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A trap for the collection of bedbugs, Cimex lectularius Linnaeus (Hemiptera: Cimicidae), is described. The trap was baited with CO2 (50-400 mL/min), heat (37.2-42.2 degrees C) and a chemical lure comprised of 33.0 microg proprionic acid, 0.33 microg butyric acid, 0.33 microg valeric acid, 100 microg octenol and 100 microg L-lactic acid, impregnated into a gel. Laboratory studies, conducted in a square arena measuring 183 cm on each side, showed that traps with and without baits captured adult bedbugs, but traps with CO2 emissions of 50-400 mL/min caught significantly (P0.05) more bedbugs than traps without CO2. In an infested unoccupied apartment, traps with heat and with or without the chemical lure were tested without CO2 on 29 trap-days and with CO2 on 9 trap-days. The numbers of bedbugs captured were 656 and 5898 in traps without and with CO2, respectively. The numbers of bedbugs of all development stages captured were significantly greater in traps with CO2 (chi2 = 15 942, d.f. = 1, P10(-9)). A non-parametric two-way analysis of variance evaluation of six different traps with or without CO2, heat or a chemical lure monitored over 19 trap-days in an infested apartment showed that trap type was highly significant (n = 2833 bedbugs collected) (P10(-7)). The trap with CO2, heat and a chemical lure captured more bedbugs than the other traps, but only caught significantly more fourth and fifth instar nymphs than all other traps. Otherwise, the catches in this trap did not differ significantly from those caught by traps that contained CO2 and heat only. The total numbers of bedbugs collected for each trapping date (pooling all six traps) followed an exponential decline over the trapping period. This type of trap, which caught bedbugs in unoccupied apartments with and without furniture, and in an occupied apartment, may have utility in studying the ecology of bedbugs, in detecting bedbug infestations and in reducing numbers of bites by trapping host-seeking bedbugs.

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-L-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161-1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider "wings" that interact with actin.

Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2Δ Arp2/3 complex) from fission yeast. An x-ray crystal structure of the ΔArp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2Δ Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2Δ Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.

Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P(i), ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the gamma-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.

ATP is required for nucleation of actin filament branches by Arp2/3 complex, but the influence of ATP binding and hydrolysis are poorly understood. We determined crystal structures of bovine Arp2/3 complex cocrystalized with various bound adenine nucleotides and cations. Nucleotide binding favors closure of the nucleotide binding cleft of Arp3, but no large scale conformational changes in the complex. Thus, ATP binding does not directly activate Arp2/3 complex, but is part of a network of interactions that contribute to nucleation. We compared nucleotide-induced conformational changes of residues lining the cleft in Arp3 and actin structures to construct a movie depicting the proposed ATPase cycle for the actin family. Chemical crosslinking stabilized subdomain 1 of Arp2, revealing new electron density for 69 residues in this subdomain. Steric clashes with Arp3 appear to be responsible for intrinsic disorder of subdomains 1 and 2 of Arp2 in inactive Arp2/3 complex.

Summary The arginine-serine (RS)-rich domain of the SR protein ASF/SF2 is phosphorylated by SR protein kinases (SRPKs) and Clk/Sty kinases. However, the mode of phosphorylation by these kinases and their coordination in the biological regulation of ASF/SF2 is unknown. Here, we report the crystal structure of an active fragment of human SRPK1 bound to a peptide derived from an SR protein. This structure led us to identify a docking motif in ASF/SF2. We find that this docking motif restricts phosphorylation of ASF/SF2 by SRPK1 to the N-terminal part of the RS domain — a property essential for its assembly into nuclear speckles. We further show that Clk/Sty causes release of ASF/SF2 from speckles by phosphorylating the C-terminal part of its RS domain. These results suggest that the docking motif of ASF/SF2 is a key regulatory element for sequential phosphorylation by SRPK1 and Clk/Sty and, thus, is essential for its subcellular localization.

Actin-related protein (Arp) 2/3 complex stimulates formation of actin filaments at the leading edge of motile cells. Nucleation of filaments depends on hydrolysis of ATP bound to Arp2. Here we report crystal structures of Arp2/3 complex with bound ATP or ADP. The nucleotides are immobilized on the face of subdomains 3 and 4 of Arp2, whereas subdomains 1 and 2 are flexible and absent from the electron density maps. This flexibility may explain why Arp2 does not hydrolyze ATP until the complex is activated. ATP stabilizes a relatively closed conformation of Arp3 with the γ-phosphate bridging loops from opposite sides of the cleft. ADP binds Arp3 in a unique conformation that favors an open cleft, revealing a conformational change that may occur in actin and Arps when ATP is hydrolyzed and phosphate dissociates. These structures provide the an opportunity to compare all nucleotide-binding states in an actin-related protein and give insights into the function of both the Arp2/3 complex and actin.

There are currently at least forty-six unique protein kinase crystal structures, twenty-four of which are available in an active state. Here we examine these structures using a structural bioinformatics approach to understand how the confromation of the activation segment controls kinase activity.

SR protein kinases (SRPKs) regulate the temporal and cell-specific selection of alternative splice sites. These enzymes are highly unique members of the protein kinase family. SRPKs contain a large domain insert (approximately 200 residues) within the kinase core, do not require phosphorylation for regulation, have an extended helix insert near the nucleotide pocket, and possess unusual substrate specificity determinants. The yeast SRPK, Sky1p, rapidly phosphorylates its natural substrate Npl3 but binds ATP with a high K(m), suggesting that some of these distinctive structural features may be correlated with nucleotide binding [Aubol et al. (2002) Biochemistry 41, 10002-10009]. To address this issue, the nucleotide binding properties of Sky1p were studied using fluorescence spectroscopy. The affinities of several nucleotides (ATP, ADP, AMP, adenosine, and AMPPNP) to Sky1p and the prototype kinase, cAMP-dependent protein kinase, were compared in the absence and presence of the metal activator, Mg(2+), using a fluorescence-based displacement assay. The data indicate that Sky1p, unlike cAMP-dependent protein kinase, potently destabilizes the gamma phosphate of ATP. This novel finding suggests that rapid phosphoryl transfer may be facilitated by unique mechanisms in both protein kinases.

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.

We summarize our two sets of controlled experiments designed to see whether single-sex classes within co-educational environments modify students' risk-taking attitudes. In Booth and Nolen (2012b), subjects are in school years 10 and 11, while in Booth et al. (2014), they are first-year university students randomly assigned to single-sex and co-educational classes. Both studies show that while on average females are significantly less likely than men to make risky choices, on exposure to single-sex environments they behave the same as the males. Thus, part of the observed gender difference in behaviour under uncertainty found in previous studies might reflect social learning rather than inherent gender traits. (JEL codes: C9, C91, C92, J16, D01, D80, J16, J24)

Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae. SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active. We have modeled a substrate peptide bound to Sky1p. The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.

We examine the effect of single?sex classes on the pass rates, grades, and course choices of students in a coeducational university. We randomly assign students to all?female, all?male, and coed classes and, therefore, get around the selection issues present in other studies on single?sex education. We find that one hour a week of single?sex education benefits females: females are 7.5% more likely to pass their first year courses and score 10% higher in their required second year classes than their peers attending coeducational classes. We find no effect of single?sex education on the subsequent probability that a female will take technical classes and there is no effect of single?sex education for males. Furthermore we are able to examine potential mechanisms and indirect effects of single?sex education. We find that the effects of single?sex education do not appear to be driven by a tracking mechanism and that there are indirect effects on class attendance and completion of optional assignments for females. However, the indirect effects cannot explain much of the effect of single-sex education for females.

The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp2 and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a "cryptic dimer" that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Here we characterize three key insertions within the actin core of Arp3 and show that each one plays a distinct role in modulating Arp2/3 function. The β4/β5 insert mediates interactions of Arp2/3 complex with actin filaments and "dampers" the nucleation activity of the complex. The Arp3 hydrophobic plug plays an important role in maintaining the integrity of the complex but is not absolutely required for formation of the daughter filament nucleus. Deletion of the αK/β15 insert did not constitutively activate the complex, as previously hypothesized. Instead, it abolished in vitro nucleation activity and caused defects in endocytic actin patch assembly in fission yeast, indicating a role for the αK/β15 insert in the activated state of the complex. Biochemical characterization of each mutant revealed steps in the nucleation pathway influenced by each Arp3-specific insert to provide new insights into the structural basis of activation of the complex.

Actin-related protein (Arp) 2/3 complex mediates the formation of actin filament branches during endocytosis and at the leading edge of motile cells. The pathway of branch formation is ambiguous owing to uncertainty regarding the stoichiometry and location of VCA binding sites on Arp2/3 complex. Isothermal titration calorimetry showed that the CA motif from the C terminus of fission yeast WASP (Wsp1p) bound to fission yeast and bovine Arp2/3 complex with a stoichiometry of 2 to 1 and very different affinities for the two sites ( K d s of 0.13 and 1.6 μM for fission yeast Arp2/3 complex). Equilibrium binding, kinetic, and cross-linking experiments showed that ( i ) CA at high-affinity site 1 inhibited Arp2/3 complex binding to actin filaments, ( ii ) low-affinity site 2 had a higher affinity for CA when Arp2/3 complex was bound to actin filaments, and ( iii ) Arp2/3 complex had a much higher affinity for free CA than VCA cross-linked to an actin monomer. Crystal structures showed the C terminus of CA bound to the low-affinity site 2 on Arp3 of bovine Arp2/3 complex. The C helix is likely to bind to the barbed end groove of Arp3 in a position for VCA to deliver the first actin subunit to the daughter filament.

Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.

Single-sex classes within coeducational environments are likely to modify students’ risk-taking attitudes in economically important ways. To test this, we designed a controlled experiment using first year college students who made choices over real-stakes lotteries at two distinct dates. Students were randomly assigned to weekly classes of three types: all female, all male, and coeducational. They were not allowed to change group subsequently. We found that women are less likely to make risky choices than men at both dates. However, after eight weeks in a single-sex class environment, women were significantly more likely to choose the lottery than their counterparts in coeducational groups. These results are robust to the inclusion of controls for IQ and for personality type, as well as to a number of sensitivity tests. Our findings suggest that observed gender differences in behaviour under uncertainty found in previous studies might partly reflect social learning rather than inherent gender traits.

Arp2/3 complex plays a central role in the de novo nucleation of filamentous actin as branches on existing filaments. The complex must bind ATP, protein activators [e.g., Wiskott-Aldrich syndrome protein (WASp)], and the side of an actin filament to form a new actin filament. Amide hydrogen/deuterium exchange coupled with mass spectrometry was used to examine the structural and dynamic properties of the mammalian Arp2/3 complex in the presence of both ATP and the activating peptide segment from WASp. Changes in the rate of hydrogen exchange indicate that ATP binding causes conformational rearrangements of Arp2 and Arp3 that are transmitted allosterically to the Arp complex (ARPC)1, ARPC2, ARPC4, and ARPC5 subunits. These data are consistent with the closure of nucleotide-binding cleft of Arp3 upon ATP binding, resulting in structural rearrangements that propagate throughout the complex. Binding of the VCA domain of WASp to ATP-Arp2/3 further modulates the rates of hydrogen exchange in these subunits, indicating that a global conformational reorganization is occurring. These effects may include the direct binding of activators to Arp3, Arp2, and ARPC1; alterations in the relative orientations of Arp2 and Arp3; and the long-range transmission of activator-dependent signals to segments proposed to be involved in binding the F-actin mother filament.

Using a controlled experiment, we investigate if individuals’ risk preferences are affected by (i) the gender composition of the group to which they are randomly assigned, and (ii) the gender mix of the school they attend. Our subjects, from eight publicly funded single-sex and coeducational schools, were asked to choose between a real-stakes lottery and a sure bet. We found that girls in an all-girls group or attending a single-sex school were more likely than their coed counterparts to choose a real-stakes gamble. This suggests that observed gender differences in behaviour under uncertainty found in previous studies might reflect social learning rather than inherent gender traits. It is well known that women are under-represented in high-paying jobs and in highlevel occupations. Recent work in experimental economics has examined to what degree this under-representation may be due to innate differences between men and women. For example, gender differences in risk aversion, feedback preferences or fondness for competition may help to explain some of the observed gender disparities. If the majority of remuneration in high-paying jobs is tied to bonuses based on a company’s performance, then, if men are less risk averse than women, women may choose not to take high-paying jobs because of the uncertainty. Differences in risk attitudes may even affect individual choices about seeking performance feedback or entering a competitive environment. Understanding the extent to which risk attitudes are innate or shaped by environment is important for policy. If risk attitudes are innate, under-representation of women in certain areas may be solved only by changing the way in which remuneration is rewarded. However, if risk attitudes are primarily shaped by the environment, changing the educational or training context could help to address under-representation. Thus, the policy prescription for dealing with under-representation of women in high-paying jobs will depend upon whether the reason for the absence is innate to an individual’s gender. Why women and men might have different preferences or risk attitudes has been discussed but not tested by economists. Broadly speaking, those differences may be due to either nurture, nature or some combination of the two. For example, boys are pushed to take risks when participating in competitive sports, whereas girls are often encouraged to remain cautious. Thus, the riskier choices made by men could be due to

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161–1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider “wings” that interact with actin.

This memo summarizes simple calculations and results of the thermal analysis on the second stripper to be used in the driver linac of Rare Isotope Accelerator (RIA). Both liquid (Sodium) and solid (Titanium and Vanadium) stripper concepts were considered. These calculations were intended to provide basic information to evaluate the feasibility of liquid (thick film) and solid (rotating wheel) second strippers. Nuclear physics calculations to estimate the volumetric heat generation in the stripper material were performed by 'LISE for Excel'. In the thermal calculations, the strippers were modeled as a thin 2D plate with uniform heat generation within the beam spot. Then, temperature distributions were computed by assuming that the heat spreads conductively in the plate in radial direction without radiative heat losses to surroundings.

Arp2/3 complex nucleates branched actin filaments responsible for powering diverse cellular processes, including cellular motility and endocytosis. WASP-mediated activation of Arp2/3 complex requires preformed filaments, ensuring that nucleation and branching are coupled, but leading to the biochemical consequence that branched network assembly cannot be initiated without a preformed seed filament. We recently discovered a class of Arp2/3 complex activators, the WISH/DIP/SPIN90 (WDS) proteins, that activate Arp2/3 complex without preformed filaments. WDS proteins have several sequence features distinct from WASP proteins, making their mechanism of activation unclear. In addition it is unknown if linear filaments produced by WDS-activated Arp2/3 complex can seed assembly of branched actin networks. Here we use single molecule TIRF microscopy to determine the mechanism of Dip1, a WDS protein from fission yeast. We directly demonstrate that linear filaments nucleated by Dip1 and Arp2/3 complex are substrates for branching by WASP-bound Arp2/3 complex. Dip1 remains bound to Arp2/3 complex at the pointed ends of the linear filaments it nucleates for hundreds of seconds, unlike WASP proteins, which are released before nucleation. Using engineered cysteine crosslinking, we show that a structural change in the complex that brings Arp2 and Arp3 into a filament-like (short pitch) conformation bypasses the need for Wsp1 but not actin filaments in activation. Therefore, we propose a model in which a step-wise set of conformational changes activate Arp2/3 complex. This model explains both how Dip1 bypasses the need for preformed actin filaments and why Wsp1 requires them, thereby providing important new insights into the molecular regulation of the assembly of actin filament networks.

Employment brings access to many types of benefits, both financial and psychological. Besides a wage, one can earn a sense of pride or a feeling of connection with her society or fellow employees. How attached one is to the labor market is not an issue that the usual unemployment rate deals with, though. Being unemployed one may not be able to gain access to health care, information about job openings or other non-pecuniary benefits. Knowing a person who is employed may mitigate some of this isolation but, as recent riots in France and the passing of the National Rural Employment Guarantee in India have shown, simply ignoring the issue of whether someone has access to employment opportunities is not necessarily a desirable option. Taking account of the distribution of employment across households as well as individuals is one way to deal with these issues and improve on the usual unemployment measure. In this paper I develop a new measure of unemployment, that, building on the insights of the literacy and social network literature, is sensitive to the distribution of unemployment across both households and individuals. With this class of measures characterized, I then show some empirical implications and test a main assumption used in creating the measure.

We discuss an object-oriented, component-based architecture for a parallel reservoir simulator. The architecture successfully balances the need for extensibility, maintainability, and reuse with the need for efficient computation. Parallelism is hidden from the application developer via a set of abstractions and a unifying framework that supports static and dynamic load balancing on unstructured and structured grids. We explain how the simulator architecture supports black oil and compositional models within a general formulation, and how it achieves parallel scalability for all the major components: property calculation, Jacobian construction, linear solver, well model, input/output etc. Some initial results are discussed for a selection of models ranging from standard benchmarks to large complex field cases.MotivationCurrent generation reservoir simulators typically have a lifespan of at least 10 years. As a result, many of the simulators in widespread use have their roots in petroleum engineering, software engineering and computing technologies that were originally targeted at structured grids and scalar architectures. Advances in software and hardware technologies, parallel computing, advanced gridding and modern software engineering techniques have created new opportunities. At the same time, evolving workflows involving complex geological models, large full-field simulations, near well studies, production and facilities optimization, and stochastic modeling have placed new demands on reservoir simulation technology.Although object-oriented design has been the main staple of software development for quite some time, its application to scientific programming has been hindered somewhat by the perceived inefficiency of run-time polymorphism. However, in recent years the emergence of compilers that are in full compliance with the C++ standard has made use of generic programming techniques commercially practical. Generic programming offers the benefits of encapsulation and code reuse without the overhead of virtual function calls1,2. Object- oriented and generic programming techniques using C++ have been shown to be useful in the development of scientific and numerical applications3,4.Large-scale object-oriented frameworks for scientific computing have been developed in many contexts5, and parallelism has been addressed for example in the POOMA project6. Object-oriented architectures for flow simulations have been developed in other industries7 and these techniques have been applied to reservoir simulators by several practitioners within academia8,9,10,11,12 and also in industry13.

SR proteins, named for their multiple arginine/serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF/SF2. By using the start–trap strategy, we monitored the progress curve for ASF/SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF/SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme–substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF/SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains “locked” onto SRPK1 during RS domain modification.

Conformational changes are thought to play a key role in the function of active protein kinases, although little is known about how these changes relate to the mechanism of phosphorylation. Here we present four high-resolution structures of a single crystal form of Sky1p, a constitutively active serine kinase implicated in yeast RNA processing, each in a different state of nucleotide binding. By comparing the apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformational changes caused by ATP binding or conversion from nucleotide reactant to product. Rotation of the small lobe of the kinase closes the cleft upon binding, allowing the nucleotide to interact with residues from both lobes of the kinase, although some interactions thought to be important for phosphotransfer are missing in the ATP-containing structure. In the apoenzyme, a kinase-conserved phosphate-anchoring loop is in a twisted conformation that is incompatible with ADP and ATP binding, providing a potential mechanism for facilitating ADP release in Sky1p. The nonhydrolyzable ATP analogue AMP-PNP binds in a unique mode that fails to induce lobe closure. This observation, along with comparisons between the two independent molecules in the asymmetric unit of each structure, has provided new molecular details about how the nucleotide binds and induces closure. Finally, we have used mutational analysis to establish the importance of a glycine within the linker that connects the two lobes of Sky1p.

Group technology (GT) seeks to exploit similarities among parts or products for efficiencies in design and manufacturing. Production Flow Analysis (PFA) seeks to exploit similarities in processes. Applications of GT in Computer Aided Process Planning (CAPP), and GT and PFA in cellular manufacturing (CM) have produced very significant, and well-publicized productivity improvements compared to traditional methods; yet a vast majority of firms are yet to use them.

Arp2/3 complex is a seven subunit assembly that nucleates actin filaments from the sides of pre-existing filaments, creating branched actin networks. Recently reported small molecule inhibitors of Arp2/3 complex, CK-666 and CK-869, block its nucleation activity. While these inhibitors have the potential to become powerful tools to investigate actin remodeling in vivo, it is currently not known how they inhibit Arp2/3 complex. To determine the mechanism of these inhibitors, we conducted biochemical/biophysical analysis and solved the crystal structures of CK-666 and CK-869 bound to Arp2/3 complex. Despite the fact that CK-869 and CK-666 bind to distinct sites on the complex, both compounds function by blocking an activating conformational change stimulated by dimeric VCA and two actin monomers. Chemical crosslinking, analytical ultracentrifugation, and small angle x-ray scattering demonstrate that this conformational change moves Arp2 and Arp3 together to adopt the short pitch conformation, mimicking two consecutive actin subunits in an actin filament and is hypothesized to form the nucleus for the daughter filament. Analysis of the crystal structures shows that CK-666 binds to a pocket between Arp3 and Arp2 formed only in the inactive conformation, stabilizing the inactive state. in contrast, binding of CK-869 locks a surface loop in Arp3 in a position, destabilizing the short pitch conformation. Neither inhibitor actively dissociates preformed branches, nor significantly affects binding of ATP or nucleation promoting factors to the complex or binding of Arp2/3 complex to the sides or ends of actin filaments. Therefore, CK-666 and CK-869 block a late step in the nucleation pathway by preventing a large-scale conformational change. These results have important implications for understanding the branching nucleation mechanism and will be critical in interpretation of the affects of the inhibitors on Arp2/3 complex activity in vivo.

To compare prothrombin time (PT), activated partial thromboplastin time (aPTT), and factor VII values in concurrent blood samples obtained by direct venipuncture and from a peripheral venous catheter.Concurrent samples obtained from catheters and by direct venipuncture were studied. In a separate crossover bioequivalence assessment of DNA-derived factor VIIa (rFVIIa) from two different batches, sample results of each technique were compared.University hospital clinical research unit.Six patients with hemophilia A under nonbleeding conditions.The patients received a single dose of rFVIIa 70 micrograms/kg administered by intravenous push over 2 minutes. Concurrent blood samples were collected at 2, 3, 4, 6, 8, 10, and 12 hours after rFVIIa administration. Catheter blood samples were drawn from a three-way stopcock attached to an 18-gauge peripheral venous catheter in the patient's forearm and connected to an intravenous solution of 5% dextrose with half normal saline maintained at a rate of 30 ml/hour. Venipuncture samples were drawn from the opposite arm.The PT and aPTT values were determined by using a BBL Fibrometer (PT) and a Coagamate X-2 with automated aPTT reagent. Blood samples were analyzed for factor VII concentration using the Novo Clot assay. The mean venipuncture-obtained PT (8.9 +/- 1.0 sec) and aPTT (48.7 +/- 13.6 sec) values were numerically equivalent to mean catheter-derived PT (9.0 +/- 1.0 sec) and aPTT (48.3 +/- 12.5 sec) results, as were mean venipuncture and catheter-obtained FVII:C values.The PT and aPTT values determined after venipuncture and through the peripheral catheter were not statistically different (p0.05) when compared by paired or unpaired analysis. Similarly, values of FVII:C measured after venipuncture were statistically equivalent to those after sampling through the peripheral catheter. All six patients preferred the catheter method of blood collection over venipuncture.

We report a measurement of the lifetime of 22S1/2 level in hydrogenlike 84Kr and the first determination of the branching ratio for the M1 decay for this Z=36 one‐electron ion. The lifetime of this state was measured by the beam‐foil time‐of‐flight method, in which the foil was moved relative to a fixed, collimated Si(Li) detector. An analysis based on the single photon spectra from a tightly collimated detector led to the measurements of the lifetime of 22S1/2, the two‐photon emission rate, the M1 emission rate and the branching ratio of M1 decay. All these exprimental results are in good agreement with the theoretical calculations of Parpia and Johnson, and Goldman and Drake.