Straightforward access to novel mitochondriotropics derived from 2-arylethanol as potent and selective antiproliferative agents.

The necessity for developing novel cytostatic agents with improved activities and reduced side-effects to tackle cancer prompted us to investigate mitochondria-targeted compounds, an approach that is gaining attention for the selective transportation of cytotoxic agents. We envisioned the possibility of conjugating a phenethyl alcohol motif, decorated with a series of phenol-based substituents on the aryl moiety, with a triphenyl phosphonium scaffold (a mitochondria-directed vector), through a hydrocarbon chain of different lengths. Thus, such compounds that incorporate the phenethyl skeleton can be considered as masked phenolic compounds derived from relevant natural counterparts found in olive tree (e.g. tyrosol, hydroxytyrosol). Title compounds exhibited very strong in vitro antiproliferative activities against the panel of six human tumor cell lines tested, with GI50 values ranging from the nanomolar (0.026 ± 0.010 µM for 36) to the submicromolar range in most of the cases; this represents an improvement of up to 350-fold compared to classical chemotherapeutic agents, like 5-fluorouracil or cisplatin. Interestingly, decrease in the linker length led to an increase of GI50 values against non-tumor cells, thus allowing a remarkable improvement of selectivity (SI up to 269). The very promising antiproliferative activities prompted us to further investigate their behaviour against multidrug resistant cell lines (MDR). The results indicated a reduced sensitivity of the multidrug resistant cells to compounds, probably due to P-gp-mediated efflux of these antiproliferative agents. Interestingly, activities were completely restored to the same levels by co-administration of tariquidar, a well-known inhibitor of P-gp. Flow cytometry analysis on sensitive cell lines revealed a decrease in the percentage of cells in G1 phase accompanied by increase in S and G2/M phases. In addition, a significant increase in subG1 area, was observed. These results are compatible with the necrotic and apoptotic cell death detected in the Annexin V assay, and with the depolarization of the mitochondria membrane. Thus, the new mitochondriotropic agents reported herein can be considered as promising antiproliferative agents, endowed with remarkable potency and selectivity, including MDR cells, upon co-administration with a pump-efflux inhibitor.

Decorated traditional cellulose with nanoscale chiral metal-organic frameworks for enhanced enantioselective capture.

Herein, a rapid approach toward the size/morphology-controlled synthesis of [Cu(L-mal)(bipy)·2H2O] (CuLBH) was developed by adjusting the concentrations of 2-methylimidazole (2-MI) and copper ions. The chiral separation efficiency test indicated that the nano-diameter CuLBH exhibited better selective potential towards (±)-1-(1-naphthyl)ethanol (NE) by providing more fully exposed recognition sites. In order to further improve the selectivity for NE enantiomers and avoid the aggregation of MOF nanoparticles, the nanosized CuLBH-decorated carboxylated cellulose (CC) composite CC-CuLBH was designed by controlling the ratio of the solvent and Cu2+, which exhibited much higher enantioselectivity than those of pristine CC and even nano CuLBH.

A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics.

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC50 values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI50 values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

Simulation of Ligand Transport in Receptors Using CaverDock.

Interactions between enzymes and small molecules lie in the center of many fundamental biochemical processes. Their analysis using molecular dynamics simulations have high computational demands, geometric approaches fail to consider chemical forces, and molecular docking offers only static information. Recently, we proposed to combine molecular docking and geometric approaches in an application called CaverDock. CaverDock is discretizing enzyme tunnel into discs, iteratively docking with restraints into one disc after another and searching for a trajectory of the ligand passing through the tunnel. Here, we focus on the practical side of its usage describing the whole method: from getting the application, and processing the data through a workflow, to interpreting the results. Moreover, we shared the best practices, recommended how to solve the most common issues, and demonstrated its application on three use cases.

Direct transfer of tri- and di-fluoroethanol units enabled by radical activation of organosilicon reagents.

Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z'.

Nrf2 transcriptional activity in the mouse affects the physiological response to tribromoethanol.

Up to date, there is no information on the influence of 2,2,2-tribromoethanol (TBE; Avertin), a commonly used anaesthetic, on mice with impaired antioxidant capacity. We aimed to analyse the effect of a single dose of Avertin on anaesthesia duration time, inflammatory response, oxidative stress and collagen deposition in the large intestine of Nrf2 transcriptional knockout mice (tNrf2-/-). The studies were performed on six-month-old female mice Nrf2+/+ and tNrf2-/- randomly assigned to Avertin (250 mg/kg b.w. single i.p. injection) or vehicle group. We observed a 2-fold increase in anaesthesia time and longer recovery time (p = 0.015) in tNrf2-/- in comparison to Nrf2+/+. However, no hepato- or nephrotoxicity was detected. Interestingly, we found severe changes in colon morphology of untreated tNrf2-/- mice associated with colon shortening (p = 0.02) and thickening (p = 0.015). Avertin treatment caused colon damage manifested with epithelial layer damage and goblet depletion in Nrf2+/+ mice but not in tNrf2-/- individuals. Additionally, Avertin did not induce oxidative stress in colon tissue, but it increased leukocyte infiltration in Nrf2+/+ mice (p = 0.02). Immunofluorescent staining also revealed enhanced deposition of collagen I and collagen III in the colon of untreated tNrf2-/- mice. Avertin contributed to increased deposition of collagen I in Nrf2+/+ mice but reduced deposition of collagen I and III in tNrf2-/- individuals. In conclusion, tNrf2-/- respond to Avertin with prolonged anaesthesia that is not associated with acute toxicity, inflammatory reaction or enhanced oxidative stress. Avertin does not impair intestine morphology in tNrf2-/- mice but can normalise the enhanced fibrosis.

Anesthesia with sevoflurane or isoflurane induces severe hypoglycemia in neonatal mice.

Sevoflurane and isoflurane are among the most commonly used general anesthetics for children including infants, but their impact on metabolism, especially on blood glucose level, in children is not well understood. We investigated the impacts of anesthesia of neonatal (7-8 days old) and adult (2-3 months old) mice with the inhalational anesthetics 2.5% sevoflurane or 1.5% isoflurane, or the injectable anesthetics propofol (150 mg/kg) or avertin (375 mg/kg), for up to 6 hours. We found that sevoflurane and isoflurane induced severe hypoglycemia in neonatal mice and that this phenomenon was specific to the inhalational anesthetics because the injectable anesthetics propofol and avertin did not induce hypoglycemia. Surprisingly, the inhalational anesthesia induced hyperglycemia instead in adult mice. We also demonstrated that the inhalational anesthesia-induced hypoglycemia was a major cause of death for the neonatal mice receiving intranasal administration of saline prior to anesthesia. These studies revealed severe hypoglycemia in neonatal mice during anesthesia with sevoflurane or isoflurane. If this phenomenon also occurs in human, our findings would warrant closely monitoring blood glucose level and maintaining it in the normal range in infants receiving inhalational anesthesia.

Avertin affects murine colitis by regulating neutrophils and macrophages.

Anesthetics are thought to be involved in immunomodulation. Avertin is one of the safest and most commonly used intravenous anesthetics in rodent experiments; it is also widely used in euthanasia of inflammatory bowel disease (IBD) models. This study aimed to define the role and mechanism of action of Avertin on murine colitis. We assessed the effects of a single Avertin injection on colitis using the disease activity index (DAI), pathology, enzyme-linked immunosorbent assay (ELISA), multiplex-ELISA, flow cytometry, and routine blood examination in wild-type (WT) and dextran sodium sulphate (DSS)-treated mice. Although Avertin caused acute cecitis in WT mice after 24 h and aggravated inflammation in the medium term, it alleviated inflammation in the late stage of DSS-induced colitis according to the DAI. Avertin upregulated MPO production and induced the accumulation of neutrophils and macrophages in intestinal mucosa of both WT and DSS-treated mice; the altered MPO might indicate a change in respiratory burst. However, it exhibited a more effective suppression of inflammatory factors secreted by macrophages as the colitis progressed. Avertin led to an increase in neutrophils and decrease in monocytes in both WT and DSS-treated mice blood. Our findings suggest that Avertin aggravates inflammation in the early and medium terms, but alleviates inflammation in the late stage of colitis by regulating neutrophils and macrophages.

Poly(2-oxazoline)s with a 2,2'-Iminodiacetate End Group Inhibit and Stabilize Laccase.

Poly(2-oxazoline)s (POxs) with 2,2'-iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC50 values were found to be in a range of 1-3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA-PEtOx30 -IDA (PEtOx: poly(2-ethyl-2-oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx-IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution.

Novel approach for the synthesis of a neutral and covalently bound capillary coating for capillary electrophoresis-mass spectrometry made from highly polar and pH-persistent N-acryloylamido ethoxyethanol.

Statically adsorbed or covalently coupled capillary coatings are of crucial importance in capillary electrophoresis-mass spectrometry for the separation of peptides and proteins. So far, published coating strategies and commercially available coated capillaries have a limited pH-stability so that the analysis at strongly acidic pH is limited, or harsh rinsing procedures for biological sample analysis cannot be applied. We here present a capillary coating based on Si-C linkages to N-acryloylamido ethoxyethanol (AAEE) with a new synthetic strategy including LiAlH4 surface reaction. We optimized the coating method with emphasis on stability and reproducibility applying harsh rinsing procedures (strong acid, strong base and organic solvent), using the electroosmotic mobility and separation efficiency of tryptic peptides as performance measure. Complete synthesis is performed in less than 2 days for up to 8 capillaries in parallel of more than 16 m total length. Intra- and inter-batch reproducibility were determined regarding electroosmotic mobility, separation efficiency and migration time precision in CE-MS separations of tryptically digested bovine serum albumin. Coating stability towards rinsing with strong acid (1 mol/L HCl), organic solvent (acetonitrile) and strong base (1 mol/L NaOH) was investigated. Outstanding performance was found for single capillaries. However, inter-capillary reproducibility is discussed critically. The new coating was successfully applied for reproducible CE-MS separation of large proteins in diluted serum, medium-sized peptides and small and highly charged polyamines in fish egg extracts using a very acidic background electrolyte containing 0.75 mol/L acetic acid and 0.25 mol/L formic acid (pH 2.2).

Anesthetic pretreatment confers thermotolerance on Saccharomyces cerevisiae yeast.

Saccharomyces cerevisiae yeast, when pretreated with elevated temperatures, undergo adaptive changes that promote survival after an otherwise lethal heat stress. The heat shock response, a cellular stress response variant, mediates these adaptive changes. Ethanol, a low-potency anesthetic, promotes thermotolerance possibly through heat shock response activation. Therefore, we hypothesized other anesthetic compounds, like ethanol, may invoke the heat shock response to promote thermotolerance. To test this hypothesis, we pretreated yeast with a series of non-volatile anesthetic and anesthetic-related compounds and quantified survival following lethal heat shock (52 °C for 5 min). Most compounds invoked thermoprotection and promoted survival with a potency proportional to hydrophobicity: tribromoethanol (5.6 mM, peak survival response), trichloroethanol (17.8 mM), dichloroethanol (100 mM), monochloroethanol (316 mM), trifluoroethanol (177.8 mM), ethanol (1 M), isopropanol (1 M), propofol (316 µM), and carbon tetrabromide (32 µM). Thermoprotection conferred by pretreatment with elevated temperatures was "left shifted" by anesthetic co-treatment from (in °C) 35.3 ± 0.1 to 32.2 ± 0.1 with trifluoroethanol (177.8 mM), to 31.2 ± 0.1 with trichloroethanol (17.8 mM), and to 29.1 ± 0.3 with tribromoethanol (5.6 mM). Yeast in postdiauxic shift growth phase, relative to mid-log, responded with greater heat shock survival; and media supplementation with tryptophan and leucine blocked thermoprotection, perhaps by reversing the amino acid starvation response. Our results suggest S. cerevisase may serve as a model organism for understanding anesthetic toxicity and anesthetic preconditioning, a process by which anesthetics promote tissue survival after hypoxic insult.

Green synthesis of enantiopure (S)-1-(benzofuran-2-yl)ethanol by whole-cell biocatalyst.

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal-based fermented beverage, was shown as a biocatalyst for the bioreduction of 1-(benzofuran-2-yl) ethanone to (S)-1-(benzofuran-2-yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1-(benzofuran-2-yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1-(benzofuran-2-yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)-1-(benzofuran-2-yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole-cell mediated biocatalytic method was performed for the enantiopure production of (S)-1-(benzofuran-2-yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.

Environmental impact of combustion of ethanolic biodiesel/diesel blends from several feedstocks on the gas emission levels in the atmosphere.

The aim of this work was to evaluate simultaneously the effect of produced ethanolic biodiesel from several feedstocks (soybean, crambe, macaw, sunflower, and waste cooking oil) and engine operational conditions (low and high engine speed) during combustion of biodiesel/diesel blends on the N2O, NOx, NO, CO2, and CO emission levels in the atmosphere. The biodiesel samples were prepared in one and/or two reaction steps, according to the acid index of each raw material, by esterification using H2SO4 and/or chemical transesterification using sodium ethoxide, both, through ethanolic route. The quality of the produced biodiesels was confirmed by ASTM/EN specifications. Then, biodiesel/diesel blends were prepared according to the following proportions: 10% (B10), 15% (B15), 25% (B25), and 50% (B50). In general way, all raw materials under combustion at low and high engine speed contributed to the formation of NOx and this effect was more drastically increased as the biodiesel concentration in the blends increases. N2O presented a similar behavior except for blends containing crambe and macaw biodiesel whose emissions were slightly reduced as a function of biodiesel content in these blends. Then, Principal component analysis (PCA) was applied to discriminate the effect of engine operating conditions, biodiesel kind, and biodiesel content in the blends during their combustion on the exhaust emissions. The attained results point to crambe and macaw as more environmentally sustainable feedstocks for biodiesel production because they generate less greenhouse gas emissions. These results are particularly attractive considering that, both, crambe and macaw are non-edible feedstocks with great potential for biodiesel production.

Cloning, expression, purification, and kinetic characterization of mitochondrial thioredoxin (TsTrx2), cytosolic thioredoxin (TsTrx1), and glutaredoxin (TsGrx1) from Taenia solium.

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.

Hydration-Shell Vibrational Spectroscopy.

Hydration-shell vibrational spectroscopy provides an experimental window into solute-induced water structure changes that mediate aqueous folding, binding, and self-assembly. Decomposition of measured Raman and infrared (IR) spectra of aqueous solutions using multivariate curve resolution (MCR) and related methods may be used to obtain solute-correlated spectra revealing solute-induced perturbations of water structure, such as changes in water hydrogen-bond strength, tetrahedral order, and the presence of dangling (non-hydrogen-bonded) OH groups. More generally, vibrational-MCR may be applied to both aqueous and nonaqueous solutions, including multicomponent mixtures, to quantify solvent-mediated interactions between oily, polar, and ionic solutes, in both dilute and crowded fluids. Combining vibrational-MCR with emerging theoretical modeling strategies promises synergetic advances in the predictive understanding of multiscale self-assembly processes of both biological and technological interest.

New evidence for refinement of anesthetic choice in procedures preceding the forced swimming test and the elevated plus-maze.

Previous studies indicated that some general anesthetics induce long-term antidepressant and/or anxiolytic-like effects. This raises the concern about the use of anesthesia in surgeries that precede psychopharmacological tests, since it may be a potential bias on results depending on the experimental design used. Thus, we evaluated whether general anesthetics used in surgeries preceding psychopharmacological tests would affect rats behavior in tests predictive of antidepressant or anxiolytic-like effects. We tested if a single exposure to sub-anesthetic or anesthetic doses of tribromoethanol, chloral hydrate, thiopental or isoflurane would change rats behavior in the forced swimming test (FST) or in the elevated plus-maze (EPM) test, at 2 h or 7 days after their administration. We also evaluated whether prior anesthesia would interfere in the detection of the antidepressant-like effect of imipramine or the anxiolytic-like effect of diazepam. Previous anesthesia with the aforementioned anesthetics did not change rats behaviors in FST per se nor it changed the antidepressant-like effect induced by imipramine treatment. Rats previously anesthetized with tribromoethanol or chloral hydrate exhibited, respectively, anxiogenic-like and anxiolytic-like behaviors in the EPM. Prior anesthesia with thiopental or isoflurane did not produce any per se effect in rats behaviors in the EPM nor disturbed the anxiolytic-like effect of diazepam. Our results suggest that, in our experimental conditions, tribromoethanol and chloral hydrate are improper anesthetics for surgeries that precede behavioral analysis in the EPM. Isoflurane or thiopental may be suitable for anesthesia before evaluation in the EPM or in the FST.

The effect of avertin anesthesia and a mixture of three types of anesthetic agents on food intakeand body weight in high fat-induced obese maleand female mice.

Basic research on obesity is becoming more important due to an increasing number of obese people. Experiments using obesity-model animals often require surgical interventions, such as gastric operation, and proper selection of anesthesia is important. Avertin, an agent mainly composed of 2,2,2-Tribromoethanol, has been used as general anesthesia for a long time, without the use of narcotic drugs. In the current study, we found that a single injection of avertin can decrease body weight (BW) in male and female C57BL/6J and ICR mice with high fat-diet (HFD)-induced obesity, but not in standard diet-fed nonobese males and females. Because the BW-reducing effect was more prominent in the female mice, we compared the effects of avertin and a mixture of three types of anesthetic agents (3MIX), which was developed in 2011, on BW reduction in HFD-induced obese female mice. Although both avertin and 3MIX decreased food intake and BW, the effects of avertin were significantly more potent than those of 3MIX. C-Fos expression, a neural activation marker, was dramatically increased in the brain regions related to the regulation of both food intake and the autonomic nervous system after avertin injection, but not after 3MIX injection. This suggests that avertin strongly stimulates the center of feeding regulation and the autonomic nervous system and therefore decreases BW. The current study suggests the advantages of using 3MIX for surgical interventions in mice in obesity research, as it is ideal to prevent anesthesia-induced BW decline.

Carbon nanotube modified glassy carbon electrode for electrochemical oxidation of alkylphenol ethoxylate.

Electrochemical oxidation of an emerging pollutant, 2-(4-methylphenoxy)ethanol (MPET), from water has been studied by cyclic voltammetry (CV). Multiwall carbon nanotubes glassy carbon electrodes (MWCNT-GCE) were used as working electrode due to their extraordinary properties. The oxidation process is irreversible, since no reduction peaks were observed in the reverse scan. The electrocatalytic effect of MWCNT was confirmed as the oxidation peak intensity increases in comparison to bare-GCE. The effect of functional groups on MWCNT was also studied by MWCNT functionalized with NH2 (MWCNT-NH2) and COOH (MWCNT-COOH) groups. The oxidation peak current decreases in the following order: MWCNT > MWCNT-NH2 > MWCNT-COOH. Taking into account the normalized peak current, MWCNT-NH2 exhibits the best results due to its strong interaction with MPET. Under optimal conditions (pH = 5.0 and volume of MWCNT = 10 µL), degradation was studied for MWCNT-GCE and MWCNT-NH2-GCE. A complete MPET removal was observed using MWCNT-GCE after four CV cycles, for a volume/area (V/A) ratio equal to 19. In the case of MWCNT-NH2-GCE, the maximum MPET removal was close to 90% for V/A = 37, higher than that obtained for MWCNT-GCE at the same conditions (≈80%). In both cases, no organic by-products were detected.

X-Ray Crystallographic Studies for Revealing Binding Sites of General Anesthetics in Pentameric Ligand-Gated Ion Channels.

X-ray crystallography is a powerful tool in structural biology and can offer insight into structured-based understanding of general anesthetic action on various relevant molecular targets, including pentameric ligand-gated ion channels (pLGICs). In this chapter, we outline the procedures for expression and purification of pLGICs. Optimization of crystallization conditions, especially to achieve high-resolution structures of pLGICs bound with general anesthetics, is also presented. Case studies of pLGICs bound with the volatile general anesthetic isoflurane, 2-bromoethanol, and the intravenous general anesthetic ketamine are revisited.

The facilitating effect of unfavorable lactation on the potentiation of electrocorticogram after spreading depression in awake and anesthetized adult rats.

OBJECTIVES: Cortical spreading depression (CSD) is a brain excitability-related phenomenon that can be affected by unfavorable conditions of lactation and by anesthetic agents. We have previously demonstrated that after CSD the electrocorticogram (ECoG) amplitude increases significantly (ECoG potentiation). Here, we investigated this potentiation in awake and anesthetized adult rats that were previously suckled among different lactation conditions. METHODS: Newborn rats were suckled in litters with 6 pups or 12 pups (L6 or L12 condition, respectively). At adulthood, we evaluated the ECoG potentiation after CSD at two cortical points (one point near, and another remote to the CSD-eliciting site). The amplitude of the ECoG waves was averaged with the support of an algorithm implemented in Matlab™ software. In both L6 and L12 condition, awake animals were compared with anesthetized groups that received either tribromoethanol- or urethane + chloralose-anesthesia. RESULTS: L12 rats presented significantly lower body- and brain weights than L6 rats (P < 0.01), indicating a nutritional deficiency. The anesthetized L6 groups presented with ECoG potentiation (P < 0.05) only in the near cortical recording point (28.0% and 32.6% increase for the tribromoethanol and urethane + chloralose groups, respectively), whereas the L12 groups displayed ECoG potentiation in both near (67.0% and 55.0%) and remote points (37.0% and 20.0%), in comparison with the baseline values (before CSD). DISCUSSION: The results suggest a facilitating effect of unfavorable lactation on the potentiation of ECoG after spreading depression in anesthetized adult rats. The potential implications for the human neurological health remain to be investigated.