A domesticated photoautotrophic microbial community as a biofilm model system for analyzing the influence of plastic surfaces on invertebrate grazers in limnic environments

The environmental fate of plastic particles in water bodies is influenced by microbial biofilm formation. Invertebrate grazers may be affected when foraging biofilms on plastics compared to biofilms on natural substrata but the mechanistic basis for these effects is unknown. For analyzing these effects in ecotoxicological assays stable and reproducible biofilm communities are required that are related to the environmental site of interest. Here, a defined biofilm community was established and used to perform grazing experiments with a freshwater snail. For this, snippets of different plastic materials were incubated in the photic zone of three different freshwater sites. Amplicon sequencing of biofilms formed on these snippets showed that the site of incubation and not the plastic material dominated the microbial community composition. From these biofilms, individual microbial strains as well as photoautotrophic consortia were isolated; these consortia consisted of heterotrophic bacteria that were apparently nourished by microalga. While biofilms formed by defined dual cultures of a microalga and an Alphaproteobacterium were not accepted by the snail P. fontinalis, a photoautotrophic consortium (Co_3) sustained growth and metabolism of this grazer. Amplicon sequencing revealed that consortium Co_3, which could be stably maintained on solid medium under photoautotrophic conditions, reproducibly formed biofilms of a defined composition on three different plastic materials and on glass surfaces. In conclusion, our study shows that the generation of domesticated photoautotrophic microbial communities is a valid novel approach for establishing laboratory ecotoxicological assays with higher environmental relevance than those based on defined microbiota

Mutations in GABRB3

Objective: To examine the role of mutations in GABRB3 encoding the b3 subunit of the GABAA receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes. Methods: We performed massive parallel sequencing of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs. Results: We identified 22 patients with heterozygous mutations in GABRB3, including 3 probands frommultiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy withmyoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies. Electrophysiologic analysis of 7 mutations in Xenopus laevis oocytes, using coexpression of wild-type or mutant beta(3), together with alpha(5) and gamma(2s) subunits and an automated 2-microelectrode voltage-clamp system, revealed reduced GABA-induced current amplitudes or GABA sensitivity for 5 of 7 mutations. Conclusions: Our results indicate that GABRB3 mutations are associated with a broad phenotypic spectrum of epilepsies and that reduced receptor function causing GABAergic disinhibition represents the relevant disease mechanism

Evidence of distinct pathways for bacterial degradation of the steroid compound cholate suggests the potential for metabolic interactions by interspecies cross-feeding

The distribution and the metabolic pathways of bacteria degrading steroid compounds released by eukaryotic organisms were investigated using the bile salt cholate as model substrate. Cholate-degrading bacteria could be readily isolated from freshwater environments. All isolated strains transiently released steroid degradation intermediates into culture supernatants before their further degradation. Cholate degradation could be initiated via two different reaction sequences. Most strains degraded cholate via a reaction sequence known from the model organism Pseudomonas sp. strain Chol1 releasing intermediates with a 3-keto-Δ1,4-diene structure of the steroid skeleton. The actinobacterium Dietzia sp. strain Chol2 degraded cholate via a different and yet unexplored reaction sequence releasing intermediates with a 3-keto-Δ4,6-diene-7-deoxy structure of the steroid skeleton such as 3,12-dioxo-4,6-choldienoic acid (DOCDA). Using DOCDA as substrate, two Alphaproteobacteria, strains Chol10–11, were isolated that produced the same cholate degradation intermediates as strain Chol2. With DOCDA as substrate for Pseudomonas sp. strain Chol1 only the side chain was degraded while the ring system was transformed into novel steroid compounds accumulating as dead-end metabolites. These metabolites could be degraded by the DOCDA-producing strains Chol10–11. These results indicate that bacteria with potentially different pathways for cholate degradation coexist in natural habitats and may interact via interspecies cross-feeding

Degradation of Bile Acids by Soil and Water Bacteria

Bile acids are surface-active steroid compounds with a C5 carboxylic side chain at the steroid nucleus. They are produced by vertebrates, mainly functioning as emulsifiers for lipophilic nutrients, as signaling compounds, and as an antimicrobial barrier in the duodenum. Upon excretion into soil and water, bile acids serve as carbon- and energy-rich growth substrates for diverse heterotrophic bacteria. Metabolic pathways for the degradation of bile acids are predominantly studied in individual strains of the genera 'Pseudomonas', 'Comamonas', 'Sphingobium', 'Azoarcus', and 'Rhodococcus'. Bile acid degradation is initiated by oxidative reactions of the steroid skeleton at ring A and degradation of the carboxylic side chain before the steroid nucleus is broken down into central metabolic intermediates for biomass and energy production. This review summarizes the current biochemical and genetic knowledge on aerobic and anaerobic degradation of bile acids by soil and water bacteria. In addition, ecological and applied aspects are addressed, including resistance mechanisms against the toxic effects of bile acids

Degradation of the acyl side chain of the steroid compound cholate in Pseudomonas sp. strain Chol1 proceeds via an aldehyde intermediate

Bacterial degradation of steroids is widespread, but the metabolic pathways have rarely been explored. Previous studies with Pseudomonas sp. strain Chol1 and the C24 steroid cholate have shown that cholate degradation proceeds via oxidation of the A ring, followed by cleavage of the C5 acyl side chain attached to C-17, with 7α,12β-dihydroxy-androsta-1,4-diene-3,17-dione (12β-DHADD) as the product. In this study, the pathway for degradation of the acyl side chain of cholate was investigated in vitro with cell extracts of strain Chol1. For this, intermediates of cholate degradation were produced with mutants of strain Chol1 and submitted to enzymatic assays containing coenzyme A (CoA), ATP, and NAD+ as cosubstrates. When the C24 steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO) was used as the substrate, it was completely transformed to 12α-DHADD and 7α-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT) as end products, indicating complete removal of the acyl side chain. The same products were formed with the C22 steroid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12β-DHADD in an NADPH-dependent reaction. When NAD+ was omitted from assays with DHOCTO, a new product, identified as 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA), was formed. This aldehyde was transformed to DHOPDC and DHOPDC-CoA in the presence of NAD+, CoA, and ATP. These results revealed that degradation of the C5 acyl side chain of cholate does not proceed via classical β-oxidation but via a free aldehyde that is oxidized to the corresponding acid. The reaction leading to the aldehyde is presumably catalyzed by an aldolase encoded by the gene skt, which was previously predicted to be a β-ketothiolase

Metagenomes Reveal Global Distribution of Bacterial Steroid Catabolism in Natural, Engineered, and Host Environments

Steroids are abundant growth substrates for bacteria in natural, engineered, and host-associated environments. This study analyzed the distribution of the aerobic 9,10-seco steroid degradation pathway in 346 publically available metagenomes from diverse environments. Our results show that steroid-degrading bacteria are globally distributed and prevalent in particular environments, such as wastewater treatment plants, soil, plant rhizospheres, and the marine environment, including marine sponges. Genomic signature-based sequence binning recovered 45 metagenome-assembled genomes containing a majority of 9,10-seco pathway genes. Only Actinobacteria and Proteobacteria were identified as steroid degraders, but we identified several alpha- and gammaproteobacterial lineages not previously known to degrade steroids. Actino- and proteobacterial steroid degraders coexisted in wastewater, while soil and rhizosphere samples contained mostly actinobacterial ones. Actinobacterial steroid degraders were found in deep ocean samples, while mostly alpha- and gammaproteobacterial ones were found in other marine samples, including sponges. Isolation of steroid-degrading bacteria from sponges confirmed their presence. Phylogenetic analysis of key steroid degradation proteins suggested their biochemical novelty in genomes from sponges and other environments. This study shows that the ecological significance as well as taxonomic and biochemical diversity of bacterial steroid degradation has so far been largely underestimated, especially in the marine environment

Biochemical and Genetic Investigation of Initial Reactions in Aerobic Degradation of the Bile Acid Cholate in Pseudomonas sp. Strain Chol1▿

Bile acids are surface-active steroid compounds with toxic effects for bacteria. Recently, the isolation and characterization of a bacterium, Pseudomonas sp. strain Chol1, growing with bile acids as the carbon and energy source was reported. In this study, initial reactions of the aerobic degradation pathway for the bile acid cholate were investigated on the biochemical and genetic level in strain Chol1. These reactions comprised A-ring oxidation, activation with coenzyme A (CoA), and β-oxidation of the acyl side chain with the C19-steroid dihydroxyandrostadienedione as the end product. A-ring oxidizing enzyme activities leading to Δ1,4-3-ketocholyl-CoA were detected in cell extracts and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cholate activation with CoA was demonstrated in cell extracts and confirmed with a chemically synthesized standard by LC-MS/MS. A transposon mutant with a block in oxidation of the acyl side chain accumulated a steroid compound in culture supernatants which was identified as 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) by nuclear magnetic resonance spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell extracts of strain Chol1 was detected by LC-MS/MS. The growth defect of the transposon mutant could be complemented by the wild-type ACAD gene located on the plasmid pBBR1MCS-5. Based on these results, the initiating reactions of the cholate degradation pathway leading from cholate to dihydroxyandrostadienedione could be reconstructed. In addition, the first bacterial gene encoding an enzyme for a specific reaction step in side chain degradation of steroid compounds was identified, and it showed a high degree of similarity to genes in other steroid-degrading bacteria

High-sensitivity cardiac troponin T for diagnosis of NSTEMI in the elderly emergency department patient: a clinical cohort study

<p><b>Purpose:</b> The aim of this study is to evaluate the impact of age on the diagnostic performance of high-sensitivity troponin T (hsTnT) under routine conditions.</p> <p><b>Materials and methods:</b> Data of 4118 consecutive emergency department (ED) patients who underwent a routine TnT measurement between 11 October 2012 and 30 November 2013 were analysed. Diagnostic accuracy of hsTnT was compared in four age categories (<50, 50–64, 65–74, ≥75 years of age) for different cut-off values. Primary endpoint was a main hospital diagnosis of NSTEMI.</p> <p><b>Results:</b> The median age of the study population (<i>n</i> = 4118) was 61 years (IQR: 45–75 years). NSTEMI was diagnosed in 3.3% (<i>n</i> = 136) of all patients. There were significant differences in hsTnT concentrations between age-groups (<i>p</i> < 0.001) in all patients, but not in NSTEMI patients (<i>p</i> = 0.297). 72.2% of all patients ≥75 years of age (583/808) without NSTEMI had hsTnT concentrations above the 99th percentile of a healthy reference population. Specificity at 14 ng/L was 93.6% (95% CI: 92.12–94.87) in patients below 50 years of age and 27.9% (95% CI: 24.78–31.08) in patients 75 years of age and older.</p> <p><b>Conclusions:</b> Patients’ age needs to be considered at least one influencing factor on hsTnT concentrations at admission and should be included in the clinical interpretation of hsTnT concentrations for further clinical workup beneath other influencing factors like comorbidities and symptom onset time. The implementation of age-specific cut-off values could be considered for single troponin testing at admission but is associated with an increased risk of underdiagnosis of NSTEMI.</p