Extracellular protein profiles from wild-type and regulatory or secretory isogenic mutants of the Pseudomonas aeruginosa exoenzyme S regulon were compared to identify proteins coordinately secreted with ExoS. Data from amino-terminal sequence analysis of purified extracellular proteins were combined with data from nucleotide sequence analysis of loci linked to exoenzyme S production. We report the identification of P. aeruginosa homologs to proteins of Yersinia spp. that function as regulators of the low calcium response, regulators of secretion, and mediators of the type III translocation mechanism.Exoenzyme S (ExoS) and ExoT are related extracellular ADP-ribosyltransferases secreted by a type III pathway in Pseudomonas aeruginosa (42,43). In previous studies we identified genes required for the regulation and secretion of ExoS (14,43). ExsA, the central regulator of the exoenzyme S regulon, controls transcription of structural, regulatory, and secretory loci (19,41,43,44). Loci, including pscB-L and pscN, encode homologs of type III secretion components (33, 43). Mutations in either regulatory or secretory loci result in a phenotype characterized by a defect in the production of ExoS, ExoT, and several additional extracellular proteins (15, 43). We postulated that proteins, coordinately regulated and secreted with ExoS, may represent additional virulence determinants.Identification of proteins coordinately secreted with ExoS and ExoT. To identify the proteins coordinately secreted with ExoS and ExoT, wild-type (PAK, PA103, and 388) and isogenic mutant (PAKexsA::⍀, PA103exsA::⍀, and 388exs1::Tn1[pscC]) strains of P. aeruginosa were grown under inducing conditions for exoenzyme S synthesis (medium containing 10 mM nitrilotriacetic acid) (38). Extracellular fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15,24). Major proteins that were common among the three wild-type strains included polypeptides that possessed molecular masses of 32.2, 32.8, 34, and 39 kDa and ExoT (53 kDa) ( Fig. 1; Table 1). ExoS (49 kDa) and a 42-kDa protein were absent from strain PA103. A 72-kDa protein (ExoU) was not produced in strains 388 and PAK. Mutations in either regulatory (exsA::⍀) or secretory (exs1::Tn1[pscC]) loci resulted in a general defect in the production of the set of proteins specific for each strain. Hybridization studies with probes for exoS and exoU confirm that the lack of ExoS synthesis in strain PA103 and the lack of ExoU synthesis in strains 388 and PAK are due to the absence of the corresponding genes (9-11).To determine if the extracellular proteins are related to previously cloned and sequenced members of the ExoS regulon, a protein sequencing approach was used. Proteins in concentrated culture supernatants were separated by SDS-PAGE, transferred to polyvinyl difluoride membranes, and stained with amido black (2). Stained bands corresponding to proteins of 32.2, 32.8, 34, 39 (isolated from the PA103 supernatant), and 42 (isolated from the PAK supernatant) kDa w...

Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii)

Background Frailty and cognitive impairment are associated with postoperative delirium, but are rarely assessed preoperatively. The study was designed to test the hypothesis that preoperative screening for frailty or cognitive impairment identifies patients at risk for postoperative delirium (primary outcome). Methods In this prospective cohort study, the authors administered frailty and cognitive screening instruments to 229 patients greater than or equal to 70 yr old presenting for elective spine surgery. Screening for frailty (five-item FRAIL scale [measuring fatigue, resistance, ambulation, illness, and weight loss]) and cognition (Mini-Cog, Animal Verbal Fluency) were performed at the time of the preoperative evaluation. Demographic data, perioperative variables, and postoperative outcomes were gathered. Delirium was the primary outcome detected by either the Confusion Assessment Method, assessed daily from postoperative day 1 to 3 or until discharge, if patient was discharged sooner, or comprehensive chart review. Secondary outcomes were all other-cause complications, discharge not to home, and hospital length of stay. Results The cohort was 75 [73 to 79 yr] years of age, 124 of 219 (57%) were male. Many scored positive for prefrailty (117 of 218; 54%), frailty (53 of 218; 24%), and cognitive impairment (50 to 82 of 219; 23 to 37%). Fifty-five patients (25%) developed delirium postoperatively. On multivariable analysis, frailty (scores 3 to 5 [odds ratio, 6.6; 95% CI, 1.96 to 21.9; P = 0.002]) versus robust (score 0) on the FRAIL scale, lower animal fluency scores (odds ratio, 1.08; 95% CI, 1.01 to 1.51; P = 0.036) for each point decrease in the number of animals named, and more invasive surgical procedures (odds ratio, 2.69; 95% CI, 1.31 to 5.50; P = 0.007) versus less invasive procedures were associated with postoperative delirium. Conclusions Screening for frailty and cognitive impairment preoperatively using the FRAIL scale and the Animal Verbal Fluency test in older elective spine surgery patients identifies those at high risk for the development of postoperative delirium. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

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At the neuromuscular junction, the acetylcholine receptor (AChR) is specifically clustered in the postsynaptic membrane via interactions with rapsyn and other scaffolding proteins. However, it remains unclear where these proteins bind on the AChR and how the interactions are regulated. Here, we define a phosphorylation-dependent binding site on the receptor that mediates agrin-induced clustering. Using chimeric proteins in which CD4 is fused to the large intracellular loop of each of the AChR subunits we found that agrin induced clustering of only chimeras containing the ␤ subunit loop. By making deletions in the ␤ loop we defined a 20 amino-acid sequence that is sufficient for clustering. The sequence contains a conserved tyrosine (Y390) whose phosphorylation is induced by agrin and whose mutation abolished clustering of ␤ loop chimeras and their ability to inhibit agrin-induced clustering of the endogenous AChR. Phosphorylation of the AChR ␤ subunit is correlated with increased rapsyn/AChR binding, suggesting that the effect of ␤Y390 phosphorylation on clustering is mediated by rapsyn. Indeed, we found that rapsyn associated with CD4-␤ loop chimeras in a phosphorylation-dependent manner, and that agrin increased the ratio of rapsyn binding to wild type AChR but not to AChR-␤ 3F/3F , which lacks ␤ loop tyrosine phosphorylation sites. Together, these findings suggest that agrin-induced phosphorylation of the ␤ subunit motif increases the stoichiometry of rapsyn binding to the AChR, thereby helping to stably cluster the receptor and anchor it at high density in the postsynaptic membrane.