New insights into the structural and functional involvement of the gate loop in AcrB export activity

AcrB is a major multidrug exporter in Escherichia coli and other Gram-negative bacteria. Its gate loop, located between the proximal and the distal pockets, have been reported to play important role in the export of many antibiotics. This loop location, rigidity and interactions with substrates have led recent reports to suggest that AcrB export mechanism operates in a sequential manner. First the substrate binds the proximal pocket in the access monomer, then it moves to bind the distal pocket in the binding monomer and subsequently it is extruded in the extrusion monomer. Recently, we have demonstrated that the gate loop is not required for the binding of Erythromycin but the integrity of this loop is important for an efficient export of this substrate. However, here we show that the antibiotic susceptibilities of the same AcrB gate loop mutants for Doxorubicin were unaffected, suggesting that this loop is not required for its export, and we demonstrate that this substrate may use principally the tunnel-1, located between transmembranes 8 and 9, more often than previously reported. To further explain our findings, here we address the gate loop mutations effects on AcrB solution energetics (fold, stability, molecular dynamics) and on the in vivo efflux of Erythromycin and Doxorubicin. Finally, we discuss the efflux and the discrepancy between the structural and the functional experiments for Erythromycin in these gate loop mutants

Hydrogen ion passivation of multicrystalline silicon solar cells

It has been recognized that hydrogen can be chosen to passivate the defects present in polycrystalline materials. Technically, the best approach is to use hydrogen ion implantation at low energy (0.5 to 5 keV) by means of a Kaufman or similar type ion source in order to reduce the processing time. For our multiple beam ion source, we have determined the effective concentration profile of the introduced hydrogen, the modification of the optical properties of the implanted wafers and the conditions under which two multicrystalline materials (POLYX and SILSO) will give the greatest improvement in solar cell performance

On the Ca2 + binding and conformational change in EF-hand domains: Experimental evidence of Ca2 +-saturated intermediates of N-domain of calmodulin

Double mutation of Q41L and K75I in the N-domain of calmodulin (N-Cam) stabilizes the closed form of N-Cam such that binding of Ca2 + in solution no longer triggers a conformational change to the open form, and its Ca2 + binding affinity decreases dramatically. To further investigate the solvation effects on the structure, Ca2 + binding affinity and conformational dynamics of this N-Cam double mutant in the Ca2 + saturated state, we solved its X-ray structure. Surprisingly, the structure revealed an open conformation of the domain which contradicts its closed conformation in solution. Here we provide evidence that crystallization conditions were responsible for this Ca2 +-saturated domain open conformation in the crystal. Importantly, we demonstrate that the presence of the crystallization co-precipitant and alcohols were able to induce a progressive opening of the closed form of this domain, in Ca2 + saturated state, in solution. However, in the Ca2 + depleted state, addition of alcohols was unable to induce any opening of this domain in solution. In addition, in the Ca2 + saturated state, the molecular dynamics simulations show that while N-Cam can populate the open and closed conformation, the N-Cam double mutant exclusively populates the closed conformation. Our results provide experimental evidence of intermediate conformations of Ca2 +-saturated N-Cam in solution. We propose that conformational change of Ca2 + sensor EF-hand domains depends on solvation energetics, Ca2 + binding to promote the full open form, Ca2 + depleted state conformational dynamics, and the chemical properties of the molecules nearby key residues such as those at positions 41 and 75 in N-Cam

EFFECT OF SALINITY ON THE PHYSIOLOGICAL BEHAVIOR OF THE OLIVE TREE (VARIETY SIGOISE

Salinity is a major problem directly affecting the ecological balance and the development of agriculture in the Mediterranean basin, particularly North Africa. This phenomenon is considered as the most important abiotic factor limiting crops growth and productivity, degrading and polluting soils in arid and semi-arid. In order to study the influence of salinity, on the physiological parameters and to assess the potential of adaptation of the olive tree in a saline environment, three parcels containing the Sigoise variety and subject to different degrees of salinity were selected: Parcel 1 (non-saline); Parcel 2 (saline); Parcel 3 (very saline). Under a saline constraint, the results showed two contrasting tendencies, an intense increase in the content of proline, sodium (Na+) and chlorophyll (b), while water content, potassium and chlorophyll (a) decreased strongly with increasing salinity

The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase δ

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL δ (hPOL δ). The hPOL δ enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL δ strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL δ. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL δ

Two-dimensional NMR lineshape analysis

NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions

From Profile to Surface Monitoring: SPC for Cylindrical Surfaces Via Gaussian Processes

Quality of machined products is often related to the shapes of surfaces that are constrained by geometric tolerances. In this case, statistical quality monitoring should be used to quickly detect unwanted deviations from the nominal pattern. The majority of the literature has focused on statistical profile monitoring, while there is little research on surface monitoring. This paper faces the challenging task of moving from profile to surface monitoring. To this aim, different parametric approaches and control-charting procedures are presented and compared with reference to a real case study dealing with cylindrical surfaces obtained by lathe turning. In particular, a novel method presented in this paper consists of modeling the manufactured surface via Gaussian processes models and monitoring the deviations of the actual surface from the target pattern estimated in phase I. Regardless of the specific case study in this paper, the proposed approach is general and can be extended to deal with different kinds of surfaces or profiles

A Method for Efficient Calculation of Diffusion and Reactions of Lipophilic Compounds in Complex Cell Geometry

A general description of effects of toxic compounds in mammalian cells is facing several problems. Firstly, most toxic compounds are hydrophobic and partition phenomena strongly influence their behaviour. Secondly, cells display considerable heterogeneity regarding the presence, activity and distribution of enzymes participating in the metabolism of foreign compounds i.e. bioactivation/biotransformation. Thirdly, cellular architecture varies greatly. Taken together, complexity at several levels has to be addressed to arrive at efficient in silico modelling based on physicochemical properties, metabolic preferences and cell characteristics. In order to understand the cellular behaviour of toxic foreign compounds we have developed a mathematical model that addresses these issues. In order to make the system numerically treatable, methods motivated by homogenization techniques have been applied. These tools reduce the complexity of mathematical models of cell dynamics considerably thus allowing to solve efficiently the partial differential equations in the model numerically on a personal computer. Compared to a compartment model with well-stirred compartments, our model affords a more realistic representation. Numerical results concerning metabolism and chemical solvolysis of a polycyclic aromatic hydrocarbon carcinogen show good agreement with results from measurements in V79 cell culture. The model can easily be extended and refined to include more reactants, and/or more complex reaction chains, enzyme distribution etc, and is therefore suitable for modelling cellular metabolism involving membrane partitioning also at higher levels of complexity