Kinetics and Yields of Pesticide Biodegradation at Low Substrate Concentrations and under Conditions Restricting Assimilable Organic Carbon

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter⁻¹. We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.     

Genetic and metabolic analysis of the carbofuran catabolic pathway in Novosphingobium sp. KN65.2

The widespread agricultural application of carbofuran and concomitant contamination of surface and ground waters has raised health concerns due to the reported toxic effects of this insecticide and its degradation products. Most bacteria that degrade carbofuran only perform partial degradation involving carbamate hydrolysis without breakdown of the resulting phenolic metabolite. The capacity to mineralize carbofuran beyond the benzofuran ring has been reported for some bacterial strains, especially sphingomonads, and some common metabolites, including carbofuran phenol, were identified. In the current study, the catabolism of carbofuran by Novosphingobium sp. KN65.2 (LMG 28221), a strain isolated from a carbofuran-exposed Vietnamese soil and utilizing the compound as a sole carbon and nitrogen source, was studied. Several KN65.2 plasposon mutants with diminished or abolished capacity to degrade and mineralize carbofuran were generated and characterized. Metabolic profiling of representative mutants revealed new metabolic intermediates, in addition to the initial hydrolysis product carbofuran phenol. The promiscuous carbofuran-hydrolyzing enzyme Mcd, which is present in several bacteria lacking carbofuran ring mineralization capacity, is not encoded by the Novosphingobium sp. KN65.2 genome. An alternative hydrolase gene required for this step was not identified, but the constitutively expressed genes of the unique cfd operon, including the oxygenase genes cfdC and cfdE, could be linked to further degradation of the phenolic metabolite. A third involved oxygenase gene, cfdI, and the transporter gene cftA, encoding a TonB-dependent outer membrane receptor with potential regulatory function, are located outside the cfd cluster. This study has revealed the first dedicated carbofuran catabolic genes and provides insight in the early steps of benzofuran ring degradation.     

Amphipols in G Protein-Coupled Receptor Pharmacology: What Are They Good For?

G protein-coupled receptors are at a central node of all cell communications. Investigating their molecular functioning is therefore crucial for both academic purposes and drug design. However, getting the receptors as isolated, stable and purified proteins for such studies still stumbles over their instability out of the membrane environment. Different membrane-mimicking environments have been developed so far to increase the stability of purified receptors. Among them are amphipols. These polymers not only preserve the native fold of receptors purified from membrane fractions but they also allow specific applications such as folding receptors purified from inclusion bodies back to their native state. Of importance, amphipol-trapped G protein-coupled receptors essentially maintain their pharmacological properties so that they are perfectly adapted to further investigate the molecular mechanisms underlying signaling processes. We review here how amphipols have been used to refold and stabilize detergent-solubilized purified receptors and what are the main subsequent molecular pharmacology analyses that were performed using this strategy.     

The lightning heart: a case report and brief review of the cardiovascular complications of lightning injury

Lightning strike is a rare natural phenomenon, which carries a risk of dramatic medical complications to multiple organ systems and a high risk of fatality. The known complications include but are not limited to: myocardial infarction, arrhythmia, cardiac contusion, stroke, cutaneous burns, respiratory disorders, neurological disorders, acute kidney injury and death. We report a case of a healthy young man who suffered a lightning injury and discuss the cardiovascular complications of lightning injury, ranging from ECG changes to death. The patient in our case, a 27-year old previously healthy male, developed a syndrome of rhabdomyolysis and symptomatic cardiogenic pulmonary edema. Electrocardiographic findings included transient T-wave inversions, late transition shift and long QT. His clinical condition improved with supportive measures.Early recognition of lightning injury syndromes and anticipation of complications may help us improve outcomes for these patients. Evaluation of patients having experienced a lightning injury should include a minimum of a detailed history and physical examination, 12-lead ECG and drawing of baseline troponins. Prolonged electrocardiographical monitoring (for monitoring of ventricular arrhythmias) and assessment for signs and symptoms of hemodynamic compromise may be warranted.     

Identification of endocrine cells of the stomach that express acid-sensitive background potassium (K<Subscript>2P</Subscript>9.1/TASK3) channels

The family of two-pore domain potassium (K_2P) channels is important in setting and controlling the background potassium current of excitable cells. This study examines the localisation of the acid-sensitive channel, K_2P9.1 (TASK3), in cells of the gastric mucosa. We observed K_2P9.1 immunoreactivity in endocrine cells of the mucosal glands of the guinea-pig, rat and mouse but the channels were not detected in parietal, chief, or mucous cells. K_2P9.1 channel immunoreactivity was consistently co-localised with histidine decarboxylase immunopositive enterochromaffin-like (ECL) cells, and with the majority of ghrelin immunoreactive X/A cells. Localisation in somatostatin immunoreactive D cells was rare in the guinea-pig, and did not occur in the stomach of rat, but, in the mouse, K_2P9.1 channels were observed in the majority of somatostatin-immunoreactive D cells. Conversely, sections taken from the guinea-pig and mouse stomachs, but not rat stomach, revealed K_2P9.1 in gastrin-containing G cells. These results demonstrate the presence of K_2P9.1 channels in the entero-endocrine ECL, G and D cell populations of the stomach that regulate acid secretion through the release of histamine, gastrin and somatostatin. K_2P9.1 channels were located in the ghrelin X/A cells that regulate food intake.     

Transiently reorganized microtubules are essential for zippering during dorsal closure in Drosophila melanogaster

There is emerging evidence that microtubules in nondividing cells can be employed to remodel the intracellular space. Here, we demonstrate an essential role for microtubules in dorsal closure, which occurs toward the end of Drosophila melanogaster embryogenesis. Dorsal closure is a morphogenetic process similar to wound healing, whereby a gap in the epithelium is closed through the coordinated action of different cell types. Surprisingly, this complex process requires microtubule function exclusively in epithelial cells and only for the last step, the zippering, which seals the gap. Preceding zippering, the epithelial microtubules reorganize to attain an unusual spatial distribution, which we describe with subcellular resolution in the intact, living organism. We provide a clearly defined example where cells of a developing organism transiently reorganize their microtubules to fulfill a specialized morphogenetic task.     

Schistosoma mansoni: biochemical characterization of lactate transporters or similar proteins

While in medium containing glucose, schistosomes exhibit homolactic fermentation. Accumulation of lactate acid in tissue fluid causes lowering of pH and a resultant inhibition of metabolic pathways. This requires lactate transporter protein in homolactic fermentors to facilitate the translocation of lactate(-) and [H(+)] across their plasma membrane. The ex-vivo experiment assessed lactic acid secretion by adult worms in absence and the presence of lactic acid transporter protein inhibitors. Phloretin and alpha-cyano-4-hydroxycinnamate caused a combined 25-35% inhibition of lactic acid secretion and probenecid increased this inhibition to 65% of control values. The removal of inhibitors resulted in 80% recovery of lactic acid secretion. In the in-vitro studies using vesicles isolated from adult worms and from schistosomula, the effects of phloretin and alpha-cyano-4-hydroxycinnamate were greater, each causing approximately 80% inhibition independently. The data obtained in this study demonstrate the presence of lactic acid transporters or similar proteins in Schistosoma mansoni.     

The RNA helicase DHX9 establishes nucleolar heterochromatin,and this activity is required for embryonic stem cell differentiation

Long non‐coding RNAs (lncRNAs) have been implicated in the regulation of chromatin conformation and epigenetic patterns. lncRNA expression levels are widely taken as an indicator for functional properties. However, the role of RNA processing in modulating distinct features of the same lncRNA is less understood. The establishment of heterochromatin at rRNA genes depends on the processing of IGS‐rRNA into pRNA, a reaction that is impaired in embryonic stem cells (ESCs) and activated only upon differentiation. The production of mature pRNA is essential since it guides the repressor TIP5 to rRNA genes, and IGS‐rRNA abolishes this process. Through screening for IGS‐rRNA‐binding proteins, we here identify the RNA helicase DHX9 as a regulator of pRNA processing. DHX9 binds to rRNA genes only upon ESC differentiation and its activity guides TIP5 to rRNA genes and establishes heterochromatin. Remarkably, ESCs depleted of DHX9 are unable to differentiate and this phenotype is reverted by the addition of pRNA, whereas providing IGS‐rRNA and pRNA mutants deficient for TIP5 binding are not sufficient. Our results reveal insights into lncRNA biogenesis during development and support a model in which the state of rRNA gene chromatin is part of the regulatory network that controls exit from pluripotency and initiation of differentiation pathways.