Complete genome sequence of Xanthomonas phage RiverRider, a novel N4-like bacteriophage that infects the strawberry pathogen Xanthomonas fragariae.

Xanthomonas phage RiverRider is a novel N4-like bacteriophage and the first phage isolated from the plant pathogen Xanthomonas fragariae. Electron microscopy revealed a Podoviridae morphology consisting of isometric heads and short noncontractile tails. The complete genome of RiverRider is 76,355 bp in length, with 90 open reading frames and seven tRNAs. The genome is characteristic of N4-like bacteriophages in both content and organization, having predicted proteins characterized into the functional groups of transcription, DNA metabolism, DNA replication, lysis, lysis inhibition, structure and DNA packaging. Amino acid sequence comparisons for proteins in these categories showed highest similarities to well-characterized N4-like bacteriophages isolated from Achromobacter xylosoxidans and Erwinia amylovora. However, the tail fiber proteins of RiverRider are clearly distinct from those of other N4-like phages. RiverRider was able to infect seven different strains of X. fragariae and none of the other species of Xanthomonas tested.

[Discontinuation of SSRIs and SNRIs]. / Het afbouwen van SSRI's en SNRI's.

Interruption or abrupt discontinuation of the use of antidepressants may lead to withdrawal symptoms. These are most common with selective serotonin reuptake inhibitors (SSRIs) and serotonin-noradrenaline reuptake inhibitors (SNRIs).There is insufficient scientific evidence about the prevalence of antidepressant withdrawal symptoms and how to optimally discontinue antidepressants. The multidisciplinary document 'Discontinuation of SSRIs & SNRIs' offers a rationale and suggestions for the gradual tapering of these antidepressants. The following factors are consistently named as risk factors for the occurrence of withdrawal symptoms: (a) the patient experiences withdrawal symptoms in case of non-compliance or skipped doses; (b) a previous attempt to stop was unsuccessful; and (c) the patient is being treated with higher doses than the smallest effective dose of SSRIs or SNRIs. In patients with one or more risk factors, a tapering schedule with non-linear dose-reduction steps should be considered. The speed at which these steps are taken, should be adjusted depending on occurrence of withdrawal symptoms. Shared decision-making by patient and physician is the best way to select a tapering schedule.

A quick guide for student-driven community genome annotation.

High quality gene models are necessary to expand the molecular and genetic tools available for a target organism, but these are available for only a handful of model organisms that have undergone extensive curation and experimental validation over the course of many years. The majority of gene models present in biological databases today have been identified in draft genome assemblies using automated annotation pipelines that are frequently based on orthologs from distantly related model organisms and usually have minor or major errors. Manual curation is time consuming and often requires substantial expertise, but is instrumental in improving gene model structure and identification. Manual annotation may seem to be a daunting and cost-prohibitive task for small research communities but involving undergraduates in community genome annotation consortiums can be mutually beneficial for both education and improved genomic resources. We outline a workflow for efficient manual annotation driven by a team of primarily undergraduate annotators. This model can be scaled to large teams and includes quality control processes through incremental evaluation. Moreover, it gives students an opportunity to increase their understanding of genome biology and to participate in scientific research in collaboration with peers and senior researchers at multiple institutions.

Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community.

Database URL: https://citrusgreening.org/.

Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily.

Naively one might have expected an early division between phosphate monoesterases and diesterases of the alkaline phosphatase (AP) superfamily. On the contrary, prior results and our structural and biochemical analyses of phosphate monoesterase PafA, from Chryseobacterium meningosepticum, indicate similarities to a superfamily phosphate diesterase [Xanthomonas citri nucleotide pyrophosphatase/phosphodiesterase (NPP)] and distinct differences from the three metal ion AP superfamily monoesterase, from Escherichia coli AP (EcAP). We carried out a series of experiments to map out and learn from the differences and similarities between these enzymes. First, we asked why there would be independent instances of monoesterases in the AP superfamily? PafA has a much weaker product inhibition and slightly higher activity relative to EcAP, suggesting that different metabolic evolutionary pressures favored distinct active-site architectures. Next, we addressed the preferential phosphate monoester and diester catalysis of PafA and NPP, respectively. We asked whether the >80% sequence differences throughout these scaffolds provide functional specialization for each enzyme's cognate reaction. In contrast to expectations from this model, PafA and NPP mutants with the common subset of active-site groups embedded in each native scaffold had the same monoesterase:diesterase specificities; thus, the >107-fold difference in native specificities appears to arise from distinct interactions at a single phosphoryl substituent. We also uncovered striking mechanistic similarities between the PafA and EcAP monoesterases, including evidence for ground-state destabilization and functional active-site networks that involve different active-site groups but may play analogous catalytic roles. Discovering common network functions may reveal active-site architectural connections that are critical for function, and identifying regions of functional modularity may facilitate the design of new enzymes from existing promiscuous templates. More generally, comparative enzymology and analysis of catalytic promiscuity can provide mechanistic and evolutionary insights.

Using Caffeine Pills for Performance Enhancement. An Experimental Study on University Students' Willingness and Their Intention to Try Neuroenhancements.

Recent research has indicated that university students sometimes use caffeine pills for neuroenhancement (NE; non-medical use of psychoactive substances or technology to produce a subjective enhancement in psychological functioning and experience), especially during exam preparation. In our factorial survey experiment, we manipulated the evidence participants were given about the prevalence of NE amongst peers and measured the resulting effects on the psychological predictors included in the Prototype-Willingness Model of risk behavior. Two hundred and thirty-one university students were randomized to a high prevalence condition (read faked research results overstating usage of caffeine pills amongst peers by a factor of 5; 50%), low prevalence condition (half the estimated prevalence; 5%) or control condition (no information about peer prevalence). Structural equation modeling confirmed that our participants' willingness and intention to use caffeine pills in the next exam period could be explained by their past use of neuroenhancers, attitude to NE and subjective norm about use of caffeine pills whilst image of the typical user was a much less important factor. Provision of inaccurate information about prevalence reduced the predictive power of attitude with respect to willingness by 40-45%. This may be because receiving information about peer prevalence which does not fit with their perception of the social norm causes people to question their attitude. Prevalence information might exert a deterrent effect on NE via the attitude-willingness association. We argue that research into NE and deterrence of associated risk behaviors should be informed by psychological theory.

[Multidisciplinary guideline irritable bowel syndrome]. / Multidisciplinaire richtlijn prikkelbaredarmsyndroom.

The multidisciplinary guideline 'Diagnostics and treatment of irritable bowel syndrome (IBS)' provides the basis for a properly coordinated collaboration between the patient suffering from IBS and all healthcare providers involved in his or her treatment, such as the general practitioner, gastroenterologist, internist, occupational-health physician, dietitian and psychologist. The diagnosis 'IBS' is often made in accordance with diagnostic criteria, such as the Rome III criteria, but a somatic condition needs to be excluded first. If there are no indications for this, additional diagnostic tests are not necessary. Management of the condition consists primarily of advice on life-style plus non-pharmacological interventions, in addition to explaining the condition and providing information. Drug treatment is rarely indicated. If the IBS symptoms have a significant impact on quality of life and patients do not respond to the measures taken in accordance with this general policy, there are three options for psychotherapeutic treatment. When the symptoms result in absenteeism or other work-related problems, the doctor can advise the patient to contact the occupational-health physician and to search for specific solutions in consultation with the employer. When individual advice is required or if the patient's diet is not well-balanced, a referral to the dietitian will follow.

Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >106-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 104-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH3). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²âº bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²âº bimetallo core.

Targeted screening for Coeliac Disease among irritable bowel syndrome patients: analysis of cost-effectiveness and value of information.

OBJECTIVES: A high prevalence of Coeliac Disease (CD) is found among patients with a clinical diagnosis of irritable bowel syndrome (IBS) compared to the general population. Symptoms of CD are quite similar to IBS, but its treatment is different. The aim of this study was to evaluate the cost-effectiveness of screening for CD in patients with diarrhoea/mixed type IBS (IBS-D/mix) in terms of cost per quality adjusted life year (QALY) in the Netherlands. METHODS: A decision model was constructed to evaluate the costs and health benefits of serological testing followed by confirmatory endoscopy with biopsy. Probabilistic sensitivity analysis (PSA) was performed to examine the effect of parameter uncertainty. Finally, the budget impact of implementing the screening process was also computed for implementation over a 10-year time horizon. RESULTS: Screening resulted in an increase of about 0.07 quality life years (QALYs) per patient over a lifetime horizon. The incremental cost effectiveness ratio was about 6,200 euro/QALY compared to no screening. The PSA showed that the uncertainty in cost effectiveness results is not considerable. The value of information analysis confirmed the robustness of the results. Screening all current patients with diarrhea/mixed type IBS would require a total budget of about 25 million Euros over a 10 year time period. CONCLUSION: Screening patients with IBS-D or IBS-mix for CD is almost certainly cost-effective. The screening program would improve the quality of life of those patients with IBS symptoms who actually have CD at a relatively low cost.

The effect of watchful waiting compared to immediate test ordering instructions on general practitioners' blood test ordering behaviour for patients with unexplained complaints; a randomized clinical trial (ISRCTN55755886).

BACKGROUND: Immediate blood testing for patients presenting with unexplained complaints in family practice is superfluous from a diagnostic point of view. However, many general pracitioners (GPs) order tests immediately. Watchful waiting reduces the number of patients to be tested and the number of false-positive results. The objectives of this study are: to determine the feasibility of watchful waiting compared to immediate test ordering; to determine if a special quality improvement strategy can improve this feasibility; and to determine if watchful waiting leads to testing at a later time. METHODS: The study is a cluster-randomized clinical trial with three groups, on blood test ordering strategies in patients with unexplained complaints. GPs in group one were instructed to order tests immediately and GPs in group two to apply a watchful waiting approach. GPs in group three received the same instruction as group two, but they were supported by a systematically designed quality improvement strategy. A total of 498 patients with unexplained complaints from 63 practices of Dutch GPs participated. We measured: the percentage of patients for whom tests were ordered and number of tests ordered at the first consultation; performance on the strategy's performance objectives (i.e., ordering fewer tests and specific communication skills); the number of tests ordered after four weeks; and GP and patient characteristics. RESULTS: Immediate test ordering proved feasible in 92% of the patients; watchful waiting in 86% and 84%, respectively, for groups two and three. The two watchful waiting groups did not differ significantly in the achievement of any of the performance objectives. Of the patients who returned after four weeks, none from group one and six from the two watchful waiting groups had tests ordered for them. CONCLUSIONS: Watchful waiting is a feasible approach. It does not lead to testing immediately afterwards. Furthermore, watchful waiting was not improved by the quality improvement strategy. CLINICAL TRIAL REGISTRATION: ISRCTN55755886.

High-resolution analysis of Zn(2+) coordination in the alkaline phosphatase superfamily by EXAFS and x-ray crystallography.

Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally related enzymes, Escherichia coli alkaline phosphatase (AP) and Xanthomonas axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP), have nearly identical binuclear Zn(2+) catalytic centers but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn(2+) coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray crystallographic data. We report a 1.29-Å crystal structure of AP with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure for AP and NPP in the free-enzyme forms, with AMP and inorganic phosphate ground-state analogs and with vanadate transition-state analogs. These studies yielded average zinc-ligand distances in AP and NPP free-enzyme forms and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal-ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the results suggest that the binuclear Zn(2+) catalytic site remains very similar between AP and NPP during the course of a reaction cycle.

Ordering blood tests for patients with unexplained fatigue in general practice: what does it yield? Results of the VAMPIRE trial.

BACKGROUND: Unexplained fatigue is frequently encountered in general practice. Because of the low prior probability of underlying somatic pathology, the positive predictive value of abnormal (blood) test results is limited in such patients. AIM: The study objectives were to investigate the relationship between established diagnoses and the occurrence of abnormal blood test results among patients with unexplained fatigue; to survey the effects of the postponement of test ordering on this relationship; and to explore consultation-related determinants of abnormal test results. DESIGN OF STUDY: Cluster randomised trial. SETTING: General practices of 91 GPs in the Netherlands. METHOD: GPs were randomised to immediate or postponed blood-test ordering. Patients with new unexplained fatigue were included. Limited and expanded sets of blood tests were ordered either immediately or after 4 weeks. Diagnoses during the 1-year follow-up period were extracted from medical records. Two-by-two tables were generated. To establish independent determinants of abnormal test results, a multivariate logistic regression model was used. RESULTS: Data of 325 patients were analysed (71% women; mean age 41 years). Eight per cent of patients had a somatic illness that was detectable by blood-test ordering. The number of false-positive test results increased in particular in the expanded test set. Patients rarely re-consulted after 4 weeks. Test postponement did not affect the distribution of patients over the two-by-two tables. No independent consultation-related determinants of abnormal test results were found. CONCLUSION: Results support restricting the number of tests ordered because of the increased risk of false-positive test results from expanded test sets. Although the number of re-consulting patients was small, the data do not refute the advice to postpone blood-test ordering for medical reasons in patients with unexplained fatigue in general practice.

What makes general practitioners order blood tests for patients with unexplained complaints? A cross-sectional study.

BACKGROUND: Approximately 13% of consultations in general practice involve patients with unexplained complaints (UCs). These consultations often end with general practitioners (GPs) ordering blood tests of questionable diagnostic informativeness. OBJECTIVE: We studied factors potentially associated with GPs' decisions to order blood tests. METHODS: Cross-sectional study. Twenty-seven GPs completed registration forms after each consultation concerning newly presented UCs. RESULTS: Of the 100 analysable patients, 59 had at least one blood test ordered. The median number of ordered tests was 10 (interpercentile range [IPR-90] 2-15). Compared to abdominal complaints, the blood test ordering (BTO) probability for fatigue was five times higher (relative risk [RR] 5.2). Duration of complaints for over 4 weeks also increased this probability (RR 1.6). Factors associated with a lower BTO probability were: likelihood of background psychosocial factors (RR 0.4) and GPs having a syndrome rather than symptom type of working hypothesis (RR 0.5). CONCLUSION: We found a high rate of BTO among GPs confronted with patients with UCs. Furthermore, a considerable number of tests were ordered. The selectivity in BTO behaviour of GPs can be improved upon.

Influence of watchful waiting on satisfaction and anxiety among patients seeking care for unexplained complaints.

PURPOSE: We undertook a study to determine whether test-ordering strategy and other consultation-related factors influence satisfaction with and anxiety after a consultation among patients seeking care for unexplained complaints. METHODS: A cluster-randomized clinical trial was conducted in family medicine practices in the Netherlands. Participants were 498 patients with unexplained complaints seen by 63 primary care physicians. Physicians either immediately ordered a blood test for patients or followed a 4-week watchful waiting approach. Physicians and patients completed questionnaires asking about their characteristics, satisfaction with care, and anxiety, and aspects of the consultation. The main outcomes were patient satisfaction and anxiety. Data were analyzed by multilevel logistic regression analysis. RESULTS: Patients were generally satisfied with their consultation and had moderately low anxiety afterward (mean scores on 11-point scales, 7.3 and 3.1, respectively), with no difference between the immediate testing and watchful waiting groups (chi(2) = 2.4 and 0.3, respectively). The factors associated with higher odds of satisfaction were mainly related to physician-patient communication: patients' satisfaction with their physician generally, feeling taken seriously, and knowing the seriousness of complaints afterward; physicians' discussing testing and not considering complaints bearable; and older physician age. The same was true for factors associated with higher odds of anxiety: patients expecting testing or referral, patients not knowing the seriousness of their complaints afterward, and physicians not seeing a cause for alarm. CONCLUSIONS: Test-ordering strategy does not influence patients' satisfaction with and anxiety after a consultation. Instead, specific aspects of physician-patient communication are important. Apparently, primary care physicians underestimate how much they can contribute to the well-being of their patients by discussing their worries.

Exploring the black box of change in improving test-ordering routines.

BACKGROUND: The effects of quality improvement strategies are sometimes limited in spite of a systematic development approach. What elements play a role in the change process is not yet fully understood. OBJECTIVE: To explore this 'black box' of change, by analysing the barriers and facilitators GPs perceive during the change process. METHODS: Qualitative study among GPs who participated in the quality improvement strategy arm of a randomized clinical trial on blood test ordering for unexplained complaints (UCs). The strategy was based on a national guideline that advocates delayed test ordering in patients presenting with UCs. Each GP's change process was assessed by means of a semi-structured interview about barriers to and facilitators of change. RESULTS: Twenty-four interviews were analysed. Important themes identified in the interviews were lack of problem awareness, the time and effort it takes to change, influence of patients and the pros and cons of the changed behaviour. CONCLUSION: The themes can be summarized into one comprehensive issue: the GPs lack a sense of urgency to change. An important explanation seems to be that two questions from the problem analysis prior to the development of the strategy had not been adequately answered: "Is the GPs' current behaviour a problem and does the problem have consequences for patients?" and if so, "What is the extent of the problem?." As a result, insufficient attention was given to applicability issues, such as time investment, costs and patient and practitioner satisfaction and anxiety.

Why do patients want to have their blood tested? A qualitative study of patient expectations in general practice.

BACKGROUND: General practitioners often take their impression of patients' expectations into account in their decision to have blood tests done. It is commonly recommended to involve patients in decision-making during consultations. The study aimed to obtain detailed information on patients' expectations about blood tests. METHODS: Qualitative study among patients in waiting rooms of general practices. Each patient was presented with a short questionnaire about their preferences in terms of diagnostics. Patients who would like blood tests to be done were interviewed. RESULTS: Fifty-seven (26%) of the 224 respondents wanted blood tests. Twenty-two were interviewed. Patients overestimated the qualities of blood tests. Favourable test results were regarded as proof of good health. Patients regarded blood tests as a useful instrument to screen for serious disorders, and were confirmed in this belief by people in their social environment and by the media. Many patients expected their GP to take an active test ordering approach, though some indicated that they might be convinced if their GP proposed a wait-and-see policy. CONCLUSIONS: GPs' perceptions about patient expectations seem justified: patients appear to have high hopes for testing as a diagnostic tool. They expect diagnostic certainty without mistakes and a proof of good health. The question is whether it would be desirable to remove patients' misconceptions, allowing them to participate in policy decisions on the basis of sound information, or whether it would be better to leave the misconceptions uncontested, in order to retain the 'magic' of additional tests and reassure patients. We expect that clarifying the precise nature of patients' expectations by the GP may be helpful in creating a diagnostic strategy that satisfies both patients and GPs. GPs will have to balance the benefits of reassuring their patients by means of blood tests which may be unnecessary against the benefits of avoiding unnecessary tests. Further research is needed into the effects of different types of patient information and the effects of testing on satisfaction and anxiety.