated intervention to accelerate rehabilitation has been established. We found that a small compound, edonerpic maleate, facilitated experience-driven synaptic glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic-acid) receptor delivery and resulted in the acceleration of motor function recovery after motor cortex cryoinjury in mice in a training-dependent manner through cortical reorganization. Edonerpic bound to collapsin-response-mediator-protein 2 (CRMP2) and failed to augment recovery in CRMP2-deficient mice. Edonerpic maleate enhanced motor function recovery from internal capsule hemorrhage in nonhuman primates. Thus, edonerpic maleate, a neural plasticity enhancer, could be a clinically potent small compound with which to accelerate rehabilitation after brain damage., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Insecticide resistance is a major challenge in successful insect pest control as the insects have the ability to develop resistance to various widely used insecticides. Butene-fipronil is a novel compound with high toxicity to insects and less toxicity to the non-target organisms. In the present study, the effect of butene-fipronil alone and in combination with three enzyme inhibitors, piperonyl butoxide (PBO), diethyl maleate (DEM), and triphenyl phosphate (TPP), was carried out on larvae and adults of Drosophilia melanogaster. Our results indicated that the co-toxicity indices of butene-fipronil + PBO, butene-fipronil + TPP, and butene-fipronil + DEM mixtures were 437.3, 335.0, and 210.3, respectively, in the second-instar larvae, while 186.6, 256.2, and 238.5, respectively, in the adults, indicating synergistic effects. Interestingly, butene-fipronil increased the expression of CYP28A5 in the larvae; CYP9F2, CYP304A1, CYP28A5, and CYP318A1 in the female adults; and CYP303A1 and CYP28A5 in the male adults. Furthermore, high-level expression of Est-7 was observed in the female adults compared to larvae and male adults. Our results suggest that there is no difference in butene-fipronil metabolism in larvae and male and female adults of D. melanogaster.

Objective: To evaluate the smear layer removal and wettability of AH Plus sealer on root canal dentin treated with MA (maleic acid), MA + CTR (cetrimide) and MA + CTR + CHX (chlorhexidine) as final irrigating regimens., Material and Methods: For smear layer removal, 40 teeth were instrumented to size F4 and divided into four groups: (1) 7% MA, (2) 7% MA + 0.2% CTR, (3) 7% MA + 0.2% CTR + 2% CHX, (4) distilled water (control). After irrigation, teeth were subjected to SEM analysis. For contact angle analysis, 20 teeth were split longitudinally and divided into four groups similar to smear layer analysis. AH plus sealer was placed on each specimen and contact angle was analysed., Results: In both smear layer (p = .393) and contact angle analysis (p = .961), there was no significant difference between the groups MA and MA + CTR. However, MA + CTR + CHX removed smear layer less effectively (p = .023) and increased the contact angle of the sealer (p = .005). In smear layer analysis, specimens in negative control group were heavily smeared. In case of contact angle analysis, samples in the control group had least contact angle., Conclusion: MA alone or in combination with CTR removed smear layer effectively and increased the wettability of AH plus sealer to root canal dentin.

Here it is demonstrated a novel approach in disinfection regimes where specific molecular acid resistance systems are inhibited aiming to eliminate microorganisms under acidic conditions. Despite the importance of the Glutamate Decarboxylase (GAD) system for survival of Listeria monocytogenes and other pathogens under acidic conditions, its potential inhibition by specific compounds that could lead to its elimination from foods or food preparation premises has not been studied. The effects of maleic acid on the acid resistance of L. monocytogenes were investigated and found that it has a higher antimicrobial activity under acidic conditions than other organic acids, while this could not be explained by its pKa or Ka values. The effects were found to be more pronounced on strains with higher GAD activity. Maleic acid affected the extracellular GABA levels while it did not affect the intracellular ones. Maleic acid had a major impact mainly on GadD2 activity as also shown in cell lysates. Furthermore, it was demonstrated that maleic acid is able to partly remove biofilms of L. monocytogenes. Maleic acid is able to inhibit the GAD of L. monocytogenes significantly enhancing its sensitivity to acidic conditions and together with its ability to remove biofilms, make a good candidate for disinfection regimes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Resistance to oncolytic virotherapy is frequently associated with failure of tumor cells to get infected by the virus. Dimethyl fumarate (DMF), a common treatment for psoriasis and multiple sclerosis, also has anticancer properties. We show that DMF and various fumaric and maleic acid esters (FMAEs) enhance viral infection of cancer cell lines as well as human tumor biopsies with several oncolytic viruses (OVs), improving therapeutic outcomes in resistant syngeneic and xenograft tumor models. This results in durable responses, even in models otherwise refractory to OV and drug monotherapies. The ability of DMF to enhance viral spread results from its ability to inhibit type I interferon (IFN) production and response, which is associated with its blockade of nuclear translocation of the transcription factor nuclear factor κB (NF-κB). This study demonstrates that unconventional application of U.S. Food and Drug Administration-approved drugs and biological agents can result in improved anticancer therapeutic outcomes., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The ring-opening copolymerization of maleic anhydride and propylene oxide, using a functionalized primary alcohol initiator and magnesium 2,6-di-tert-butyl phenoxide as a catalyst, was investigated in order to produce high end-group fidelity poly(propylene maleate). Subsequent isomerization of the material into 3D printable poly(propylene fumarate) was utilized to produce thin films and scaffolds possessing groups that can be modified with bioactive groups postpolymerization and postprinting. The surface concentration of these modifiable groups was determined to be 30.0 ± 3.3 pmol·cm -2 , and copper-mediated azide-alkyne cycloaddition was used to attach a small molecule dye and cell adhesive GRGDS peptides to the surface as a model system. The films were then studied for cytotoxicity and found to have high cell viability before and after surface modification.

Fumaric acid ester-based drugs are used for the treatment of psoriasis and multiple sclerosis. All licensed fumaric acid ester drugs contain dimethylfumarate (DMF) as the main active component. Due to the expanding use of oral DMF there is growing scientific interest in determining its as-yet-unknown mechanism of action. However, the pharmacology and chemistry of DMF are often not fully considered in the design and interpretation of experiments; namely, that while DMF is plasma-membrane permeable and has strong effects on many cell types in vitro, it is rapidly metabolized into membrane-impermeable monomethylfumarate (MMF) in vivo. This can lead to significant biological effects being erroneously assigned to DMF. Understanding the pharmacology of DMF means that future work can more closely reflect the state in vivo., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ralstonia pseudosolanacearum Ps29 was repelled by maleate. Screening of a complete collection of Ps29 single-methyl-accepting chemotaxis protein (mcp) gene mutants identified the RSp0303 homolog (McpP) as a chemotaxis sensor mediating negative chemotaxis to maleate. Interestingly, the mcpP-deletion mutant was attracted to maleate, indicating that this bacterium expresses a MCP(s) for both positive and negative chemotaxis to maleate. We constructed a Ps29 derivative (designated POC14) harboring deletions in 14 individual mcp genes, including mcpP, to characterize McpP. Introduction of a plasmid harboring the mcpP gene (pPS16) restored the ability to negatively respond to maleate, confirming that McpP is a MCP for negative chemotaxis to maleate. We thought that maleate might be applied to controlling plant infection by R. pseudosolanacearum. To evaluate this possibility, we measured chemotactic responses of seven other virulent R. pseudosolanacearum strains to maleate. We confirmed that they harbored functional mcpP orthologues, but they showed no chemotactic responses to maleate. Quantitative RT-PCR analysis revealed that these seven R. pseudosolanacearum strains did not show negative chemotaxis to maleate because of negligible transcription of the mcpP genes. We compared the chemotactic responses of POC14 and POC14[pPS16] toward various chemicals and found that McpP senses inorganic phosphate as a chemoattractant., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The acid-labile behavior of di-substituted maleamic acid (DMA) and its equilibrium with di-substituted maleimide (DMI) are exploited to build an ultra acid-sensitive, small molecule prodrug that can be activated by tumor extracellular pH (pHe) in the range of 6.5-6.9. Such a DMA prodrug reversed the unfavorable pH-profile of doxorubicin (Dox), which may improve its therapeutic window.