Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

HIV-1 reverse transcriptase connection domain mutations: dynamics of emergence and implications for success of combination antiretroviral therapy.

BACKGROUND: Factors promoting the emergence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) connection domain mutations and their effect on antiretroviral therapy (ART) are still largely undetermined. We investigated this matter by analyzing genotypic resistance tests covering 400 amino acid positions in the RT of HIV-1 subtype B viruses and corresponding treatment histories and laboratory measurements. METHODS: The emergence of connection domain mutations was studied in 334 patients receiving monotherapy or dual therapy with thymidine analogues at the time of the genotypic resistance test. Response to subsequent combination ART (cART) was analyzed using Cox regression for 291 patients receiving unboosted protease inhibitors. Response was defined by ever reaching an HIV RNA level <50 copies/mL during the first cART. RESULTS: The connection domain mutations N348I, R356K, R358K, A360V, and A371V were more frequently observed in ART-exposed than ART-naive patients, of which only N348I and A360V were nonpolymorphic (with a prevalence of <1.5% in untreated patients). N348I correlated with M184V and predominantly occurred in patients receiving lamivudine and zidovudine concomitantly. A360V was not associated with specific drug combinations and was found to emerge later than M184V or thymidine analogue mutations. Nonpolymorphic connection domain mutations were rarely detected in the absence of established drug resistance mutations in ART-exposed individuals (prevalence, <1%). None of the 5 connection domain mutations associated with treatment showed a statistically significant effect on response to cART. CONCLUSIONS: Despite their frequent emergence, connection domain mutations did not show large detrimental effects on response to cART. Currently, routine implementation of connection domain sequencing seems unnecessary for developed health care settings.

Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers.

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.

Examining the information content of time-lapse crosshole GPR data collected under different infiltration conditions to estimate unsaturated soil hydraulic properties

Time-lapse geophysical data acquired during transient hydrological experiments are being increasingly employed to estimate subsurface hydraulic properties at the field scale. In particular, crosshole ground-penetrating radar (GPR) data, collected while water infiltrates into the subsurface either by natural or artificial means, have been demonstrated in a number of studies to contain valuable information concerning the hydraulic properties of the unsaturated zone. Previous work in this domain has considered a variety of infiltration conditions and different amounts of time-lapse GPR data in the estimation procedure. However, the particular benefits and drawbacks of these different strategies as well as the impact of a variety of key and common assumptions remain unclear. Using a Bayesian Markov-chain-Monte-Carlo stochastic inversion methodology, we examine in this paper the information content of time-lapse zero-offset-profile (ZOP) GPR traveltime data, collected under three different infiltration conditions, for the estimation of van Genuchten-Mualem (VGM) parameters in a layered subsurface medium. Specifically, we systematically analyze synthetic and field GPR data acquired under natural loading and two rates of forced infiltration, and we consider the value of incorporating different amounts of time-lapse measurements into the estimation procedure. Our results confirm that, for all infiltration scenarios considered, the ZOP GPR traveltime data contain important information about subsurface hydraulic properties as a function of depth, with forced infiltration offering the greatest potential for VGM parameter refinement because of the higher stressing of the hydrological system. Considering greater amounts of time-lapse data in the inversion procedure is also found to help refine VGM parameter estimates. Quite importantly, however, inconsistencies observed in the field results point to the strong possibility that posterior uncertainties are being influenced by model structural errors, which in turn underlines the fundamental importance of a systematic analysis of such errors in future related studies.

A pseudo-spectral method for the simulation of poro-elastic seismic wave propagation in 2D polar coordinates using domain decomposition

We present a novel numerical approach for the comprehensive, flexible, and accurate simulation of poro-elastic wave propagation in 2D polar coordinates. An important application of this method and its extensions will be the modeling of complex seismic wave phenomena in fluid-filled boreholes, which represents a major, and as of yet largely unresolved, computational problem in exploration geophysics. In view of this, we consider a numerical mesh, which can be arbitrarily heterogeneous, consisting of two or more concentric rings representing the fluid in the center and the surrounding porous medium. The spatial discretization is based on a Chebyshev expansion in the radial direction and a Fourier expansion in the azimuthal direction and a Runge-Kutta integration scheme for the time evolution. A domain decomposition method is used to match the fluid-solid boundary conditions based on the method of characteristics. This multi-domain approach allows for significant reductions of the number of grid points in the azimuthal direction for the inner grid domain and thus for corresponding increases of the time step and enhancements of computational efficiency. The viability and accuracy of the proposed method has been rigorously tested and verified through comparisons with analytical solutions as well as with the results obtained with a corresponding, previously published, and independently bench-marked solution for 2D Cartesian coordinates. Finally, the proposed numerical solution also satisfies the reciprocity theorem, which indicates that the inherent singularity associated with the origin of the polar coordinate system is adequately handled.

The role of MMP-9 and its fibronectin-like domain in tumor growth & invasion : certainties, controversies & discrepancies

Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-g revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-g in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-g activity in Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-9 revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-9 in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-9 activity in those cells and therefore prevent MMP-9-induced activation of TGF-b, which results in increased invasion. Curiously, xenografts of SW480 colorectal adenocarcinoma cells stably expressing the FN domain of MMP-9 displayed reduced growth at both the primary (subcutaneous) injection site and the lungs of NOD/SCID mice, in experimental metastasis assays, whilst the same cells overexpressing wt MMP-9 showed enhanced growth and dissemination. Gelatin zymography of conditioned medium revealed that these effects may be due to the FN domain, which displaces MMP-9 from SW480 cell surface. These observations suggest a dual role of MMP-9 and its FN domain in primary tumor growth and metastasis, underscoring the notion that the effect of MMP-9 on tumor cells may depend on the cell type and highlighting possible protective effects of MMPs in tumor progression.

Domain growth in binary mixtures at low temperatures

We have studied domain growth during spinodal decomposition at low temperatures. We have performed a numerical integration of the deterministic time-dependent Ginzburg-Landau equation with a variable, concentration-dependent diffusion coefficient. The form of the pair-correlation function and the structure function are independent of temperature but the dynamics is slower at low temperature. A crossover between interfacial diffusion and bulk diffusion mechanisms is observed in the behavior of the characteristic domain size. This effect is explained theoretically in terms of an equation of motion for the interface.

Front and domain growth in the presence of gravity

Front and domain growth of a binary mixture in the presence of a gravitational field is studied. The interplay of bulk- and surface-diffusion mechanisms is analyzed. An equation for the evolution of interfaces is derived from a time-dependent Ginzburg-Landau equation with a concentration-dependent diffusion coefficient. Scaling arguments on this equation give the exponents of a power-law growth. Numerical integrations of the Ginzburg-Landau equation corroborate the theoretical analysis.

The sandstone-hosted Beverley uranium Deposit, Lake Frome Basin, South Australia: Mineralogy, geochemistry, and a time-constrained model for its genesis

The sandstone-hosted Beverley uranium deposit is located in terrestrial sediments in the Lake Frome basin in the North Flinders Ranges, South Australia. The deposit is 13 km from the U-rich Mesoproterozoic basement of the Mount Painter inlier, which is being uplifted 100 to 200 m above the basin by neotectonic activity that probably initiated in the early Pliocene. The mineralization was deposited mainly in organic matter-poor Miocene lacustrine sands and partly in the underlying reductive strata comprising organic matter-rich clays and silts. The bulk of the mineralization consists of coffinite and/or uraninite nodules, growing around Co-rich pyrite with an S isotope composition (delta S-34 = 1.0 +/- 0.3 parts per thousand), suggestive of an early diagenetic lacustrine origin. In contrast, authigenic sulfides in the bulk of the sediments have a negative S isotope signature (delta S-34 ranges from -26.2 to -35.5 parts per thousand), indicative of an origin via bacterially mediated sulfate reduction. Minor amounts of Zn-bearing native copper and native lead also support the presence of specific, reducing microenvironments in the ore zone. Small amounts of carnotite are associated with the coffinite ore and also occur beneath a paleosoil horizon overlying the uranium deposit. Provenance studies suggest that the host Miocene sediments were derived from the reworking of Early Cretaceous glacial or glaciolacustrine sediments ultimately derived from Paleozoic terranes in eastern Australia. In contrast, the overlying Pliocene strata were in part derived from the Mesoproterozoic basement inlier. Mass-balance and geochemical data confirm that granites of the Mount Painter domain were the ultimate source of U and BEE at Beverley. U-Pb dating of coffinite and carnotite suggest that the U mineralization is Pliocene (6.7-3.4 Ma). The suitability of the Beverley deposit for efficient mining via in situ leaching, and hence its economic value, are determined by the nature of the hosting sand unit, which provides the permeability and low reactivity required for high fluid flow and low chemical consumption. These favorable sedimentologic and geometrical features result from a complex conjunction of factors, including deposition in lacustrine shore environment, reworking of angular sands of glacial origin, deep Pliocene weathering, and proximity to an active fault exposing extremely U rich rocks.

n=1/4 domain-growth universality class: Crossover to the n=1/2 class

The kinetic domain-growth exponent is studied by Monte Carlo simulation as a function of temperature for a nonconserved order-parameter model. In the limit of zero temperature, the model belongs to the n=(1/4 slow-growth unversality class. This is indicative of a temporal pinning in the domain-boundary network of mixed-, zero-, and finite-curvature boundaries. At finite temperature the growth kinetics is found to cross over to the Allen-Cahn exponent n=(1/2. We obtain that the pinning time of the zero-curvature boundary decreases rapidly with increasing temperature.

Magnetic properties and domain-wall motion in single-crystal BaFe10.2Sn0.74Co0.66O19

The magnetic properties of BaFe12O19 and BaFe10.2Sn0.74Co0.66O19 single crystals have been investigated in the temperature range (1.8 to 320 K) with a varying field from -5 to +5 T applied parallel and perpendicular to the c axis. Low-temperature magnetic relaxation, which is ascribed to the domain-wall motion, was performed between 1.8 and 15 K. The relaxation of magnetization exhibits a linear dependence on logarithmic time. The magnetic viscosity extracted from the relaxation data, decreases linearly as temperature goes down, which may correspond to the thermal depinning of domain walls. Below 2.5 K, the viscosity begins to deviate from the linear dependence on temperature, tending to be temperature independent. The near temperature independence of viscosity suggests the existence of quantum tunneling of antiferromagnetic domain wall in this temperature range.

Dynamics of transient domain structures in nematic liquid crystals: The splay Fréedericksz transition

We discuss the dynamics of the transient pattern formation process corresponding to the splay Fréedericksz transition. The emergence and subsequent evolution of the spatial periodicity is here described in terms of the temporal dependence of the wave numbers corresponding to the maxima of the structure factor. Situations of perpendicular as well as oblique field-induced stripes relative to the initial orientation of the director are both examined with explicit indications of the time scales needed for their appearance and posterior development.

Distinct levels in Pom1 gradients limit Cdr2 activity and localization to time and position division.

Where and when cells divide are fundamental questions. In rod-shaped fission yeast cells, the DYRK-family kinase Pom1 is organized in concentration gradients from cell poles and controls cell division timing and positioning. Pom1 gradients restrict to mid-cell the SAD-like kinase Cdr2, which recruits Mid1/Anillin for medial division. Pom1 also delays mitotic commitment through Cdr2, which inhibits Wee1. Here, we describe quantitatively the distributions of cortical Pom1 and Cdr2. These reveal low profile overlap contrasting with previous whole-cell measurements and Cdr2 levels increase with cell elongation, raising the possibility that Pom1 regulates mitotic commitment by controlling Cdr2 medial levels. However, we show that distinct thresholds of Pom1 activity define the timing and positioning of division. Three conditions-a separation-of-function Pom1 allele, partial downregulation of Pom1 activity, and haploinsufficiency in diploid cells-yield cells that divide early, similar to pom1 deletion, but medially, like wild-type cells. In these cells, Cdr2 is localized correctly at mid-cell. Further, Cdr2 overexpression promotes precocious mitosis only in absence of Pom1. Thus, Pom1 inhibits Cdr2 for mitotic commitment independently of regulating its localization or cortical levels. Indeed, we show Pom1 restricts Cdr2 activity through phosphorylation of a C-terminal self-inhibitory tail. In summary, our results demonstrate that distinct levels in Pom1 gradients delineate a medial Cdr2 domain, for cell division placement, and control its activity, for mitotic commitment.

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.