Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa.

The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)-a 3'→5' exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria-as the primary enzyme responsible for pGpG degradation in Pseudomonas aeruginosa cells. We found that a P. aeruginosa Δorn mutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorn cells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorn cells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.

Seclusion room vs. physical restraint in an adolescent inpatient setting: patients' attitudes.

BACKGROUND: The use of physical restraints or a seclusion room for the treatment of adolescents in a psychiatric inpatient setting raises ethical dilemmas. We investigated the attitudes of adolescents towards these two means of confinement. METHOD: We used a structured questionnaire to collect data on the attitudes of 50 adolescent patients, hospitalized in a closed psychiatric ward, towards the use of physical restraint versus a seclusion room. RESULTS: Seventy per cent of the participants in the study preferred seclusion in the seclusion room over bed restraint, whereas 22% preferred physical restraint. Eighty-two percent described seclusion in the seclusion room as less frightening than restraint. Seventy-four per cent reported that seclusion in the seclusion room improved their mental state to a larger extent than restraint. The inpatient adolescents reported feeling the time they needed to reach a state of calm was shorter when they were confined to the seclusion room than when they were physically restrained (p>.001). CONCLUSIONS: The use of a seclusion room may be preferable compared to physical restraint for inpatient adolescents.

Identification of a novel nanoRNase in Bartonella.

In Escherichia coli, only one essential oligoribonuclease (Orn) can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). In Bacillus subtilis, NrnA and NrnB, which do not show any sequence similarity to Orn, have been identified as functional analogues of Orn. Sequence comparisons did not identify orn, nrnA or nrnB homologues in the genomes of the Chlamydia/Cyanobacteria and Alphaproteobacteria family members. Screening a genomic library from Bartonella birtlesii, a member of the Alphaproteobacteria, for genes that can complement a conditional orn mutant in E. coli, we identified BA0969 (NrnC) as a functional analogue of Orn. NrnC is highly conserved (more than 80 % identity) in the Bartonella genomes sequenced to date. Biochemical characterization showed that this protein exhibits oligo RNA degradation activity (nanoRNase activity). Like Orn from E. coli, NrnC is inhibited by micromolar amounts of 3'-phosphoadenosine 5'-phosphate in vitro. NrnC homologues are widely present in genomes of Alphaproteobacteria. Knock down of nrnC decreases the growth ability of Bartonella henselae, demonstrating the importance of nanoRNase activity in this bacterium.

Evaluation of the Clearview® Malaria pLDH Malaria Rapid Diagnostic Test in a non-endemic setting.

BACKGROUND: Malaria Rapid Diagnostic Tests (RDTs) are widely used to diagnose malaria. The present study evaluated a new RDT, the Clearview® Malaria pLDH test targeting the pan-Plasmodium antigen lactate dehydrogenase (pLDH). METHODS: The Clearview® Malaria pLDH test was evaluated on fresh samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included samples were Plasmodium falciparum (139), Plasmodium vivax (22), Plasmodium ovale (20), Plasmodium malariae (7), and 102 negative. RESULTS: Overall sensitivity for the detection of Plasmodium spp was 93.2%. For P. falciparum, the sensitivity was 98.6%; for P. vivax, P. ovale and P. malariae, overall sensitivities were 90.9%, 60.0% and 85.7% respectively. For P. falciparum and for P. vivax, the sensitivities increased to 100% at parasite densities above 100/µl. The specificity was 100%. The test was easily to perform and the result was stable for at least 1 hour. CONCLUSION: The Clearview® Malaria pLDH was efficient for the diagnosis of malaria. The test was very sensitive for P. falciparum and P. vivax detection. The sensitivities for P. ovale and P. malariae were better than other RDTs.

Level of training of nursing staff and the autonomy given to psychiatric inpatients: a multicenter study.

This research examines the influence of the level of professional training of the caretaking staff in psychiatric wards, the type of wards in which a patient is treated and the patient's age compared with the level of limitation put on patient autonomy. Detailed questionnaires were administered to 296 nurses from five mental health centers who met inclusion criteria for the study. The level of autonomy restriction was measured using six representative cases from fieldwork of the interviewees. These cases were analyzed by the authors based on Collopy's theory, by categorizing the data according to the six polarities of autonomy presented in his work. Our findings suggest a positive correlation between the level of professional training of the nursing staff, patient's age and the level of autonomy given. Our findings did not show a significant relationship between the type of ward and level of autonomy, although there could be a tendency for higher autonomy within closed wards.

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene.

UNLABELLED: Hepatitis B virus (HBV) causes liver diseases from acute hepatitis to cirrhosis and liver cancer. Currently, more than 350 million people are chronic HBV carriers, with devastating prognosis. HBV is a small enveloped noncytopathic virus, containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells. Infected individuals (acute and chronic) secrete about 10(7) to 10(11) virions per day to the bloodstream, with each infected cell releasing 50-300 viruses per day. HBV infects nondividing hepatocytes and replicates by reverse-transcribing the pregenomic RNA to DNA in the host cells. The level of deoxyribonucleotide triphosphates (dNTPs) in nondividing cells is too low to support viral replication and enable the high yield of secreted virions. Here, we report production of dNTPs by viral-dependent transcription activation of R2, the key component of ribonucleotide reductase (RNR), and show that this process is critical for the HBV life-cycle. This was found in an established HBV-positive cell line and was reproduced by HBV DNA-transduced cells, in both culture and mice. Furthermore, the viral hepatitis B X protein is essential in activating R2 expression by blocking access of Regulatory factor x1, a repressor of the R2 gene. CONCLUSION: Our findings demonstrate that the hepatitis B X protein is critical in infecting nonproliferating hepatocytes, which contain a low dNTP level. In addition, we provide molecular evidence for a new mechanism of HBV-host cell interaction where RNR-R2, a critical cell-cycle gene, is selectively activated in nonproliferating cells. This mechanism may set the stage for formulating a new category of anti-HBV drugs.

The symptom experience of oncology outpatients has a different impact on quality-of-life outcomes.

The aims of this replication study were to determine if subgroups of oncology outpatients receiving active treatment could be identified based on their experience with the symptoms of fatigue, sleep disturbance, depression, and pain; whether patients in these subgroups differed on selected demographic, disease, and treatment characteristics; and if patients in these subgroups differed on functional status and quality of life (QOL). A convenience sample of 228 oncology outpatients was recruited from seven outpatient settings in Israel. Patients completed a demographic questionnaire, a Karnofsky Performance Status score, the Multidimensional Quality of Life Scale-Cancer, the Lee Fatigue Scale, the General Sleep Disturbance Scale, the Center for Epidemiological Studies-Depression Scale, and a numeric rating scale of worst pain intensity. Cluster analysis was used to identify the patient subgroups based on their symptom experience. Four relatively distinct patient subgroups were identified based on their experiences with the above symptoms (i.e., low levels of all four symptoms (32.9%), low levels of pain and high levels of fatigue (18.0%), high levels of pain and moderate levels of fatigue (42.5%), and high levels of all four symptoms (6.6%). No differences were found among the four subgroups on any demographic, disease, or treatment characteristics. The subgroup of patients who reported high levels of all four symptoms reported the worst functional status and poorest QOL. In conclusion, differences in the symptom experience of oncology outpatients suggest that patients may harbor different phenotypic characteristics (e.g., environmental or physiologic) or genetic determinants for experiencing symptoms that are independent of demographic, disease, and treatment characteristics.

GLUT4 repression in response to oxidative stress is associated with reciprocal alterations in C/EBP alpha and delta isoforms in 3T3-L1 adipocytes.

Insulin responsiveness of adipocytes is acquired during normal adipogenesis, and is essential for maintaining whole-body insulin sensitivity. Differentiated adipocytes exposed to oxidative stress become insulin resistant, exhibiting decreased expression of genes like the insulin-responsive glucose transporter GLUT4. Here we assessed the effect of oxidative stress on DNA binding capacity of C/EBP isoforms known to participate in adipocyte differentiation, and determine the relevance for GLUT4 gene regulation. By electrophoretic mobility shift assay, nuclear proteins from oxidized adipocytes exhibited decreased binding of C/EBPalpha-containing dimers to a DNA oligonucleotide harboring the C/EBP binding sequence from the murine GLUT4 promoter. C/EBPdelta-containing dimers were increased, while C/EBPbeta-dimers were unchanged. These alterations were mirrored by a 50% decrease and a 2-fold increase in the protein content of C/EBPalpha and C/EBPdelta, respectively. In oxidized cells, GLUT4 protein and mRNA levels were decreased, and a GLUT4 promoter segment containing the C/EBP binding site partially mediated oxidative stress-induced repression of a reported gene. The antioxidant lipoic acid protected against oxidation-induced decrease in GLUT4 and C/EBPalpha mRNA, but did not prevent the increase in C/EBPdelta mRNA. We propose that oxidative stress induces adipocyte insulin resistance partially by affecting the expression of C/EBPalpha and delta, resulting in altered C/EBP-dimer composition potentially occupying the GLUT4 promoter.