Termination of pre-mRNA splicing requires that the ATPase and RNA unwindase Prp43p acts on the catalytic snRNA U6.

The termination of pre-mRNA splicing functions to discard suboptimal substrates, thereby enhancing fidelity, and to release excised introns in a manner coupled to spliceosome disassembly, thereby allowing recycling. The mechanism of termination, including the RNA target of the DEAH-box ATPase Prp43p, remains ambiguous. We discovered a critical role for nucleotides at the 3' end of the catalytic U6 small nuclear RNA in splicing termination. Although conserved sequence at the 3' end is not required, 2' hydroxyls are, paralleling requirements for Prp43p biochemical activities. Although the 3' end of U6 is not required for recruiting Prp43p to the spliceosome, the 3' end cross-links directly to Prp43p in an RNA-dependent manner. Our data indicate a mechanism of splicing termination in which Prp43p translocates along U6 from the 3' end to disassemble the spliceosome and thereby release suboptimal substrates or excised introns. This mechanism reveals that the spliceosome becomes primed for termination at the same stage it becomes activated for catalysis, implying a requirement for stringent control of spliceosome activity within the cell.

Spliceosome activation: U4 is the path, stem I is the goal, and Prp8 is the keeper. Let's cheer for the ATPase Brr2!

During pre-mRNA splicing, the spliceosome is activated for catalysis by unwinding base-paired U4/U6 small nuclear RNAs, a step that must be precisely timed. We know that unwinding requires the ATPase Brr2, but the mechanism and regulation of unwinding have been understood poorly. In the November 1, 2012, issue of Genes & Development, Hahn and colleagues (pp. 2408-2421) and Mozaffari-Jovin and colleagues (pp. 2422-2434) defined a pathway for U4/U6 unwinding. Moreover, Mozaffari-Jovin and colleagues suggested a mechanism for regulating Brr2.

The treatment in the Danish suicide prevention clinics: a clinician perspective.

Background: Few qualitative studies have focused on clinicians' perspectives regarding treatment of suicidal people. Despite limited evidence and imperfect risk-assessment tools, the psychosocial therapy at the Danish suicide prevention clinics has been linked to reductions in numbers of repeated self-harm, deaths by suicide, and other causes. This merits an investigation into how clinicians describe their practice.Methods: Using a qualitative design, 10 semi-structured interviews were conducted and analyzed to describe the psychosocial therapy.Results: The practices that the therapists described could be categorized along four dichotomous continuums. These illustrated dilemmas encountered during treatment of suicidal patients: 1) intuitive vs. specific risk assessment, 2) meaningful vs. formal, 3) patient-oriented vs. therapist-oriented and 4) direct vs. indirect approach to suicide prevention.Conclusions: Treatment in the Danish Suicide Prevention Clinics is characterized by methodological flexibility and diversity and with an emphasis on a patient-oriented approach. Furthermore, clinicians balance knowledge available by switching between a direct and an indirect approach according to the perceived suicide risk. If suicide risk was perceived as high, they would administer a direct approach and if low, an indirect approach. Finally, there seems to be differences as to how effective therapeutic methodologies work in the practice of suicide prevention.

Structure of the DEAH/RHA ATPase Prp43p bound to RNA implicates a pair of hairpins and motif Va in translocation along RNA.

Three families of nucleic acid-dependent ATPases (DEAH/RHA, Ski2-like, and NS3/NPH-II), termed the DExH ATPases, are thought to execute myriad functions by processive, ATP-dependent, 3' to 5' translocation along single-stranded nucleic acid. While the mechanism of translocation of the viral NS3/NPH-II family has been studied extensively, it has not been clear if or how the principles that have emerged for this family extend to the other two families. Here we report the crystal structure of the yeast DEAH/RHA family ATPase Prp43p, which functions in splicing and ribosome biogenesis, in complex with poly-uracil and a nonhydrolyzable ATP analog. The structure reveals a conserved DEAH/RHA-specific variation of motif Ib within the RecA1 domain of the catalytic core, in which the motif elongates as a ß-hairpin that bookends the 3' end of a central RNA stack, a function that in the viral and Ski-2 families is performed by an auxiliary domain. Supporting a fundamental role in translocation, mutations in this hairpin abolished helicase activity without affecting RNA binding or ATPase activity. While the structure reveals differences with viral ATPases in the RecA1 domain, our structure demonstrates striking similarities with viral ATPases in the RecA2 domain of the catalytic core, including both a prominent ß-hairpin that bookends the 5' end of the RNA stack and a dynamic motif Va that is implicated in mediating translocation. Our crystal structure, genetic, and biochemical experiments, as well as comparisons with other DExH ATPases, support a generalized mechanism for the DExH class of helicases involving a pair of bookends that inchworm along RNA.

The efficacy of psychoeducation on recurrent depression: a randomized trial with a 2-year follow-up.

BACKGROUND: The efficacy of psychoeducation is well documented in the treatment of relapse prevention of schizophrenia, and recently also in bipolar disorder; however, for recurrent depression only few controlled studies focusing on the efficacy of psychoeducation have been conducted. AIMS: This randomized study tests the efficacy of treatment-as-usual supplemented with a psychoeducative programme for patients with recurrent depression, treated at Community Mental Health Centres (CMHC) in Denmark. The primary outcome measurements concern was decline in consumption of psychiatric inpatient services and decline in Beck's Depression Inventory (BDI). METHODS: Eighty patients were randomized, either to the psychoeducative programme (consisting of eight sessions, each of 2 hours duration) and 2-year outpatient follow-up (42 cases), or only to 2-year outpatient follow-up (38 controls). The patients were monitored during 2 years after randomization. Data were collected from interviews including BDI, drug treatment and social measurements, and register data concerning use of psychiatric services. RESULTS: At 2-year follow-up, a significant reduction in the consumption of psychiatric inpatient services and in BDI was found; however, it was uniform for case and control patients. Drop-out/non-compliance was significantly more frequent among patients randomized to the control group. Furthermore, during follow-up the case group got a significant stronger attachment to the Labour market than the control group. CONCLUSIONS: The primary hypothesis could not be confirmed. Secondary outcome measurements concerning drop-out/non-compliance and attachment to the Labour market were significantly in favour of cases.

Adapting and Implementing Open Dialogue in the Scandinavian Countries: A Scoping Review.

Open Dialogue is a resource-oriented mental health approach, which mobilises a crisis-struck person's psychosocial network resources. This scoping review 1) identifies the range and nature of literature on the adoption of Open Dialogue in Scandinavia in places other than the original sites in Finland, and 2) summarises this literature. We included 33 publications. Most studies in this scoping review were published as "grey" literature and most grappled with how to implement Open Dialogue faithfully. In the Scandinavian research context, Open Dialogue was mainly described as a promising and favourable approach to mental health care.

Synergistic activation of eIF4A by eIF4B and eIF4G.

eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants KD of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A-eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A-eIF4B-RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.

Ribosome recycling step in yeast cytoplasmic protein synthesis is catalyzed by eEF3 and ATP.

After each round of protein biosynthesis, the posttermination complex (PoTC) consisting of a ribosome, mRNA, and tRNA must be disassembled into its components for a new round of translation. Here, we show that a Saccharomyces cerevisiae model PoTC was disassembled by ATP and eukaryotic elongation factor 3 (eEF3). GTP or ITP functioned with less efficiency and adenosine 5gamma'-(beta,gamma-imido)triphosphate did not function at all. The k(cat) of eEF3 was 1.12 min(-1), which is comparable to that of the in vitro initiation step. The disassembly reaction was inhibited by aminoglycosides and cycloheximide. The subunits formed from the yeast model PoTC remained separated under ionic conditions close to those existing in vivo, suggesting that they are ready to enter the initiation process. Based on our experimental techniques used in this paper, the release of mRNA and tRNA and ribosome dissociation took place simultaneously. No 40S*mRNA complex was observed, indicating that eEF3 action promotes ribosome recycling, not reinitiation.

Access to Development Opportunities in Biomedical and Health Informatics.

In between users and trained informaticians, we find a group of people carrying out important work in implementing and further developing health information technology, without access to formal biomedical and health informatics (BMHI) training. Study findings show what is required of novices in BMHI to gain access to communities of practice through which expertise can be developed.

Knocking on Doors - How Healthcare Workers Informally Develop Expertise in Biomedical and Health Informatics.

With digital systems permeating the healthcare sector, the healthcare workforce (clinical and administrative) need insight in biomedical health informatics (BMHI) to some degree. This study shows how novices in BMHI had to knock hard on several doors to find and become part of a community of practice to gain such expertise within BMHI. While it may be generally understood that gaining access to expertise is important, our findings suggest that more attention is needed to how such access is gained. The study exemplifies that the needed skills and competencies are difficult to identify in the individual projects and are highly situated - not least because it requires access to various experts in communities of practices. Therefore, there is a continued need to reframe the necessary education and training. Knowing when to knock on doors, which doors to knock on, and keeping doors open is central to becoming - and keep on being - a part of a community of practice centring on health information technology and BMHI.

Structural basis for the function of DEAH helicases.

DEAH helicases participate in pre-messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p-ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide-binding motif. A beta-hairpin from the second RecA domain is wedged between two carboxy-terminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C-terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides the framework for functional and genetic analysis of all DEAH helicases.

Serologic response in bottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA.

Marine-origin Brucella infections and serologic evidence of exposure have been documented in multiple cetacean species. A dolphin-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen bottlenose dolphin sera for anti-Brucella antibodies. A total of 131 serum samples collected over a 2 to 18 yr period from 6 bottlenose dolphins Tursiops truncatus with confirmed Brucella infections were analyzed for the presence and magnitude of antibody titers against marine-origin Brucella to compare individual antibody responses to various disease manifestations. Additionally, an epidemiologic serologic survey of a managed population of 64 bottlenose dolphins was performed to evaluate for the presence of antibodies and to determine whether there were any clinical pathology predictors for exposure or infection. The serologic results revealed that the dolphins with Brucella-associated abortions were seronegative for 7 to 18 yr until after the abortion and maintained positive titers for several years, with 2 of 3 animals returning to seronegative status. In contrast, the dolphins with Brucella-associated pulmonary or bone lesions maintained persistent positive titers for 2 to 18 yr. The population serosurvey revealed no significant differences in antibody levels among males and females, and dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older. Seropositive dolphins did not have significant inflammation compared to seronegative dolphins but were more likely to have higher levels of aspartate aminotransferase and gamma-glutamyl transpeptidase. Among 16 dolphins that tested seropositive, 13 (81.3%) had previously been seropositive for at least 3 to 5 yr.

Airway management in unconscious non-trauma patients.

BACKGROUND: Tracheal intubation is recommended in unconscious trauma patients to protect the airway from pulmonary aspiration of gastric contents and also to ensure ventilation and oxygenation. Unconsciousness is often defined as a Glasgow Coma Scale (GCS) score below 9. In non-trauma patients, however, there are no such firm recommendations regarding airway management and the GCS score may be less useful. The aim of this study was to describe the authors' experience with airway management in unconscious non-trauma patients in the prehospital setting with a physician-manned Mobile Emergency Care Unit (MECU). The main focus of the study was on the need for subsequent tracheal intubation during hospitalisation after initial treatment. METHODS: The study was based on an analysis of data prospectively collected from the MECU database in Copenhagen, Denmark. All unconscious (GCS scores below 9) non-trauma patients registered in the database during 2006 were included. The ambulance patient charts and medical records were scrutinised to assess outcome and the need for tracheal intubation during the first 24 h after admittance into hospital. RESULTS: A total of 557 unconscious non-trauma patients were examined and 129 patients (23%) were tracheally intubated by the MECU physician before or during transport to the hospital. Intubation was done in most patients with cardiac arrest, severe stroke or respiratory failure. Of the remaining 428 patients, 364 (85%) regained consciousness before being transported to the hospital, whereas 64 patients remained unconscious during transport and 12 (19%) of these were intubated in the emergency department. CONCLUSIONS: The majority of unconscious non-trauma patients were not intubated in the prehospital setting. Unconscious non-trauma patients may not all need tracheal intubation before being transferred to hospital.

Mechanism of ATP turnover inhibition in the EJC.

The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF(3), the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC-ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5-RNA-ADP-AlF(3), suggesting that the use of ADP-AlF(3) may be a valuable tool for examining DEAD-box ATPases in general.

Clinical outcome of assertive community treatment (ACT) in a rural area in Denmark: a case-control study with a 2-year follow-up.

AIM: The aim of the present study was to evaluate the effect of assertive community treatment (ACT) in the Tønder Region, South Jutland, where the first Danish ACT team was established to treat patients with severe and persistent mental illness (SMI). METHODS: The study compares outcome over a 2-year period between recipients of ACT and standard community mental healthcare. RESULTS: The study included 86 cases and 88 controls. At the time of recruitment, the cases and the controls did not differ significantly in demographic details and eligibility criteria. At the 2-year follow-up, the ACT patients showed a significant reduction in admissions, bed days and day hospital days, and a significant increase in the number of consultations compared with the controls. Adherence to outpatient services was higher in the ACT group. No significant improvements in psychopathology were found after 2 years, but a significant improvement in met needs and fewer unmet needs, indicating better functioning, occurred. Clients' satisfaction with care (Client Satisfaction Questionnaire, CSQ) was significantly higher among ACT patients than among controls. CONCLUSION: The treatment of these patients in this ACT service has yielded promising results, suggesting that ACT treatment may be a useful intervention for SMI patients. However, large, rigorous, randomized control trials with ACT are needed in Europe as the existing evidence mainly comes from American studies.

Review of detection of Brucella spp. by polymerase chain reaction.

Here we present a review of most of the currently used polymerase chain reaction (PCR)-based methods for identification of Brucella bacteria in biological samples. We focused in particular on methods using single-pair primers, multiplex primers, real-time PCRs, PCRs for marine Brucella, and PCRs for molecular biotyping. These methods are becoming very important tools for the identification of Brucella, at the species level and recently also at the biovar level. These techniques require minimum biological containment and can provide results in a very short time. In addition, genetic fingerprinting of isolates aid in epidemiological studies of the disease and its control. PCR-based methods are more useful and practical than conventional methods used to identify Brucella spp., and new methods for Brucella spp. identification and typing are still being developed. However, the sensitivity, specificity, and issues of quality control and quality assurance using these methods must be fully validated on clinical samples before PCR can be used in routine laboratory testing for brucellosis.

Diagnosis of brucellosis in livestock and wildlife.

AIM: To describe and discuss the merits of various direct and indirect methods applied in vitro (mainly on blood or milk) or in vivo (allergic test) for the diagnosis of brucellosis in animals. METHODS: The recent literature on brucellosis diagnostic tests was reviewed. These diagnostic tests are applied with different goals, such as national screening, confirmatory diagnosis, certification, and international trade. The validation of such diagnostic tests is still an issue, particularly in wildlife. The choice of the testing strategy depends on the prevailing brucellosis epidemiological situation and the goal of testing. RESULTS: Measuring the kinetics of antibody production after Brucella spp. infection is essential for analyzing serological results correctly and may help to predict abortion. Indirect ELISAs help to discriminate 1) between false positive serological reactions and true brucellosis and 2) between vaccination and infection. Biotyping of Brucella spp. provides valuable epidemiological information that allows tracing an infection back to the sources in instances where several biotypes of a given Brucella species are circulating. Polymerase chain reaction and new molecular methods are likely to be used as routine typing and fingerprinting methods in the coming years. CONCLUSION: The diagnosis of brucellosis in livestock and wildlife is complex and serological results need to be carefully analyzed. The B. abortus S19 and B. melitensis Rev. 1 vaccines are the cornerstones of control programs in cattle and small ruminants, respectively. There is no vaccine available for pigs or for wildlife. In the absence of a human brucellosis vaccine, prevention of human brucellosis depends on the control of the disease in animals.

Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast.

We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts(-) phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts(-) phenotype is exacerbated by tif32-Delta6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Delta impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo.

Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast.

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.

Agrobacterium-mediated transformation of meadow fescue (Festuca pratensis Huds.).

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and beta-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene.