Lithium-ion battery degradation: how to model it.

Predicting lithium-ion battery degradation is worth billions to the global automotive, aviation and energy storage industries, to improve performance and safety and reduce warranty liabilities. However, very few published models of battery degradation explicitly consider the interactions between more than two degradation mechanisms, and none do so within a single electrode. In this paper, the first published attempt to directly couple more than two degradation mechanisms in the negative electrode is reported. The results are used to map different pathways through the complicated path dependent and non-linear degradation space. Four degradation mechanisms are coupled in PyBaMM, an open source modelling environment uniquely developed to allow new physics to be implemented and explored quickly and easily. Crucially it is possible to see 'inside the model and observe the consequences of the different patterns of degradation, such as loss of lithium inventory and loss of active material. For the same cell, five different pathways that can result in end-of-life have already been found, depending on how the cell is used. Such information would enable a product designer to either extend life or predict life based upon the usage pattern. However, parameterization of the degradation models remains as a major challenge, and requires the attention of the international battery community.

Solubilization of a stereospecific opiate-macromolecular complex from rat brain.

A [3H]etorphine-macromolecular complex has been solubilized from rat brain synaptosomal fraction by extraction with the nonionic detergent Brij 36T. Stereospecificity of binding to this solubilized complex was demonstrated by the finding that radioactivity in the complex was virtually eliminated when binding had occurred in the presence of excess levorphanol, an active narcotic analgesic, while it was unaffected by its inactive enantiomorph dextrorphan. Bound radioactivity was dissociated by proteolytic enzymes, sulfhydryl reagents, and heat, suggesting the presence of protein. The bound solubilized macromolecular moiety may be the opiate receptor.

Multiple opiate receptors: alcohol selectively inhibits binding to delta receptors.

The addition of ethanol or other aliphatic alcohols to rat brain membranes strongly inhibits binding of enkephalins at concentrations at which little inhibition of opiate alkaloids is seen. Inhibition is reversible, and potency increases with chain length of the alcohol. The results suggest that delta receptors are considerably more sensitive to alcohols than mu receptors. This is the first demonstration of selective inhibition of one of the postulated classes of opiate receptors by a reagent that is not a ligand for the receptor.

The effects of the morphine analogue levorphanol on leukocytes. Metabolic effects at rest and during phagocytosis.

Studies on bacteria have suggested that morphine-like drugs have effects on the cell membrane. To determine the effect of this class of drugs on a mammalian cell, we selected the rabbit peritoneal exudate granulocyte, which undergoes striking membrane changes during phagocytosis. We examined the effect in vitro of the morphine analogue, levorphanol on phagocytosis and metabolism by granulocytes incubated with and without polystyrene particles or live Escherichia coli. Levorphanol (1 or 2 mmoles/liter) decreased: (a) acylation of lysolecithin or lysophosphatidylethanolamine in the medium (which is stimulated about two-fold during phagocytosis) both at rest (40%) and during phagocytosis (60%); (b) uptake of latex particles and Escherichia coli, as judged by electron microscopy; (c) killing of live Escherichia coli (10-fold); (d) (14)CO(2) production from glucose-1-(14)C during phagocytosis by at least 80%; (e) K(+) content of granulocytes (35%); (f) oxidation of linoleate-1-(14)C by 50%, and its incorporation into triglyceride by more than 80%. However, levorphanol stimulated 2 to 3-fold the incorporation of linoleate-1-(14)C or palmitate-1-(14)C into several phospholipids. Glucose uptake, lactate production, and adenosine triphosphate (ATP) content are not affected by the drug. Thus, levorphanol does not appear to exert its effects through generalized metabolic suppression. Removal of levorphanol by twice resuspending the granulocytes completely reverses all inhibition. In line with observations on bacteria, it appears that the complex effects of levorphanol on granulocytes may be due at least in part to an effect on the cell membrane.

A member of the heat shock protein 40 family, hlj1, binds to the carboxyl tail of the human mu opioid receptor.

A yeast two-hybrid screen, using the carboxyl tail of the human mu opioid receptor as bait and a human brain cDNA library as target, indicated that the carboxyl terminal portion of hlj1, a member of the human heat shock protein 40 family, interacts with the carboxyl tail of the human mu opioid receptor. To determine if direct in vitro binding occurs between these two proteins, we performed overlay experiments. Results from the overlay experiments showed that binding occurs between the His fusion protein of hlj1 and the GST fusion protein of the carboxyl tail of the human mu opioid receptor. In contrast, no binding with the His fusion protein of hlj1 occurred with GST alone or the GST fusion protein of the third cytoplasmic loop of the human mu opioid receptor. Results from co-immunoprecipitation studies, carried out in whole HEK cell lysates, confirmed in vivo binding between these two proteins. Immunofluorescent studies, using laser scanning confocal microscopy, showed significant co-localization between hlj1 and the human mu opioid receptor in the cell membrane. The function of this protein-protein interaction and its physiological relevance in animal and human brain is yet to be determined.

Cardio-pulmonary resuscitation training, knowledge and attitudes of newly-qualified doctors in New Zealand in 2003.

OBJECTIVE: To assess the resuscitation knowledge and confidence of newly-qualified doctors in New Zealand (NZ) in 2003. DESIGN: Anonymous questionnaires were distributed to all newly-qualified doctors in NZ (n=279). PARTICIPANTS: Two hundred and thirty-three respondents from hospitals throughout NZ (84% response rate). MAIN OUTCOME MEASURES: Resuscitation training received during medical school and use of recommended text (Level 7 of NZ resuscitation manual), confidence in resuscitation skills and core knowledge of basic and advanced resuscitation. RESULTS: 98.3% of doctors received advanced resuscitation training during their final year of medical school, of these 64.6% had received training in the previous 6 months. The mean knowledge score was 56.6% and 45% of doctors made 'fatal errors'. Eighty-four percentage of doctors had read the Level 7 manual and 72.6% found it very or extremely useful. Those who had read the manual had higher scores and were less likely to make a 'fatal error'. Having attended a cardiac arrest and having received training within the last 6 months improved doctor confidence in resuscitation. CONCLUSIONS: The resuscitation knowledge and confidence of newly-qualified doctors in NZ are sub-optimal, with some doctors displaying dangerous deficiencies. Our results suggest mandatory attainment of an advanced resuscitation certificate, six-monthly practical resuscitation sessions and increased exposure to real resuscitation situations should be implemented to improve undergraduate resuscitation training.

Inactivation and solubilization of opiate receptors by phospholipases A2.

(1) As previously shown, stereospecific binding of opiates to membrane bound receptors is inhibited by treatment with small amounts of phospholipase A2 from Vipera russelli. This effect is quantified and compared with the enzymes from the venoms of Naja Naja siamensis, Apis Mellifica and from porcine pancreas. All enzymes are equally effective. The inhibition is due to partial phospholipid hydrolysis leading to inactivation of membrane-bound receptor. (2) Bee venom phospholipase A2 together with the synergistically acting peptide, melittin, causes receptor solubilization up to 80% of preformed receptor-ligand complex can be solubilized in this manner. (3) Lysophosphatidylcholine, a product of phospholipid hydrolysis, solubilizes performed receptor-ligand complex to a similar extent. Several other detergents were tested for their ability to solubilize receptor-ligand complex. Digitonin appears to be most effective in solubilizing such a complex.

Effects of naloxone and its quarternary analogue on stimulation-induced feeding.

Feeding was induced in rats by electrical stimulation in the lateral hypothalamus. Naloxone (0.2 and 1.0 mg/kg) produced a dose-related elevation of the frequency threshold for stimulation-induced feeding while quarternary naloxone (2.0 and 10.0 mg/kg) had no effect. Since quarternary naloxone does not readily penetrate the blood-brain barrier, we conclude that the opiate receptors at which naloxone exerts its anorectic action are located in the brain rather than in potential peripheral tissues such as gastrointestinal tract, pancreas or adrenal medulla. The threshold-elevating effect of naloxone only became marked after rats had engaged in one or two 5-sec bouts of feeding. The effect continued to increase following each subsequent bout of feeding. Naloxone therefore appears to inhibit feeding by interacting with post-ingestive factors.

mu and delta-opioid receptor agonists induce mitogen-activated protein kinase (MAPK) activation in the absence of receptor internalization.

Agonist-promoted internalization (endocytosis) of G-protein-coupled receptors (GPCRs), including all three opioid receptor types (mu, delta and kappa), has been shown to occur via the clathrin endosomal pathway in response to receptor phosphorylation and the actions of the proteins, beta-arrestin and dynamin. Many members of the GPCR family stimulate mitogen-activated protein kinases (MAPK or ERK) activity and, in several cases, it appears that MAPK activation is dependent on receptor internalization. We have reinvestigated the question of whether internalization is obligatory for MAPK activation by opioid receptors, using cell lines expressing the cloned mu or delta receptor. Morphine, which is known to activate both mu and delta receptors, does not induce their rapid internalization into clathrin-coated endosomes. However, morphine produced a robust stimulation of MAPK in both cell lines, as demonstrated by the appearance of phosphorylated MAPK. Moreover, pre-exposure of cells to the internalization inhibitors, concanavalin A or hypertonic sucrose, totally blocked DAMGO mu-selective agonist) and DTLET (delta-selective agonist)-mediated receptor internalization, yet neither treatment affected MAPK phosphorylation induced by these peptides. Our results provide evidence that receptor internalization is not an obligatory requirement for MAPK activation by mu and delta opioid receptors. Hypotheses are presented to explain the seemingly contradictory results obtained from different laboratories.

Electrophilic alpha-methylene-gamma-lactone and isothiocyanate opioid ligands related to etorphine.

Isothiocyanate and alpha-methylene-gamma-lactone analogues of 6,14-endo-ethenotetrahydrothebaine and -oripavine were prepared with the electrophilic groups being located at C-19 in the C-7 alpha-side chain. Isothiocyanates were prepared in the N-Me and N-CPM (N-cyclopropylmethyl) series, both as the phenols and 3-O-methyl ethers from the diastereomeric amines formed from reductive amination of thevinone (2) and N-(cyclopropylmethyl)northevinone (13). Although addition of the organozinc reagent from methyl alpha-bromomethacrylate to 25 failed, addition to 3-O-protected aldehydes 27 and 35 produced, after subsequent deprotection, alpha-methylene-gamma-lactones 29 and 37, respectively. In the opioid receptor displacement assays against [3H]bremazocine as the radiolabeled ligand, the phenolic compounds were most potent with N-CPM isothiocyanates 20 and 21 showing IC50s of 0.32 and 0.76 nM, respectively, and N-CPM alpha-methylene-gamma-lactone 37 having an IC50 = 1.0 nM. Compound 37 showed irreversible effects in the binding assay which were mu-selective, as demonstrated by analogous experiments using [3H]DAGO, and naloxone was found to protect against the irreversible effects. This observation suggests that a receptor-bound nucleophile is located at a position where it can readily reach the alpha-methylene group of lactone 37.

Potential affinity labels for the opiate receptor based on fentanyl and related compounds.

Derivatives of fentanyl, 3-methylfentanyl, sufentanil, and lofentanil, possessing chemo- or photoaffinity functionalities, were synthesized as potential affinity reagents for the opiate receptor. Opiate receptor binding constants (IC50) were determined in competition experiments with [3H]naloxone and [3H]naltrexone. Affinity-labeling experiments were generally unsuccessful, although some irreversible attachment was achieved with alpha-diazoamide 17 and aryl azide 23.

Opioid agonists and antagonists. 6-Desoxy-6-substituted lactone, epoxide, and glycidate ester derivatives of naltrexone and oxymorphone.

Synthesis and opioid radioreceptor assay data on analogues closely related to 6-desoxy-6-spiro-alpha-methylene-gamma-lactone 5a, a compound with irreversible activity in this assay, are reported. Saturated lactones (7a,b), endocyclic alpha, beta-unsaturated gamma-lactones (8a,b and 9a), and 6 alpha,7 alpha-fused alpha-methylene-gamma-lactones (10a and 11a) were prepared. Related 6-desoxy-6-methylene 6 beta- and 6 alpha-oxides (12a,b and 13a) and glycidate esters 14a,b and 15a,b were also prepared with use of naltrexone (1a) and oxymorphone (1b) as starting material. Compounds in the N-cyclopropylmethyl (N-CPM) series were more potent than those in the N-Me series in displacing [3H]naltrexone in the opioid radioreceptor assay, usually by 2-16-fold in the absence of Na ion. The most potent N-CPM analogues were epoxides 12a and 13a and glycidate esters 14a and 15a, showing IC50's of 2-6 nM, similar to that of 5a. Of the N-Me analogues, 6 beta-oxide 12b was most active, with an IC50 of 8 nM in the absence of Na ion. For the N-CPM analogues, the Na ion ratios were generally less than 1, with two exceptions. The N-Me analogues showed expected larger Na ion effects of 7 or greater. None of the lactone analogues had irreversible effects when preincubated in the rat brain membrane preparation, even at 37 degrees C for 30 min, i.e., washing restored [3H]naltrexone binding to control levels. These results clearly show that the alpha-methylene-gamma-lactone moiety of 5a is required for irreversible effects, consistent with it serving as a conjugate addition acceptor of a nucleophilic group from a ligand at or near the receptor. The epoxides and glycidate esters also had no irreversible activity, indicating more electrophilic functional groups are needed and/or these electrophiles are not properly aligned to react with nucleophilic groups at or near the opioid receptor.

Diastereomeric 6-desoxy-6-spiro-alpha-methylene-gamma-butyrolactone derivatives of naltrexone and oxymorphone. Selective irreversible inhibition of naltrexone binding in an opioid receptor preparation by a conformationally restricted michael acceptor ligand.

The diastereomeric 6-desoxy-6-spiro-alpha-methylene-gamma-butyrolactone derivatives of naltrexone (4a and 5a) and of oxymorphone (4b and 5b) were prepared from their parent ketones. Diastereomers 4a and 4b were obtained from the 3,14-diacetate derivatives of naltrexone (6a) and oxymorphone (6b) by reaction with the Reformatsky reagent prepared from methyl alpha-(bromomethyl)acrylate. Deacetylation with methanol completed the synthesis. Diastereomers 5a and 5b were obtained from oxiranes 8a and 8b, respectively. The oxiranes were allowed to react with the sodium salt of ethyl acetoacetate, followed by methenation and deprotection to complete the synthesis of 5a and 5b, respectively. Compound 5a was the most potent agent tested in competition against [3H]naltrexone in the opioid radioreceptor assay. At a concentration of 5 nM this compound produced a 50% inhibition of binding. The majority of this inhibition (30%) was irreversible, i.e., it remained even after extensive washing of the membrane preparation in the presence and absence of Na+. Naloxone protected against this irreversible effect. The data suggest a receptor nucleophile, perhaps a sulfhydryl group, is located where it can add to the alpha, beta-unsaturated carbonyl system of 5a.

Conjugate addition ligands of opioid antagonists. Methacrylate esters and ethers of 6 alpha- and 6 beta-naltrexol.

Alpha- and beta-naltrexol derived esters 9 and 10 and ethers 11 and 12, each containing the alpha, beta-unsaturated ester functionality, were prepared as conformationally more flexible analogues of spiro-alpha-methylene-gamma-lactones 5 and 6. All were active in the opioid radioreceptor binding assay against [3H]bremazocine and more active against [3H]DAGO, indicating mu-subtype selectivity, but only ether 12 showed significant irreversible activity. We conclude that small structural changes, made in very closely related electrophilic opioids, lead to changes in receptor binding. All four compounds were long-acting antagonists to morphine in mice, with ester 10 being approximately equipotent with naltrexone.

Hybromet: a ligand for purifying opioid receptors.

Condensation of the Grignard reagent derive from 2-[4-(allyloxy)phenyl]ethyl bromide (4b) with 7 alpha-acetyl-6,14-endo-ethenotetrahydrothebaine (5) furnished the (R) tertiary carbinol, 7, which upon methoxymercuration followed by treatment with the KBr gave the bromomercurio compound 10 (Hybromet). The corresponding N-cyclopropylmethyl analogue, 11, was prepared also. The bromomercurio compound, 1, and the mercaptobenzothiazole derivative, 3, gave allyl phenyl ether when treated with BAL at room temperature. Similar treatment of 10 with BAL gave 7 in high yield. Binding studies using rat brain homogenates indicated that 7, 13, and 14 have moderately high affinities for mu rather than delta binding sites. Although much weaker, 10 showed preferential mu binding also. These results along with the fact that 10 reacted smoothly with sulfhydryl groups suggest that Hybromet would be a suitable ligand for use in affinity chromatography.

Effects of stress on opioid receptor binding in the rat central nervous system.

The endogenous opioid peptides are known to play a significant role in the modulation and/or mediation of numerous environmental or experimental stressors. However, the specific opioid peptide(s) and receptor type(s) involved, under what physiologic conditions they are engaged and within which regions of the CNS is not well understood. We therefore examined the effects of both a chronic and an acute stressor-90-h water deprivation and a single 20-min foot shock on opioid receptor binding in 17 specific rat brain nuclei. [3H]DSTLE (Tyr-D-Ser-Gly-Phe-Leu-Thr) and [3H]DAGO(Tyr-D-Gly-Phe-NMe-Phe-Gly-ol) were used to label delta and mu receptors, respectively. Foot shock induced profound antinociception as measured by tail-flick latency which outlasted the stressor by several minutes. However, only the septum responded with a decrease in [3H]DAGO binding to this type of stress-induced analgesia. No other alterations in either [3H]DAGO or [3H]DSTLE binding were seen in response to foot shock. In contrast, water deprivation induced increases in [3H-DAGO] binding in the septum as well as increases in [3H]DSTLE binding in the caudate and accumbens nuclei. Moreover, the presumptive mild stress of handling in the foot shock control group was sufficient to decrease mu or delta receptor binding in seven out of 17 brain regions investigated (including the frontal cortex and olfactory tubercle where both mu and delta binding were increased) when compared to unhandled deprivation control animals. These changes in opioid receptor binding may have been the result of alterations in treatment-induced peptide release, receptor regulation, or interactions with other released neurotransmitter ligand/receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in kappa opioid receptors in the guinea-pig brain: a quantitative autoradiographic study.

Investigation into the effect of aging on kappa opioid receptors in the brains of guinea-pigs was carried out in animals aged one, six, 24 and 36 months. Quantitative autoradiography was used to monitor the concentration of kappa receptors in various anatomic regions at five rostrocaudal levels in each age-group. Areas of high binding were found in the deep layers (laminae V, VI) of the neocortex and in the internal band of the periallocortical, dorsal agranular insular cortex. Among non-cortical areas examined, the nucleus accumbens and the substantia nigra possessed kappa binding levels equal to those seen in the deep neocortical layers. In all cases where an age-related change in the level of kappa receptors was detected, the direction of the change was one of decreased binding with advancing age. Statistical analysis of the binding data revealed that the one-month-old animal possessed the highest levels of kappa binding among all age groups examined. The vast majority of age-related changes in kappa binding levels occurred in laminae V and VI of neocortical regions. The per cent decreases (18-42%), as well as their age of onset (six to 36 months) varied in different anatomical regions. Possible mechanisms to explain the age-related decreases in kappa opioid binding are presented. The majority of the age-related decreases in kappa opioid binding are found in areas of the neocortex which are characterized by their motor, sensory and associative functions. It is within these three areas of function that diminutions in performance are most apparent in senescence.

Immunohistochemical localization of mu-opioid receptors in rat brain using antibodies generated against a peptide sequence present in a purified mu-opioid binding protein.

Light-microscope visualization in rat brain of a pattern of distribution of immunoreactivity, which included immunolabeled perikarya and beaded processes, was achieved using an immunoaffinity purified polyclonal antibody, Ab165, which recognizes the amino acid sequence, IRNLRQDRSKYY, found in the mu-opioid binding protein purified in our laboratory. Immunohistochemical staining with Ab165 was carried out by the avidin-biotin procedure. Antibody, preabsorbed with antigen, served as control. Extensive immunoreactivity was seen in the hippocampal formation, the amygdaloid complex, the striatal complex, cortical regions, select areas of the thalamus and hypothalamus and in laminae I and II of the dorsal horn in spinal cord. The distribution of immunoreactivity in the rat brain of antibody 165, which recognizes a purified mu-opioid binding protein, is concordant with the distribution of mu-opioid binding sites as determined by other laboratories in autoradiographic, electrophysiological and immunocytochemical studies. These findings have enabled us to distinguish areas possessing large fields of mu-opioid receptor containing cell bodies from areas possessing dense networks of immunolabeled neuronal processes or mixtures of both.

Tyrosine phosphorylation of the delta-opioid receptor. Evidence for its role in mitogen-activated protein kinase activation and receptor internalization*.

The internalization of G-protein-coupled receptors (GPCRs), including the delta opioid receptor (delta-OR), has been shown to involve the phosphorylation of serine and threonine residues. However, recent studies suggest that these residues may not be the only ones phosphorylated in response to prolonged opioid exposure. Tyrosines also appear important for delta-OR signalling, but it remains unclear whether they undergo phosphorylation. We examined whether the delta-OR, stably expressed in Chinese hamster ovary (CHO-K1) cells, was tyrosine-phosphorylated during prolonged agonist treatment. The epitope-tagged delta-OR was purified by immunoprecipitation, and the presence of phosphorylated tyrosines was detected using anti-phosphotyrosine antibodies. Tyrosine residues in the delta-OR were phosphorylated after exposure to the high-affinity agonist [d-Thr(2)]-Leu-enkephalin-Thr (DTLET) in a time- and concentration-dependent manner. Tyrosine phosphorylation of the delta-OR appeared to require the actions of a Src-like protein tyrosine kinase, since the Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-[3,4-d]-pyrimidine (PP1) attenuated this response. PP1 also attenuated the DTLET-mediated activation of mitogen-activated protein kinase, as well as rapid delta-OR internalization, but not receptor down-regulation. Finally, only opioid agonists that induce receptor internalization via the clathrin-dependent endosomal pathway stimulated significant tyrosine phosphorylation of the delta-OR protein. Evidence is presented that the delta-OR is tyrosine-phosphorylated, and we suggest how this may have an active role in opioid receptor signalling and regulation.

Identification of a neurorestrictive suppressor element (NRSE) in the human mu-opioid receptor gene.

Analysis of the DNA sequence of the human mu-opioid receptor gene (MOR) revealed that a region overlapping the start codon was substantially homologous to a DNA element named the neurorestrictive suppressor element (NRSE) or restrictive element 1 (RE-1). Transient transfection experiments in the L929 and HEK non-neural cell lines showed that expression of a MOR promoter/reporter gene construct was suppressed in non-neural cell lines by inclusion of this MOR NRSE. Expression from a thymidine kinase promoter was also suppressed when the MOR NRSE was inserted upstream or downstream of the reporter gene. The MOR NRSE did not suppress expression of the reporter gene in neural derived cell lines, IMR-32 and Neuro 2a. The transcription factor REST which binds NRSE thereby enacting the suppression of transcription, was encoded in a plasmid and co-transfected into the IMR-32 cells. The REST co-transfected neuronal derived (IMR-32) cells became sensitive to the MOR NRSE mediated suppression of reporter gene expression. Electrophoretic mobility shift experiments revealed that oligonucleotides containing the MOR NRSE were bound by a factor from nuclear extracts of non-neural cell lines, HeLa and Jurkat. This binding was specifically competed by oligonucleotides containing NRSE sequences previously shown to suppress transcription through REST. Thus an NRSE element overlapping the human MOR start codon suppresses gene expression in non-neural cell lines and may help direct neural tissue specific expression of MOR.