Can Simpson's paradox explain co-operation in Pseudomonas aeruginosa biofilms?

Co-operative behaviours, such as the production of public goods, are commonly displayed by bacteria in biofilms and can enhance their ability to survive in environmental or clinical settings. Non-co-operative cheats commonly arise and should, theoretically, disrupt co-operative behaviour. Its stability therefore requires explanation, but no mechanisms to suppress cheating within biofilms have yet been demonstrated experimentally. Theoretically, repeated aggregation into groups, interleaved with dispersal and remixing, can increase co-operation via a 'Simpson's paradox'. That is, an increase in the global proportion of co-operators despite a decrease in within-group proportions, via differential growth of groups. We investigate the hypothesis that microcolony formation and dispersal produces a Simpson's paradox that explains bacterial co-operation in biofilms. Using the production of siderophores in Pseudomonas aeruginosa as our model system for co-operation, we use well-documented co-operator and siderophore-deficient cheat strains to measure the frequency of co-operating and cheating individuals, in-situ within-microcolony structures. We detected significant within-type negative density-dependant effects that vary over microcolony development. However, we find no evidence of Simpson's paradox. Instead, we see clear within-microcolony spatial structure (cheats occupying the interior portions of microcolonies) that may violate the assumption required for Simpson's paradox that group members share equally in the public good.     

Centralized mouse repositories

Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world.     

Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis

Siah proteins are ubiquitin-protein isopeptide ligases (E3) that have been implicated in a variety of cellular actions, including promotion of apoptotic death. Here, we show that Siah1 is a binding partner for POSH (plenty of SH3s), a scaffold component of the apoptotic JNK pathway, and that Siah contributes to death of neurons and other cell types by activating the JNK pathway. Such proapoptotic activity requires the E3 ligase activity of Siah1. Moreover, apoptotic stimuli markedly elevate cellular Siah1 levels by a mechanism reliant on Siah1 protein stabilization. This stabilization requires JNK pathway activation and interaction with POSH and is enhanced by phosphorylation of SIAH1 at tyrosines 100 and 126. Depletion of intracellular Siah proteins via small interference RNA partially protects cells from death evoked by apoptotic stimuli such as trophic factor deprivation and DNA damage. These findings thus reveal a "loop" mechanism in which the JNK pathway promotes SIAH1 stabilization and in which SIAH1 in turn activates the JNK pathway and, ultimately, contributes to cell death.     

The polycystin 1-C-terminal fragment stimulates ERK-dependent spreading of renal epithelial cells

Polycystin 1, the product of the PKD1 gene, is mutated in autosomal dominant polycystic kidney disease, a disease characterized by renal cyst formation and progressive renal failure. We show that expression of the C-terminal domain of human polycystin-1 (PKD1-CT) triggers spreading of isolated inner medullary collecting duct cells, a process mediated by Erk. As inner medullary collecting duct cells spread, PKD1-CT localizes to cell-extracellular matrix contacts, interacts with focal adhesion proteins Fak and paxillin, and stimulates Fak phosphorylation, paxillin phosphorylation, Fak-paxillin association, and formation of small focal complexes. PKD1-CT-mediated spreading requires membrane localization and the integrity of the C-terminal protein binding sites. We additionally show that Pkd1 null proximal tubule cells generated from Pkd1(flox/-):TSLargeT mice by in vitro Cre recombinase transfection demonstrate diminished spreading when compared with Pkd(flox/-) heterozygous parental cells. These findings suggest that membrane-bound PC1 has a central role in regulating morphogenic protein signaling at cell-matrix interfaces in non-confluent cells.     

A hypothesis for the function of braking forces during running turns

We examined the functional role of braking forces observed when humans execute turning maneuvers. Deceleration caused by braking forces contributes to changing the movement direction of the center of mass (COM) and maintaining constant velocity. We argue that braking forces also prevent over-rotation of the body about the vertical axis during maneuvers. We analyzed data from sidestep and crossover cuts at average initial running velocities of 3 m s(-1). Absent braking, lateral forces would result in body rotations 1.4-3 times the change in COM movement direction, causing the orientation of the body to be substantially mis-aligned with the direction of movement at the end of the step. A simple model based on the hypothesis that body rotation should match COM deflection can explain 70% of the variance in braking forces employed during running turns.     

Structure-activity profiles of Ab-derived TNF fusion proteins

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD(50) of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.     

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.     

Modulation of the turnover of formamidopyrimidine DNA glycosylase

In recent years, significant progress has been made in determining the catalytic mechanisms by which base excision repair (BER) DNA glycosylases and glycosylase-abasic site (AP) lyases cleave the glycosyl bond. While these investigations have identified active site residues and active site architectures, few investigations have analyzed postincision turnover events. Previously, we identified a critical residue (His16) in the T4-pyrimidine dimer glycosylase (T4-Pdg) that, when mutated, interferes with enzyme turnover [Meador et al. (2004) J. Biol. Chem. 279, 3348-3353]. To test whether comparable residues and mechanisms might be operative for other BER glycosylase:AP-lyases, molecular modeling studies were conducted comparing the active site regions of T4-Pdg and the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg). These analyses revealed that His71 in Fpg might perform a similar function to His16 in T4-Pdg. Site-directed mutagenesis of the Fpg gene and analyses of the reaction mechanism of the mutant enzyme revealed that the H71A enzyme retained activity on a DNA substrate containing an 8-oxo-7,8-dihydroguanine (8-oxoG) opposite cytosine and DNA containing an AP site. The H71A Fpg mutant was severely compromised in enzyme turnover on the 8-oxoG-C substrate but had turnover rates comparable to that of wild-type Fpg on AP-containing DNA. The similar mutant phenotypes for these two enzymes, despite a complete lack of structural or sequence homology between them, suggest a common mechanism for the rate-limiting step catalyzed by BER glycosylase:AP-lyases.     

Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis

Previous work [Sharp, K. H., et al. (2003) Nat. Struct. Biol. 10, 303-307] has revealed the location of the ascorbate binding site in ascorbate peroxidase and has identified hydrogen-bonding interactions to Arg172, Lys30, and the heme 6-propionate as important in formation of the enzyme-substrate complex. In this work, the individual and collective contributions of these hydrogen bond interactions have been dissected using site-directed mutagenesis, steady-state and pre-steady-state kinetics, X-ray crystallography, and modified substrate analogues. Steady-state and pre-steady-state kinetic data reveal that the hydrogen bonds to Arg172 and the heme 6-propionate play a major part in stabilization of the bound ascorbate but that the interaction with Lys30 plays only a minor role. Binding of aromatic substrates is not affected by substitutions at Arg172/Lys30. Neutralization or removal of electrostatic charge at (Lys30) or adjacent to (Lys31) the ascorbate site does not substantially disrupt the binding interaction. Substrate oxidation and reduction of Compounds I and II is still possible in the absence of Arg172, but at a much reduced level. Crystallographic data (to 1.8 A) for the R172A variant indicate that the molecular structure of the proposed [Sharp, K. H., et al. (2004) Biochemistry 43, 8644-8651] proton transfer pathway from the ascorbate to the heme is conserved, which accounts for the residual activity. The results are discussed in terms of our wider understanding of the structural features that control substrate binding specificity in other peroxidase enzymes.