Analysing the Cyanobacterial PipX Interaction Network Using NanoBiT Complementation in Synechococcus elongatus PCC7942.

The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding to them, in vivo studies are required to understand the physiological significance and contribution of environmental factors to the regulation of PipX complexes. Here, we have used the NanoBiT complementation system to analyse the regulation of complex formation in Synechococcus elongatus PCC 7942 between PipX and each of its two best-characterized partners, PII and NtcA. Our results confirm previous in vitro analyses on the regulation of PipX-PII and PipX-NtcA complexes by 2-oxoglutarate and on the regulation of PipX-PII by the ATP/ADP ratio, showing the disruption of PipX-NtcA complexes due to increased levels of ADP-bound PII in Synechococcus elongatus. The demonstration of a positive role of PII on PipX-NtcA complexes during their initial response to nitrogen starvation or the impact of a PipX point mutation on the activity of PipX-PII and PipX-NtcA reporters are further indications of the sensitivity of the system. This study reveals additional regulatory complexities in the PipX interaction network, opening a path for future research on cyanobacteria.

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions.

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a "false positive", the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.

The Best and the Worst Things About the Hospital for Children with Cancer: A Qualitative, Interview-Based Study in a Spanish Hospital.

The comprehensive psychosocial assessment of children with cancer requires listening to them and understanding their point of view. The objective of this work was to know what the participating children with cancer thought about the hospital, which they considered to be the best and worst aspects of their experience. We conducted a qualitative study using semi-structured interviews with 27 children with cancer. The interviewer asked the participating children what they considered to be the best thing about the hospital and what they considered to be the worst. These conversations with them were recorded and transcribed, and a thematic analysis of these transcripts was conducted. Among the aspects of the hospital that the children rated most highly was the treatment they received from the health care staff. Among what the children considered the worst aspect of their experience was pain, particularly pain caused by medical procedures such as injections. The obtained results lead us to conclude that children have a complex view of the hospital and are capable of a detailed analysis that must be taken into account.

Regulatory Connections Between the Cyanobacterial Factor PipX and the Ribosome Assembly GTPase EngA.

Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to important environmental challenges, like those imposed by the succession of days and nights. Not surprisingly, certain regulatory proteins are found exclusively in this phylum. One of these unique proteins, PipX, provides a mechanistic link between signals of carbon/nitrogen and of energy, transduced by the signaling protein PII, and the control of gene expression by the global nitrogen regulator NtcA. PII, required for cell survival unless PipX is inactivated or downregulated, functions by protein-protein interactions with transcriptional regulators, transporters, and enzymes. PipX also functions by protein-protein interactions, and previous studies suggested the existence of additional interacting partners or included it into a relatively robust six-node synteny network with proteins apparently unrelated to the nitrogen regulation system. To investigate additional functions of PipX while providing a proof of concept for the recently developed cyanobacterial linkage network, here we analyzed the physical and regulatory interactions between PipX and an intriguing component of the PipX synteny network, the essential ribosome assembly GTPase EngA. The results provide additional insights into the functions of cyanobacterial EngA and of PipX, showing that PipX interacts with the GD1 domain of EngA in a guanosine diphosphate-dependent manner and interferes with EngA functions in Synechococcus elongatus at a low temperature, an environmentally relevant context. Therefore, this work expands the PipX interaction network and establishes a possible connection between nitrogen regulation and the translation machinery. We discuss a regulatory model integrating previous information on PII-PipX with the results presented in this work.

Pharmacological treatment of social anxiety disorder: a meta-analysis.

Placebo-controlled trials have evaluated the efficacy of several medications in the treatment of social anxiety disorder but information regarding their relative efficacy is lacking. We compared the efficacy of medications systematically studied for the treatment of social anxiety disorder using meta-analytic techniques. The methodology included a database search of articles published between January 1980 and June 2001 and manual searches of bibliographies in published manuscripts. Trials were included if they reported outcome data on the Liebowitz Social Anxiety Scale (LSAS) or a categorical measure of responder status. Data were extracted independently by two authors. The Q statistic was used to assess homogeneity across trials. All analyses were conducted using intent-to-treat data. There was substantial heterogeneity across trials. The medications with largest effect sizes were phenelzine [effect size, 1.02; 95% Confidence Interval (CI), 0.52-1.52], clonazepam (effect size, 0.97; 95% CI, 0.49-1.45), gabapentin (effect size, 0.78; 95% CI, 0.29-1.27), brofaromine (effect size, 0.66; 95% CI, 0.38-0.94), and the selective serotonin reuptake inhibitors (SSRIs; effect size, 0.65; 95% CI, 0.50-0.81). There were no statistically significant differences between medications or medication groups. However, formal methods of interim monitoring adapted for meta-analyses suggested strongest evidence of efficacy for SSRIs and brofaromine. Several medications are efficacious for the treatment of social anxiety disorder. The stability of the SSRI effect size estimate in conjunction with other evidence for safety and tolerability and their ability to treat comorbid conditions supports the use of SSRIs as the first-line treatment. Direct comparisons of SSRIs vs. other promising medications deserve consideration.