Variability of Clinical Presentation in Patients Heterozygous for the F508del Cystic Fibrosis Variant: A Series of Three Cases and a Review of the Literature.

Cystic fibrosis (CF) is a genetic disease that affects the lung, pancreas, and other organs caused by the presence of biallelic CF-causing variants in the cystic fibrosis conductance regular gene (CFTR). CFTR variants can also be found in CFTR-related disorders (CFTR-RD), which present milder symptoms. Increasing access to next-generation sequencing has demonstrated that both CF and CFTR-RD have a broader array of genotypes than formerly thought. Here we present three patients who carry the most common CFTR pathogenic variant - F508del - but express a wide array of phenotypes. These cases open discussion on the role of concurrent variants in CFTR, the importance of early diagnosis and treatment, and the contribution of lifestyle factors in CF and CFTR-RD presentation.

Sentinel Riparian Spiders Predict Mercury Contamination of Riverine Fish.

Mercury (Hg) is a widespread and toxic environmental contaminant. It is challenging to determine the level of Hg contamination of food chains and fish within the millions of water bodies in the United States. Mercury contamination can vary 10-fold between ecosystems, even those in the same region. Therefore, aquatic ecosystems need to be individually monitored for Hg contamination to determine which ecosystems are most contaminated and pose the greatest risk to human and wildlife health. One approach to monitoring Hg is to use sentinel species, defined as biological monitors that accumulate a contaminant in their tissues without significant adverse effects. Riparian spiders such as long-jawed orb weavers (Tetragnathidae) have been proposed as sentinels of persistent bioavailable contaminants, like Hg, in aquatic systems. Long-jawed orb weavers feed on emergent aquatic insects and have concentrations of Hg that reflect levels of Hg contamination in the aquatic food web. Previous studies have documented elevated contaminant concentrations in long-jawed orb weavers from shorelines of aquatic ecosystems, suggesting that they could be used as sentinels of chemical contaminants in aquatic ecosystems. We demonstrate for the first time that long-jawed orb weavers can be used as sentinels to identify aquatic systems that contain fish with elevated concentrations of Hg. Environ Toxicol Chem 2022;41:1297-1303. © 2022 SETAC.

Identification of the natural product 2,3,4,5-tetrabromo-1-methylpyrrole in Pacific biota, passive samplers and seagrass from Queensland, Australia.

Halogenated natural products (HNPs) are frequently detected in marine organisms. High HNP concentrations have previously been found in marine mammals from the Great Barrier Reef, Australia, including in the blubber of herbivorous dugongs (Dugong dugon). To identify the source of HNPs we initially focused on the analysis of Australian seagrass (Halophila ovalis) which serves as the principal food source for dugongs. GC/MS analysis of the seagrass indicated the presence of several organobromine compounds. One compound was identified as 2,3,4,5-tetrabromo-1-methylpyrrole (TBMP) by synthesis. Subsequent analysis of semipermeable membrane devices demonstrated that the photo-sensitive TBMP is widespread in the Great Barrier Reef (Queensland, Australia). The detection of larger TBMP concentrations in fish fillets from Chile and traces in mussels from New Zealand indicated that this potential HNP may be distributed throughout the Southern Pacific Ocean.

Production of mixed halogenated congeners of the natural product heptachloro-1'-methyl-1,2'-bipyrrole (Q1) by photolysis in the presence of bromine.

Halogenated 1'-methyl-1,2'-bipyrroles (MBPs) are a class of marine halogenated natural products that have been detected in biota from all over the world. However, structures and standards of many mixed chlorinated/brominated MBPs are not available. For this reason, the known 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1 or MBP-79) was UV-irradiated in the presence of bromine with the goal of inducing a chlorine-->bromine exchange. A few drops of bromine were added to a solution of Q1 and 10 mL of either CH2Br2, CH2Cl2, or CHCl3. The experiments were performed both at room temperature and elevated temperature for 30 min. At least four out of five possible bromohexachloro-1'methyl-1,2'-bipyrroles (BrCl6-MBPs), at least seven out of 13 possible Br(2)Cl(5)-MBPs, as well as traces of Br(3)Cl(4)-MBPs and Br(4)Cl(3)-MBPs were obtained in this way. Selective fragment ions in the GC/ECNI-MS spectra as well as electrophilic bromination of hexachloro-MBP solutions were used to verify the structures of the BrCl6-MBP isomers. The BrCl6-MBPs eluted from DB-5-like columns in the order of 4'-bromo-2,3,3',4,5,5'-hexachloro-1'-methyl-1,2'-bipyrrole (Br-MBP-76), which co-eluted with 3'-bromo-2,3,4,4',5,5'-hexachloro-1'-methyl-1,2'-bipyrrole (Br-MBP-78), followed by 2-bromo-3,3',4,4',5,5'-hexachloro-1'-methyl-1,2'-bipyrrole (Br-MBP-75), 3-bromo-2,3',4,4',5,5'-hexachloro-1'-methyl-1,2'-bipyrrole (Br-MBP-77), and 5'-bromo-2,3,3',4,4',5-hexachloro-1'-methyl-1,2'-bipyrrole (Br-MBP-74). These BrCl6-MBPs were also detected in a sample of cetacean blubber from Australia, but the abundance pattern was different. While Br-MBP-76/Br-MBP-78 dominated in the cetacean, irradiation of Q1 (MBP-79) in the presence of bromine led to high proportions of Br-MBP-75. The suitability of the UV-induced Cl-->Br exchange was confirmed by the Br-assisted UV-irradiation of pentachloroanisole (PCA). This experiment produced at least two bromotetrachloro- and three dibromotrichloroanisoles, the last eluting in each case being the most relevant. Thus, this method is most likely generally suited for the production of mixed-halogenated aromatic organohalogen compounds which are not readily obtainable by synthesis.

Stable carbon isotope ratios of POPs--a tracer that can lead to the origins of pollution.

An elemental analyzer in combination with stable isotope ratio mass spectrometry (EA-IRMS) was used to demonstrate that two samples of the chloropesticide Melipax discovered in two countries varied only by 0.1 per thousand in their delta 13C values. Gas chromatography interfaced to a stable isotope mass spectrometer (GC-IRMS) was used to study the delta 13C values of individual congeners in two technical polybrominated diphenyl ether products, i. e. DE-71 and DE-79, at three different concentration levels. Injections of 4 microg and 5.2 microg of DE-71 and DE-79 were most suited for the determinations. In both products, the delta 13C values were usually the more negative the higher the degree of bromination was. The delta 13C values of three hexabromo isomers BDE 138, BDE 153, and BDE 154 could be determined in both products. In all cases the delta 13C values of the three hexaBDEs were more depleted in delta 13C in the higher brominated technical octabromo diphenyl ether DE-79.

Photolytic dehalogenation of the marine halogenated natural product Q1.

The marine halogenated natural product 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1) has been detected in high-trophic level biota throughout the world. In this study we UV-irradiated Q1 in order to produce hexahalogenated 1'-methylbipyrroles (Cl(6)-MBPs). Q1 was transformed with half-lives of <5 min. Already after 5 min, all of the five existing Cl(6)-MBPs (H1-H5) were detected in the irradiated sample. Only one Cl(6)-MBP (2,3,3',4',5,5'-hexachloro-1'-methyl-1,2'-bipyrrole (MBP-77, H2) has been previously described in the literature. H5 was identified as 2,3,3',4,4',5'-hexachloro-1'-methyl-1,2'-bipyrrole (MBP-75) by a specific fragment ion detected by GC/ECNI-MS. Fractionations of the irradiation mixture by reversed-phase HPLC followed by (1)H NMR analysis led to the structure of H4, i.e. 2,3,3',4,4',5-hexachloro-1'-methyl-1,2'-bipyrrole (MBP-74). H1 and H3 showed virtually identical (1)H NMR data. Therefore, it could not determined which of either isomers is 2,3,3',4,5,5'-hexachloro-1'-methyl-1,2'-bipyrrole (MBP-76) and which is 2,3,4,4',5,5'-hexachloro-1'-methyl-1,2'-bipyrrole (MBP-78). In addition, two pentachloro-MBPs (P1 and P3) could be traced back to MBP-62 and MBP-69. Cl(6)-MBPs were analyzed in whale blubber from Australia and skua adipose tissue from Antarctica. The marine mammals contained all Cl(6)-MBPs except for the most abundant in the irradiation experiment. The concentrations of the Cl(6)-MBPs amounted to 0.04-1.76% of the concentration of Q1. The highest concentrations of Cl(6)-MBP isomers in the biota samples were found for MBP-76, MBP-77, and MBP-78. These congeners appeared to be the most lipophilic ones owing to the highest retention time in RP-HPLC. Nevertheless, it remained unclear whether the Cl(6)-MBPs were actual halogenated natural products or environmental metabolites of Q1.

Stable carbon isotope ratios of methyl-branched fatty acids are different to those of straight-chain fatty acids in dairy products.

Methyl-branched fatty acids (MBFAs) are the dominant form of fatty acid found in many bacteria. They are also found at low levels in a range of foodstuffs, where their presence has been linked to bacterial sources. In this study we evaluated the potential of compound-specific isotope analysis to obtain insights into the stable carbon isotope ratios (delta(13)C values in per thousand) of individual MBFAs and to compare them to the stable carbon isotope ratios of straight-chain fatty acids in food. Due to their low abundance in foodstuffs, the MBFAs were enriched prior to gas chromatography coupled to isotope ratio mass spectrometric (GC-IRMS) analysis. After transesterification, urea complexation was used to suppress the 16:0 and 18:0 methyl esters that were dominant in the samples. Following that, silver-ion high performance liquid chromatography was used to separate the saturated from the unsaturated fatty acids. The resulting solutions of saturated fatty acids obtained from suet, goat's milk, butter, and human milk were studied by GC-IRMS. The delta(13)C values of fatty acids with 12-17 carbons ranged from -25.4 per thousand to -37.6 per thousand. In all samples, MBFAs were most depleted in carbon-13, followed by the odd-chain fatty acids 15:0 and 17:0. 14:0 and 16:0 contained the highest proportions of carbon-13. The results from this study illustrate that MBFAs have distinctive delta(13)C values and must originate from other sources and/or from very different substrates. These measurements support the initial hypothesis that delta(13)C values can be used to attribute MBFAs to particular sources.

Improved quality control in gas chromatography interfaced to stable isotope ratio mass spectrometry by application of derivative chromatography.

Compound-specific isotope analysis using gas chromatography interfaced to isotope ratio mass spectrometry (GC-IRMS) is a versatile technique for applications ranging from source appointment and the elucidation of biochemical pathways. When delta(13)C values are going to be determined, the sample is combusted to CO(2) and the resulting gas is analyzed relative to a standard with known stable carbon isotope ratio. With the combustion step any information on the identity of a peak is lost. Co-eluting compounds can no more be identified which can lead to significant alterations of the delta(13)C value of the analyte. For improvement of the QA/QC protocols in GC-IRMS, we used first, second, and third order derivative chromatography. The suitability of the technique was studied using mixtures of 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1) and 2,2',4,5,5'-pentachlorobiphenyl (PCB 101). By application of different GC oven programs four scenarios ranging from baseline separation to full co-elution were obtained. Derivative chromatography enabled identification of the interference of Q1 with PCB 101 even when both peaks fully co-eluted. Although the delta(13)C values could not be determined from interfered scenarios, the use of derivative spectroscopy will help to prevent acceptance of incorrect data due to co-elutions. Derivative chromatography was finally used to study the peak purity of 2,2',3,4,4'-pentabromodiphenyl ether (BDE 85) in technical pentabromo diphenyl ether (DE-71). Already the first order derivative demonstrated that this key-BDE congener was interfered by a compound identified as 2,2',4,4',6,6'-hexabromodiphenyl ether (BDE 155).

The neuroactive steroids alphaxalone and pregnanolone increase the conductance of single GABAA channels in newborn rat hippocampal neurons.

The effects of the neuroactive steroids alphaxalone and pregnanolone on single GABA(A) receptor channels were tested in cell-attached and inside-out patches from cultured newborn rat hippocampal neurons. The conductance of these single channels ranged between 10 and 80 pS when exposed to low (0.5-3 microM) GABA concentrations. These GABA concentrations activated low-conducting channels (<40 pS) in 78% of the patches, 22% of patches had channels with a maximum conductance above 40 pS. Alphaxalone at concentrations above 1 microM, and pregnanolone at concentrations above 0.1 microM, significantly increased the conductance of initially low-conducting single channels activated by GABA up to seven-fold and at all concentrations tested, both drugs increased open probability and mean open time and decreased closed probability and mean closed time of channels. Both steroids at higher concentrations could directly activate high conductance (>40 pS) chloride channels. Both the directly activated channels and those channels that had been previously affected by alphaxalone were modulated by diazepam, a benzodiazepine drug that is known to specifically modulate GABA(A) channels. The present study is the first one to show that neurosteroids can significantly increase single GABA(A) channel conductance, thus enlarging our current knowledge on the molecular mechanism of action of these compounds.

Monobromo and higher brominated congeners of the marine halogenated natural product 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1).

The marine halogenated natural product 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1) is widely distributed in the environment. In this study, we screened samples which have previously been found to contain remarkably high residues of Q1 (blubber of marine mammals from Australia, samples from Antarctica, human milk from the Faroe Island) for the additional presence of mixed chlorinated and brominated congeners. Using GC/ECNI-MS, all samples tested were positive and many contained four out of five possible bromohexachloro congeners (BrCl6-MBPs), five out of 14 possible dibromopentachloro congeners (Br2Cl5-MBPs), five of 21 possible tribromotetrachloro-congeners (Br3Cl4-MBPs), as well as several higher brominated congeners. About 20 heptahalo congeners of Q1 are described for the first time in the scientific literature. Isomers eluted within about one minute, respectively. Hence it is possible, that the peak clusters identified may be composed of more, co-eluting congeners. Similarities in the GC/ECNI-MS mass spectra with polychlorinated biphenyls (PCBs) were addressed. We also suggest an acronym system similar to that in use for polychlorinated biphenyls that may simplify the use of this substance class in scientific papers. In the samples from Australia, BrCl6-MBPs and Br2Cl5-MBPs amounted for 7-27.5% and 0.4-4.2% of Q1, respectively whereas Br3Cl4-MBPs and higher brominated MBPs were found in the range of <1% of Q1 or less.

Anaerobic transformation of a technical brominated diphenyl ether mixture by super-reduced vitamin B12 and dicyanocobinamide.

The anaerobic transformation of the technical octabromo diphenyl ether mixture, DE-79, was investigated by incubation with two super-reduced corrinoids, dicyanocobinamide and cyanocobalamin (vitamin B12). The transformation produced 33 brominated diphenyl ethers (BDEs), 23 of which could be assigned to known structures. Within 1 d, the hepta- to nona-BDEs in DE-79 were almost quantitatively transformed with dicyanocobinamide to lower-brominated homologs. Along with the decrease of high-brominated congeners, concentrations of some tetra- to hexabromo congeners initially increased, followed by a decreasing or constant concentration during longer incubations. Super-reduced cyanocobalamin also was able to transform BDE congeners of DE-79. Whereas the transformation rates of hepta- to nona-BDEs of DE-79 by both corrinoids were comparable, tri- to hexa-BDEs were susceptible to further transformation by super-reduced cyanocobalamin. Incubation of DE-79 with either corrinoid produced significant amounts of BDE 49. Because this congener is found in small quantities in both penta- and octabromo technical BDE mixtures, it may be a suitable indicator for reductive debromination of higher-brominated BDEs. Isolated BDE 196 was incubated as well with super-reduced dicyanocobalamine. After 1 d, 13 BDEs could be detected, with BDE 153, BDE 100, and BDE 99 dominating. After 7 d, only tetra-BDEs or lower-brominated BDEs were detectable. It could be shown that reductive debromination by super-reduced corrinoids is a possible source for lower-brominated BDEs (i.e., BDE 47, BDE 99, BDE 153, and BDE 154). The patterns observed with our bench-scale study design demonstrate that anaerobic transformation in the presence of reducing agents can help to explain the occurrence of environmentally relevant BDE congeners (e.g., BDE 47, BDE 99, and BDE 153).

HPLC isolation and NMR structure elucidation of the most prominent octabromo isomer in technical octabromo diphenyl ether.

The major octabromo isomer of technical octabromo diphenyl ether mixture (technical octaBDE) DE-79 was isolated by RP-HPLC. Three serially coupled columns (each 250 mm long) enabled a good separation of the target compound from other congeners using 100% ACN as eluent. Approximately 100 microg of the target compound was isolated with a purity of >90% and investigated by MS for confirmation of the degree of bromination. 1H-NMR and 2-D 1H-13C correlation NMR spectra unequivocally clarified that the octaBDE in question is 2,2',3,3',4,4',6,6'-octabromodiphenyl ether (BDE 197). Based on annual production rates of technical BDE products (1999/2001), approximately 380 tons of BDE 197 were annually produced which, on the long term, may enter the environment. Compared with other individual BDE congeners, BDE 197 has the seventh highest application rate. Reductive debromination of BDE 197 can lead to four hepta-, 15 hexa-, 23 penta-, and 28 tetra-BDE isomers, respectively. This variety includes all known major BDEs of environmental concern (BDE 47, 85, 99, 100, 153, 154, and 183). The identification of BDE 197 in technical octaBDE DE-79 strongly suggests that research on the environmental fate of BDEs should include this key-BDE congener.

Tetra- and tribromophenoxyanisoles in marine samples from Oceania.

Some methoxylated polybrominated diphenyl ethers (MeO-BDEs) are known halogenated natural products (HNPs) and are frequently detected in higher organisms of the marine environment. In this study we demonstrate that a prominent MeO-BDE, previously detected in marine mammals from Australia, is identical to 3,5-dibromo-2-(2',4'dibromo)phenoxyanisole (BC-3, 6-MeO-BDE 47). Up to 1.9 mg/ kg of 6-MeO-BDE 47 was present in cetaceans from Australia, 0.2-0.3 mg/kg in two crocodile eggs from Australia, but concentrations of 1 or 2 orders of magnitude lower were found in shark liver oil from NewZealand and in marine mammals from Africa and the Antarctic. Concentrations of 6-MeO-BDE 47 in samples from Australia were in the same range as anthropogenic pollutants such as PCB 153 and p,p'-DDE. Along with 6-MeO-BDE 47 and the known HNP 4,6-dibromo-2-(2',4'-dibromo)phenoxyanisole (BC-2, 2'-MeO-BDE 68), several tribromophenoxyanisoles (MeO-triBDE) were present in tissue of Australian cetaceans. To determine their structure, abiotic debromination experiments were performed using 6-MeO-BDE 47 and 2'-MeO-BDE 68 and superreduced dicyanocobalamine. These experiments resulted in formation of eight MeO-triBDEs, all of which were detected in the cetacean samples. Five of these eight MeO-triBDEs could be identified based on two standard compounds as well as gas chromatographic and mass spectrometric features. It was also shown that the first eluting isomer (compound 1), 6-MeO-BDE 17 (compound 2), and 2-MeO-BDE 39 (compound 5) were the most prominent MeO-triBDEs in the Australian cetacean samples. The concentrations of the MeO-triBDEs in two cetacean samples were 0.20 and 0.36 mg/kg, respectively. Although the reductive debromination with dicyanocobalamine resulted in a different congener pattern than was found in the marine mammals, it could not be excluded that the tribromo

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons.

The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca(2+)](I), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca(2+)](i) transients was 28 microM, close to the estimated clinical EC(50) (clinically relevant (half-maximal) effective concentration) of thiopental. In fura-2-loaded neurons, voltage clamped at -60 mV to eliminate any contribution of voltage-gated Ca(2+) channels, thiopental (25 microM) simultaneously inhibited nAChR-induced increases in [Ca(2+)](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by approximately 40% at -120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC(50) were also shown to inhibit nAChR-induced increases in [Ca(2+)](i) by approximately 40%. Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca(2+)](i), indicating that inhibition of Ca(2+) release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. Depolarization-activated Ca(2+) channel currents were unaffected in the presence of thiopental (25 microM), pentobarbital (50 microM) and ketamine (10 microM). In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca(2+)](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.