Electrochemical Acid-Base Transport Limitation Principle for Low Electroactive Analyte Sensing in Wastewater Monitoring.

Electrochemical sensing (ES) is crucial for improving data acquisition in wastewater treatment, but obtaining the signal for a low electroactive analyte is challenging. Here, we propose an electrochemical acid-base transport limitation (eABTL) principle for inertness-based sensing, offering a new insight into generating ES signals from an interfacial transport process rather than electron transfer. This principle enables potential ES application for various weak acids and bases (WABs) without reactions themselves. We established an eABTLP method for detecting orthophosphate in solutions as a proof of concept, demonstrating commendable accuracy and precision, and a wide detection range from 10 µM to over 300 mM. Endogenous interferences were identified using 23 weak acids, indicating no significant endogenous interfering factors in typical wastewaters. Of them, volatile fatty acids are the main interference, but their effect can be eliminated by adjusting pH above 6.0. Exogenous factors like anions, cations, ion strength, temperature, organic load, and dissolved oxygen were examined, and most of their effects can be ignored by maintaining consistent analytical procedures between calibration curve and sample. Furthermore, measurement of wastewater samples confirmed the applicability toward domestic wastewater and demonstrated its wide applicability when combined with digestion pretreatment. Given the merits of inertness-based sensing, the eABTL-based methods have the potential to be a crucial part of ES techniques for environmental and industrial monitoring.

The Influence of Heteroresistance on Minimum Inhibitory Concentration, Investigated Using Weak-Acid Stress in Food Spoilage Yeasts.

Populations of microbial cells may resist environmental stress by maintaining a high population-median resistance (IC50) or, potentially, a high variability in resistance between individual cells (heteroresistance); where heteroresistance would allow certain cells to resist high stress, provided the population was sufficiently large to include resistant cells. This study sets out to test the hypothesis that both IC50 and heteroresistance may contribute to conventional minimal inhibitory concentration (MIC) determinations, using the example of spoilage-yeast resistance to the preservative sorbic acid. Across a panel of 26 diverse yeast species, both heteroresistance and particularly IC50 were positively correlated with predicted MIC. A focused panel of 29 different isolates of a particular spoilage yeast was also examined (isolates previously recorded as Zygosaccharomyces bailii, but genome resequencing revealing that several were in fact hybrid species, Z. parabailii and Z. pseudobailii). Applying a novel high-throughput assay for heteroresistance, it was found that IC50 but not heteroresistance was positively correlated with predicted MIC when considered across all isolates of this panel, but the heteroresistance-MIC interaction differed for the individual Zygosaccharomyces subspecies. Z. pseudobailii exhibited higher heteroresistance than Z. parabailii whereas the reverse was true for IC50, suggesting possible alternative strategies for achieving high MIC between subspecies. This work highlights the limitations of conventional MIC measurements due to the effect of heteroresistance in certain organisms, as the measured resistance can vary markedly with population (inoculum) size. IMPORTANCE Food spoilage by fungi is a leading cause of food waste, with specialized food spoilage yeasts capable of growth at preservative concentrations above the legal limit, in part due to heteroresistance allowing small subpopulations of cells to exhibit extreme preservative resistance. Whereas heteroresistance has been characterized in numerous ecological contexts, measuring this phenotype systematically and assessing its importance are not encompassed by conventional assay methods. The development here of a high-throughput method for measuring heteroresistance, amenable to automation, addresses this issue and has enabled characterization of the contribution that heteroresistance may make to conventional MIC measurements. We used the example of sorbic acid heteroresistance in spoilage yeasts like Zygosaccharomyces spp., but the approach is relevant to other fungi and other inhibitors, including antifungals. The work shows how median resistance, heteroresistance, and inoculum size should all be considered when selecting appropriate inhibitor doses in real-world antimicrobial applications such as food preservation.

General mechanisms of weak acid-tolerance and current strategies for the development of tolerant yeasts.

Yeast cells are often subjected to various types of weak acid stress in the process of industrial production, food processing, and preservation, resulting in growth inhibition and reduced fermentation performance. Under acidic conditions, weak acids enter the near-neutral yeast cytoplasm and dissociate into protons and anions, leading to cytoplasmic acidification and cell damage. Although some yeast strains have developed the ability to survive weak acids, the complexity and diversity of stresses during industrial production still require the application of appropriate strategies for phenotypes improvement. In this review, we summarized current knowledge concerning weak acid stress response and resistance, which may suggest important targets for further construction of more robust strains. We also highlight current feasible strategies for improving the weak acid resistance of yeasts, such as adaptive laboratory evolution, transcription factors engineering, and cell membrane/wall engineering. Moreover, the challenges and perspectives associated with improving the competitiveness of industrial strains are also discussed. This review provides effective strategies for improving the industrial phenotypes of yeast from multiple dimensions in future studies.

Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH.

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pHc) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pHc by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pHc causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pHc in regulating the metabolic program of yeast cells.

Electrified Ion Exchange Enabled by Water Dissociation in Bipolar Membranes for Nitrogen Recovery from Source-Separated Urine.

Ion exchange (IX) is a promising technology for selective nitrogen recovery from urine; however, IX requires chemical-intensive regeneration that escalates energy consumption and carbon emissions. To overcome this barrier, we demonstrated and investigated a novel electrified IX stripping process (EXS) enabling electrochemical in situ IX regeneration with simultaneous ammonia stripping. EXS combines a weak acid cation exchange resin (WAC) to concentrate ammonia, a bipolar membrane to produce protons for WAC regeneration, and membrane stripping to recover the eluted ammonium from WAC. We observed over 80% regeneration (elution from resin) and recovery (membrane stripping) efficiencies during multiple adsorption-recovery cycles with synthetic and real urine. Comparing WAC with a strong acid cation exchange resin illustrated the critical role of the proton affinity of resin moieties in regulating resin regenerability and conductivity in EXS, which we distinguished from the rationale for material choice in electrodeionization. Compared to other electrochemical recovery methods using unamended wastewater as an electrolyte, EXS enabled control of electrolyte composition during recovery by separating and equalizing influent ammonium via WAC-mediated removal. This electrolyte engineering facilitated tunable EXS energy efficiency (100-300 MJ/kg N). This study informs the design of electrified, intensified systems that enable decentralized nitrogen recovery from urine.

Adsorption studies of a multi-metal system within acetate media, with a view to sustainable phosphate recovery from sewage sludge.

Phosphate shortages and the ensuing pressures on food security have led to an interest in processed sewage sludge as a substitute for commercial fertilisers. The presence of heavy metals in this nutrient source causes concerns around environmental release and pollution. This work builds towards a resin-in-pulp sludge detoxification process. It showcases the kinetic and thermodynamic adsorption capabilities of the ion-exchange resins C107E (carboxylic acid functionality), MTS9301 (iminodiacetic acid) and TP214 (thiourea), with respect to Cu(II), Fe(II), Pb(II) and Zn(II), within a simulated sewage sludge weak acid (acetate) leachate. The isotherms produced in this complex system were quite different to those generated when single metals were investigated in isolation, with desorption of lower affinity species clearly observed at higher equilibrium concentration values. Mixed-metal isotherm data were fitted to common two-parameter isotherm models and also a novel modified Langmuir model, which better accounted for the effects of desorption and competition. Kinetic data were also fit to common two-parameter models; results suggesting the system was likely film diffusion-controlled and followed pseudo-2nd-order kinetics. C107E displayed rapid adsorption of lead (t1/2 = 26 ± 3min), and significant uptake of all metals. MTS9301 showed high affinity for copper ions, with concurrent desorption of all the other metals, and also displayed the fastest kinetics (t1/2 = 14.1 ± 0.9, 130 ± 20, 25 ± 5 and 49 ± 6 min for copper, iron(II), lead and zinc, respectively). C107E and MTS9301 showed far slower adsorption for iron(II) than the other three metals, which invited the possibility of kinetic separations. TP214 had reasonable effectiveness in removal of copper, but poor affinity for all other metals. The greatest difficulty in modelling the multi-metal system was the two-stage trends observed in equilibrium experiments, as metal-proton exchanges become metal-metal exchanges. While not having the highest capacity, MTS9301 was recommended as the most appropriate resin for rapid and efficient removal of Cu, Pb and Zn from the acetate medium.

Dissolution of a Biopharmaceutics Classification System Class II Free Acid from Immediate Release Tablets Containing a Microenvironmental pH Modulator: Comparison of a Biorelevant Bicarbonate Buffering System with Phosphate Buffers.

Poor water dissolution of active pharmaceutical ingredients (API) limits the rate of absorption from the gastrointestinal tract. Increasing the pH of a solid form microenvironment can enhance the dissolution of weakly acidic drugs, but data on this phenomenon in a physiologically relevant bicarbonate media are lacking. In this paper, we examined the effect of a microenvironmental pH modulator (Na2HPO4) on the dissolution of a Biopharmaceutics Classification System (BCS) class II free weak acid (ibuprofen) at biorelevant conditions, including an automatic bicarbonate buffering system, as well as in compendial (50 mM) and low-concentration (10 mM) phosphate buffers with no external pH control. The tablets of 200 mg ibuprofen with either Na2HPO4 (phosphate formulation, PF) or NaCl (reference formulation, RF) were manufactured using a compression method. In a pH 2 simulated gastric fluid, only PF produced a transient supersaturation of ibuprofen, dissolving a fourfold higher drug amount than RF. In a bicarbonate-buffered simulated intestinal fluid with a dynamically controlled pH (5.7, 7.2, and 5.8 to 7.7 gradient), PF dissolved more drug within 30 min than RF (p ≤ 0.019). Of note, the use of a 50 mM phosphate buffer pH 7.2 provided opposite results-RF dissolved the API much faster than PF. Moreover, 10 mM phosphate buffers of pH 5.6 and 7.2 could neither maintain a constant pH nor mimic the bicarbonate buffer performance. In conclusion, the use of a bicarbonate-buffered intestinal fluid, instead of phosphate buffers, may be essential in dissolution tests of BCS class II drugs combined with pH modulators.

High level production of itaconic acid at low pH by Ustilago maydis with fed-batch fermentation.

The metabolically engineered plant pathogen Ustilago maydis MB215 Δcyp3 Petefria1 has been cultivated to produce more than 80 g/L itaconate in 16 L scale pH and temperature controlled fermentation, in fed-batch mode with two successive feedings. The effect of pH as well as successive rounds of feeding has been quantified via elemental balances. Volumetric itaconic acid productivity gradually decreased with successive glucose feedings with increasing itaconic titers, with nearly constant product yield. Extracellular pH was decreased from 6 down to 3.5 and the fermentation was characterized in specific uptake, production, and growth rates. Notable is that the biomass composition changes significantly from growth phase to itaconic acid production phase, carbon content increases from 42% to around 62%. Despite the gradual decrease in itaconic acid levels with decreasing pH (nearly 50% decrease in itaconic acid at pH 3.5, compared to pH 6), significant itaconate production is still observed at pH 4 (around 63 g/L). Biomass yield remained nearly constant until pH 4. Taken together, these results strongly illustrate the potential of engineered Ustilago maydis in itaconate production at commercial levels.

Disinfection efficiency of hospital infectious disease wards with chlorine dioxide and hypochlorous acid.

The disinfection efficiencies of two chemical disinfectants, chlorine dioxide and weak acid hypochlorous water (WAHW), were examined in the soiled room and dishwashing room of a hospital infectious disease ward in Taiwan. The investigations were conducted in two seasons, namely winter and summer, in order to examine the correlation between the bioaerosol concentration and the environmental factors. In addition, a single-daily disinfection mode (SM) and a twice-daily disinfection mode (TM) were applied in this study. The results showed that the bacteria and fungi colony counts were strongly correlated with the temperature. Both disinfectants reduced the bacteria and fungi concentrations in the considered rooms. However, of the two disinfectants, the ClO2 showed a stronger disinfection effect than the WAHW. It means that when using ClO2 as the disinfectant, the disinfection efficiency of the TM treatment mode is significantly better than that of the SM treatment mode. But, when using WAHW as the disinfectant, no significant difference is found between the disinfection efficiencies of the two methods. Overall, the results showed that the application of ClO2 twice daily provided the most effective means of satisfying the Taiwan EPA guidelines for the indoor air quality of hospital medical wards.

Physiological Genomics of the Highly Weak-Acid-Tolerant Food Spoilage Yeasts of Zygosaccharomyces bailii sensu lato.

Zygosaccharomyces bailii and two closely related species, Z. parabailii and Z. pseudobailii ("Z. bailii species complex", "Z. bailii sensu lato" or simply "Z. bailii (s.l.)"), are frequently implicated in the spoilage of acidified preserved foods and beverages due to their tolerance to very high concentrations of weak acids used as food preservatives. The recent sequencing and annotation of these species' genomes have clarified their genomic organization and phylogenetic relationship, which includes events of interspecies hybridization. Mechanistic insights into their adaptation and tolerance to weak acids (e.g., acetic and lactic acids) are also being revealed. Moreover, the potential of Z. bailii (s.l.) to be used in industrial biotechnological processes as interesting cell factories for the production of organic acids, reduction of the ethanol content, increase of alcoholic beverages aroma complexity, as well as of genetic source for increasing weak acid resistance in yeast, is currently being considered. This chapter includes taxonomical, ecological, physiological, and biochemical aspects of Z. bailii (s.l.). The focus is on the exploitation of physiological genomics approaches that are providing the indispensable holistic knowledge to guide the effective design of strategies to overcome food spoilage or the rational exploitation of these yeasts as promising cell factories.

Wireless 24, 48, and 96 Hour or Impedance or Oropharyngeal Prolonged pH Monitoring: Which Test, When, and Why for GERD?

PURPOSE OF REVIEW: pH monitoring technologies are routinely utilized in practice to further evaluate symptoms of gastro-esophageal reflux disease and laryngopharyngeal reflux (LPR). This is a review of the recent literature of the available pH monitoring technology and creates an algorithm in the diagnostic work up of a patient with GERD or LPR. RECENT FINDINGS: The catheter-free wireless pH monitor traditionally collects data for 48 h. Recent studies have found that extending to 96 h can be helpful in patients with conflicting results on the first 2 days of the study. In addition, 96 h can allow for testing both on and off of PPI therapy. The oropharyngeal monitoring device is a newer technology that is designed to aid in the diagnoses of LPR. There are limitations with this technology as there is no universal abnormal cutoff and some studies have suggested a poor correlation between multichannel intraluminal impedance-pH (MII-pH) and the oropharyngeal monitoring device. MII-pH has recently developed two additional parameters, the measurement of three 10-min nighttime periods and the post-reflux swallow-induced peristaltic wave (PSPW) index, both of which may increase accuracy of testing. Each of these technologies can provide unique data regarding acid reflux exposure in the esophagus and oropharynx. The wireless pH monitor performed off of PPI therapy can help to establish or exclude the diagnosis of GERD. For those patients with refractory symptoms on PPI, MII-pH study can be performed while on therapy and provides data regarding the response to treatment. Oropharyngeal pH monitoring is being utilized in some practices to aid in diagnosis of LPR, but the scientific validity is controversial.

Mechanism of the effect of pH and biochar on the phytotoxicity of the weak acid herbicides imazethapyr and 2,4-D in soil to rice (Oryza sativa) and estimation by chemical methods.

The existing form of an ionizable organic compound can simultaneously affect its soil adsorption and plant bioactivity. In this experiment, the adsorption and bioactivity of two weak acid herbicides (WAHs), imazethapyr and 2,4-D, were studied to explore the predominant mechanism by which the soil pH and the addition of biochar can influence the phytotoxicity of WAHs in soil. Then, the WAH concentration extracted by hollow fiber-based liquid-phase microextraction (CHF-LPME), the in situ pore water concentration (CIPW) and the added concentration (CAC) were employed to estimate the phytotoxicity. The results showed that with increased pH from 5.5 to 8.5, the phytotoxicity of the WAHs to rice increased about 1-fold in the soil, but decreased in aqueous solutions, the IC50 values for imazethapyr and 2,4-D at pH 5.0 were 3- and 2-fold higher than that at pH 8.0. In addition, the soil adsorption decreased, indicating that the adsorption process was the dominant factor for the variation of the phytotoxicity of the WAHs in the tested soil instead of the decreasing bioactivity. The concentration that inhibits plant growth by 50% (IC50) calculated by the CAC in different pH and biochar soils ranged from 0.619 to 3.826 mg/kg for imazethapyr and 1.871-72.83 mg/kg for 2,4-D. The coefficient of variation (CV) of the IC50 values reached 65.61% for imazethapyr and 130.0% for 2,4-D. However, when IC50 was calculated by CIPW and CHF-LPME, the CVs of the IC50 values decreased to 23.51% and 36.23% for imazethapyr and 40.21% and 50.93% for 2,4-D, respectively. These results suggested that CIPW and CHF-LPME may be more appropriate than CAC for estimating the phytotoxicity of WAHs.

Transfer Behavior of the Weakly Acidic BCS Class II Drug Valsartan from the Stomach to the Small Intestine During Fasted and Fed States.

The objective of this study was to investigate the transfer behavior of the weakly acidic BCS class II drug valsartan from the stomach to the small intestine during fasted and fed states. An in vitro transfer model previously introduced by Kostewicz et al. (J Pharm Pharmacol 56(1):43-51, 2004) based on a syringe pump and a USP paddle apparatus was used to determine the concentration profiles of valsartan in the small intestine. Donor phases of simulated gastric fluid during fasted (FaSSGF) and fed (FeSSGF) states were used to predisperse Diovan® tablets (160 mg valsartan). The initial concentrations of valsartan in FaSSGF and FeSSGF were 6.2 and 91.8%, respectively. Valsartan dispersions were then transferred to acceptor phases that simulate intestinal fluid and cover the physiological properties (pH, buffer capacity, and ionic strength) of the gastrointestinal fluid at a flow rate of 2 mL/min. The pH measurements were reported at time intervals corresponded to those of the transfer experiments to investigate the effect of percent dissolved of valsartan in the donor phase on lowering the pH of the acceptor phases. The f2 similarity test was used to compare the concentration profiles in the acceptor phases. In fasted state, the concentration of valsartan in the acceptor phases ranged between 33.1 and 89.4% after 240 min. Whereas in fed state, valsartan was fully dissolved in all acceptor phases within a range of 94.5-104.9% after 240 min. Therefore, the transfer model provides a useful screen for the concentrations of valsartan in the small intestine during fasted and fed states.

H+ translocation by weak acid uncouplers is independent of H+ electrochemical gradient.

According to the common view, weak acid uncouplers increase proton conductance of biological (and phospholipid bilayer) membranes, thus effecting H+ fluxes driven by their electrochemical gradients. Under certain conditions, however, uncouplers can induce unexpected effects opposite to the dissipation of H+ gradients. Results are presented here demonstrating CCCP-induced proton influx into Saccharomyces cerevisiae cytosol driven by the electrochemical potentials of CCCP and its CCCP- anions, independent of electrochemical H+-gradient. Another view of week acid uncouplers' action is proposed that is logically consistent with these observations.

Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic manipulations and physiological studies.

Zygosaccharomyces bailii is one of the most problematic spoilage yeast species found in the food and beverage industry particularly in acidic products, due to its exceptional resistance to weak acid stress. This article describes the annotation of the genome sequence of Z. bailii IST302, a strain recently proven to be amenable to genetic manipulations and physiological studies. The work was based on the annotated genomes of strain ISA1307, an interspecies hybrid between Z. bailii and a closely related species, and the Z. bailii reference strain CLIB 213T. The resulting genome sequence of Z. bailii IST302 is distributed through 105 scaffolds, comprising a total of 5142 genes and a size of 10.8 Mb. Contrasting with CLIB 213T, strain IST302 does not form cell aggregates, allowing its manipulation in the laboratory for genetic and physiological studies. Comparative cell cycle analysis with the haploid and diploid Saccharomyces cerevisiae strains BY4741 and BY4743, respectively, suggests that Z. bailii IST302 is haploid. This is an additional trait that makes this strain attractive for the functional analysis of non-essential genes envisaging the elucidation of mechanisms underlying its high tolerance to weak acid food preservatives, or the investigation and exploitation of the potential of this resilient yeast species as cell factory.

Yeast response and tolerance to benzoic acid involves the Gcn4- and Stp1-regulated multidrug/multixenobiotic resistance transporter Tpo1.

The action of benzoic acid in the food and beverage industries is compromised by the ability of spoilage yeasts to cope with this food preservative. Benzoic acid occurs naturally in many plants and is an intermediate compound in the biosynthesis of many secondary metabolites. The understanding of the mechanisms underlying the response and resistance to benzoic acid stress in the eukaryotic model yeast is thus crucial to design more suitable strategies to deal with this toxic lipophilic weak acid. In this study, the Saccharomyces cerevisiae multidrug transporter Tpo1 was demonstrated to confer resistance to benzoic acid. TPO1 transcript levels were shown to be up-regulated in yeast cells suddenly exposed to this stress agent. This up-regulation is under the control of the Gcn4 and Stp1 transcription factors, involved in the response to amino acid availability, but not under the regulation of the multidrug resistance transcription factors Pdr1 and Pdr3 that have binding sites in TPO1 promoter region. Benzoic acid stress was further shown to affect the intracellular pool of amino acids and polyamines. The observed decrease in the concentration of these nitrogenous compounds, registered upon benzoic acid stress exposure, was not found to be dependent on Tpo1, although the limitation of yeast cells on nitrogenous compounds was found to activate Tpo1 expression. Altogether, the results described in this study suggest that Tpo1 is one of the key players standing in the crossroad between benzoic acid stress response and tolerance and the control of the intracellular concentration of nitrogenous compounds. Also, results can be useful to guide the design of more efficient preservation strategies and the biotechnological synthesis of benzoic acid or benzoic acid-derived compounds.

Sphingolipid biosynthesis upregulation by TOR complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress.

Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.

PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs.

Analysis of trace inorganic anions in weak acid salts by single pump cycling-column-switching ion chromatography.

The application of ion chromatography with the single pump cycling-column-switching technique was described for the analysis of trace inorganic anions in weak acid salts within a single run. Due to the hydrogen ions provided by an anion suppressor electrolyzing water, weak acid anions could be transformed into weak acids, existing as molecules, after passing through the suppressor. Therefore, an anion suppressor and ion-exclusion column were adopted to achieve on-line matrix elimination of weak acid anions with high concentration for the analysis of trace inorganic anions in weak acid salts. A series of standard solutions consisting of target anions of various concentrations from 0.005 to 10 mg/L were analyzed, with correlation coefficients r ≥ 0.9990. The limits of detection were in the range of 0.67 to 1.51 µg/L, based on the signal-to-noise ratio of 3 and a 25 µL injection volume. Relative standard deviations for retention time, peak area, and peak height were all less than 2.01%. A spiking study was performed with satisfactory recoveries between 90.3 and 104.4% for all anions. The chromatographic system was successfully applied to the analysis of trace inorganic anions in five weak acid salts.

Hypersalinity reduces the risk of cyanide toxicosis to insectivorous bats interacting with wastewater impoundments at gold mines.

Wildlife and livestock that ingest bioavailable cyanide compounds in gold mining tailings dams are known to experience cyanide toxicosis. Elevated levels of salinity in open impoundments have been shown to prevent wildlife cyanide toxicosis by reducing drinking and foraging. This finding appears to be consistent for diurnal wildlife interacting with open impoundments, however the risks to nocturnal wildlife of cyanide exposure are unknown. We investigated the activity of insectivorous bats in the airspace above both fresh (potable to wildlife) and saline water bodies at two gold mines in the goldfields of Western Australian. During this study, cyanide-bearing solutions stored in open impoundments at both mine sites were hypersaline (range=57,000-295,000 mg/L total dissolved solids (TDS)), well above known physiological tolerance of any terrestrial vertebrate. Bats used the airspace above each water body monitored, but were more active at fresh than saline water bodies. In addition, considerably more terminal echolocation buzz calls were recorded in the airspace above fresh than saline water bodies at both mine sites. However, it was not possible to determine whether these buzz calls corresponded to foraging or drinking bouts. No drinking bouts were observed in 33 h of thermal video footage recorded at one hypersaline tailings dam, suggesting that this water is not used for drinking. There is no information on salinity tolerances of bats, but it could be assumed that bats would not tolerate salinity in drinking water at concentrations greater than those documented as toxic for saline-adapted terrestrial wildlife. Therefore, when managing wastewater impoundments at gold mines to avoid wildlife mortalities, adopting a precautionary principle, bats are unlikely to drink solutions at salinity levels ≥50,000 mg/L TDS.