Thermostable Enzymes Important For Industrial Biotechnology

The use of enzymes in technology is of increasing commercial interest due to their high catalytic efficiency and specificity and the lowering of manufacturing costs. Enzymes are also becoming more widely utilised because they are more environmentally friendly compared to chemical methods. Firstly, they carry out their reactions at ambient temperatures requiring less energy to achieve the high temperatures and pressures that many chemical methods require. Secondly, they can substitute for toxic chemical catalysts which need careful disposal. In this project two classes of enzymes of industrial interest from thermophiles were investigated, lactonase enzymes and 1-deoxy-D-xylulose 5-phosphate (DXP) synthases. A quorum sensing lactonase from Vulcanisaeta moutnovskia, a thermoacidophilic anaerobic crenarchaeon, was expressed in high levels in an Escherichia coli host, then purified and characterised with a range of industrially relevant substrates. These enzymes are of industrial interest for water treatment and bioreactors for their ability to prevent biofilm formation in bacteria. This enzyme showed different specificity to another well characterised quorum sensing lactonase from a thermophilic crenarchaeon, Sulfolobus solfataricus. Crystals of the native enzyme were obtained. Structural examination revealed that the V. moutnovskia lactonase possesses an α-helix obstructing a hydrophobic channel near the active site, whereas the S. solfataricus lactonase has a flexible loop leaving the hydrophobic channel unrestricted. As a result the acyl chains of substrates interact with surface residues of the α-helix in V. moutnovskia lactonase rather than sitting in the channel, so its activity is no longer restricted to substrates with long acyl chains. A gluconolactonase encoded by a thermophilic Planctomyces genome was cloned and expressed at high levels in an E. coli host, purified and successfully crystallised. The crystals had a space group of P 3 2 1 and diffracted to a resolution of 2.41 Å. This enzyme was intended to be used by an industrial partner for synthesis of metabolite standards for mass spectrometry and diagnostics. Attempts were made to clone, purify and express two other lactonases from thermophilic metagenomes obtained from terrestrial hot springs: an enol lactonase and a second quorum sensing lactonase. Homology modelling was used to create predicted structures for both of these enzymes. The quorum sensing lactonase showed a 2.5 Å difference in the position of a catalytic serine and a 3.1 Å difference in a catalytic histidine in comparison to a mesophilic homologue. The enol lactonase contained an aspartic acid in place of a catalytic serine found in a mesophilic homologue. A DXP synthase from an anaerobic Gram-negative bacterium Thermovibrio ammonificans was successfully cloned, over-expressed and purified. Crystals were successfully produced although these diffracted only to low resolution. A DXP synthase from an anaerobic Gram-positive bacterium Carboxydothermus hydroformans was successfully cloned, however the protein was expressed primarily in the insoluble fraction. Homology models were made for these two enzymes. Both enzymes showed strong similarity with mesophilic DXP synthases in terms of tertiary structure and positions of active site residues. Visual analysis revealed an increase of 15-20 % in the number of hydrophobic interactions within the enzymes and a high proportion of charged residues at the dimer interface, which would confer increased thermostability. The hope was to obtain high resolution diffraction data to assist in understanding what allows these enzymes to utilise pyruvate as a substrate compared to transketolase, a related enzyme, which uses hydroxypyruvate

Verifying Transcription Factor Regulation of ADV-1 Protein Rhythms through Complementation

Roughly half of the eukaryotic genome is under the control of a circadian clock at the level of transcript abundance, with rhythmic transcripts peaking at all possible phases of the day. However, the mechanism for phase regulation is not well understood. To determine how phase is regulated in Neurospora crassa, ChIP-seq was performed on the morning active transcription factor (TF) and core oscillator component, the White Collar Complex (WCC). The direct targets of WCC were found to be enriched for TFs. Further investigation of these first-tiered TFs revealed that they form a large and complex network made up of multiple feedback and feedforward loops. We hypothesized that this intricate TF network plays a role in phase regulation. To test this hypothesis, we focused our experiments on the network surrounding the first-tier TF, ADV-1. In preliminary data, we found that TF knockouts of CSP-1, CSP-2, SUB-1, and ZNF-21, altered the phase and/or period of ADV-1 protein rhythms. To confirm that these 4 TF knockouts, and not second site mutations, cause the changes in ADV-1 rhythms, genetic complementation will be performed. If the TF knockout causes the defect, then supplying the wild type gene back should rescue the ADV-1 rhythm back to wild type. This information will help us to validate which TFs in the network are necessary for generating proper phase control of ADV-1 protein rhythms

The role of behaviour problems in screening for mental ill-health in adults with intellectual disability

Depression and anxiety are common conditions in adults with intellectual disabilities (ID) and often coexist with behaviour problems. We examined whether behaviour problems can be used to screen for depression and anxiety in ID. Clinical prediction models (CPM) generated from independent databases supported the utility of the depression screen, especially in severe/profound ID. CPM did not support the utility of the anxiety screen at any ID level. Given the paucity of screening tools to improve ascertainment of mental ill-health in ID, the short depression screen would be clinically useful in identifying those who need to undergo a full diagnostic evaluation

Verifying Transcription Factor Regulation of ADV-1 Protein Rhythms through Complementation

Roughly half of the eukaryotic genome is under the control of a circadian clock at the level of transcript abundance, with rhythmic transcripts peaking at all possible phases of the day. However, the mechanism for phase regulation is not well understood. To determine how phase is regulated in Neurospora crassa, ChIP-seq was performed on the morning active transcription factor (TF) and core oscillator component, the White Collar Complex (WCC). The direct targets of WCC were found to be enriched for TFs. Further investigation of these first-tiered TFs revealed that they form a large and complex network made up of multiple feedback and feedforward loops. We hypothesized that this intricate TF network plays a role in phase regulation. To test this hypothesis, we focused our experiments on the network surrounding the first-tier TF, ADV-1. In preliminary data, we found that TF knockouts of CSP-1, CSP-2, SUB-1, and ZNF-21, altered the phase and/or period of ADV-1 protein rhythms. To confirm that these 4 TF knockouts, and not second site mutations, cause the changes in ADV-1 rhythms, genetic complementation will be performed. If the TF knockout causes the defect, then supplying the wild type gene back should rescue the ADV-1 rhythm back to wild type. This information will help us to validate which TFs in the network are necessary for generating proper phase control of ADV-1 protein rhythms

Saturn Plasma Sources and Associated Transport Processes

This article reviews the different sources of plasma for Saturn’s magnetosphere, as they are known essentially from the scientific results of the Cassini-Huygens mission to Saturn and Titan. At low and medium energies, the main plasma source is the H2OH2O cloud produced by the “geyser” activity of the small satellite Enceladus. Impact ionization of this cloud occurs to produce on the order of 100 kg/s of fresh plasma, a source which dominates all the other ones: Titan (which produces much less plasma than anticipated before the Cassini mission), the rings, the solar wind (a poorly known source due to the lack of quantitative knowledge of the degree of coupling between the solar wind and Saturn’s magnetosphere), and the ionosphere. At higher energies, energetic particles are produced by energy diffusion and acceleration of lower energy plasma produced by the interchange instabilities induced by the rapid rotation of Saturn, and possibly, for the highest energy range, by contributions from the CRAND process acting inside Saturn’s magnetosphere. Discussion of the transport and acceleration processes acting on these plasma sources shows the importance of rotation-induced radial transport and energization of the plasma, and also shows how much the unexpected planetary modulation of essentially all plasma parameters of Saturn’s magnetosphere remains an unexplained mystery

Energetic charged particle fluxes relevant to Ganymede's polar region

The JEDI instrument made measurements of energetic charged particles near Ganymede during a close encounter with that moon. Here we find ion flux levels are similar close to Ganymede itself but outside its magnetosphere and on near wake and open field lines. But energetic electron flux levels are more than a factor of 2 lower on polar and near-wake field lines than on nearby Jovian field lines at all energies reported here. Flux levels are relevant to the weathering of the surface, particularly processes that affect the distribution of ice, since surface brightness has been linked to the open-closed field line boundary. For this reason, we estimate the sputtering rates expected in the polar regions due to energetic heavy ions. Other rates, such as those related to radiolysis by plasma and particles that can reach the surface, need to be added to complete the picture of charged particle weathering

Patterns of adversity and post-traumatic stress among children adopted from care

Background Children adopted from care are more likely to have experienced early adversity, but little is known about the impact of early adversity on later post-traumatic stress (PTS) symptoms. Objective To investigate sub-groups of adversity in a sample of adopted children and examine the association with later PTS symptoms. Participants and setting A study of British children adopted from care using social worker records (N = 374) and questionnaire-based longitudinal study of n = 58 children over 4-years post adoptive placement. Methods We used latent class analysis to identify subgroups of children based on commonalities in perinatal and postnatal adversity experienced prior to adoption and examined differences in PTS symptoms at 4-years post-placement between subgroups. Results Nearly one in five (19 %) children were in the clinical or borderline ranges for symptoms of PTS arousal, 14 % for PTS avoidance and 8 % for PTS intrusion. The 5-class solution fitted the data best, with one class characterized by children with a low probability of experiencing any adversity, one perinatal adversity class and three classes capturing different patterns of adversity. The multiple complex adversity class involving both perinatal and postnatal adversity had significantly higher symptoms of PTS avoidance and arousal than other sub-groups. Conclusions The prevalence and complexity of PTS symptoms among adoptive children highlights the need for effective interventions considering different profiles of early adversity

Electronic structure, phase stability and chemical bonding in Th2_2Al and Th2_2AlH4_4

We present the results of theoretical investigation on the electronic structure, bonding nature and ground state properties of Th$_2$Al and Th$_2$AlH$_4$ using generalized-gradient-corrected first-principles full-potential density-functional calculations. Th$_2$AlH$_4$ has been reported to violate the "2 \AA rule" of H-H separation in hydrides. From our total energy as well as force-minimization calculations, we found a shortest H-H separation of 1.95 {\AA} in accordance with recent high resolution powder neutron diffraction experiments. When the Th$_2$Al matrix is hydrogenated, the volume expansion is highly anisotropic, which is quite opposite to other hydrides having the same crystal structure. The bonding nature of these materials are analyzed from the density of states, crystal-orbital Hamiltonian population and valence-charge-density analyses. Our calculation predicts different nature of bonding for the H atoms along $a$ and $c$. The strongest bonding in Th$_2$AlH$_4$ is between Th and H along $c$ which form dumb-bell shaped H-Th-H subunits. Due to this strong covalent interaction there is very small amount of electrons present between H atoms along $c$ which makes repulsive interaction between the H atoms smaller and this is the precise reason why the 2 {\AA} rule is violated. The large difference in the interatomic distances between the interstitial region where one can accommodate H in the $ac$ and $ab$ planes along with the strong covalent interaction between Th and H are the main reasons for highly anisotropic volume expansion on hydrogenation of Th$_2$Al.Comment: 14 pages, 9 figure