Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

Responses of the endangered pygmy bluetongue lizard to conspecific scats

Many animals use chemical signals for communication between conspecifics and for territory marking. The pygmy bluetongue lizard is normally solitary, focussing activity around the entrance of its burrow, from where it ambushes prey, and rarely contacts other individuals. In this paper we examined whether lizards in laboratory experiments alter their behaviour in the presence of scats from conspecifics. In the first experiment, when lizards were offered a choice of two vacant burrows with or without a scat close to the entrance, they tongue flicked more often at the burrow entrance when the scat was present, and more often chose to occupy the burrow with the scat. An interpretation is that lizards use scat signals to recognise burrows that may be suitable because they have previously been occupied by a conspecific, but that they approach those burrows cautiously in case a resident is still present and likely to resist a takeover. Scats from male lizards were inspected (by both sexes) for longer than scats of female lizards. In the second experiment, when resident lizards were presented with scats outside of their burrows, they inspected and tongue flicked at those scats more often if the scat came from a male than a female lizard, but there was no definitive evidence from our experiments that lizards differentiated in their response to scats from lizards that were found close to or far from the test lizard. The results were consistent with a communication system in which lizards use scats to advertise their presence, independent of any direct contact.     

Contemplating the future: Acting now on long‐term monitoring to answer 2050's questions

In 2050, which aspects of ecosystem change will we regret not having measured? Long‐term monitoring plays a crucial part in managing Australia's natural environment because time is a key factor underpinning changes in ecosystems. It is critical to start measuring key attributes of ecosystems – and the human and natural process affecting them – now, so that we can track the trajectory of change over time. This will facilitate informed choices about how to manage ecological changes (including interventions where they are required) and promote better understanding by 2050 of how particular ecosystems have been shaped over time. There will be considerable value in building on existing long‐term monitoring programmes because this can add significantly to the temporal depth of information. The economic and social processes driving change in ecosystems are not identical in all ecosystems, so much of what is monitored (and the means by which it is monitored) will most likely target specific ecosystems or groups of ecosystems. To best understand the effects of ecosystem‐specific threats and drivers, monitoring also will need to address the economic and social factors underpinning ecosystem‐specific change. Therefore, robust assessments of the state of Australia's environment will be best achieved by reporting on the ecological performance of a representative sample of ecosystems over time. Political, policy and financial support to implement appropriate ecosystem‐specific monitoring is a perennial problem. We suggest that the value of ecological monitoring will be demonstrable, when plot‐based monitoring data make a unique and crucial contribution to Australia's ability to produce environmental accounts, environmental reports (e.g. the State of the Environment, State of the Forests) and to fulfilling reporting obligations under international agreements, such as the Convention on Biological Diversity. This paper suggests what must be done to meet Australia's ecological information needs by 2050.     

Unilateral duplication of the cerebellar hemisphere and internal, middle, and external ear: a clinical case study

This article presents the unique congenital anomaly complex of ipsilateral cerebellar hemisphere duplication and internal, middle, and external ear duplication. Diagnostic techniques included a head CT scan, a three-dimensional head CT scan, and a head MRI. An aberrant notochordal split was proposed as the embryologic mechanism leading to the development of such anomalies.     

Evading resistance to gene drives

Gene drives offer the possibility of altering and even suppressing wild populations of countless plant and animal species, and CRISPR technology now provides the technical feasibility of engineering them. However, population-suppression gene drives are prone to select resistance, should it arise. Here, we develop mathematical and computational models to identify conditions under which suppression drives will evade resistance, even if resistance is present initially. Previous models assumed resistance is allelic to the drive. We relax this assumption and show that linkage between the resistance and drive loci is critical to the evolution of resistance and that evolution of resistance requires (negative) linkage disequilibrium between the two loci. When the two loci are unlinked or only partially so, a suppression drive that causes limited inviability can evolve to fixation while causing only a minor increase in resistance frequency. Once fixed, the drive allele no longer selects resistance. Our analyses suggest that among gene drives that cause moderate suppression, toxin-antidote systems are less apt to select for resistance than homing drives. Single drives of moderate effect might cause only moderate population suppression, but multiple drives (perhaps delivered sequentially) would allow arbitrary levels of suppression. The most favorable case for evolution of resistance appears to be with suppression homing drives in which resistance is dominant and fully suppresses transmission distortion; partial suppression by resistance heterozygotes or recessive resistance are less prone to resistance evolution. Given that it is now possible to engineer CRISPR-based gene drives capable of circumventing allelic resistance, this design may allow for the engineering of suppression gene drives that are effectively resistance-proof.     

Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn's disease cases in the city of Cardiff

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Design and synthesis of 4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one derivatives as a new series of Phosphodiesterase 4 (PDE4) inhibitors

Phosphodiesterase 4 (PDE4) inhibitors have attractive therapeutic potential in respiratory, inflammatory, metabolic and CNS disorders. The present work details the design, chemical exploration and biological profile of a novel PDE4 inhibitor chemotype. A diazepinone ring was identified as an under-represented heterocyclic system fulfilling a set of PDE4 structure-based design hypotheses. Rapid exploration of the structure activity relationships for the series was enabled by robust and scalable two/three-steps parallel chemistry protocols. The resulting compounds demonstrated PDE4 inhibitory activity in cell free and cell-based assays comparable to the Zardaverine control used, suggesting potential avenues for their further development.