14-3-3γ mediates the long-term inhibition of peripheral kappa opioid receptor antinociceptive signaling by norbinaltorphimine.

Long-term inhibition of kappa opioid receptor (KOR) signaling in peripheral pain-sensing neurons is a potential obstacle for development of peripherally-restricted KOR agonists that produce analgesia. Such a long-term inhibitory mechanism is invoked from activation of c-Jun N-terminal kinase (JNK) that follows a single injection of the KOR antagonist norbinaltorphimine (norBNI). This effect requires protein synthesis of an unknown mediator in peripheral pain-sensing neurons. Using 2D difference gel electrophoresis with tandem mass spectrometry, we have identified that the scaffolding protein 14-3-3γ is upregulated in peripheral sensory neurons following activation of JNK with norBNI. Knockdown of 14-3-3γ by siRNA eliminates the long-term reduction in KOR-mediated cAMP signaling by norBNI in peripheral sensory neurons in culture. Similarly, knockdown of 14-3-3γ in the rat hind paw abolished the norBNI-mediated long-term reduction in peripheral KOR-mediated antinociception. Further, overexpression of 14-3-3γ in KOR expressing CHO cells prevented KOR-mediated inhibition of cAMP signaling. These long-term effects are selective for KOR as heterologous regulation of other receptor systems was not observed. These data suggest that 14-3-3γ is both necessary and sufficient for the long-term inhibition of KOR by norBNI in peripheral sensory neurons.

Age-related changes in peripheral nociceptor function.

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4-5 months) and aged (26-27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Influence of inoculum selection on the utilisation of volatile fatty acid and glucose in sulfate reducing reactors.

The capacity of three inocula (sewer biofilm, mangrove and estuary sediment) to utilise typical fermentation products of municipal solid waste for biological sulfate reduction was investigated. Each inoculum was used in two reactors, one fed a mixture of volatile fatty acids and another fed glucose to provide a suite of fermentation products via naturally occurring fermentation. Following 228 days of reactor operation, reactors inoculated with mangrove and estuary sediments exhibited higher sulfate reducing efficiencies (80-88%) compared to the biofilm-inoculated reactors (32-49%). Minimal use of acetate and its accumulation in the biofilm-inoculated reactors pointed to the high abundance of incomplete-oxidising sulfate reducing bacteria (SRB), Desulfovibrio and Desulfobulbus (90-96% of the sulfate reducing population). Although Desulfovibrio was also prominent in reactors inoculated with mangrove and estuary sediments, Desulfobacter, a known acetoclastic sulfate reducer, emerged from trace levels in these sediment (0.01% abundance in the estuary sediments and below detection in the mangrove sediments) to comprise 14%-70% of the sulfate reducing population at the end of reactor operation.

Long-term antagonism and allosteric regulation of mu opioid receptors by the novel ligand, methocinnamox.

Opioid overdose is a leading cause of death in the United States. The only treatment available currently is the competitive antagonist, naloxone (Narcan® ). Although naloxone is very effective and has saved many lives, as a competitive antagonist it has limitations. Due to the short half-life of naloxone, renarcotization can occur if the ingested opioid agonist remains in the body longer. Moreover, because antagonism by naloxone is surmountable, renarcotization can also occur in the presence of naloxone if a relatively larger dose of opioid agonist is taken. In such circumstances, a long-lasting, non-surmountable antagonist would offer an improvement in overdose treatment. Methocinnamox (MCAM) has been reported to have a long duration of antagonist action at mu opioid receptors in vivo. In HEK cells expressing the human mu opioid receptor, MCAM antagonism of mu agonist-inhibition of cAMP production was time-dependent, non-surmountable and non-reversible, consistent with (pseudo)-irreversible binding. In vivo, MCAM injected locally into the rat hindpaw antagonized mu agonist-mediated inhibition of thermal allodynia for up to 96 h. By contrast, antagonism by MCAM of delta or kappa agonists in HEK cells and in vivo was consistent with simple competitive antagonism. Surprisingly, MCAM also shifted the concentration-response curves of mu agonists in HEK cells in the absence of receptor reserve in a ligand-dependent manner. The shift in the [D-Ala2 ,N-MePhe4 ,Gly-ol5 ]-enkephalin (DAMGO) concentration-response curve by MCAM was insensitive to naloxone, suggesting that in addition to (pseudo)-irreversible orthosteric antagonism, MCAM acts allosterically to alter the affinity and/or intrinsic efficacy of mu agonists.

Transition of microbial communities and degradation pathways in anaerobic digestion at decreasing retention time.

Tuning of operational variables is a common practice to control the anaerobic digestion process and, in advanced applications, to promote the accumulation of fermentation products. However, process variables are interrelated. In this study, the hydraulic retention time (HRT) was decoupled from the organic loading rate (OLR) in order to isolate the effect of HRT as a selective pressure on: process performance, metabolic rates (hydrolytic, acetogenic, and methanogenic) and the microbial community. Four mesophilic anaerobic digesters were subjected to a sequential decrease in HRT from 15 to 8, 4 and 2 days while keeping the OLR constant at chemical oxygen demand of 1 gCOD L r-1 d-1. The results showed that HRT alone was insufficient to washout methanogens from the digesters, which in turn prevented the accumulation of volatile fatty acids (VFA). Methanosaeta was the dominant genus in the four digesters at all HRTs. Metabolic rates showed that process performance was controlled by hydrolysis, with a clear shift in acetogenic rates, from butyrate and propionate degradation to ethanol degradation at 4 and 2d HRT. The change in acetogenic pathways was attributed to a shift in the fermentation pathways co-current with changes in fermentative bacteria. At 2d HRT, biofilm was formed on the walls and paddles of the digesters, probably as a survival strategy. Most of the taxa in the biofilm were also present in the digester media. Overall, it is the combination of HRT with other operational parameters which promotes the washout of methanogens and the accumulation of VFA.

Organic waste biorefineries: Looking towards implementation.

The concept of biorefinery expands the possibilities to extract value from organic matter in form of either bespoke crops or organic waste. The viability of biorefinery schemes depends on the recovery of higher-value chemicals with potential for a wide distribution and an untapped marketability. The feasibility of biorefining organic waste is enhanced by the fact that the biorefinery will typically receive a waste management fee for accepting organic waste. The development and implementation of waste biorefinery concepts can open up a wide array of possibilities to shift waste management towards higher sustainability. However, barriers encompassing environmental, technical, economic, logistic, social and legislative aspects need to be overcome. For instance, waste biorefineries are likely to be complex systems due to the variability, heterogeneity and low purity of waste materials as opposed to dedicated biomasses. This article discusses the drivers that can make the biorefinery concept applicable to waste management and the possibilities for its development to full scale. Technological, strategic and market constraints affect the successful implementations of these systems. Fluctuations in waste characteristics, the level of contamination in the organic waste fraction, the proximity of the organic waste resource, the markets for the biorefinery products, the potential for integration with other industrial processes and disposal of final residues are all critical aspects requiring detailed analysis. Furthermore, interventions from policy makers are necessary to foster sustainable bio-based solutions for waste management.

Signaling characteristics and functional regulation of delta opioid-kappa opioid receptor (DOP-KOP) heteromers in peripheral sensory neurons.

Receptor heteromers often display distinct pharmacological and functional properties compared to the individual receptor constituents. In this study, we compared the properties of the DOP-KOP heteromer agonist, 6'-guanidinonaltrindole (6'-GNTI), with agonists for DOP ([D-Pen2,5]-enkephalin [DPDPE]) and KOP (U50488) in peripheral sensory neurons in culture and in vivo. In primary cultures, all three agonists inhibited PGE2-stimulated cAMP accumulation as well as activated extracellular signal-regulated kinase 1/2 (ERK) with similar efficacy. ERK activation by U50488 was Gi-protein mediated but that by DPDPE or 6'-GNTI was Gi-protein independent (i.e., pertussis toxin insensitive). Brief pretreatment with DPDPE or U50488 resulted in loss of cAMP signaling, however, no desensitization occurred with 6'-GNTI pretreatment. In vivo, following intraplantar injection, all three agonists reduced thermal nociception. The dose-response curves for DPDPE and 6'-GNTI were monotonic whereas the curve for U50488 was an inverted U-shape. Inhibition of ERK blocked the downward phase and shifted the curve for U50488 to the right. Following intraplantar injection of carrageenan, antinociceptive responses to either DPDPE or U50488 were transient but could be prolonged with inhibitors of 12/15-lipoxgenases (LOX). By contrast, responsiveness to 6'-GNTI remained for a prolonged time in the absence of LOX inhibitors. Further, pretreatment with the 12/15-LOX metabolites, 12- and 15- hydroxyeicosatetraenoic acid, abolished responses to U50488 and DPDPE but had no effect on 6'-GNTI-mediated responses either in cultures or in vivo. Overall, these results suggest that DOP-KOP heteromers exhibit unique signaling and functional regulation in peripheral sensory neurons and may be a promising therapeutic target for the treatment of pain.

Making Sense of Pharmacology: Inverse Agonism and Functional Selectivity.

Constitutive receptor activity/inverse agonism and functional selectivity/biased agonism are 2 concepts in contemporary pharmacology that have major implications for the use of drugs in medicine and research as well as for the processes of new drug development. Traditional receptor theory postulated that receptors in a population are quiescent unless activated by a ligand. Within this framework ligands could act as agonists with various degrees of intrinsic efficacy, or as antagonists with zero intrinsic efficacy. We now know that receptors can be active without an activating ligand and thus display "constitutive" activity. As a result, a new class of ligand was discovered that can reduce the constitutive activity of a receptor. These ligands produce the opposite effect of an agonist and are called inverse agonists. The second topic discussed is functional selectivity, also commonly referred to as biased agonism. Traditional receptor theory also posited that intrinsic efficacy is a single drug property independent of the system in which the drug acts. However, we now know that a drug, acting at a single receptor subtype, can have multiple intrinsic efficacies that differ depending on which of the multiple responses coupled to a receptor is measured. Thus, a drug can be simultaneously an agonist, an antagonist, and an inverse agonist acting at the same receptor. This means that drugs have an additional level of selectivity (signaling selectivity or "functional selectivity") beyond the traditional receptor selectivity. Both inverse agonism and functional selectivity need to be considered when drugs are used as medicines or as research tools.

Methodology to determine the extent of anaerobic digestion, composting and CH4 oxidation in a landfill environment.

An examination of the processes contributing to the production of landfill greenhouse gas (GHG) emissions is required, as the actual level to which waste degrades anaerobically and aerobically beneath covers has not been differentiated. This paper presents a methodology to distinguish between the rate of anaerobic digestion (rAD), composting (rCOM) and CH4 oxidation (rOX) in a landfill environment, by means of a system of mass balances developed for molecular species (CH4, CO2) and stable carbon isotopes (δ13C-CO2 and δ13C-CH4). The technique was applied at two sampling locations on a sloped area of landfill. Four sampling rounds were performed over an 18 month period after a 1.0 m layer of fresh waste and 30-50 cm of silty clay loam had been placed over the area. Static chambers were used to measure the flux of the molecular and isotope species at the surface and soil gas probes were used to collect gas samples at depths of approximately 0.5, 1.0 and 1.5 m. Mass balances were based on the surface flux and the concentration of the molecular and isotopic species at the deepest sampling depth. The sensitivity of calculated rates was considered by randomly varying stoichiometric and isotopic parameters by ±5% to generate at least 500 calculations of rOX, rAD and rCOM for each location in each sampling round. The resulting average value of rAD and rCOM indicated anaerobic digestion and composting were equally dominant at both locations. Average values of rCOM: ranged from 9.8 to 44.5 g CO2 m-2 d-1 over the four sampling rounds, declining monotonically at one site and rising then falling at the other. Average values of rAD: ranged from 10.6 to 45.3 g CO2 m-2 d-1. Although the highest average rAD value occurred in the initial sampling round, all subsequent rAD values fell between 10 and 20 g CO2 m-2 d-1. rOX had the smallest activity contribution at both sites, with averages ranging from 1.6 to 8.6 g CO2 m-2 d-1. This study has demonstrated that for an interim cover, composting and anaerobic digestion of shallow landfill waste can occur simultaneously.

Allosterism within δ Opioid-κ Opioid Receptor Heteromers in Peripheral Sensory Neurons: Regulation of κ Opioid Agonist Efficacy.

There is abundant evidence for formation of G protein-coupled receptor heteromers in heterologous expression systems, but little is known of the function of heteromers in native systems. Heteromers of δ and κ opioid receptors (DOR-KOR heteromers) have been identified in native systems. We previously reported that activation of DOR-KOR heteromers expressed by rat pain-sensing neurons (nociceptors) produces robust, peripherally mediated antinociception. Moreover, DOR agonist potency and efficacy is regulated by KOR antagonists via allosteric interactions within the DOR-KOR heteromer in a ligand-dependent manner. Here we assessed the reciprocal regulation of KOR agonist function by DOR antagonists in adult rat nociceptors in culture and in a behavioral assay of nociception. Naltrindole enhanced the potency of the KOR agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-pyrrolidin-1-ylethyl]acetamide (ICI-199441) 10- to 20-fold, but did not alter responses to 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50488). By contrast, the potency of U50488 was enhanced 20-fold by 7-benzylidenenaltrexone. The efficacy of 6'-guanidinonaltrindole (6'-GNTI) to inhibit nociceptors was blocked by small interfering RNA knockdown of DOR or KOR. Replacing 6'-GNTI occupancy of DOR with either naltrindole or 7-benzylidenenaltrexone abolished 6'-GNTI efficacy. Further, peptides derived from DOR transmembrane segment 1 fused to the cell membrane-penetrating HIV transactivator of transcription peptide also blocked 6'-GNTI-mediated responses ex vivo and in vivo, suggesting that 6'-GNTI efficacy in nociceptors is due to its positive allosteric regulation of KOR via occupancy of DOR in a DOR-KOR heteromer. Together, these results provide evidence for the existence of functional DOR-KOR heteromers in rat peripheral sensory neurons and that reciprocal, ligand-dependent allosteric interactions occur between the DOR and KOR protomers.

Pilot scale evaluation of a model to distinguish the rates of simultaneous anaerobic digestion, composting and methane oxidation in static waste beds.

The aim of this paper was to apply and validate a model for measuring the rate and extent of anaerobic digestion, composting and CH4 oxidation in laboratory scale beds. Degradation studies were performed in four reactors each packed with shredded unsorted municipal solid waste, with one bed covered with a 100 mm layer of soil. The rates of production of CH4, CO2, 13C-CO2 and the rate of consumption of O2 were measured and used as inputs to a mass balance expressions for these components to calculate the rates of anaerobic digestion, composting and CH4 oxidation. The results showed that anaerobic digestion, composting and CH4 oxidation occurred simultaneously in both the covered and uncovered beds. The analysis showed that 50 ±â€¯4% of the solids (COD basis) in the uncovered beds degraded anaerobically, with the generated CH4 subsequently oxidized, and that 32 ±â€¯4% of the solids degraded aerobically in the covered bed.

Pharmacological augmentation of nicotinamide phosphoribosyltransferase (NAMPT) protects against paclitaxel-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) arises from collateral damage to peripheral afferent sensory neurons by anticancer pharmacotherapy, leading to debilitating neuropathic pain. No effective treatment for CIPN exists, short of dose-reduction which worsens cancer prognosis. Here, we report that stimulation of nicotinamide phosphoribosyltransferase (NAMPT) produced robust neuroprotection in an aggressive CIPN model utilizing the frontline anticancer drug, paclitaxel (PTX). Daily treatment of rats with the first-in-class NAMPT stimulator, P7C3-A20, prevented behavioral and histologic indicators of peripheral neuropathy, stimulated tissue NAD recovery, improved general health, and abolished attrition produced by a near maximum-tolerated dose of PTX. Inhibition of NAMPT blocked P7C3-A20-mediated neuroprotection, whereas supplementation with the NAMPT substrate, nicotinamide, potentiated a subthreshold dose of P7C3-A20 to full efficacy. Importantly, P7C3-A20 blocked PTX-induced allodynia in tumored mice without reducing antitumoral efficacy. These findings identify enhancement of NAMPT activity as a promising new therapeutic strategy to protect against anticancer drug-induced peripheral neurotoxicity.

A mass balance model to estimate the rate of composting, methane oxidation and anaerobic digestion in soil covers and shallow waste layers.

Although CH4 oxidation in landfill soil covers is widely studied, the extent of composting and CH4 oxidation in underlying waste layers has been speculated but not measured. The objective of this study was to develop and validate a mass balance model to estimate the simultaneous rates of anaerobic digestion (rAD), CH4 oxidation (rOX) and composting (rCOM) in environments where O2 penetration is variable and zones of aerobic and anaerobic activity are intermingled. The modelled domain could include, as an example, a soil cover and the underlying shallow waste to a nominated depth. The proposed model was demonstrated on a blend of biogas from three separate known sources of gas representing the three reaction processes: (i) a bottle of laboratory grade 50:50% CH4:CO2 gas representing anaerobic digestion biogas; (ii) an aerated 250mL bottle containing food waste that represented composting activity; and (iii) an aerated 250mL bottle containing non-degradable graphite granules inoculated with methanotrophs and incubated with CH4 and O2 to represent methanotrophic activity. CO2, CH4, O2 and the stable isotope 13C-CO2 were chosen as the components for the mass balance model. The three reaction rates, r (=rAD, rOX, rCOM) were calculated as fitting parameters to the overdetermined set of 4mass balance equations with the net flux of these components from the bottles q (= [Formula: see text] , [Formula: see text] , [Formula: see text] and [Formula: see text] ) as inputs to the model. The coefficient of determination (r2) for observed versus modelled values of r were 1.00, 0.97, 0.98 when the stoichiometry of each reaction was based on gas yields measured in the individual bottles and q was calculated by summing yields from the three bottles. r2 deteriorated to 0.95, 0.96, 0.87 when using an average stoichiometry from 11 incubations of each of the composting and methane oxidation processes. The significant deterioration in the estimation of rCOM showed that this output is highly sensitive to the evaluated stoichiometry coefficients for the reactions. r2 deteriorated further to 0.86, 0.77, 0.74 when using the average stoichiometry and experimental measurement of the composition and volume of the blended biogas to determine q. This was primarily attributed to average errors of 8%, 7%, 11% and 14% in the measurement of [Formula: see text] , [Formula: see text] , [Formula: see text] and [Formula: see text] relative to the measurement of the same quantities from the individual bottles.

Constitutive Desensitization of Opioid Receptors in Peripheral Sensory Neurons.

Opioid receptors expressed by peripheral pain-sensing neurons are functionally inactive for antinociceptive signaling under most basal conditions; however, tissue damage or exposure to inflammatory mediators (e.g., bradykinin) converts these receptors from a nonresponsive state to a functionally competent state. Here we tested the hypothesis that the basal, nonresponsive state of the mu- and delta-opioid receptors (MOR and DOR, respectively) is the result of constitutive receptor activity that activates desensitization mechanisms, resulting in MOR and DOR receptor systems that are constitutively desensitized. Consistent with our previous findings, under basal conditions, neither the MOR agonist [d-Ala2,N-MePhe4,Gly-ol5]-enkephalin nor the DOR agonist [d-Pen2,5]-enkephalin, inhibited prostaglandin E2 (PGE2)-stimulated cAMP accumulation in peripheral sensory neurons in culture (ex vivo) or inhibited PGE2-stimulated thermal allodynia in the rat hind paw in vivo. Prolonged treatment with naloxone induced MOR and DOR responsiveness both in vivo and ex vivo to a similar magnitude as that produced by bradykinin. Also similar to bradykinin, the effect of naloxone persisted for 60 minutes after washout of the ligand. By contrast, prolonged treatment with 6ß-naltrexol, did not induce functional competence of MOR or DOR but blocked the effect of naloxone. Treatment with siRNA for ß-arrestin-2, but not ß-arrestin-1, also induced MOR and DOR functional competence in cultured peripheral sensory neurons. These data suggest that the lack of responsiveness of MOR and DOR to agonist for antinociceptive signaling in peripheral sensory neurons is due to constitutive desensitization that is likely mediated by ß-arrestin-2.

Long-Term Reduction of Kappa Opioid Receptor Function by the Biased Ligand, Norbinaltorphimine, Requires c-Jun N-Terminal Kinase Activity and New Protein Synthesis in Peripheral Sensory Neurons.

A single administration of the κ opioid receptor (KOR) antagonist, norbinaltorphimine (norBNI), produces long-term reduction in KOR function in heterologous expression systems and brain that is mediated by activation of c-Jun N-terminal kinase (JNK). In this study, we examined the long-term effects of norBNI on adult rat peripheral sensory neurons in vivo and ex vivo. Following a single intraplantar (i.pl.) injection of norBNI into the hind paw, peripheral KOR-mediated antinociception in the ipsilateral, but not the contralateral, hindpaw was abolished for at least 9 days. By contrast, the antinociceptive response to mu and delta opioid receptor agonists was unaltered. The long-term inhibitory effect on antinociception produced by pretreatment with norBNI required occupancy of peripheral KOR and was completely blocked by i.pl. injection of the JNK inhibitor, SP600125. In cultures of peripheral sensory neurons, norBNI activated JNK for at least 30 minutes. Furthermore, norBNI blocked KOR-mediated inhibition of adenylyl cyclase activity measured 24 hours later in a JNK-dependent manner, but did not block activation of extracellular signal-regulated kinase (ERK). The long-term inhibitory effect of norBNI on KOR function in vivo and ex vivo was blocked by inhibitors of mRNA translation, cycloheximide and rapamycin. These data suggest that in peripheral sensory neurons norBNI is a KOR-biased ligand for activation of JNK signaling, resulting in long-term blockade of some (antinociception, inhibition of adenylyl cyclase activity), but not all (ERK), KOR signaling. Importantly, norBNI elicits de novo protein synthesis in sensory neuron terminals that produces selective long-term regulation of KOR.

Critical analysis of hydrogen production from mixed culture fermentation under thermophilic condition (60 °C).

Bio-hydrogen production from mixed culture fermentation (MCF) of glucose was studied by conducting a comprehensive product measurement and detailed mass balance analysis of their contributions to the final H2 yield. The culture used in this study was enriched on glucose at 60 °C through a sequential batch operation consisting of daily glucose feeds, headspace purging and medium replacement every third day in serum bottles for over 2 years. 2-Bromoethanesulfonate (BES) was only required during the first three 3-day cycles to permanently eliminate methanogenic activity. Daily glucose feeds were fully consumed within 24 h, with a persistent H2 yield of 2.7 ± 0.1 mol H2/mol glucose, even when H2 was allowed to accumulate over the 3-day cycle. The measured H2 production exceeded by 14 % the theoretical production of H2 associated with the fermentation products, dominated by acetate and butyrate. Follow-up experiments using acetate with a (13)C-labelled methyl group showed that the excess H2 production was not due to acetate oxidation. Chemical formula analysis of the biomass showed a more reduced form of C5H11.8O2.1N1.1 suggesting that the biomass formation may even consume produced H2 from fermentation.

The use of food waste as a carbon source for on-site treatment of nutrient-rich blackwater from an office block.

Wastewater from office blocks is typically dominated by blackwater and is therefore concentrated and nutrient-rich. A pilot plant was operated for 260 days, receiving 300 L d(-1) of wastewater directly from an office building to determine whether nutrient removal could be achieved using food waste (FW) as a supplemental carbon source. The pilot plant consisted of a 600 L prefermenter and a 600 L membrane bioreactor that was operated as a sequential batch reactor in order to cycle through anoxic, anaerobic and aerobic phases. The influent wastewater Chemical Oxygen Demand (COD)/N/P was, on average, 1438/275/40 mg L(-1), considerably higher than typical municipal wastewater. Treatment trials on the wastewater alone showed that the COD was only marginally sufficient to exhaust nitrate, and initiate anaerobic conditions required for phosphate removal. The addition of 15 kg d(-1) of macerated FW increased the average influent COD/N/P concentrations to 20,072/459/66 mg L(-1). The suitability of FW as a carbon source was demonstrated by denitrification to NOx-N concentration of <1 mg L(-1) during the biological nutrient removal (BNR) cycles. N removal was limited by nitrification. FW also induced the anaerobic phase within the BNR cycles necessary for P removal. The final average COD (non-recalcitrant)/N/P effluent concentrations under FW supplementation were 7/50/13 mg L(-1) which equates to 99%, 89% and 80% COD/N/P removal, respectively, meeting the highest nutrient removal efficiency standards stipulated by state jurisdictions for on-site systems in the USA.

Rapid digestion of shredded MSW by sequentially flooding and draining small landfill cells.

This paper compares the digestion of a packed bed of shredded municipal waste using a flood and drain regime against a control digestion of similarly prepared material using a trickle flow regime. All trials were performed on shallow (2m) beds of the sub-8cm fraction of shredded mixed MSW, encapsulated in a polyethylene bladder. The control cell (Cell 1) was loaded with 1974 tonnes shredded municipal waste and produced 76±9m(3) CH4dryt(-1) (45±2m(3) CH4 'as received't(-1)) over 200days in response to a daily recirculation of the leachate inventory which was maintained at 60m(3). The flood and drain operation was performed on two co-located cells (Cell 2 and Cell 3) that were loaded simultaneously with 1026 and 915 tonnes of the sub-8cm fraction of shredded mixed MSW, with a third empty cell used as a reservoir for 275m(3) of mature landfill leachate. Cell 2 was first digested in isolation by flooding and draining once per week to avoid excessive souring. Gas production from Cell 2 peaked and declined to a steady residual level in 150days. Cell 3 was flooded and drained for the first time 186days after the commencement of Cell 2, using the same inventory of leachate which was now exchanged between the cells, such that each cell was flooded and drained twice per week. Biogas production from Cell 3 commenced immediately with flooding, peaking and reducing to a residual level within 100days. The average CH4 yield from Cells 2 and 3 was 123±15m(3)dryt(-1) (92±2m(3) 'as received't(-1), equal to 95% of the long term (2month) BMP yield.

Functional selectivity of kappa opioid receptor agonists in peripheral sensory neurons.

Activation of kappa opioid receptors (KORs) expressed by peripheral sensory neurons that respond to noxious stimuli (nociceptors) can reduce neurotransmission of pain stimuli from the periphery to the central nervous system. We have previously shown that the antinociception dose-response curve for peripherally restricted doses of the KOR agonist (-)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50488) has an inverted U shape. Here, we found that the downward phase of the U50488 dose-response curve was blocked by an inhibitor of extracellular signal-regulated kinase (ERK) activation U0126. Local administration of the selective KOR agonist salvinorin A (Sal-A), also resulted in an inverted U-shaped curve; however, the downward phase was insensitive to U0126. By contrast, inhibition of c-Jun N-terminal kinase (JNK) partially blocked the downward phase of the dose-response curve to Sal-A, suggesting a role for JNK. In cultures of peripheral sensory neurons, U50488 and Sal-A inhibited adenylyl cyclase activity with similar efficacies; however, their ability to activate ERK and JNK differed. Whereas U50488 activated ERK but not JNK, Sal-A activated JNK but not ERK. Moreover, although both U50488 and Sal-A produced homologous desensitization, desensitization to U50488 was blocked by inhibition of ERK activation, whereas desensitization to Sal-A was blocked by inhibition of JNK. Substitution of an ethoxymethyl ether for the C2 position acetyl group of Sal-A reduced stimulation of JNK, prevented desensitization by ethoxymethyl ether for the C2 position acetyl group of Sal-A, and resulted in a monotonic antinociception dose-response curve. Collectively, these data demonstrate the functional selectivity of KOR ligands for signaling in peripheral sensory neurons, which results in differential effects on behavioral responses in vivo.

Atypical antipsychotics and inverse agonism at 5-HT2 receptors.

It is now well accepted that receptors can regulate cellular signaling pathways in the absence of a stimulating ligand, and inverse agonists can reduce this ligand-independent or "constitutive" receptor activity. Both the serotonin 5-HT2A and 5-HT2C receptors have demonstrated constitutive receptor activity in vitro and in vivo. Each has been identified as a target for treatment of schizophrenia. Further, most, if not all, atypical antipsychotic drugs have inverse agonist properties at both 5-HT2A and 5-HT2C receptors. This paper describes our current knowledge of inverse agonism of atypical antipsychotics at 5-HT2A/2C receptor subtypes in vitro and in vivo. Exploiting inverse agonist properties of APDs may provide new avenues for drug development.