The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

Catheter-associated infections are difficult to treat with available antimicrobial agents because of their biofilm etiology. We examined the effect of low-amperage direct electrical current (DC) exposure on established bacterial and fungal biofilms in a novel experimental in vitro catheter model. Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida parapsilosis biofilms were grown on the inside surfaces of polyvinyl chloride (PVC) catheters, after which 0, 100, 200, or 500 A of DC was delivered via intraluminally placed platinum electrodes. Catheter biofilms and intraluminal fluid were quantitatively cultured after 24 h and 4 days of DC exposure. Time-and dose-dependent biofilm killing was observed with all amperages and durations of DC administration. Twenty-four hours of 500 A of DC sterilized the intraluminal fluid for all bacterial species studied; no viable bacteria were detected after treatment of S. epidermidis and S. aureus biofilms with 500 A of DC for 4 days. Catheter-associated infections, including catheter-associated urinary tract infection (CAUTI) and catheter-related bloodstream infection (CRBSI), are associated with morbidity, mortality, and expense, often requiring catheter removal. The pathogenesis of these infections relates to the presence of biofilms on the surface of the catheters.Compared with planktonic (i.e., free-floating) forms, microorganisms in biofilms exhibit increased resistance to host immunity and antimicrobial therapy (1). Proposed mechanisms underlying biofilm-associated antimicrobial resistance include limited penetration through or neutralization of antimicrobials within biofilms (2, 3); subpopulations of resistant phenotypes, referred to as "persister" cells (4, 5); and dormant stationary-phase zones within biofilms (4, 6, 7). As a result, most conventional systemically administered antimicrobial agents have little ability to cure catheter-associated infections. Catheter removal is necessary in the majority of cases, typically in conjunction with systemic antimicrobial treatment. Strategies to control biofilms, such as coating catheters with silver ions, chlorhexidine or minocycline plus rifampin, have been proposed (8-12), and catheter lock solutions, using conventional antimicrobial agents or antiseptics, have shown activity against catheter-associated biofilms (13-19). However, none of these strategies has solved the clinical challenge of catheter-associated infections, underscoring the need for new approaches.We previously described an antibiofilm strategy that we termed the electricidal effect. Biofilms of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa on Teflon discs were exposed to 20, 200, or 2,000 A direct current (DC) for up to 7 days, which resulted in time-and dose-dependent antibiofilm effects, as measured by decreases in numbers of viable cells (20). Subsequent studies confirmed the microbicidal activity of continuously and intermittently applied electrical current against estab...

LAMTOR2 (p14), a part of the larger LAMTOR/Ragulator complex, plays a crucial role in EGF-dependent activation of p42/44 mitogen-activated protein kinases (MAPK, ERK1/2). In this study, we investigated the role of LAMTOR2 in nerve growth factor (NGF)-mediated neuronal differentiation. Stimulation of PC12 (rat adrenal pheochromocytoma) cells with NGF is known to activate the MAPK. Pharmacological inhibition of MEK1 as well as siRNA–mediated knockdown of both p42 and p44 MAPK resulted in inhibition of neurite outgrowth. Contrary to expectations, siRNA–mediated knockdown of LAMTOR2 effectively augmented neurite formation and neurite length of PC12 cells. Ectopic expression of a siRNA-resistant LAMTOR2 ortholog reversed this phenotype back to wildtype levels, ruling out nonspecific off-target effects of this LAMTOR2 siRNA approach. Mechanistically, LAMTOR2 siRNA treatment significantly enhanced NGF-dependent MAPK activity, and this effect again was reversed upon expression of the siRNA-resistant LAMTOR2 ortholog. Studies of intracellular trafficking of the NGF receptor TrkA revealed a rapid colocalization with early endosomes, which was modulated by LAMTOR2 siRNA. Inhibition of LAMTOR2 and concomitant destabilization of the remaining members of the LAMTOR complex apparently leads to a faster release of the TrkA/MAPK signaling module and nuclear increase of activated MAPK. These results suggest a modulatory role of the MEK1 adapter protein LAMTOR2 in NGF-mediated MAPK activation required for induction of neurite outgrowth in PC12 cells.