TAL Effectors Specificity Stems from Negative Discrimination

Transcription Activator-Like (TAL) effectors are DNA-binding proteins secreted by phytopathogenic bacteria that interfere with native cellular functions by binding to plant DNA promoters. The key element of their architecture is a domain of tandem-repeats with almost identical sequences. Most of the polymorphism is located at two consecutive amino acids termed Repeat Variable Diresidue (RVD). The discovery of a direct link between the RVD composition and the targeted nucleotide allowed the design of TAL-derived DNA-binding tools with programmable specificities that revolutionized the field of genome engineering. Despite structural data, the molecular origins of this specificity as well as the recognition mechanism have remained unclear. Molecular simulations of the recent crystal structures suggest that most of the protein-DNA binding energy originates from non-specific interactions between the DNA backbone and non-variable residues, while RVDs contributions are negligible. Based on dynamical and energetic considerations we postulate that, while the first RVD residue promotes helix breaks - allowing folding of TAL as a DNA-wrapping super-helix - the second provides specificity through a negative discrimination of matches. Furthermore, we propose a simple pharmacophore-like model for the rationalization of RVD-DNA interactions and the interpretation of experimental findings concerning shared affinities and binding efficiencies. The explanatory paradigm presented herein provides a better comprehension of this elegant architecture and we hope will allow for improved designs of TAL-derived biotechnological tools

The Lipopolysaccharide from Capnocytophaga canimorsus Reveals an Unexpected Role of the Core-Oligosaccharide in MD-2 Binding

Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a “hybrid backbone” lacking the 4′ phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 – lipid A complex in case the 4′ phosphate is not present

Modelling estuarine wetlands under climate change and infrastructure pressure

Abstract: Estuarine wetlands are an extremely valuable resource in terms of biotic diversity, flood attenuation, storm surge protection, groundwater recharge, filtering of surface flows and carbon sequestration. The survival of these systems depends on a balance between the slope of the land, and the rates of accretion and sea-level rise. Climate change predictions for most of Australia include both an accelerated sea level rise and an increase on the frequency of extraordinary river floods, which will endanger estuarine wetlands. Furthermore, coastal infrastructure poses an additional constraint on the adaptive capacity of these ecosystems. In recent years a number of numerical models have been developed in order to assess wetland dynamics and to help manage some of these situations. In this paper we present a wetland evolution model that is based on computed values of hydroperiod and tidal range that drive vegetation preference. Results from a 2D spatially distributed model of wetland dynamics in area E of Kooragang Island (Hunter estuary, NSW) are presented as an example of a system heavily constricted by infrastructure undergoing the effects of sea level rise. Area E presents a vegetation zonation sequence mudflats - mangrove - saltmarsh from the seaward margin and up to the topographic gradient and is compartmentalized by the presence of internal culverts. The model includes a detailed hydrodynamic module (CTSS8), which is able to handle man-made flow controls and spatially varying roughness. The model continually simulates tidal inputs into the wetland and computes annual values of hydroperiod and tidal range to update vegetation distribution based on preference to hydrodynamic conditions of the different vegetation types. It also computes soil accretion and carbon sequestration rates and updates roughness coefficient values according to evolving vegetation types. In order to further explore the magnitude of flow attenuation due to roughness and its effects on the computation of tidal range and hydroperiod, numerical experiments were carried out simulating floodplain flow on the side of a tidal creek using different roughness values. Even though the values of roughness that produce appreciable changes in hydroperiod and tidal range are relatively high, they are within the range expected for some of the wetland vegetation. Both applications of the model show that flow attenuation plays a major role in wetland hydrodynamics and that its effects must be considered when predicting wetland evolution under climate change scenarios, particularly in situations where existing infrastructure affects the flow.School of Engineering, University of Newcastle, Callaghan, NSW, AustraliaDepartamento de Hidráulica, Escuela de Ingenieria Civil, Fac. de Cs. Exactas, Ingenieria y Agrimensura, Universidad Nacional de RosarioCentro Universitario Rosario de Investigaciones Hidroambientales (CURIHAM)Consejo de Investigaciones de la Universidad Nacional de Rosari

Computational Evidence for the Catalytic Mechanism of Human Glutathione S-Transferase A3-3: A QM/MM Investigation

A Quantum Mechanics/Molecular Mechanics (QM/MM) computational investigation of the catalytic mechanism of the human glutathione transferase A3-3 (hGSTA3-3) has been carried out. The results demonstrate that the isomerization reaction is concerted, but highly asynchronous: in the first reaction phase the glutathione (GSH) negative sulfur (thiolate) acts as a base and deprotonates carbon C4 of the substrate \u3945-androstene-3,17-dione (\u3945-AD); in the second reaction phase the hydroxyl proton of the tyrosine fragment Y9 is transferred to C6 affording the \u3944-androstene-3,17-dione product (\u3944-AD). The initial state of the enzyme is subsequently restored by transferring a proton from the GSH sulfur to the tyrosine negative oxygen. There is no evidence for a \u201cgenuine\u201d stepwise mechanism involving the formation of a real dienolate intermediate as suggested in previous papers. Furthermore, our computations have evidenced that, when we consider the whole process (including the restoring of the enzyme), GSH behaves as a base/acid catalyst (as hypothesized by some authors), but it requires the participation of the tyrosine Y9 acting as a proton shuttle. A \u201cfingerprint analysis\u201d has been used to rank the electrostatic effects on the catalysis of the various residues surrounding the active site. This analysis highlights the role played by the arginine residue R15 in stabilizing the initial complex in agreement with previous suggestions based on crystal structures

Coevolution of hydraulic, soil and vegetation processes in estuarine wetlands.

Estuarine wetlands of south eastern Australia, typically display a vegetation zonation with a sequence mudflats - mangrove forest - saltmarsh plains from the seaward margin and up the topographic gradient. Estuarine wetlands are among the most productive ecosystems in the world, providing unique habitats for fish and many terrestrial species. They also have a carbon sequestration capacity that surpasess terrestrial forest. Estuarine wetlands respond to sea-level rise by vertical accretion and horizontal landward migration, in order to maintain their position in the tidal frame. In situations in which buffer areas for landward migration are not available, saltmarsh can be lost due to mangrove encroachment. As a result of mangrove invasion associated in part with raising estuary water levels and urbanisation, coastal saltmarsh in parts of south-eastern Australia has been declared an endangered ecological community. Predicting estuarine wetlands response to sea-level rise requires modelling the coevolving dynamics of water flow, soil and vegetation. This paper presents preliminary results of our recently developed numerical model for wetland dynamics in wetlands of the Hunter estuary of NSW. The model simulates continuous tidal inflow into the wetland, and accounts for the effect of varying vegetation types on flow resistance. Coevolution effects appear as vegetation types are updated based on their preference to prevailing hydrodynamic conditions. The model also considers that accretion values vary with vegetation type. Simulations are driven using local information collected over several years, which includes estuary water levels, accretion rates, soil carbon content, flow resistance and vegetation preference to hydraulic conditions. Model results predict further saltmarsh loss under current conditions of moderate increase of estuary water levels.School of Engineering, The University of Newcastle, Callaghan 2308, AustraliaDepartamento de Hidráulica, Escuela de Ingenieria Civil, Fac. de Cs. Exactas, Ingenieria y Agrimensura, Universidad Nacional de RosarioConsejo de Investigaciones de la Universidad Nacional de Rosari

A tunable QM/MM approach to chemical reactivity, structure and physico-chemical properties prediction

This contribution describes a new implementation of a general hybrid approach with a modular structure (called COBRAMM: Computations at Bologna Relating Ab‐initio and Molecular Mechanic Methods) that is able to integrate some specialized programs and acts as a flexible computational environment, thus increasing the flexibility/efficiency of both QM, and MM, and QM/MM calculations. Specifically, QM/MM ground and excited states geometry optimizations, frequency calculations, conical intersection searches and adiabatic/non‐adiabatic molecular dynamics can be performed on a large molecular system, that can be split up to three different layers corresponding to different levels of accuracy

Estuarine wetland evolution including sea-level rise and infrastructure effects.

Estuarine wetlands are an extremely valuable resource in terms of biotic diversity, flood attenuation, storm surge protection, groundwater recharge, filtering of surface flows and carbon sequestration. On a large scale the survival of these systems depends on the slope of the land and a balance between the rates of accretion and sea-level rise, but local man-made flow disturbances can have comparable effects. Climate change predictions for most of Australia include an accelerated sea level rise, which may challenge the survival of estuarine wetlands. Furthermore, coastal infrastructure poses an additional constraint on the adaptive capacity of these ecosystems. Numerical models are increasingly being used to assess wetland dynamics and to help manage some of these situations. We present results of a wetland evolution model that is based on computed values of hydroperiod and tidal range that drive vegetation preference. Our first application simulates the long term evolution of an Australian wetland heavily constricted by infrastructure that is undergoing the effects of predicted accelerated sea level rise. The wetland presents a vegetation zonation sequence mudflats - mangrove - saltmarsh from the seaward margin and up the topographic gradient but is also affected by compartmentalization due to internal road embankments and culverts that effectively attenuates tidal input to the upstream compartments. For this reason, the evolution model includes a 2D hydrodynamic module which is able to handle man-made flow controls and spatially varying roughness. It continually simulates tidal inputs into the wetland and computes annual values of hydroperiod and tidal range to update vegetation distribution based on preference to hydrodynamic conditions of the different vegetation types. It also computes soil accretion rates and updates roughness coefficient values according to evolving vegetation types. In order to explore in more detail the magnitude of flow attenuation due to roughness and its effects on the computation of tidal range and hydroperiod, we performed numerical experiments simulating floodplain flow on the side of a tidal creek using different roughness values. Even though the values of roughness that produce appreciable changes in hydroperiod and tidal range are relatively high, they are within the range expected for some of the wetland vegetation. Both applications of the model show that flow attenuation can play a major role in wetland hydrodynamics and that its effects must be considered when predicting wetland evolution under climate change scenarios, particularly in situations where existing infrastructure affects the flow.School of Engineering, University of Newcastle, Callaghan, NSW, AustraliaCentro Universitario Rosario de Investigaciones Hidroambientales (CURIHAM)Consejo de Investigaciones de la Universidad Nacional de Rosari

Short term prognosis of stroke in a clinical series of 94 patients

We conducted a study on the factors predictive of early mortality (within 30 days of onset of symptoms) in a clinical series of 94 patients at their first stroke. Irrespective of the type of stroke, ischemic or hemorrhagic, early mortality proved to correlate with clinical parameters, such as coma at onset, presence of paralysis, changes in ocular motility, and neuroradiological parameters (lesion size on the CT scan) indicative of stroke severity

A Rational Approach Towards a New Ferrocenyl Pyrrolidine for Stereoselective Enamine Catalysis

Proline and pyrrolidine derivatives (Hayashi– Jørgensen catalysts) are considered “work horses” in organocatalysis. This report describes a new effective ferrocenyl pyrrolidine catalyst that is able to perform well in benchmark organocatalytic reactions (see figure). The ferrocene moiety controls the conformational space and a simple alkyl group effectively covers a face of the derived enamine. This new framework can find applications in organocatalysis, and in general, in new ligand desig