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Bioretention basins are a prominent type of vegetated stormwater infrastructure that provides various ecosystem services, such as carbon (C) sequestration. Despite the key role of organic matter in the performance of bioretention basins, there is little understanding of their C accumulation properties. Using detailed field studies, we investigated the spatial, temporal and vertical variation of C capture in the soil of 25 subtropical bioretention basins in Australia. A thirteen-year soil chronosequence was used to estimate C sequestration rate. It was observed that the bioretention basins displayed a spatially uniform depositional pattern of C in their ponding area. The mean areal C density of soil in the upper 20 cm was 3.8 ± 0.3 kg C m−2, from which 32% was associated with the top 5 cm of soil. There was a strong influence of age on C density only throughout the first top 20 cm of the soil profile with a C sequestration rate of 0.31 kg C m−2 yr−1. Carbon quickly accumulates in the top 5 cm layer while in the lower depths it accumulated at a more gradual rate. The results show that bioretention systems could be designed for the enhancement of their C sequestration potential, and amendments in their design, such as addition of a carbon source layer, are important for better managing carbon availability in the basins.

Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells. Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

Accurate and effective analysis of microplastics depends on the purification of any biogenic matter present in the environmental sample. Within the soil environment, the presence of biogenic materials (living and non-living) obstruct reliable identification of microplastics. However, while conventional acidic, alkaline, and wet peroxidation methods are often used for microplastics purification, they can result in significant alteration to the polymer integrity. To overcome this issue of polymer damage, we have developed a more efficient protocol using enzymes to eliminate organic materials from soil media without affecting the synthetic polymers. This method describes a simple enzymatic protocol that eliminates the organic matter based on the application of enzymes that target the three natural components of lignocellulosic biomass (cellulose, hemicellulose, and lignin). The enzymatic protocol introduced in this study allows for the use of an oxidizing agent for the pre-treatment of samples and density separation step. Additionally, this method was used to determine the enzymatic digestion efficiencies of soil organic matter and recovery rates of microplastics. Finally, the recovered microplastics were characterized by techniques including stereomicroscopy, FT-IR, and SEM. This method proved to be effective in reducing approximately >90% of organic materials in soil samples. After showing a high digestion efficiency the method was applied to spiked microplastic soil samples where high recovery rates were established and plastic characteristics were conserved. Despite being a time consuming and expensive method when compared to other purification methods. The key benefits of this methodology are:•Minimal sample preparation•Representative purification of microplastic from diverse soil media; and•Most importantly, preservation of polymer integrity aiding requirements needed for particle identification (e.g.,% mass polymer remaining, SEM images revealing no obvious polymer morphology change after enzyme treatment)., Graphical abstract Image, graphical abstract

Green stormwater infrastructure is a common feature of urban cities which is mostly designed for hydrological and water quality purposes. The last decade has seen a rise in research on the environmental impact assessment of vegetated water sensitive urban design (WSUD) technologies. However, the added ecosystem benefits of these systems, such as carbon sequestration, have received less attention. In this study, the life cycle net carbon footprint of various vegetated WSUD technologies namely green roofs, rain gardens, bioretention basins, vegetated swales and stormwater ponds, have been reviewed and analysed including their carbon sequestration potential. The carbon footprint of each vegetated WSUD technology was evaluated through the four phases of the life cycle assessment (LCA): material production, construction, operation and maintenance and end-of-life phases. The results of this study show that the initial embodied carbon associated with production, transportation and construction phases is the major contributor to the carbon footprint for most of the vegetated WSUD technologies. Rain gardens are shown to provide the highest carbon sequestration potential which offsets its carbon footprint. Carbon sequestration of bioretention basins, green roofs, vegetated swales and stormwater ponds can mitigate approximately 70%, 68%, 45% and 8% of their carbon footprint respectively. This study demonstrates the significant role of carbon sequestration in mitigating the carbon footprint from the assigned life time of the vegetated WSUD technologies. The results presented in this study will allow designers and policymakers to include the carbon implication in their WSUD strategies.

Patterns in white mica (muscovite, paragonite, and phengite) Al-Si chemistry (Tschermak substitution) were examined for the Archean Kanowna Belle and Sunrise Dam gold deposits in the Eastern Goldfields of Western Australia, using short-wave infrared (SWIR) spectrometry of drill core samples. Both of these deposits represent greenschist-facies, structurally controlled, >10-Moz gold systems with broadly similar host rocks and superimposed metasomatic hydrothermal alteration halos that extend for up to 3 km from the economic zone. However, zonation toward gold mineralization at Kanowna Belle is associated with increasingly longer wavelength white mica, whereas Sunrise Dam comprises the opposite pattern. The study also found that gold mineralization at Kanowna Belle is associated with quartz, chalcopyrite, and pyrite with negative δ 34 S values and a relative lack of carbonate, chlorite, and paragonite (Na-rich white mica), whereas gold mineralization at Sunrise Dam is associated with paragonite as well as carbonate, Fe-rich chlorite and pyrite with positive δ 34 S values and a relative lack of quartz and chalcopyrite. Thermodynamic numerical modeling revealed that these contrasting patterns in white mica Tschermak substitution (and associated alteration mineralogy) can be explained by two different types of ore fluids, namely (1) oxidized, alkaline, and silica-rich at Kanowna Belle; and (2) reduced, acid, Fe-rich and silica-poor at Sunrise Dam, though the sources of these contrasting fluids are less clear. Importantly for explorers, spectral measurement (using drill core, field, airborne, and potentially satellite hyperspectral SWIR data) of gradients (especially within a given geologic unit) in white mica absorption wavelength (Tschermak substitution) represents a potential vectoring tool for the discovery of new Archean hydrothermal gold systems.

Most metastatic melanoma patients exhibit poor and variable response to radiotherapy and targeted therapies, including immune checkpoint inhibitors. There is a need for therapeutics that can potentiate existing treatments to positively impact clinical outcomes of metastatic melanoma patients. We reanalyzed melanoma TCGA transcriptomes and identified, as linked to previously defined molecular subgroups, enhanced expression of genes coding for subunits of the Type A GABA receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Using whole-cell patch clamp electrophysiology, we find that melanoma cells possess GABAARs that control membrane permeability to anions. Select benzodiazepines, by enhancing GABAAR mediated anion transport, depolarize melanoma cell mitochondrial membrane potential and impair cell viability in vitro. Using a syngeneic melanoma mouse model, we find that a benzodiazepine promotes reduction in tumor volume when administered alone and potentiated radiation or immune checkpoint inhibitor α-PD-L1. When a benzodiazepine is combined with concurrent α-PD-L1 and a sub-lethal radiation dose, there is near complete loss of tumor, beyond what is observed for benzodiazepine with radiation or α-PD-L1. Mechanistically, benzodiazepine with radiation or α-PD-L1 results in ipsilateral and an abscopal tumor volume reduction commensurate with enhanced infiltration into the tumor milieu of polyfunctional CD8 T-cells. There is also an increased expression of genes with roles in the cytokine-cytokine receptor and p53 signaling pathways. This study provides evidence for melanoma cell GABAARs as a therapeutic vulnerability with benzodiazepines promoting both direct and immune-mediated anti-tumor activity.

This study presents an assessment of below-ground carbon dynamics of green infrastructure using artificial intelligence, targeting sub-tropical bioretention basins in South East Queensland, Australia. This extended abstract describes the context for the study and the significance of the work, which was recognised and enabled through the international Microsoft Artificial Intelligence (AI) for Earth Grants (2018 Grant winner). Four different scenarios were tested with three different approaches for modelling of the regression values. The three different machine learning methods were applied to predict belowground carbon and nitrogen, based on soil physical characteristics data entry. The neural network model performed better in predicting both the carbon and nitrogen concentration in all the scenarios. The implication of this study provides a profound shift in the type of platform that can be used, wherein machine learning methods can assist decision-makers in finding low-cost proxies for measuring carbon and nitrogen capture in bioretention basins.

Medulloblastoma is the most common childhood malignant brain tumor. Children with highest morbidity have tumors that are TP53 wild-type and express high levels of MYC and GABRA5, which codes for the α-5 subunit of the ligand (chloride) gated GABA(A) receptor. Previously, we have established that the α-5 subunit of the GABA(A) receptor contributes to the assembly of a functional receptor in a subset of medulloblastoma cells and benzodiazepines, synthesized to function as α-5 selective ligands with psychotropic activity, impair viability(1,2). Utilizing a TP53 wild-type medulloblastoma cell line with amplified MYC, high GABRA5 expression, and a functional α-5 GABA(A) receptor, we provide insight into how benzodiazepines impair cell viability by positively modulating the native GABA(A) receptors. We screened benzodiazepines to assess impact of varying chemical group identity on cell viability. The most potent benzodiazepine binds with specificity to ~1000 native receptors per cell with EC50 and IC50 values of ~0.8 micromolar. This binding evokes a 2 x 10(9) ions.sec-1 chloride flux, which morphologically elicits mitochondrial fragmentation, nuclei distention, and cellular blebbing. The cascade of events culminates in a caspase-mediated activation of apoptosis through the intrinsic pathway and a localization of pro-apoptotic Bcl-2-associated death promoter (BAD) protein. Benzodiazepines may be efficacious as anti-cancer therapeutics for medulloblastoma patients exhibiting a GABA(A) receptor expression signature by driving a chloride imbalance that leads to cell apoptosis. References: (1)Sengupta, et al. α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth. Acta Neuropathol. 2014; 127(4): 593–603. (2)Jonas, et al. First in vivo testing of compounds targeting Group 3 medulloblastomas using an implantable microdevice as a new paradigm for drug development. J. Biomed. Nanotechnol. 2016; 12(6): 1297–1302.

Pediatric brain cancer medulloblastoma standard-of-care results in numerous comorbidities. Medulloblastoma is comprised of four molecular subgroups. Group 3 subgroup patients have the highest relapse rates and 20% survival after standard-of-care. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. We performed analysis of GABR and MYC expression in medulloblastoma tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells. Analysis of expression of 763 medulloblastoma tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2x109 ions/sec. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 μM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter (BAD) protein localization. GABR expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis. Citation Format: Laura Kallay, Havva Keskin, Alexandra Ross, Manali Rupji, Olivia A. Moody, Xin Wang, Guanguan Li, Taukir Ahmed, Farjana Rashid, Michael Rajesh Stephen, Kirsten A. Cottrill, Austin Nuckois, Maxwell Xu, Deborah E. Martinson, Frank Tranghese, Yanxin Pei, James M. Cook, Jeanne Kowalski, Michael D. Taylor, Andrew Jenkins, Daniel Pomeranz Krummel, Soma Sengupta. Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2623.

International audience; We have simulated the coupled electron and nuclear dynamics using the Ehrenfest method upon valence ionisation of modified bismethylene-adamantane (BMA) molecules where there is an electron transfer between the two π bonds. We have shown that the nuclear motion significantly affects the electron dynamics after a few fs when the electronic states involved are close in energy. We have also demonstrated how the non-stationary electronic wave packet determines the nuclear motion, more precisely the asymmetric stretching of the two π bonds, illustrating "charge-directed reactivity". Taking into account the nuclear wave packet width results in the dephasing of electron dynamics with a half-life of 8 fs; this eventually leads to the equal delocalisation of the hole density over the two methylene groups and thus symmetric bond lengths.

International audience; We simulate electron dynamics following ionization in 2-phenyl-ethyl-amine and 2-phenylethyl-N,N-dimethylamine as examples of systems where 3 coupled cationic states are involved. We study two nuclear effects on electron dynamics: (i) coupled electron-nuclear motion and (ii) nuclear spatial delocalization as a result of the zero-point energy in the neutral molecule. Within the Ehrenfest approximation, our calculations show that the coherent electron dynamics in these molecules is not lost as a result of coupled electron-nuclear motion. In contrast, as a result of nuclear spatial delocalization, dephasing of the oscillations occurs on a time scale of only a few fs, long before any significant nuclear motion can occur. The results have been rationalized using a semi-quantitative model based upon the gradients of the potential energy surfaces.

Background Fetal alcohol syndrome is an important clinical problem. Human embryonic stem cells (hESC) have not been widely used to study developmental alcohol toxicity. Here we document the phenotype of hESC exposed to clinically-relevant, low dose ethanol (20 mM). Methods All cultures were maintained in 3% O2 to reflect normal physiologic conditions. Undifferentiated hESC were expanded with basic fibroblast growth factor (bFGF), with or without ethanol, then differentiated without ethanol. Proliferation and apoptosis in response to ethanol were assayed, and PCR used to examine expression of GABA receptor subunits. Whole cell patch clamping was used to examine GABAA receptor function in undifferentiated hESC. Immunocytochemistry and western blotting were used to follow differentiation of early neurons, astrocytes, and oligodendrocytes, Principal findings Exposure to 20 mM ethanol resulted in larger colonies of undifferentiated hESC despite an increase in apoptosis, because proliferation of the undifferentiated cells (and neuroblasts) was significantly increased. Differentiation of hESC (following a week of ethanol exposure) resulted in decreased expression of GFAP (by western) compared to unexposed cells, suggesting that astrocyte differentiation was reduced, while markers of oligodendrocyte and neuron differentiation were unchanged. At the message level, undifferentiated hESC express all GABAA receptor subunits, but functional receptors were not found by whole cell patch clamping. Conclusion Our results in hESC suggest a complex mix of ethanol-induced phenotypic changes when ethanol exposure occurs very early in development. Not only increased apoptosis, but inappropriate proliferation and loss of trophic astrocytes could result from low-dose ethanol exposure very early in development. More generally, these studies support a role for hESC in developing hypotheses and focusing questions to complement animal studies of developmental toxicities.

The GABA type A receptor (GABA(A)R) is expressed ubiquitously throughout the brain and is a target for many therapeutic agents, including general anesthetics and benzodiazepines, which enhance receptor function by increasing the open probability (P(o)) of the ion channel. It is commonplace for in vitro studies of receptor pharmacological characteristics to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification, which results in chloride flow in the opposite direction, compared with in vivo conditions. This critical difference is usually overlooked because the GABA(A)R has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is nonlinear with respect to P(o). Specifically, we found that currents were outwardly rectifying at low P(o) and linear at high P(o). We confirmed the correlation between P(o) and rectification with a partial agonist, piperidine-4-sulfonic acid, and a gating-impaired mutation, α1(L277A); both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted under altered chloride gradient conditions, which suggests that rectification is linked to the direction of chloride flux. Finally, our results showed that the degree of potentiation by general anesthetics (etomidate, propofol, and isoflurane) was greater at negative membrane potentials. Traditional in vitro experiments thus overestimate the action of positive allosteric modulators of the GABA(A)R. Our results show that the direction of the driving force on the permeant ion, as well as P(o), must be considered together for a complete understanding of drug actions on ligand-gated ion channels.

In gamma-aminobutyric acid type A (GABA(A)) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the beta2-subunit may be involved in GABA(A) receptor activation. Specifically, residues Gly(170)-Gln(185) of the beta2-subunit were mutated to alanine, co-expressed with wild-type alpha1- and gamma2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC(50) whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC(50). None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC(50). Taken together, these results indicate that beta2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that beta2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABA(A) receptor.

M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of alpha and beta subunits of type "A" GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the gamma subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engineered gamma2(L287A) mutation on GABA activation and propofol modulation. In the macroscopic analysis, we found that the gamma2(L287A) mutation decreased GABA potency but increased the ability of propofol to enhance both GABA potency and efficacy compared with wild-type receptors. Indeed, although propofol had significant effects on GABA potency in wild-type receptors, we found that propofol produced no corresponding increase in GABA efficacy. At the single-channel level, mutant receptors showed a loss in the longest of three open-time components compared with wild-type receptors under GABA activation. Furthermore, propofol reduced the duration of one closed-time component, increased the duration of two open-time components, and generated a third open component with a longer lifetime in mutant compared with wild-type receptors. Taken together, we conclude that although the gamma subunit is not required for the binding of GABA or propofol, the M2-M3 linker of this subunit plays a critical role in channel gating by GABA and allosteric modulation by propofol. Our results also suggest that in wild-type receptors, propofol exerts its enhancing effects by mechanisms extrinsic to channel gating.

How general anesthetics change neurotransmitter receptor function is not fully understood. In this short review, we describe the key molecular elements governing GABAA receptor activation and modulation by general anesthetics.

Ligand-gated ion channels (LGICs) mediate rapid chemical neurotransmission. This gene superfamily includes the nicotinic acetylcholine, GABAA/C, 5-hydroxytryptamine type 3, and glycine receptors. A signature disulfide loop (Cys loop) in the extracellular domain is a structural motif common to all LGIC member subunits. Here we report that a highly conserved aspartic acid residue within the Cys loop at position 148 (Asp-148) of the glycine receptor alpha1 subunit is critical in the process of receptor activation. Mutation of this acidic residue to the basic amino acid lysine produces a large decrease in the potency of glycine, produces a decrease in the Hill slope, and converts taurine from a full agonist to a partial agonist; these data are consistent with a molecular defect in the receptor gating mechanism. Additional mutation of Asp-148 shows that alterations in the EC50 for agonists are dependent upon the charge of the side chain at this position and not molecular volume, polarity, or hydropathy. This study implicates negative charge at position Asp-148 as a critical component of the process in which agonist binding is coupled to channel gating. This finding adds to an emerging body of evidence supporting the involvement of the Cys loop in the gating mechanism of the LGICs.

Previous studies have shown that amino acid residues in trans-membrane (TM) segments 1, 2 and 3 of the alpha subunit are critical for the enhancement of GABA(A) receptor function by inhaled anesthetics. In this study we used tryptophan (Trp) scanning mutagenesis between Ile 406 and Asn 417 in the alpha1 subunit to determine the effects of Trp substitution in the fourth transmembrane segment (TM4) on receptor gating and anesthetic modulation. Wild-type and mutant alpha1 subunits were transiently expressed in HEK 293 cells with wild-type beta2 and gamma2s subunits and GABA-activated currents were recorded using whole-cell voltage clamp. The potentiation by three inhaled anesthetics (isoflurane, halothane and chloroform) of responses elicited by a submaximal concentration of GABA were also examined.EC(50) values for GABA at the mutant receptors were in the range 4-60 microM (wild-type=20 microM), indicating that Trp substitution can alter the apparent affinity of the receptor for GABA positively or negatively, dependent on position. The variation of the calculated EC(50) value for GABA exhibited an interesting periodicity, with the cycle length for each repeat corresponding to approximately 3.6 amino acids. These data are consistent with an alpha-helical structure for the TM4 segment of the alpha subunit. Several of these Trp point mutations altered the ability of one or more of the three inhaled anesthetics to modulate receptor function; four of the 12 mutations abolished receptor modulation by one or more of the anesthetics tested. These data are consistent with a role for these residues at the extracellular end of TM4 in anesthetic modulation of GABA(A) receptors.

Gamma-aminobutyric acid type A (GABA(A) )receptors are targets for allosteric modulation by general anesthetics. Mutation of Ser270 within the second transmembrane domain of the GABA(A) receptor alpha subunit can ablate the modulation of the receptor by the anesthetic ether isoflurane. To investigate further the function of this critical amino acid residue, we made multiple amino acid substitutions at Ser270 and analyzed the concentration-dependent gating by GABA and regulation by isoflurane in each mutant receptor. There is a strong negative correlation between the EC(50) for GABA and the molecular volume of the amino acid residue at position 270. Replacement of Ser by large residues such as His and Trp produced a shift of the GABA concentration-response curve to the left, whereas replacement of Ser with Gly had the opposite effect. There also was a strong negative association between the molecular volume of the amino acid residue at 270 and the degree of enhancement of submaximal GABA responses by isoflurane. These results indicate the significance of the amino acid at position alpha270 in gating of the GABA(A) receptor. In addition, the data on isoflurane are consistent with the existence of a cavity of finite size in the region of alpha270 that may be filled by the anesthetic molecule or by the side chain of a larger residue at alpha270. The introduction of isoflurane, or of a large residue, into this cavity may stabilize the open state of the GABA(A) receptor relative to the closed state.

The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-Å resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.

Using multiple PCR primer sets, we tried to optimize the detection of human papillomavirus (HPV) in DNA samples isolated from 361 frozen biopsy specimens from patients with invasive cervical carcinomas. The HPVs detected were placed into three distinct groups, including group I/Inex at Telelab (Skien, Norway) and group Ineg and group II at the Norwegian Radium Hospital (Oslo, Norway). The consensus primer sets were Oli-1b-oli-2i, My09-My11, Gp5-Gp6, and Gp(5+)-Gp6+ from the HPV L1 gene and CpI-CpIIG from the E1 gene. Using these consensus primers together with the type-specific primers from E6-E7, we found that 355 patients (98%) were HPV positive. Type-specific primers for HPV types 11, 16, 18, 31, 33, and 35 detected more HPV-infected patients than the most sensitive consensus primer set, while the three consensus primer sets My, Gp/Gp+, and Cp together detected more HPV-positive patients than the type-specific primers. Testing of sensitivity of the PCR with SiHa cells serially diluted in lymphocytes (HPV-negative cells) indicated a detection limit of 6,300 HPV type 16 DNA copies with consensus primers (My, Gp+, and Cp) and 126 original HPV type 16 DNA copies with type-specific primers. Comparison of the amplification results for consensus L1 primers and type-specific E6-E7 primers indicated the presence of L1 deletions in 23 of 56 samples. The conclusion is that in PCR detection systems, multiple consensus primers and type-specific primers should be used in order to detect all patients harboring HPV.

AIMS--To investigate the integration of human papillomavirus (HPV)16 in 13 HPV16 positive cervical squamous carcinomas. METHODS--Samples were investigated by Southern blot analysis of the Pst I digestion pattern, two-dimensional gel-electrophoresis, and in situ hybridisation. RESULTS--Integration of HPV16 was found in all cases. In 12 biopsy specimens episomal HPV16 DNA and integrated HPV16 DNA were seen. The episomal DNA occurred as dimers and multimers. In situ hybridisation showed that both integrated and episomal HPV16 DNA were present in the same cell in most tumour cell nuclei. CONCLUSIONS--An intact episomal E2 gene is present in most cases of these cervical cancers, and could therefore replace the regulatory function of an integrated defective E2 gene.

BACKGROUND: We hypothesized that pairs of inhaled anesthetics having divergent potencies [one acting weakly at minimum alveolar anesthetic concentration (MAC); one acting strongly at MAC] on specific receptors/channels might act synergistically, and that such deviations from additivity would support the notion that anesthetics act on multiple sites to produce anesthesia. METHODS: Accordingly, we studied the additivity of MAC for 11 anesthetic pairs divergently (one weakly, one strongly) affecting a specific receptor/channel at MAC. By “divergently,” we usually meant that at MAC the more strongly acting anesthetic enhanced or blocked the in vitro receptor or channel at least twice (and usually more) as much as did the weakly acting anesthetic. The receptors/channels included: TREK-1 and TASK-3 potassium channels; and -aminobutyric acid type A, glycine, N-methyl-d-aspartic acid, and acetylcholine receptors. We also studied the additivity of cyclopropane-benzene because the N-methyl-d-aspartic acid blocker MK-801 had divergent effects on the MACs of these anesthetics. We also studied four pairs that included nitrous oxide because nitrous oxide had been reported to produce infraadditivity (antagonism) when combined with isoflurane. RESULTS: All combinations produced a result within 10% of that which would be predicted by additivity except for the combination of isoflurane with nitrous oxide where infraadditivity was found. CONCLUSIONS: Such results are consistent with the notion that inhaled anesthetics act on a single site to produce immobility in the face of noxious stimulation. (Anesth Analg 2008;107:479‐85)

Gamma-aminobutyric acid type A receptors (GABAA-R) containing α1β2γ2 subunits are weakly inhibited by Zn2+, whereas receptors containing only the α1β2 subunits are strongly inhibited. We built homology models of the ion pores of α1β2 and α1β2γ2 GABAA-R using coordinates of the nicotinic acetylcholine receptor as a template. Threading the GABAA-R β2 sequence onto this template placed the 17‘ histidine and the 20‘ glutamate residues at adjacent locations in the mouth of the pore, such that a nearly ideal tetradentate site for Zn2+ was formed from two histidine and two glutamate residues between adjacent β subunits in the α1β2 GABAA-R. Following optimization with CHARMM, the distance between the α-carbons of the adjacent histidine residues was approximately 9.2 A, close to the ideal distance for a Zn2+ binding site. Loss of inhibition by Zn2+ in α1β2γ2 GABAA-R can be explained by the geometry of these residues in the arrangement α1β2γ2α1β2, in which the nearest C-α-C-α distance between the histidine residues...

Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM)isopulegolisomentholalpha-terpineolcyclohexanol. We studied menthol's actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor.

Gamma-aminobutyric acid type A receptors (GABA(A)-R) containing alpha1beta2gamma2 subunits are weakly inhibited by Zn2+, whereas receptors containing only the alpha1beta2 subunits are strongly inhibited. We built homology models of the ion pores of alpha1beta2 and alpha1beta2gamma2 GABA(A)-R using coordinates of the nicotinic acetylcholine receptor as a template. Threading the GABA(A)-R beta2 sequence onto this template placed the 17' histidine and the 20' glutamate residues at adjacent locations in the mouth of the pore, such that a nearly ideal tetradentate site for Zn2+ was formed from two histidine and two glutamate residues between adjacent beta subunits in the alpha1beta2 GABA(A)-R. Following optimization with CHARMM, the distance between the alpha-carbons of the adjacent histidine residues was approximately 9.2 A, close to the ideal distance for a Zn2+ binding site. Loss of inhibition by Zn2+ in alpha1beta2gamma2 GABA(A)-R can be explained by the geometry of these residues in the arrangement alpha1beta2gamma2alpha1beta2, in which the nearest C-alpha-C-alpha distance between the histidine residues is 15.5 A, too far apart for an energetically optimal Zn2+ binding site. We then mutated the gamma subunit at the 17' and/or 20' positions. Zn2+ inhibition was not restored in alpha1beta2gamma2 (I282H) receptors. A novel finding is that the modeling shows the native 20' lysine in gamma2 can compete with Zn2+ for binding to the inserted 17' histidine. Sensitivity to Zn2+ was restored in the double mutant receptor, alpha1beta2gamma2 (I282H; K285E), in which the competition with lysine was removed and a more favorable Zn2+ binding site was formed.

Observed reductions in pollutant concentrations through stormwater treatment devices commonly display the characteristic form of exponential decay, in which the rate of decrease of pollutant concentration with distance is proportional to the concentration. The observation of an apparently irreducible or background pollutant concentration, C*, in many devices has led to development of the two-parameter “k-C*” model. It is known that this model is too simplistic because the parameters k and C* are not constant but can vary greatly with pollutant concentration and hydraulic conditions. This paper presents an alternative exponential decay model for filtration of particulate pollutants, which is based on simple mathematical descriptions of key removal processes. The model delivers a process-based method for estimating the exponential decay constant. Moreover, the need to specify a background concentration is eliminated. To test the theory, the model is applied to the removal of clay and silica particles from horizontal flow through an experimental gravel trench. Particle concentrations were measured at nine locations along a 7.2 m long flume. The model agrees very well with the observed change in suspended solids concentration for the two pollutant materials and the range of flow rates tested. A single model parameter, notionally representing the “stickiness” of pollutant particles, is required for different pollutant materials.

Liquefied Petroleum Gas (LPG) is a mixture of light hydrocarbons, gaseous at normal temperature (15°C) and pressure (101.329 kPa) and maintained in the liquid state by increased pressure or lowered temperature. LPG is the generic name for “commercial butane” and “commercial propane”. Because of its high heating values, high purity, cleanness of combustion and easy of handling, LPG finds very wide application in a large variety of industrial, commercial, domestic and leisure uses. The history of LPG goes back to the early 1900s. The first car powered by propane ran in 1913 and by 1915 propane was used in torches to cut through metal. Current global LPG consumption is over 200 million tonnes/annum. Transportation of LPG by pipelines is environmentally friendly in that it entails less energy consumption and exhaust emissions than other modes of transportation. Worldwide, there are over 220,000 miles (350,000 kilometers) of petroleum, refined products and LPG pipelines. The majority are in the United States. Some refined products pipelines carry LPG in batch form. However, there are only about 8000 kilometers of single phase pipelines, of various diameters, that transport LPG (propane or butane) fluids (Mohitpour et al, 2006). There are a number of codes that industry follows for the design, fabrication, construction and operation of LPG facilities. However, there are no regulations or legislation that specifically cite the pipeline transportation of the product. From a safety point of view, although LPG is non-toxic, it can be very dangerous if not handled properly. A partial or complete rupture of an LPG pipeline, resulting in an accidental release, will cause issues related to evaporation, vapor cloud propagation and dispersion. Response to emergencies such as rupture and leak in LPG pipelining is thus critical and must ensure rapid action with respect to containment, control, elimination and effective maintenance/repair. This paper provides an overview the code and regulatory requirements and summarizes the more significant aspects of the design, construction and safe operation pertaining to LPG pipeline systems. It covers the timeline and statistics of the global LPG business; the type of facilities that make up the industry; and the LPG properties pertinent to pipeline design. It also addresses the significant safety issues of LPG pipelining including a discussion on emergency response and associated equipment needs and repair techniques.

The GABA(A) receptor is an important target for a variety of general anesthetics, including halogenated ethers such as isoflurane and halogenated ethers such as chloroform. Bromoform is a halogenated alkane that exhibits anesthetic properties and has been shown by X-ray crystallography to bind to model anesthetic targets. In this study we report the ability of bromoform to potentiate GABA-induced current in recombinant GABA(A) receptors composed of alpha(1)beta(2)gamma(2s) subunits. Recent studies have shown that specific point mutations in the transmembrane (TM) region of the GABA(A) receptor alpha-subunit can selectively abolish the modulatory activity of specific general anesthetics, and that molecular volume is a key determinant of anesthetic activity. The action of bromoform was examined in a series of mutant receptors and compared with the activity profiles of three other volatile anesthetics. The pharmacological profile of bromoform at the mutant receptors used in this study was similar to that seen with halothane, and distinct from that observed for isoflurane and chloroform. The molecular volume of bromoform is closest to that of halothane, and therefore our data are consistent with the idea that molecular volume is an important determinant of inhaled anesthetic activity at the GABA(A) receptor.

Previous studies have suggested that two specific amino acid residues in transmembrane segments 2 and 3 of the GABA(A) receptor alpha 2 subunit, Ser270 and Ala291, are critical for the enhancement of GABA(A) receptor function by inhaled anesthetics. The aim of this study was to determine the effects of amino acid substitutions in alpha 1 beta 2 gamma 2s GABA(A) receptors at alpha 1(S270) and at the homologous beta 2(N265) on receptor gating and anesthetic potentiation of GABA-induced responses. The wild-type and mutant receptors were transiently expressed in HEK 293 cells and GABA-induced currents were recorded using whole-cell voltage clamp. Potentiation of responses to a submaximal concentration of GABA by the anesthetics halothane and isoflurane was also examined. Some of the point mutations caused shifts in the GABA dose-response curve, indicating that the mutations changed the apparent affinity of the receptor for GABA. In receptors mutated at alpha 1(S270), the GABA EC(50) is inversely correlated with the volume of the residue of 270. On the contrary, there was no clear relationship between the physical properties of the amino acid residue at 265 in the beta 2 subunit and either the GABA EC(50) or anesthetic modulation, although mutations at N265 altered both parameters in a quantitative manner. These data are consistent with the results of previous work using other subunit combinations, in confirming that alpha 1(S270) may be involved in channel gating, and also may be important in anesthetic binding; the role of beta 2(N265) is less clear.

The GABA(A) receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABA(A) receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABA(A) receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 A(3). The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABA(A) receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.

Purging of a gas pipeline is the process of displacing the air/nitrogen by natural gas in an accepted constant practice in the natural gas pipeline industry. It is done when pipelines are put into service. Gas Pipelines are also purged out of service. In this case they are filled with air or other neutral gases. Traditionally, “purging” a newly constructed pipeline system is carried out by introducing high pressure gas into one end of the pipeline section to force air out of the pipeline through the outlet until 100% gas is detected at the outlet end. While this technique will achieve the purpose of purging air out of the pipeline, it gives little or no consideration to minimizing the emission of methane gas into the atmosphere. With the advances of the pipeline simulation technology, it is possible through simulation to develop a process to minimize the gas to air interface and thereby minimize the emission of methane gas. In addition, simulation can also be used to predict the timing of purging and loading of the pipeline. Therefore, scheduling of manpower and other activities can be more accurately interfaced. In this paper a brief background to purging together with a summary of current industry practices are provided. A simplified purging calculation method is described and a simulation technique using commercially available software is provided for planning purging and loading operations of gas pipeline systems. An Example is provided of a recently constructed pipeline (Mayakan Gas Pipeline System) in Mexico to demonstrate how the planning process was developed and carried out through the use of this simulation technique. Simulation results are compared with field data collected during the actual purging and loading of the Mayakan Pipeline.

1. NIE-115 mouse neuroblastoma cells were studied under voltage clamp in the whole-cell patch-clamp configuration. Peak currents induced by bath application of 5-hydroxytryptamine (5-HT) were inwardly rectifying, reversed at 0.4 +/- 0.2 mV (mean +/- s.e.mean), and were approximately half-inhibited (at 1 microM 5-HT) by 2 nM of the 5-HT3 selective antagonist MDL-72222 (3-tropanyl-3,5-dichlorobenzoate). 2. Peak inward currents activated by a low concentration of 5-HT at a holding potential of -50 mV were potentiated by volatile general anaesthetics. At their human minimum alveolar concentrations (MACs), the degree of potentiation increased in the order isoflurane < halothane < enflurane < methoxyflurane. Potentiation by methoxyflurane was independent of membrane potential in the range -70 mV to +40 mV. The reversal potential was the same in the presence and absence of methoxyflurane. 3. Methoxyflurane shifted the 5-HT dose-response curve to lower 5-HT concentrations, without significantly changing the Hill coefficient or maximum response. The EC50 concentration for 5-HT decreased from 1.86 +/- 0.02 microM to 1.07 +/- 0.11 microM (means +/- s.e.mean) due to the presence of 1 MAC (270 microM) methoxyflurane. 4. In contrast to the volatile anaesthetics, the barbiturate anaesthetic, thiopentone, inhibited the 5-HT3 receptor. Hill analysis of thiopentone dose-response data gave an average IC50 = 117 +/- 8 microM thiopentone and Hill coefficient = 1.6 +/- 0.2 (means +/- s.e.mean). These parameters were not significantly different for data obtained at 5-HT concentrations above and below the control EC50 concentration for 5-HT, consistent with non-competitive inhibition. 5. The n-alcohols occupied an intermediate position between the volatile and barbiturate anaesthetics. The lower alcohols (butanol and hexanol) potentiated 5-HT responses at low alcohol concentrations but inhibited them at high concentrations. In contrast, the higher alcohols (octanol, decanol, dodecanol, tridecanol, tetradecanol and pentadecanol) produced no potentiation, but only inhibition, at all alcohol concentrations. 6. Inhibition of the 5-HT3 receptor by the n-alcohols exhibited a cutoff in potency similar to those previously found for tadpoles, luciferase enzymes and a neuronal nicotinic acetylcholine receptor channel.

We describe the detection of eight genital human papillomavirus (HPV) types, including HPV16 and HPV18, by PCR amplification of a 323 base-pair region of the genome within the L1 open reading frame (ORF). The primer sequences are: TGYAAATATCCWGATTWTWT and GTATCWACMACAGTAACAAA. The method will detect purified HPV16 DNA down to a concentration of as little as a single molecule in 100 microliters. The method is also applicable to purified DNA and crude lysates from tumour biopsies. Typing of the PCR product can be achieved with specific oligonucleotide probes.

During a study on the epidemiology of Staphylococcus aureus colonization in newborns, mothers, and hospital staff, S. aureus was isolated from 536 of 1,945 specimens. Ninety-three isolates of S. aureus from the three groups of individuals were included in a study to evaluate the potential of DNA fingerprinting for strain differentiation. The 93 isolates were also phage typed and their plasmid profiles were analyzed. Cleavage of DNA with BamHI resulted in 13 different DNA restriction endonuclease band patterns (DNA REBPs), one of which consisted of eight isolates whose DNA was not cleaved with BamHI. The DNAs from these eight isolates were easily cleaved with HindIII. The different DNA REBPs were stable both during in vitro and in vivo growth and allowed strain differentiation within phage groups or types. We could not show any strong association between DNA REBP classes, phage types or groups, and plasmid profiles. Of the 93 isolates, 27 (29.0%) could not be phage typed and 12 (12.9%) lacked plasmids. We therefore conclude that DNA fingerprinting is a powerful tool, in addition to phage typing and plasmid profile analysis, for strain differentiation of S. aureus.

The proton-coupled carbon-13 n.m.r. spectra in ABCDX, ABCX and ABX spin systems are shown to be non-first order when the condition 1JCH/2 = Δδ is met, where Δδ is the proton chemical shift separation between two closely coupled protons associated with adjacent carbon atoms. Solvent and temperature effects are shown to be useful in removing the spectral complexity and thus assist in complete analysis. Examples in heteroaromatic and hydrocarbon compounds are presented. For a chosen solvent system the non-first-order behaviour may be predicted from the 1H n.m.r. spectrum and used as an aid in 13C n.m.r. spectral assignment.

Cys-loop ligand-gated ion channels are pentameric transmembrane proteins that are essential for the regulation of neuronal excitability. In synaptic GABA(A) receptors, residues in the two α-β+ interfaces are known to define the GABA binding site while homologous residues in the γ-α+ interface define the classical high-affinity benzodiazepine binding site. Very little however, is known about the structure and function of the remaining two interfaces: β-α+ and β-γ+. Since the β- interface is common to both, we chose to investigate the role this interface plays in receptor function, using the tools of structure-homology modeling, site-directed mutagenesis, transient expression of receptors in HEK-293 cells and whole-cell patch-clamp electrophysiology. In this study, we focused on a non-canonical loop domain (NCLD) that links the canonical loops A and E, also known as the β-strand 5-β- strand 5’ linker. Homology models predict that the N-terminal end of this loop flanks a large inter-subunit lumen-accessible crevice. An alanine screen of the NCLD revealed 4 positions within the domain that are critical for receptor activation. Of particular interest was a highly-conserved asparagine: β2(N113). Our data indicate that mutations at this position results in reduced receptor sensitivity to GABA (increased EC50), impaired receptor gating (reduced partial agonist efficacy) and perturbed ion flow (increased rectification). Interestingly, the change in rectification was reversed by the addition of positive allosteric modulators such as propofol and pregnanolone. Taken together, our observations demonstrate the importance of the non-binding interfaces in controlling receptor function.

The γ-aminobutyric acid type A (GABAA) receptor functions as a chemical to voltage transducer in the central nervous system, converting neurotransmitter signals to changes in postsynaptic membrane potential. The kinetics of GABAergic currents are known to be affected by membrane potential, but the dependence on neurotransmitter concentration has not been fully explored. The present study investigates the current-voltage (IV) relationship of α1β2γ2s GABAA receptors across a range of GABA concentrations using whole-cell patch clamp of transfected HEK293 cells. We have determined and validated a voltage ramp protocol verses a more traditional stepwise procedure, in order to rapidly quantify rectification, hysteresis and the reversal potential at GABA concentrations ranging from 0.3 to 1000 μM. Results show that the rectification and hysteresis of the IV curves are dependent on GABA concentration, whereas the reversal potential is independent. Specifically, measures of rectification and hysteresis segregated into two distinct populations, at low (0.3-10 μM) and high (30-1000 μM) GABA concentrations. We provide evidence that the current-voltage properties of GABAA receptors are concentration dependent and, consequently, that the GABA concentration must be taken into context when designing experiments and interpreting results. Finally, these results have implications for a better understanding of synaptic responses where receptor location plays a role in the level of GABA exposure.