Validation of Plasmodium falciparum dUTPase as the target of 5'-tritylated deoxyuridine analogues with anti-malarial activity

BACKGROUND: Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world. Due to the appearance of resistance and the lack of an effective vaccine, the search of novel anti-malarials is required. Deoxyuridine 5'-triphosphate nucleotido-hydrolase (dUTPase) is responsible for the hydrolysis of dUTP to dUMP within the parasite and has been proposed as an essential step in pyrimidine metabolism by providing dUMP for thymidylate biosynthesis. In this work, efforts to validate dUTPase as a drug target in Plasmodium falciparum are reported. METHODS: To investigate the role of PfdUTPase in cell survival different strategies to generate knockout mutants were used. For validation of PfdUTPase as the intracellular target of four inhibitors of the enzyme, mutants overexpressing PfdUTPase and HsdUTPase were created and the IC50 for each cell line with each compound was determined. The effect of these compounds on dUTP and dTTP levels from P. falciparum was measured using a DNA polymerase assay. Detailed localization studies by indirect immunofluorescence microscopy and live cell imaging were also performed using a cell line overexpressing a Pfdut-GFP fusion protein. RESULTS:Different attempts of disruption of the dut gene of P. falciparum were unsuccessful while a 3' replacement construct could recombine correctly in the locus suggesting that the enzyme is essential. The four 5'-tritylated deoxyuridine analogues described are potent inhibitors of the P. falciparum dUTPase and exhibit antiplasmodial activity. Overexpression of the Plasmodium and human enzymes conferred resistance against selective compounds, providing chemical validation of the target and confirming that indeed dUTPase inhibition is involved in anti-malarial activity. In addition, incubation with these inhibitors was associated with a depletion of the dTTP pool corroborating the central role of dUTPase in dTTP synthesis. PfdUTPase is mainly localized in the cytosol. CONCLUSION: These results strongly confirm the pivotal and essential role of dUTPase in pyrimidine biosynthesis of P. falciparum intraerythrocytic stages

Hope in action—facing cardiac death: A qualitative study of patients with life-threatening disease

Coping with existential challenges is important when struck by serious disease, but apart from cancer and palliative care little is known about how patients deal with such issues and maintain hope. To explore how patients with life-threatening heart disease experience hope when coping with mortality and other existential challenges, we conducted a qualitative study with semi-structured interviews. We made a purposive sample of 11 participants (26–88 years) who had experienced life-threatening disease: eight participants with serious heart disease, two with cancer, and one with severe chronic obstructive pulmonary disease. Analysis was by systematic text condensation. The findings showed that hope could enhance coping and diminish existential distress when patients were confronted with mortality and other existential challenges. Hope was observed as three types of dynamic work: to shift perception of mortality from overwhelming horror toward suppression or peaceful acceptance, to foster reconciliation instead of uncertainty when adapting to the new phase of life, and to establish go-ahead spirit instead of resignation as their identity. Meaning of life could, hence, be sustained in spite of serious threats to the persons' future, everyday life, and self-conception. The work of hoping could be supported or disturbed by relationships with family, friends, and health care professionals. Hope can be regarded as an active, dynamic state of existential coping among patients with life-threatening disease. Physicians may support this coping and thereby provide personal growth and alleviation of existential distress by skillfully identifying, acknowledging, and participating in the work of hoping performed by the patient

Physician and Patient Predictors of Evidence-Based Prescribing in Heart Failure: A Multilevel Study

BACKGROUND: The management of patients with heart failure (HF) needs to account for changeable and complex individual clinical characteristics. The use of renin angiotensin system inhibitors (RAAS-I) to target doses is recommended by guidelines. But physicians seemingly do not sufficiently follow this recommendation, while little is known about the physician and patient predictors of adherence. METHODS: To examine the coherence of primary care (PC) physicians' knowledge and self-perceived competencies regarding RAAS-I with their respective prescribing behavior being related to patient-associated barriers. Cross-sectional follow-up study after a randomized medical educational intervention trial with a seven month observation period. PC physicians (n = 37) and patients with systolic HF (n = 168) from practices in Baden-Wuerttemberg. Measurements were knowledge (blueprint-based multiple choice test), self-perceived competencies (questionnaire on global confidence in the therapy and on frequency of use of RAAS-I), and patient variables (age, gender, NYHA functional status, blood pressure, potassium level, renal function). Prescribing was collected from the trials' documentation. The target variable consisted of ≥50% of recommended RAAS-I dosage being investigated by two-level logistic regression models. RESULTS: Patients (69% male, mean age 68.8 years) showed symptomatic and objectified left ventricular (NYHA II vs. III/IV: 51% vs. 49% and mean LVEF 33.3%) and renal (GFR<50%: 22%) impairment. Mean percentage of RAAS-I target dose was 47%, 59% of patients receiving ≥50%. Determinants of improved prescribing of RAAS-I were patient age (OR 0.95, CI 0.92-0.99, p = 0.01), physician's global self-confidence at follow-up (OR 1.09, CI 1.02-1.05, p = 0.01) and NYHA class (II vs. III/IV) (OR 0.63, CI 0.38-1.05, p = 0.08). CONCLUSIONS: A change in physician's confidence as a predictor of RAAS-I dose increase is a new finding that might reflect an intervention effect of improved physicians' intention and that might foster novel strategies to improve safe evidence-based prescribing. These should include targeting knowledge, attitudes and skills

Changing the culture of assessment: the dominance of the summative assessment paradigm

Background Despite growing evidence of the benefits of including assessment for learning strategies within programmes of assessment, practical implementation of these approaches is often problematical. Organisational culture change is often hindered by personal and collective beliefs which encourage adherence to the existing organisational paradigm. We aimed to explore how these beliefs influenced proposals to redesign a summative assessment culture in order to improve students’ use of assessment-related feedback. Methods Using the principles of participatory design, a mixed group comprising medical students, clinical teachers and senior faculty members was challenged to develop radical solutions to improve the use of post-assessment feedback. Follow-up interviews were conducted with individual members of the group to explore their personal beliefs about the proposed redesign. Data were analysed using a socio-cultural lens. Results Proposed changes were dominated by a shared belief in the primacy of the summative assessment paradigm, which prevented radical redesign solutions from being accepted by group members. Participants’ prior assessment experiences strongly influenced proposals for change. As participants had largely only experienced a summative assessment culture, they found it difficult to conceptualise radical change in the assessment culture. Although all group members participated, students were less successful at persuading the group to adopt their ideas. Faculty members and clinical teachers often used indirect techniques to close down discussions. The strength of individual beliefs became more apparent in the follow-up interviews. Conclusions Naïve epistemologies and prior personal experiences were influential in the assessment redesign but were usually not expressed explicitly in a group setting, perhaps because of cultural conventions of politeness. In order to successfully implement a change in assessment culture, firmly-held intuitive beliefs about summative assessment will need to be clearly understood as a first step

The experience of palliative patients and their families of a family meeting utilised as an instrument for spiritual and psychosocial care: A qualitative study

Background: This study explores the experience of palliative patients and their family members of a family meeting model, utilised as an instrument for the provision of spiritual and psychosocial care. In doing so the study embraces a broad understanding of spirituality which may or may not include formal religious practice and a concept of psychosocial care that includes: social and emotional well-being, communication, self esteem, mental health and adaptation to illness. The meeting of spiritual and psychosocial needs is considered to be an important aspect of palliative care. Methods. This qualitative study, philosophically underpinned by hermeneutic phenomenology, investigates the participatory experience of palliative care patients and their significant family members of such a family meeting. People registered with two large metropolitan palliative care services, who met selection criteria, were referred by medical staff. Twelve of the 66 referred took part in family meetings which also included significant others invited by the patient. A total of 36 family members participated. The number of participants of individual family meetings ranged from two to eleven. After the family meeting every participant was invited to take part in an individual in-depth interview about their experience of the meeting. Forty seven interviews were conducted. These were audio recorded and transcribed. Results: Data analysis, utilising Ricoeur's theory of interpretation, revealed seven main themes: personal experience of the meeting, personal outcomes, observation of others' experience, observation of experience and outcomes for the family unit, meeting facilitation, how it could have been different and general applicability of the family meeting. Throughout these themes were numerous references to aspects of the web of relationships which describe the concept of spirituality as it is defined for the purpose of this study. Conclusions: The findings indicate the potential of the type of family meeting reported for use in the spiritual and psychosocial care of people receiving palliative care and their families. However further research is needed to explore its application to more culturally diverse groups and its longer term impact on family members.Heather M Tan, Anne Wilson, Ian Olver and Christopher Barto