Estimating methane emissions in California's urban and rural regions using multitower observations

We present an analysis of methane (CH_4) emissions using atmospheric observations from 13 sites in California during June 2013 to May 2014. A hierarchical Bayesian inversion method is used to estimate CH_4 emissions for spatial regions (0.3° pixels for major regions) by comparing measured CH_4 mixing ratios with transport model (Weather Research and Forecasting and Stochastic Time-Inverted Lagrangian Transport) predictions based on seasonally varying California-specific CH_4 prior emission models. The transport model is assessed using a combination of meteorological and carbon monoxide (CO) measurements coupled with the gridded California Air Resources Board (CARB) CO emission inventory. The hierarchical Bayesian inversion suggests that state annual anthropogenic CH_4 emissions are 2.42 ± 0.49 Tg CH_4/yr (at 95% confidence), higher (1.2–1.8 times) than the current CARB inventory (1.64 Tg CH_4/yr in 2013). It should be noted that undiagnosed sources of errors or uncaptured errors in the model-measurement mismatch covariance may increase these uncertainty bounds beyond that indicated here. The CH_4 emissions from the Central Valley and urban regions (San Francisco Bay and South Coast Air Basins) account for ~58% and 26% of the total posterior emissions, respectively. This study suggests that the livestock sector is likely the major contributor to the state total CH_4 emissions, in agreement with CARB's inventory. Attribution to source sectors for subregions of California using additional trace gas species would further improve the quantification of California's CH_4 emissions and mitigation efforts toward the California Global Warming Solutions Act of 2006 (Assembly Bill 32)

Genomic traits of Klebsiella oxytoca DSM 29614, an uncommon metal-nanoparticle producer strain isolated from acid mine drainages

Abstract Background: Klebsiella oxytoca DSM 29614 - isolated from acid mine drainages - grows anaerobically using Fe(III)- citrate as sole carbon and energy source, unlike other enterobacteria and K. oxytoca clinical isolates. The DSM 29614 strain is multi metal resistant and produces metal nanoparticles that are embedded in its very peculiar capsular exopolysaccharide. These metal nanoparticles were effective as antimicrobial and anticancer compounds, chemical catalysts and nano-fertilizers. Results: The DSM 29614 strain genome was sequenced and analysed by a combination of in silico procedures. Comparative genomics, performed between 85 K. oxytoca representatives and K. oxytoca DSM 29614, revealed that this bacterial group has an open pangenome, characterized by a very small core genome (1009 genes, about 2%), a high fraction of unique (43,808 genes, about 87%) and accessory genes (5559 genes, about 11%). Proteins belonging to COG categories “Carbohydrate transport and metabolism” (G), “Amino acid transport and metabolism” (E), “Coenzyme transport and metabolism” (H), “Inorganic ion transport and metabolism” (P), and “membrane biogenesis-related proteins” (M) are particularly abundant in the predicted proteome of DSM 29614 strain. The results of a protein functional enrichment analysis - based on a previous proteomic analysis – revealed metabolic optimization during Fe(III)- citrate anaerobic utilization. In this growth condition, the observed high levels of Fe(II) may be due to different flavin metal reductases and siderophores as inferred form genome analysis. The presence of genes responsible for the synthesis of exopolysaccharide and for the tolerance to heavy metals was highlighted too. The inferred genomic insights were confirmed by a set of phenotypic tests showing specific metabolic capability in terms of i) Fe2+ and exopolysaccharide production and ii) phosphatase activity involved in precipitation of metal ion-phosphate salts. Conclusion: The K. oxytoca DSM 29614 unique capabilities of using Fe(III)-citrate as sole carbon and energy source in anaerobiosis and tolerating diverse metals coincides with the presence at the genomic level of specific genes that can support i) energy metabolism optimization, ii) cell protection by the biosynthesis of a peculiar exopolysaccharide armour entrapping metal ions and iii) general and metal-specific detoxifying activities by different proteins and metabolites

β-Arrestin-2 knockout prevents development of cellular μ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons

BACKGROUND AND PURPOSE: Tolerance to the behavioural effects of morphine is blunted in β-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. EXPERIMENTAL APPROACH: μ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa ) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and β-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. KEY RESULTS: MOPr inhibition of ICa in PAG neurons was unaffected by β-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT β-arrestin-2 knockouts. However, β-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. CONCLUSIONS AND IMPLICATIONS: In β-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by β-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in β-arrestin-2 knockouts. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.NHMRC Grant Numbers: 1011979 & 104596

Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine

1. The midbrain periaqueductal gray (PAG) is a major site of opioid analgesic action, and a significant site of cellular adaptations to chronic morphine treatment (CMT). We examined μ-opioid receptor (MOP) regulation of voltage-gated calcium channel currents (I(Ca)) and G-protein-activated K channel currents (GIRK) in PAG neurons from CMT mice. 2. Mice were injected s.c. with 300 mg kg(−1) of morphine base in a slow release emulsion three times over 5 days, or with emulsion alone (vehicles). This protocol produced significant tolerance to the antinociceptive effects of morphine in a test of thermal nociception. 3. Voltage clamp recordings were made of I(Ca) in acutely isolated PAG neurons and GIRK in PAG slices. The MOP agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) inhibited I(Ca) in neurons from CMT mice (230 nM) with a similar potency to vehicle (150 nM), but with a reduced maximal effectiveness (37% inhibition in vehicle neurons, 27% in CMT neurons). Inhibition of I(Ca) by the GABA(B) agonist baclofen was not altered by CMT. 4. Met-enkephalin-activated GIRK currents recorded in PAG slices were significantly smaller in neurons from CMT mice than vehicles, while GIRK currents activated by baclofen were unaltered. 5. These data demonstrate that CMT-induced antinociceptive tolerance is accompanied by homologous reduction in the effectiveness of MOP agonists to inhibit I(Ca) and activate GIRK. Thus, a reduction in MOP number and/or functional coupling to G proteins accompanies the characteristic cellular adaptations to CMT previously described in PAG neurons

Cellular actions of opioids on periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR-1

1. Patch clamp recordings were made from periaqueductal grey (PAG) neurons in vitro to investigate the cellular actions of opioids in wild-type C57B16/J mice and mutant mice lacking the first exon of the μ-opioid (MOP) receptor. 2. In wild-type mice, the κ-(KOP) agonist U-69593 (300 nM) and the mixed μ/δ-opioid agonist met-enkephalin (10 μM), but not the δ-(DOP) agonist deltorphin (300 nM), reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). Met-enkephalin and U-69593 also reduced the rate of spontaneous miniature IPSCs, but had no effect on their amplitude and kinetics. In μ-receptor-deleted mice, only U-69593 (300 nM) reduced the amplitude of evoked IPSCs. 3. In wild-type mice, the MOP agonist DAMGO (3 μM) produced an outward current in 76% of the neurons. Deltorphin and U-69593 produced outward currents in 24 and 32% of the neurons, respectively. In μ-receptor-deleted mice, deltorphin and U-69593 produced similar outward currents in 32 and 27% of the neurons, respectively, while DAMGO was without effect. All neurons in both the wild-type and μ-receptor-deleted mice responded with similar outward currents to either the GABA(B) receptor agonist baclofen (10 μM), or the opioid-like receptor ORL1 (NOP) agonist nociceptin (300 nM). 4. The DAMGO-, deltorphin-, U-69593-, baclofen- and nociceptin-induced currents displayed inward rectification and reversed polarity at −109 to −116 mV. 5. These findings indicate that μ-, δ- and κ-opioid receptor activation has complex pre- and postsynaptic actions within the mouse PAG. This differs to the rat PAG where only μ-opioid receptor actions have been observed

Estimating methane emissions in California’s urban and rural regions using multi-tower observations:

We present an analysis of methane (CH4) emissions using atmospheric observations from 36 thirteen sites in California during June 2013 – May 2014. A hierarchical Bayesian inversion 37 method is used to estimate CH4 emissions for spatial regions (0.3° pixels for major regions) by 38 comparing measured CH4 mixing ratios with transport model (WRF-STILT) predictions based 39 on seasonally varying California-specific CH4 prior emission models. The transport model is 40 assessed using a combination of meteorological and carbon monoxide (CO) measurements 41 coupled with the gridded California Air Resources Board (CARB) carbon monoxide (CO) 42 emission inventory. Hierarchical Bayesian inversion suggests that state annual anthropogenic 43 CH4 emissions are 2.42 ± 0.49 Tg CH4/yr (at 95% confidence, including transport bias 44 uncertainty), higher (1.2 - 1.8 times) than the CARB current inventory (1.64 Tg CH4/yr in 2013). 45 We note that the estimated CH4 emissions drop to 1.0 - 1.6 times the CARB inventory if we 46 correct for the 10% median CH4 emissions assuming the bias in CO analysis is applicable to 47 CH4. The CH4 emissions from the Central Valley and urban regions (San Francisco Bay and 48 South Coast Air Basins) account for ~58% and 26% of the total posterior emissions, 49 respectively. This study suggests that the livestock sector is likely the major contributor to the 50 state total CH4 emissions, in agreement with CARB’s inventory. Attribution to source sectors for 51 sub-regions of California using additional trace gas species would further improve the 52 quantification of California’s CH4 emissions and mitigation efforts towards the California Global 53 Warming Solutions Act of 2006 (AB-32)

Cellular actions of somatostatin on rat periaqueductal grey neurons in vitro

Functional studies indicate that the midbrain periaqueductal grey (PAG) is involved in the analgesic actions of somatostatin; however, the cellular actions of somatostatin in this brain region are unknown. In the present study, whole-cell patch clamp recordings were made from rat PAG neurons in vitro. In 93% of acutely isolated neurons, somatostatin inhibited Ca(2+)-channel currents. This effect was mimicked by the sst-2 selective agonist BIM-23027, but not by the sst-1 and sst-5 selective agonists CH-275 and L-362855. In brain slices, 81% of neurons responded to somatostatin (300 nM) with an increase in K(+) conductance that reversed polarity at −114 mV. A greater proportion of somatostatin-sensitive neurons (93%) than somatostatin-insensitive neurons (53%) responded to the opioid agonist met-enkephalin (10 μM). Somatostatin also reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). The actions of somatostatin in brain slices were mimicked by BIM-23027, but not by CH-275. Somatostatin had a variable effect on the rate of spontaneous miniature IPSCs in normal external potassium solutions. In high external potassium solutions, somatostatin reduced the rate of miniature IPSCs in all neurons, and this inhibition was abolished by addition of Cd(2+) (30 μM). Somatostatin had no effect on the amplitude of miniature IPSCs. These results indicate that somatostatin acts via sst-2 receptors to directly inhibit a subpopulation of PAG neurons by activating a potassium conductance and inhibits GABA release within PAG via a presynaptic Ca(2+)-dependent mechanism. Thus, like opioids, somatostatin has the potential to exert pre- and postsynaptic disinhibitory effects within the PAG