Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods

Evolution favors protein mutational robustness in sufficiently large populations

BACKGROUND: An important question is whether evolution favors properties such as mutational robustness or evolvability that do not directly benefit any individual, but can influence the course of future evolution. Functionally similar proteins can differ substantially in their robustness to mutations and capacity to evolve new functions, but it has remained unclear whether any of these differences might be due to evolutionary selection for these properties. RESULTS: Here we use laboratory experiments to demonstrate that evolution favors protein mutational robustness if the evolving population is sufficiently large. We neutrally evolve cytochrome P450 proteins under identical selection pressures and mutation rates in populations of different sizes, and show that proteins from the larger and thus more polymorphic population tend towards higher mutational robustness. Proteins from the larger population also evolve greater stability, a biophysical property that is known to enhance both mutational robustness and evolvability. The excess mutational robustness and stability is well described by existing mathematical theories, and can be quantitatively related to the way that the proteins occupy their neutral network. CONCLUSIONS: Our work is the first experimental demonstration of the general tendency of evolution to favor mutational robustness and protein stability in highly polymorphic populations. We suggest that this phenomenon may contribute to the mutational robustness and evolvability of viruses and bacteria that exist in large populations

Pseudomonas aeruginosa Eliminates Natural Killer Cells via Phagocytosis-Induced Apoptosis

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells

IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems

Salvage of contaminated osteochondral allografts: the effects of chlorhexidine on human articular chondrocyte viability.

Because chondrocyte viability is imperative for successful osteochondral allograft transplantation, sterilization techniques must provide antimicrobial effects with minimal cartilage toxicity. Chlorhexidine gluconate (CHG) is an effective disinfectant; however, its use with human articular cartilage requires further investigation.To determine the maximal chlorhexidine concentration that does not affect chondrocyte viability in allografts and to determine whether this concentration effectively sterilizes contaminated osteoarticular grafts.Controlled laboratory study.Osteochondral plugs were subjected to pulse lavage with 1-L solutions of 0.002\%, 0.01\%, 0.05\%, and 0.25\% CHG and cultured for 0, 1, 2, and 7 days in media of 10\% fetal bovine serum and antibiotics. Chondrocyte viability was determined via LIVE/DEAD Viability Assay. Plugs were contaminated with Staphylococcus aureus and randomized to 4 treatment groups. One group was not contaminated; the 3 others were contaminated and received no treatment, saline pulse lavage, or saline pulse lavage with 0.002\% CHG. Serial dilutions were plated and colony-forming units assessed.The control group and the 0.002\% CHG group showed similar cell viability, ranging from 67\% \ub1 4\% to 81\% \ub1 22\% (mean \ub1 SD) at all time points. In the 0.01\% CHG group, cell viability was reduced in comparison with control by 2-fold at day 2 and remained until day 7 (P 0.002\% significantly decreases chondrocyte viability within 1 to 2 days after exposure and should therefore not be used for disinfection of osteochondral allograft. Pulse lavage does not affect chondrocyte viability but cannot be used in isolation to sterilize contaminated fragments. Overall, 0.002\% CHG was shown to effectively decontaminate osteoarticular fragments.This study offers a scientific protocol for sterilizing osteochondral fragments that does not adversely affect cartilage viability

Pseudomonas aeruginosa type III-secreted toxin ExoT inhibits host-cell division by targeting cytokinesis at multiple steps

Pseudomonas aeruginosa is an opportunistic pathogen that requires preexisiting epithelial injury to cause acute infections. We report that P. aeruginosa inhibits mammalian cytokinesis in a type III secretion system and exotoxin T (ExoT)-dependent manner. ExoT is a bifunctional type III secretion system effector protein that contains an N-terminal GTPase-activating protein domain and a C-terminal ADP-ribosyl transferase domain. Each of its domains inhibits cytokinesis in a kinetically, morphologically, and mechanistically distinct manner. The GTPase-activating protein-mediated inhibition of cytokinesis occurs early, likely as a consequence of its inhibitory effect on RhoA. The ADP-ribosyl transferase domain inhibits late steps of cytokinesis by blocking syntaxin-2 localization to the midbody, an event essential for completion of cytokinesis. These findings provide an example of a bacterial pathogen targeting cytokinesis

The ADP Ribosyltransferase Domain of Pseudomonas aeruginosa ExoT Contributes to Its Biological Activities

ExoT is a type III secreted effector protein found in almost all strains of Pseudomonas aeruginosa and is required for full virulence in an animal model of acute pneumonia. It is comprised of an N-terminal domain with GTPase activating protein (GAP) activity towards Rho family GTPases and a C-terminal ADP ribosyltransferase (ADPRT) domain with minimal activity towards a synthetic substrate in vitro. Consistent with its activity as a Rho family GTPase, ExoT has been shown to inhibit P. aeruginosa internalization into epithelial cells and macrophages, disrupt the actin cytoskeleton through a Rho-dependent pathway, and inhibit wound repair in a scrape model of injured epithelium. We have previously shown that mutation of the invariant arginine of the GAP domain to lysine (R149K) results in complete loss of GAP activity in vitro but only partially inhibits ExoT anti-internalization and cell rounding activity. We have constructed in-frame deletions and point mutations within the ADPRT domain in order to test whether this domain might account for the residual activity observed in ExoT GAP mutants. Deletion of a majority of the ADPRT domain (residues 234 to 438) or point mutations of the ADPRT catalytic site (residues 383 to 385) led to distinct changes in host cell morphology and substantially reduced the ability of ExoT to inhibit in vitro epithelial wound healing over a 24-h period. In contrast, only subtle effects on the efficiency of ExoT-induced bacterial internalization were observed in the ADPRT mutant forms. Expression of each domain individually in Saccharomyces cerevisiae was toxic, whereas expression of each of the catalytically inactive mutant domains was not. Collectively, these data demonstrate that the ADPRT domain of ExoT is active in vivo and contributes to the pathogenesis of P. aeruginosa infections