DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site- bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site- bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.

Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

Building economic capability to improve the management of marine resources in Australia: Final report

FRDC project 2008/306 Building economic capability to improve the management of marine resources in Australia was developed and approved in response to the widespread recognition and acknowledgement of the importance of incorporating economic considerations into marine management in Australia and of the persistent undersupply of suitably trained and qualified individuals capable of providing this input. The need to address this shortfall received broad based support and following widespread stakeholder consultation and building on previous unsuccessful State-based initiatives, a collaborative, cross-jurisdictional cross-institutional capability building model was developed. The resulting project sits within the People Development Program as part of FRDC’s ‘investment in RD&E to develop the capabilities of the people to whom the industry entrusts its future’, and has addressed its objectives largely through three core activities: 1. The Fisheries Economics Graduate Research Training Program which provides research training in fisheries/marine economics through enrolment in postgraduate higher degree studies at the three participating Universities; 2. The Fisheries Economics Professional Training Program which aims to improve the economic literacy of non-economist marine sector stakeholders and was implemented in collaboration with the Seafood Cooperative Research Centre through the Future Harvest Masterclass in Fisheries Economics; and, 3. The Australian Fisheries Economics Network (FishEcon) which aims to strengthen research in the area of fisheries economics by creating a forum in which fisheries economists, fisheries managers and Ph.D. students can share research ideas and results, as well as news of upcoming research opportunities and events. These activities were undertaken by a core Project team, comprising economic researchers and teachers from each of the four participating institutions (namely the University of Tasmania, the University of Adelaide, Queensland University of Technology and the Commonwealth Scientific and Industrial Research Organisation), spanning three States and the Commonwealth. The Project team reported to and was guided by a project Steering Committee. Commensurate with the long term nature of the project objectives and some of its activities the project was extended (without additional resources) in 2012 to 30th June 2015.

Soy supplementation and role of equol production capability in postmenopausal women using tibolone: effects on cardiovascular risk markers

Tibolone, a synthetic steroid, is effective in the treatment of postmenopausal symptoms. Its cardiovascular safety profile has been questioned, because tibolone reduces the levels of high-density lipoprotein (HDL) cholesterol. Soy-derived isoflavones may offer health benefits, particularly as regards lipids and also other cardiovascular disease (CVD) risk factors. The soy-isoflavone metabolite equol is thought to be the key as regards soy-related beneficial effects. We studied the effects of soy supplementation on various CVD risk factors in postmenopausal monkeys and postmenopausal women using tibolone. In addition, the impact of equol production capability was studied. A total of 18 monkeys received casein/lactalbumin (C/L) (placebo), tibolone, soy (a woman s equivalent dose of 138 mg of isoflavones), or soy with tibolone in a randomized order for 14 weeks periods, and there was a 4-week washout (C/L) in between treatments. Postmenopausal women using tibolone (N=110) were screened by means of a one-week soy challenge to find 20 women with equol production capability (4-fold elevation from baseline equol level) and 20 control women, and treated in a randomized cross-over trial with a soy powder (52 g of soy protein containing 112 mg of isoflavones) or placebo for 8 weeks. Before and after the treatments lipids and lipoproteins were assessed in both monkeys and women. In addition, blood pressure, arterial stiffness, endothelial function, sex steroids, sex hormone-binding globulin (SHBG), and vascular inflammation markers were assessed. A 14% increase in plasma low-density lipoprotein (LDL) + very low-density lipoprotein (VLDL) cholesterol was observed in tibolone-treated monkeys vs. placebo. Soy treatment resulted in a 18% decrease in LDL+VLDL cholesterol, and concomitant supplementation with tibolone did not negate the LDL+VLDL cholesterol-lowering effect of soy. A 30% increase in HDL cholesterol was observed in monkeys fed with soy, whereas HDL cholesterol levels were reduced (48%) after tibolone. Interestingly, Soy+Tibolone diet conserved HDL cholesterol levels. Tibolone alone increased the total cholesterol (TC):HDL cholesterol ratio, whereas it was reduced by Soy or Soy+Tibolone. In postmenopausal women using tibolone, reductions in the levels of total cholesterol and LDL cholesterol were seen after soy supplementation compared with placebo, but there was no effect on HDL cholesterol, blood pressure, arterial stiffness or endothelial function. Soy supplementation decreased the levels of estrone in equol producers, and those of testosterone in the entire study population. No changes were seen in the levels of androstenedione, dehydroepiandrosterone sulfate, or SHBG. The levels of vascular cell adhesion molecule-1 increased, and platelet-selectin decreased after soy treatment, whereas C-reactive protein and intercellular adhesion molecule-1 remained unchanged. At baseline and unrelated to soy treatment, equol producers had lower systolic, diastolic and mean arterial pressures, less arterial stiffness and better endothelial function than non-producers. To conclude, soy supplementation reversed the tibolone-induced fall in HDL cholesterol in postmenopausal monkeys, but this effect was not seen in women taking tibolone. Equol production capability was associated with beneficial cardiovascular changes and thus, this characteristic may offer cardiovascular benefits, at least in women using tibolone.

Seismic Tomography and Earthquake Mechanism Beneath the Central Fennoscandian Shield

The aim of this thesis was to study the seismic tomography structure of the earth s crust together with earthquake distribution and mechanism beneath the central Fennoscandian Shield, mainly in southern and central Finland. The earthquake foci and some fault plane solutions are correlated with 3-D images of the velocity tomography. The results are discussed in relation to the stress field of the Shield and with other geophysical, e.g. geomagnetic, gravimetric, tectonic, and anisotropy studies of the Shield. The earthquake data of the Fennoscandian Shield has been extracted from the Nordic earthquake parameter data base which was founded at the time of inception of the earthquake catalogue for northern Europe. Eight earlier earthquake source mechanisms are included in a pilot study on creating a novel technique for calculating an earthquake fault plane solution. Altogether, eleven source mechanisms of shallow, weak earthquakes are related in the 3-D tomography model to trace stresses of the crust in southern and central Finland. The earthquakes in the eastern part of the Fennoscandian Shield represent low-active, intraplate seismicity. Earthquake mechanisms with NW-SE oriented horizontal compression confirm that the dominant stress field originates from the ridge-push force in the North Atlantic Ocean. Earthquakes accumulate in coastal areas, in intersections of tectonic lineaments, in main fault zones or are bordered by fault lines. The majority of Fennoscandian earthquakes concentrate on the south-western Shield in southern Norway and Sweden. Onwards, epicentres spread via the ridge of the Shield along the west-coast of the Gulf of Bothnia northwards along the Tornio River - Finnmark fault system to the Barents Sea, and branch out north-eastwards via the Kuusamo region to the White Sea Kola Peninsula faults. The local seismic tomographic method was applied to find the terrane distribution within the central parts of the Shield the Svecofennian Orogen. From 300 local explosions a total of 19765 crustal Pg- and Sg-wave arrival times were inverted to create independent 3-D Vp and Vs tomographic models, from which the Vp/Vs ratio was calculated. The 3-D structure of the crust is presented as a P-wave and for the first time as an S-wave velocity model, and also as a Vp/Vs-ratio model of the SVEKALAPKO area that covers 700x800 km2 in southern and central Finland. Also, some P-wave Moho-reflection data was interpolated to image the relief of the crust-mantle boundary (i.e. Moho). In the tomography model, the seismic velocities vary smoothly. The lateral variations are larger for Vp (dVp =0.7 km/s) than for Vs (dVs =0.4 km/s). The Vp/Vs ratio varies spatially more distinctly than P- and S-wave velocities, usually from 1.70 to 1.74 in the upper crust and from 1.72 to 1.78 in the lower crust. Schist belts and their continuations at depth are associated with lower velocities and lower Vp/Vs ratios than in the granitoid areas. The tomography modelling suggests that the Svecofennian Orogen was accreted from crustal blocks ranging in size from 100x100 km2 to 200x200 km2 in cross-sectional area. The intervening sedimentary belts have ca. 0.2 km/s lower P- and S-wave velocities and ca. 0.04 lower Vp/Vs ratios. Thus, the tomographic model supports the concept that the thick Svecofennian crust was accreted from several crustal terranes, some hidden, and that the crust was later modified by intra- and underplating. In conclusion, as a novel approach the earthquake focal mechanism and focal depth distribution is discussed in relation to the 3-D tomography model. The schist belts and the transformation zones between the high- and low-velocity anomaly blocks are characterized by deeper earthquakes than the granitoid areas where shallow events dominate. Although only a few focal mechanisms were solved for southern Finland, there is a trend towards strike-slip and oblique strike-slip movements inside schist areas. The normal dip-slip type earthquakes are typical in the seismically active Kuusamo district in the NE edge of the SVEKALAPKO area, where the Archean crust is ca. 15-20 km thinner than the Proterozoic Svecofennian crust. Two near vertical dip-slip mechanism earthquakes occurred in the NE-SW junction between the Central Finland Granitoid Complex and the Vyborg rapakivi batholith, where high Vp/Vs-ratio deep-set intrusion splits the southern Finland schist belt into two parts in the tomography model.

Leading Digital Transformation: Building Technology and Information Governance Capability in Boards of Directors and the C-Suite

Information and technology and its use in organisation transformation presents unprecedented opportunities and risks. Increasingly, the Governance of Enterprise Information and Technology (GEIT) competency in the board room and executive is needed. Whether your organization is small or large, public, private or not for profit or whether your industry is not considered high-tech, IT is impacting your sector – no exceptions. But there is a skill shortage in boards: GEIT capability is concerningly low. This capability is urgently needed across the board, including those directors who come from finance, legal, marketing, operations and HR backgrounds. Digital disruption also affects all occupations. Putting in place a vision will help ensure emergency responses will meet technology-related duty of care responsibilities. When GEIT-related forward thinking and planning is carried out at the same time that you put your business strategy and plan in place, your organization has a significantly increased chance of not only surviving, but thriving into the future. Those organizations that don’t build GEIT capability risk joining the growing list of once-leading firms left behind in the digital ‘cloud of smoke’. Those organizations that do will be better placed to reap the benefits and hedge against the risks of a digital world. This chapter provides actionable, research-based considerations and processes for boards to use, to build awareness, knowledge and skills in governing technology-related organization strategy, risk and value creation.

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway THE MECHANISM OF EXPANSION OF THE ACYL CARRIER PROTEIN CORE

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C-alpha and C-beta of Ser-37 in tetradecanoyl-ACP. C-13, N-15-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C-8)- and dodecanoyl (C-12)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C-14)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK(a) value for the carboxylate, similar to 1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.

Synthesis, Structure, Spirocyclization Mechanism, and Glutathione Peroxidase-like Antioxidant Activity of Stable Spirodiazaselenurane and Spirodiazatellurane

The first examples of stable spirodiazaselenurane and spirodiazatellurane were synthesized by oxidative spirocyclization of the corresponding diaryl selenide and telluride and were structurally characterized. X-ray crystal structures of the spirodiazaselenurane and spirodiazatellurane suggest that the structures are distorted trigonal bipyramidal (TBP) with the electronegative nitrogen atoms occupying the apical positions and two carbon atoms and the lone pair of Se/Te occupying the equatorial positions. Interestingly, the spirodiazatellurane underwent spontaneous chiral resolution during crystallization, and the absolute configurations of its enantiomers were confirmed by single-crystal X-ray analyses. A detailed mechanistic study indicates that the cyclization to spirodiazaselenurane and spirodiazatellurane occurs via selenoxide and telluroxide intermediates. The chalcogenoxides cyclize to the corresponding spiro compounds in a stepwise manner via the involvement of hydroxyl chalcogenurane intermediates, and the activation energy for them spirocyclization reaction decreases in the order S > Se > Te. In addition to the synthesis, characterization, and mechanism of cyclization, the glutathione peroxidase (GPx) mimetic activity of the newly synthesized compounds was evaluated. These studies suggest that the tellurium compounds are more effective as GPx mimics than their selenium counterparts due to the fast oxidation of the tellurium center in the presence of peroxide and the involvement of an efficient redox cycle between the telluride and telluroxide intermediate.

Design of mechanism-based inhibitors of transthyretin amyloidosis: Studies with biphenyl ethers and new structural templates

Transthyretin (TTR), a tetrameric thyroxine (T4) carrier protein, is associated with a variety of amyloid diseases. In this study, we explore the potential of biphenyl ethers (BPE), which are shown to interact with a high affinity to its T4 binding site thereby preventing its aggregation and fibrillogenesis. They prevent fibrillogenesis by stabilizing the tetrameric ground state of transthyretin. Additionally, we identify two new structural templates (2-(5-mercapto-[1,3,4]oxadiazol-2-yl)-phenol and 2,3,6-trichloro-N-(4H-[1,2,4]triazol-3-yl) represented as compounds 11 and 12, respectively, throughout the manuscript) exhibiting the ability to arrest TTR amyloidosis. The dissociation constants for the binding of BPEs and compound 11 and 12 to TTR correlate with their efficacies of inhibiting amyloidosis. They also have the ability to inhibit the elongation of intermediate fibrils as well as show nearly complete (> 90%) disruption of the preformed fibrils. The present study thus establishes biphenyl ethers and compounds 11 and 12 as very potent inhibitors of TTR fibrillization and inducible cytotoxicity.

Solution structures of conformationally equilibrium forms of holo-acyl carrier protein (PfACP) from Plasmodium falciparum provides insight into the mechanism of activation of ACPs

Acyl Carrier Protein (ACP) from the malaria parasite, Plasmodium falciparum (PfACP) in its holo form is found to exist in two conformational states in solution. Unique 3D solution structures of holo-PfACP have been determined for both equilibrium conformations, using high-resolution NMR methods. Twenty high-resolution solution structures for each of the two forms of holo-PfACP have been determined on the basis of 1226 and 1218 unambiguously assigned NOEs (including NOEs between 4 '-phosphopantetheine prosthetic group (4 '-PP) and protein), 55 backbone dihedral angles and 26 hydrogen bonds. The atomic rmsd values of the determined structures of two equilibrium forms, about the mean coordinates of the backbone and heavy atoms, are 0.48 +/- 0.09 and 0.92 +/- 0.10 and 0.49 +/- 0.08 and 0.97 +/- 0.11 angstrom, respectively. The interaction of 4 '-PP with the polypeptide backbone is reported here for the first time for any of the ACPs. The structures of holo-PfACP consist of three well-defined helices that are tightly packed. The structured regions of the molecule are stabilized by extensive hydrophobic interactions. The difference between the two forms arises from a reorientation of the 4 '-PP group. The enthalpy difference between the two forms, although small, implies that a conformational switch is essential for the activation of holo-ACP. Sequence and structures of holo-PfACP have been compared with those of the ACPs from type I and type II fatty acid biosynthesis pathways (FAS), in particular with the ACP from rat and the butyryl-ACP from E. coli. The PfACP structure, thus determined has several novel features hitherto not seen in other ACPs.

Kinetics and the Mechanism of Interaction of the Endoplasmic Reticulum Chaperone, Calreticulin, with Monoglucosylated (Glc1Man9GlcNAc2) Substrate

Calreticulin is a lectin-like molecular chaperone of the endoplasmic reticulum in eukaryotes. Its interaction with N-glycosylated polypeptides is mediated by the glycan, Glc(1)Man(9)GlcNAc(2), present on the target glycoproteins. In this work, binding of monoglucosyl IgG (chicken) substrate to calreticulin has been studied using real time association kinetics of the interaction with the biosensor based on surface plasmon resonance (SPR). By SPR, accurate association and dissociation rate constants were determined, and these yielded a micromolar association constant. The nature of reaction was unaffected by immobilization of either of the reactants. The Scatchard analysis values for K-a agreed web crith the one obtained by the ratio k(1)/k(-1). The interaction was completely inhibited by free oligosaccharide, Glc(1)Man(9)GlcNAc(2), whereas Man(9)GlcNAc(2) did not bind to the calreticulin-substrate complex, attesting to the exquisite specificity of this interaction. The binding of calreticulin to IgG was used for the development of immunoassay and the relative affinity of the lectin-substrate association was indirectly measured. The values are in agreement with those obtained with SPR. Although the reactions are several orders of magnitude slower than the diffusion controlled processes, the data are qualitatively and quantitatively consistent with single-step bimolecular association and dissociation reaction. Analyses of the activation parameters indicate that reaction is enthalpically driven and does not involve a highly ordered transition state. Based on these data, the mechanism of its chaperone activity is briefly discussed.

Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl galactosides to the basic agglutinin from winged bean (Psophocarpus tetragonolobus)

The binding of winged bean basic agglutinin (WBA I) to 4-methylumbelliferyl (MeUmb) galactosides was examined by extrinsic fluorescence titration and stopped-flow spectrofluorimetry. Upon binding to WBA I, MeUmb alpha-galactosides show quenching in fluorescence intensity, decrease in UV absorbance with a concomitant blue shift, and decrease in fluorescence excited-state lifetimes. However, their beta-analogues show enhancement in fluorescence intensity, increase in UV absorbance with a red shift, and an increase in fluorescence excited-state lifetimes. This implies that the umbelliferyl groups of alpha- and beta-galactosides experience non-polar and polar microenvironments, respectively, upon binding to WBA I. Replacement of the anomeric hydroxyl group of galactose by 4-methylumbelliferyl moiety increases the affinity of resulting saccharides. Substitution of C-2 hydroxyl of galactose by an acetamido group leads to increased affinity due to a favorable entropy change. This suggests that acetamido group of MeUmb-alpha/beta-GalNAc binds to a relatively non-polar subsite of WBA I. Most interestingly, this substitution also reduces the association rate constants dramatically. Inspection of the activation parameters reveals that the enthalpy of activation is the limiting factor for the differences in the forward rate constants for these saccharides and the entropic contribution to the activation energy is small

Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl glycosides to jacalin

The binding of Artocarpus integrifolia lectin (jacalin) to 4-methylumbelliferyl (Meumb)-glycosides, Gal alpha Meumb, Gal beta Meumb, GalNAc alpha Meumb, GalNAc beta-Meumb, and Gal beta 3GalNAc beta Meumb was examined by extrinsic fluorescence quenching titration and stopped flow spectrofluorimetry. The binding was characterized by 100% quenching of fluorescence of Meumb-glycosides. Their association constants range from 2.0 x 10(4) to 1.58 x 10(6) M-1 at 15 degrees C. Entropic contribution is the major stabilizing force for avid binding of Meumb-glycosides indicating the existence of a hydrophobic site that is complementary to their methylumbelliferyl group. The second order association rate constants for interaction of these sugars with lectin at 15 degrees C vary from 8.8 x 10(5) to 3.24 x 10(6) M-1 S-1, at pH 7.2. The first order dissociation rate constants range from 2.30 to 43.0 S-1 at 15 degrees C. Despite the differences in their association rate constants, the overall values of association constants for these saccharides are determined by their dissociation rate constants. The second order rate constant for the association of Meumb-glycosides follows a pattern consistent with the magnitude of the activation energies involved therin. Activation parameters for association of all ligands illustrate that the origin of the barrier between binding of jacalin to Meumb-glycosides is entropic, and the enthalpic contribution is small. A correlation between these parameters and the structure of the ligands on the association rates underscores the importance of steric factors in determining protein saccharide recognitions.

Mechanism of lectin-cell interactions: thermodynamic and kinetic analysis of the binding of winged bean acidic lectin (WBA II) to human erythrocytes

In order to identify the forces involved in the binding and to understand the mechanism involved, equilibrium and kinetic studies were performed on the binding of the winged bean acidic lectin to human erythrocytes. The magnitudes of delta S and delta H were positive and negative respectively, an observation differing markedly from the lectin-simple sugar interactions where delta S and delta H are generally negative. Analysis of the sign and magnitudes of these values indicate that ionic and hydrogen bonded interactions prevail over hydrophobic interactions resulting in net -ve delta H (-37.12 kJ.mol-1) and +ve delta S (14.4 J.mole-1 K-1 at 20 degrees C), thereby suggesting that this entropy driven reaction also reflects conformational changes in the lectin and/or the receptor. Presence of two kinds of receptors for WBA II on erythrocytes, as observed by equilibrium studies, is consistent with the biexponential dissociation rate constants (at 20 degrees C K1 = 1.67 x 10(-3) M-1 sec-1 and K2 = 11.1 x 10(-3) M-1 sec-1). These two rate constants differed by an order of magnitude accounting for the difference in the association constants of the two receptors of WBA II. However, the association process remains monoexponential suggesting no observable difference in the association rates of the lectin molecule with both the receptors, under the experimental conditions studied. The thermodynamic parameters calculated from kinetic data correlate well with those observed by equilibrium. A two-step binding mechanism is proposed based on the kinetic parameters for WBA II-receptor interaction