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Microbially induced carbonate precipitation (MICP) has been proposed as a sustainable approach to solve various environmental, structural, geotechnical and architectural issues. In the last decade, a ubiquitous microbial metabolism, nitrate reduction (also known as denitrification) got attention in MICP research due to its unique added benefits such as simultaneous corrosion inhibition in concrete and desaturation of porous media. The latter even upgraded MICP into a more advanced concept called microbially induced desaturation and precipitation (MIDP) which is being investigated for liquefaction mitigation. In this paper, we present the findings on MICP through denitrification by covering applications under two main titles: (i) applications solely based on MICP, such as soil reinforcement, development of microbial self-healing concrete, restoration of artwork and historical monuments, and industrial wastewater treatment, (ii) an application based on MIDP: liquefaction mitigation. After explaining the denitrification process in detail and describing the MICP and MIDP reaction system occurring through denitrification metabolism, the most recent advances in each potential field of application are collected, addressing the novel findings and limitations, to provide insights toward the practical applications in situ. Finally, the research needs required to deal with the defined challenges in application-oriented upscaling and optimization of MICP through denitrification are suggested. Overall, collected research findings revealed that MICP through denitrification possesses a great potential to replace conventionally used petrochemical-based, labour intensive, destructive and economically unfeasible techniques used in construction industry with a bio-based, labourless, low-carbon technology. This worldwide applicable bio-based technology will facilitate the sustainable development and contribute to the carbon-emission-reduction.

Physical modelling under the gravitational field between 0 and ng plays a key role in both terrestrial- and aerospace-geotechnical engineering. A geotechnical magnetic–similitude–gravity model testing (GMMT) method is newly proposed to provide an alternative approach to the current physical modelling methods. The theoretical criteria for achieving a magnetic field to simulate the gravitational field are presented first, based on which an exact simulation of the stress field is achieved between the physical model and its corresponding prototype. A GMMT equipment is then designed and manufactured, and a related procedure for obtaining its control and input parameters is presented. Accordingly, the preparation of the model material is demonstrated utilising a kind of magnetically sensitive granular (MSG) media. The relationship between mechanical properties, magnetisation, magnetic–similitude–gravity and magnetic powder content of the MSG media are further investigated, and the influences of both magnetising field and magnetisation process are shown to be negligible in terms of mechanical behaviour. A series of physical modelling of the bearing capacity of granular media under low-gravity conditions is finally performed using the GMMT equipment. The results show good agreement with previous data obtained by the parabolic flight aircraft.

At signalized intersection areas, bicycle traffic presents a dispersion feature which may influence the movements of vehicles during peak period. The primary objective of this study is to simulate the dispersion effect in through-movement bicycle traffic at intersection areas and evaluate its influence on through-movement traffic. A cellular automata (CA) model is developed and validated to simulate the operations of through-movement bicycle traffic departing from two types of intersection approaches. Simulation results show that bicycles benefit from the dispersion effect when they depart from the approach with an exclusive right-turn vehicle lane. But when bicycles travel from the approach with a shared right-turn and through vehicle lane, the dispersion effect will result in friction interference and block interference on through-movement vehicles. Bicycle interferences reduce the vehicle speed and increase the delay of through-movement vehicles. The policy implications in regard to the dispersion effect from two types of approaches are discussed to improve the performance of through-movement traffic operations at signalized intersections.

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.

The analysis and design of single piles subjected to lateral load are very important. The traditional expression for calculating rotational stiffness coefficient is too complex, and it needs large amount of computation. This paper proposes a new approach for getting rotational stiffness coefficient of pile hinged on top under moment load. It has been proved that the new method is the equivalent of traditional method of calculating rotational stiffness coefficient using power series. A case study shows that the same answer can be gotten in calculating rotational stiffness coefficient by the two methods. However, a large computational cost can be reduced by the new method.

A series of triaxial compression tests for frozen clay were performed by K0DCGF (freezing with non-uniform temperature under loading after K0 consolidation) method and GFC (freezing with non-uniform temperature without experiencing K0 consolidation) method at various confining pressures and thermal gradients. The experimental results indicate that the triaxial compression strength for frozen clay in K0DCGF test increases with the increase of confining pressure, but it decreases as the confining pressure increases further in GFC test. In other words, the compression strength for frozen clay with identical confining pressure decreases with the increase in thermal gradient both in K0DCGF test and GFC test. The strength of frozen clay in K0DCGF test is dependent of pore ice strength, soil particle strength and interaction between soil skeleton and pore ice. The decrease of water content and distance between soil particles leads to the decrease of pore size and the increase of contact area between particles in K0DCGF test, which further results in a higher compression strength than that in GFC test. The compression strength for frozen clay with thermal gradient can be descried by strength for frozen clay with a uniform temperature identical to the temperature at the height of specimen where the maximum tensile stress appears.

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aβ) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aβ levels was detected.

This article describes the application of Contour to the design and discovery of a novel, potent, orally efficacious liver X receptor β (LXRβ) agonist (17). Contour technology is a structure-based drug design platform that generates molecules using a context perceptive growth algorithm guided by a contact sensitive scoring function. The growth engine uses binding site perception and programmable growth capability to create drug-like molecules by assembling fragments that naturally complement hydrophilic and hydrophobic features of the protein binding site. Starting with a crystal structure of LXRβ and a docked 2-(methylsulfonyl)benzyl alcohol fragment (6), Contour was used to design agonists containing a piperazine core. Compound 17 binds to LXRβ with high affinity and to LXRα to a lesser extent, and induces the expression of LXR target genes in vitro and in vivo. This molecule served as a starting point for further optimization and generation of a candidate which is currently in human clinical trials for treating atopic dermatitis.

In order to obtain the earth pressure coefficient at rest (K0) at higher consolidation pressures during secondary compression, a series of K0 tests for saturated reconstituted clay were conducted. The results indicate that the measured K0 in secondary compression can be described by equations related to internal friction angle, secondary compression coefficient, compression index, recompression index, and sediment time. Effects of consolidation pressures and sediment time on K0 during secondary compression can be attributed to cementation (part of cohesion) increase and internal friction angle decrease. Cementation increase leads to nonlinear variation for K0 and internal friction angle decrease results in increase of K0. K0 computed by equations associated with internal friction angle is overestimated at apparent lower consolidation pressures with different sediment time, which agrees with the measured values well at apparent higher consolidation pressures.

In order to study the deformation characteristics of artificial frozen soil with thermal gradient, such as the stress–strain relationship, a series of triaxial compression tests for frozen clay had been conducted by K 0 DCGF (K 0 consolidation, freezing with non-uniform temperature under loading) method and GFC (freezing with non-uniform temperature, isotropic consolidation) method at various consolidation pressures and thermal gradients. Stress–strain curves in K 0 DCGF test present strain softening during shearing process and the elastic strain is approximately 0.001;but which present the strain hardening characteristics in GFC tests and the elastic strain is approximately 0.01. The elastic modulus and peak stress for frozen clay decrease as the thermal gradient increased at different consolidation pressure both in K 0 DCGF test and GFC test. The peak stress and elastic modulus in K 0 DCGF test are significant independent on the pressure melting and crushing phenomena occurring in GFC test. To describe the shear deformation characteristics for frozen clay with thermal gradient, the exponent and power equations considering the correction equation on thermal gradient and model parameters from frozen clay with uniform temperature are developed .The results indicated that the proposed equations can reproduce the shear deformation well both in K 0 DCGF test and GFC test.

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ~60 Å, twice the ~30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multi-domain proteins.

Many Gram-negative pathogens utilize type III secretion systems (T3SS) to translocate effector proteins into eukaryotic host cells. Expression of T3SS genes is highly regulated and is often coupled to type III secretory activity. Transcription of the Pseudomonas aeruginosa T3SS genes is coupled to secretion by a cascade of interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is an activator of type III gene transcription, ExsD binds ExsA to inhibit transcription, ExsC inhibits ExsD activity, and ExsE inhibits ExsC activity. The entire process is coupled to secretion by virtue of the fact that ExsE is a secreted substrate of the T3SS. Changes in the intracellular concentration of ExsE are thought to govern formation of the ExsC-ExsE, ExsC-ExsD, and ExsD-ExsA complexes. Whereas formation of the ExsC-ExsE complex allows ExsD to bind ExsA and transcription of the T3SS is repressed, formation of the ExsC-ExsD complex sequesters ExsD from ExsA and transcription of the T3SS is induced. In this study, we characterized the self-association states of ExsC, ExsD, and ExsE and the binding interactions of ExsC with ExsE and ExsD. ExsC exists as a homodimer and binds one molecule of ExsE substrate. Dimeric ExsC also interacts directly with ExsD to form a heterotetrameric complex. The difference in binding affinities between the ExsC-ExsE (K(d) 1 nm) and ExsC-ExsD (K(d) 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

The flagellum and injectisome of gram-negative bacteria are large multiprotein organelles spanning the inner and outer membranes (12). Whereas flagella are involved in motility, injectisomes function by injecting toxins into eukaryotic host cells. The biogenesis of both organelles requires a dedicated protein export system referred to as a type III secretion system (T3SS). Many of the individual protein constituents of flagella and injectisomes are first secreted via the T3SS before assembly into the complete organelles (10). The flagellar T3SS is located at the base of the basal body in the inner membrane, where it secretes proteins required for assembly of the flagellar rod, hook, and filament (12). The T3SS associated with the injectisome is also located at the base of a basal body-like structure and secretes proteins required for injectisome assembly as well as the toxins destined for delivery to host cells (10). An interesting regulatory feature of both the flagellum and injectisome systems is the direct coupling of transcription with secretion (16). The prototypical example of this regulatory mechanism is seen in Salmonella spp., where genes required for assembly of the flagellar basal body, hook, and filament are expressed in a temporal hierarchy (1). The late genes, required for the final stage of flagellar biogenesis, are not expressed until the basal body is completely assembled. Prior to completion of the basal body complex, the FlgM anti-σ factor accumulates in the cytoplasm, where it binds to and inhibits the FliA σ factor required for late gene transcription. Upon completion of the basal body complex, however, there is a switch in the substrate specificity of the T3SS, FlgM is secreted from cells, and FliA-dependent transcription of late genes ensues. The coupling of transcription to secretion provides an elegant means of coordinating gene expression with the different stages of the flagellar assembly process. Secretion competence also serves as an inducing signal for transcription in the injectisome system (16). Unlike the case in the flagellar system, however, where FlgM secretion is linked to basal body assembly, secretion competence in many injectisome systems is controlled by environmental signals (2, 14, 15, 18, 19, 27). Through a poorly understood mechanism, these environmental signals convert the type III secretion machinery from a closed (secretion incompetent) conformation to an open (secretion competent) conformation. Conversion to the open state triggers a regulatory cascade resulting in transcriptional activation. Mechanistically, these regulatory cascades fall into one of the following three general categories: (i) secretion of a negative regulatory factor, seen in Yersinia spp.; (ii) sequestration of a coactivator, as reported for Salmonella and Shigella spp.; and (iii) sequestration of a negative regulatory factor, as recently described for Pseudomonas aeruginosa (6, 7, 13, 14, 19-21). Transcription of the P. aeruginosa T3SS is induced under Ca2+-limiting growth conditions or following contact of the bacterium with a host cell (9, 22). Although the mechanism of activation by host cell contact is unclear, the low-Ca2+ signal converts the T3SS machinery from a secretion-incompetent to a secretion-competent state (14). Recent studies have demonstrated that transcription of the T3SS is intimately linked to the activity of the type III secretion machinery. Transcription is repressed when the T3SS machinery is secretion incompetent and is derepressed when the machinery is secretion competent (7, 14, 21). The mechanism of coupling transcription to secretion involves a cascade of four interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is a positive activator of T3SS transcription, while ExsD functions as an antiactivator by binding to and inhibiting ExsA activity (11, 14, 25, 26). ExsC functions as an anti-antiactivator by binding to and inhibiting the negative regulatory activity of ExsD and also as a type III-specific chaperone for ExsE (7, 21). Finally, ExsE binds to ExsC and inhibits its activity (20, 21). A key feature of the system is that ExsE is secreted from cells under low-Ca2+ conditions. Based on these findings, the following model has been proposed to account for the coupling of transcription with secretion (7, 21). Under high-Ca2+ conditions, intracellular ExsE binds to and sequesters ExsC, and ExsD is preferentially bound to ExsA, resulting in the inhibition of ExsA-dependent transcription. Under low-Ca2+ conditions, however, ExsE is secreted from cells. This results in a shift in the binding equilibrium whereby ExsD is preferentially bound to ExsC, and liberated ExsA is made available to activate transcription of the T3SS. In the present study, we characterize the ExsC-ExsD binding interaction. Both ExsC and ExsD were found to form self-associated multimeric complexes. ExsC and ExsD readily form a complex when coexpressed in vivo or when individually purified and mixed in vitro. Biochemical analyses suggest that formation of the complex is thermodynamically favorable and consists of multiple copies of ExsC and ExsD. Finally, an ExsD mutant unable to interact with ExsC was isolated and found to have a hyperrepressive phenotype.

The quorum-sensing transcriptional activator TraR of Agrobacterium tumefaciens, which controls the replication and conjugal transfer of the tumour-inducing (Ti) virulence plasmid, is inhibited by the TraM antiactivator. The crystal structure of TraM reveals this protein to form a homodimer in which the monomer primarily consists of two long coiled alpha-helices, and one of the helices from each monomer also bundles to form the dimeric interface. The importance of dimerization is addressed by mutational studies in which disruption of the hydrophobic dimer interface leads to aggregation of TraM. Biochemical studies confirm that TraM exists as a homodimer in solution in equilibrium with the monomeric form, and also establish that the TraM-TraR complex is a heterodimer. Thus, the TraM homodimer undergoes dissociation in forming the antiactivation complex. Combined with the structure of TraR (Zhang et al., 2002, Nature 417: 971-974; Vannini et al., 2002, EMBO J 21: 4393-4401), our structural analysis suggests overlapping interactive surfaces in homodimeric TraM with those in the TraM-TraR complex and a mechanism for TraM inhibition on TraR.

Bacteria can communicate via diffusible signal molecules they generate and release to coordinate their behavior in response to the environment. Signal molecule concentration is often proportional to bacterial population density, and when this reaches a critical concentration, reflecting a bacterial quorum, specific behaviors including virulence, symbiosis, and horizontal gene transfer are activated. Quorum-sensing regulation in many Gram-negative bacteria involves acylated homoserine lactone signals that are perceived through binding to LuxR-type, acylated-homoserine-lactone-responsive transcription factors. Bacteria of the rhizobial group employ the LuxR-type transcriptional activator TraR in quorum sensing, and its activity is further regulated through interactions with the TraM antiactivator. In this study, we have crystallographically determined the 3D structure of the TraR–TraM antiactivation complex from Rhizobium sp. strain NGR234. Unexpectedly, the antiactivator TraM binds to TraR at a site distinct from its DNA-binding motif and induces an allosteric conformational change in the protein, thereby preventing DNA binding. Structural analysis reveals a highly conserved TraR–TraM interface and suggests a mechanism for antiactivation complex formation. This structure may inform alternative strategies to control quorum-sensing-regulated microbial activity including amelioration of infectious disease and antibiotic resistance. In addition, the structural basis of antiactivation presents a regulatory interaction that provides general insights relevant to the field of transcription regulation and signal transduction.

Quorum sensing is a community behavior that bacteria utilize to coordinate a variety of population density-dependent biological functions. In Agrobacterium tumefaciens , quorum sensing regulates the replication and conjugative transfer of the tumor-inducing (Ti) plasmid from pathogenic strains to nonpathogenic derivatives. Most of the quorum-sensing regulatory proteins are encoded within the Ti plasmid. Among these, TraR is a LuxR-type transcription factor playing a key role as the quorum-sensing signal receptor, and TraM is an antiactivator that antagonizes TraR through the formation of a stable oligomeric complex. Recently, a second TraM homologue called TraM2, not encoded on the Ti plasmid of A. tumefaciens A6, was identified, in addition to a copy on the Ti plasmid. In this report, we have characterized TraM2 and its interaction with TraR and solved its crystal structure to 2.1 Å. Like TraM, TraM2 folds into a helical bundle and exists as homodimer. TraM2 forms a stable complex ( K d = 8.6 nM) with TraR in a 1:1 binding ratio, a weaker affinity than that of TraM for TraR. Structural analysis and biochemical studies suggest that protein stability may account for the difference between TraM2 and TraM in their binding affinities to TraR and provide a structural basis for L54 in promoting structural stability of TraM.

Agrobacterium tumefaciens wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the traM gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated traM was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second traM homologue. The homologue, designated traM2 , has 64% and 65% identities with traM at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the tra gene promoters. Deletion of traM2 in strain A6 harboring the mutated traM confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.

The quorum-sensing transcriptional activator TraR of Agrobacterium tumefaciens, which controls the replication and conjugal transfer of the tumour-inducing (Ti) virulence plasmid, is inhibited by the TraM antiactivator. The crystal structure of TraM reveals this protein to form a homodimer in which the monomer primarily consists of two long coiled alpha-helices, and one of the helices from each monomer also bundles to form the dimeric interface. The importance of dimerization is addressed by mutational studies in which disruption of the hydrophobic dimer interface leads to aggregation of TraM. Biochemical studies confirm that TraM exists as a homodimer in solution in equilibrium with the monomeric form, and also establish that the TraM-TraR complex is a heterodimer. Thus, the TraM homodimer undergoes dissociation in forming the antiactivation complex. Combined with the structure of TraR (Zhang et al., 2002, Nature 417: 971-974; Vannini et al., 2002, EMBO J 21: 4393-4401), our structural analysis suggests overlapping interactive surfaces in homodimeric TraM with those in the TraM-TraR complex and a mechanism for TraM inhibition on TraR.