SummaryUropathogenic Escherichia coli, the predominant causative agent of urinary tract infections, use type 1 pili to bind and invade bladder epithelial cells. Upon entry, the bacteria rapidly replicate and enter a complex developmental pathway ultimately forming intracellular bacterial communities (IBCs), a niche with biofilm-like properties protected from innate defences and antibiotics. Paradoxically, bacteria within IBCs produce type 1 pili, an organelle thought only to be an extracellular colonization factor. Thus, we investigated the function of type 1 pili in IBC development. The cystitis isolate, UTI89, was genetically manipulated for conditional fim expression under control of the tet promoter. In this strain, UTI89-tetR/P tet fim, piliation is constitutively inhibited by the tetracycline repressor, TetR. Repression is relieved by anhydrotetracycline (AHT) treatment. UTI89-tetR/ Ptet fim and the isogenic control strain, UTI89-tetR, grown in the presence of AHT, colonized the bladder and invaded the superficial umbrella cells at similar levels at early times in a murine model of infection. However, after invasion UTI89-tetR/Ptet fim became non-piliated and was unable to form typical IBCs comprised of tightly packed, coccoid-shaped bacteria in contrast to the control strain, UTI89-tetR. Thus, this work changes the extracellular colonization functional paradigm of pili by demonstrating their intracellular role in biofilm formation.

SUMMAR Y Given the suggestion of a reciprocal relationship between sleep and pain and the recognition of sleep as an important parameter in determining quality of life, there is increasing research interest in sleep disturbance linked to chronic pain. The present study aimed to provide an estimate of the prevalence of Ôclinical insomniaÕ in patients attending a specialist pain clinic and identify factors associated with it. Seventy chronic back pain patients and 70 gender-and age-matched pain-free controls completed a set of questionnaires measuring sleep (Insomnia Severity Index; ISI), pain (Short-Form McGill Pain Questionnaire) and a selection of general and specific psychological variables (Hospital Anxiety and Depression Scale, Short Health Anxiety Inventory). Scores suggestive of clinical insomnia (ISI ‡ 15) were noted in 53% of chronic pain patients, when compared with only 3% in pain-free controls. Significant positive correlations with insomnia severity were detected for all six variables of interest (pain intensity, sensory pain ratings, affective pain ratings, general anxiety, general depression and health anxiety). Affective pain ratings and health anxiety were the best predictors of insomnia severity in this sample, accounting for 30% of the total variance, even when present pain intensity was controlled for. Affective pain remained as a significant predictor of insomnia severity when both the effect of pain intensity and the effects of anxiety and depression were controlled for. Future research should consider investigating the role of pain appraisal and health anxiety in the development and manifestation of insomnia concomitant to chronic pain.k e y w o r d s anxiety, chronic pain, depression, health anxiety, insomnia, pain appraisal

In the murine model of urinary tract infections (UTI), cystitis by uropathogenic Escherichia coli (UPEC) occurs through an intimate relationship with the bladder superficial umbrella cell entailing cycles of adherence, invasion, intracellular bacterial community (IBC) formation, and dispersal (fluxing) from the intracellular environment. IBC dispersal is a key step that results in the spread of bacteria over the epithelial surface to initiate additional rounds of IBC formation. We investigated the role of flagella in mediating adherence and motility during UTI, hypothesizing that the dispersion of the IBC would be incomplete in the absence of motility, thus interrupting the IBC pathway and attenuating the infection. Using gfp reporter fusions, the expression of the flagellar class I flhDC and class III fliC genes was monitored to track key points of regulation throughout the pathogenic cascade. In vitro, growth under conditions promoting motility resulted in the robust expression of both fusions. In contrast, only the class I fusion produced significant expression throughout early stages of IBC development including the dispersion stage. Thus, unlike in vitro modeling of motility, the regulatory cascade appeared incomplete in vivo. Throughout IBC formation, nonmotile ⌬fliC mutants achieved the same number of IBCs as the wild-type (wt) strain, demonstrating that flagella are neither essential nor required for first-or second-generation IBC formation. However, in competition experiments between wt and ⌬fliC strains, the wt was shown to have a fitness advantage in persisting throughout the urinary tract for 2 weeks, demonstrating a subtle but measurable role for flagella in virulence.

Although chronic pain and depression commonly co-occur, causal relationships have yet to be established. A reciprocal relationship, with depression increasing pain and vice versa, is most frequently suggested, but experimental evidence is needed to validate such a view. The most straightforward approach would be a demonstration that increasing or decreasing depressed mood predictably modifies pain responses. The current experiment tested whether experimentally induced depressed and happy mood have differential effects on pain ratings and tolerance in 55 patients suffering from chronic back pain. Participants were randomly assigned to depressed, neutral (control) or elated mood induction conditions. They completed a physically passive baseline task prior to receiving mood induction, then a clinically relevant physically active task (holding a heavy bag) to elicit pain responses and tolerance. Measures were taken immediately after the baseline task and immediately after the mood induction to assess the changes in mood, pain ratings and tolerance before and after the experimental manipulation. Results indicate that the induction of depressed mood resulted in significantly higher pain ratings at rest and lower pain tolerance, whilst induced happy mood resulted in significantly lower pain ratings at rest and greater pain tolerance. Correlations between changes in mood on the one hand and changes in pain response and pain tolerance on the other hand were consistent with these findings. It is concluded that, in chronic back pain patients, experimentally induced negative mood increases self-reported pain and decreases tolerance for a pain-relevant task, with positive mood having the opposite effect.

Monovalent-cation-activated enzymes are abundantly represented in plants and in the animal world. Most of these enzymes are specifically activated by K ؉ , whereas a few of them show preferential activation by Na ؉ . The monovalent cation specificity of these enzymes remains elusive in molecular terms and has not been reengineered by site-directed mutagenesis. Here we demonstrate that thrombin, a Na ؉ -activated allosteric enzyme involved in vertebrate blood clotting, can be converted into a K ؉ -specific enzyme by redesigning a loop that shapes the entrance to the cation-binding site. The conversion, however, does not result into a K ؉ -activated enzyme.