Purinergic ligands induce extracellular acidification and increased ATP turnover in HepG2 cells.

Nucleosides and nucleotides at µM concentrations stimulated a 300% increase in acid secretion in HepG2 cells, which was quantitatively accounted for as increased export of lactate generated by glycogenolysis. Agonist selectivity encompassed nucleosides and nucleotides for all 5 natural nucleobases and, along with antagonist profiles, was inconsistent with a role for purinergic receptors in mediating this activity. Agonist catabolism did not contribute significantly to either low selectivity or lactate production. Lactate production was driven by an increase in ATP turnover of as much as 56%. For some agonists, especially adenosine, ATP turnover decreased precipitously at mM concentrations, correlating with known adenosine-stimulated apoptosis. We propose that nucleoside/nucleotide agonists induce a futile energy cycle via a novel mechanism, which results in increased ATP turnover and initiates a continuum of events that for some agonists culminates in apoptosis.

"Your child needs surgery": A survey-based evaluation of simulated expert consent conversations by key stakeholders.

BACKGROUND: Consent conversations in pediatric surgery are essential components of pre-operative care which, when inadequate, can lead to significant adverse consequences for the child, parents, surgeon, and others in the healthcare system. The aim of this study is to explore expert consenting practice from the key stakeholders' perspective. METHODS: Four senior attending pediatric surgeons obtained consent from a standardized mother of a child requiring surgery in two scenarios: a low-risk elective surgery (inguinal hernia repair - Video 1), and a high-risk emergency surgery (intestinal atresia - Video 2). All sessions were recorded. Families of children who had undergone minor or major surgery, families without medical or surgical background, and healthcare professionals were invited to view and evaluate the videos using a semi-structured questionnaire. RESULTS: Out of 251 distributed surveys, 56 complete responses were received. Thirty two participants (57.1%) evaluated video 1 and 24 (42.9%) evaluated. Overall, 22 (69%) respondents to video 1 and 20 (84%) respondents to video 2 were "very satisfied" with the recorded consent conversation. Qualitative responses shared common themes of valuing surgeon empathy, good surgeon communication, patient engagement, and adequate time and information. Suggestions for improvement included additional resources and visual aids, improved patient engagement, and discussion of post-operative expectations. CONCLUSION: Our data identifies strengths and gaps in the current consent process from the perspective of patient families and providers. Identified areas for improvement in the informed consent process based on multi-stakeholder input will guide the planned development of a consenting educational video resource. LEVEL OF EVIDENCE: IV.

Understanding the effectiveness of consent processes and conversations in pediatric surgery: A systematic-scoping review.

BACKGROUND: The consent conversation in pediatric surgery is an essential part of pre-operative care which, when inadequate, can lead to significant adverse consequences for the child, parents, surgeon, other healthcare workers and the healthcare system. We reviewed the published literature on what key stakeholders perceive are the components of effective and ineffective consenting processes in pediatric surgery. METHODS: A medical librarian searched seven databases to retrieve articles looking at the informed consenting process in surgical care for the pediatric population. Two independent reviewers screened all publications and categorized them by stakeholder perspectives (patient/family, surgical team, other healthcare team, and hospital administration or policy maker). General study characteristics, interventions to improve consent and features of effective and ineffective consent conversations were extracted. RESULTS: 5079 titles and abstracts were screened, resulting in 88 full-text studies and 43 articles included in the final review. Most publications (51%) discussed informed consent only from the patient/family perspective, while 21% added surgeon's perspective. No study approached the consenting process from the perspective of all stakeholder groups. Effective consent components identified included use of multimedia, presence of multiple conversations prior to surgery, and individualized communication catered to unique family knowledge and needs. In contrast, ineffective conversations did not include a clear assessment of parental understanding, delivered too much information, and did not address parental anxiety. CONCLUSIONS: The literature on the consenting process in pediatric surgery is narrow in stakeholder perspectives. Our findings highlight gaps in the literature and opportunities to improve the informed consent processes prior to pediatric surgery.

Japanese medical learners' achievement emotions: Accounting for culture in translating Western medical educational theories and instruments into an asian context.

Medical learners' achievement emotions during educational activities have remained unexamined in Asian cultural contexts. The Medical Emotion Scale (MES) was previously developed to assess achievement emotions experienced by North American medical learners during learning activities. The goal of this study was to create and validate a Japanese version of the Medical Emotion Scale (J-MES). We translated the MES into Japanese and conducted two initial validation studies of the J-MES. In the first pilot study, we asked five, native-Japanese, second-year medical students to assess their emotions with the J-MES during a computer-based clinical reasoning activity. Each participant was then interviewed to assess the clarity and suitability of the items. In a second, larger study, 41 Japanese medical students were recruited to assess the psychometric properties of the J-MES. We also conducted individual, semi-structured interviews with ten of these participants to explore potential cultural features in the achievement emotions of Japanese students. The first pilot study demonstrated that the J-MES descriptions were clear, and that the scale captured an appropriate range of emotions. The second study revealed that the J-MES scale's profiles and internal structure were largely consistent with control-value theory. The achievement emotions of pride, compassion, and surprise in the J-MES were found to be susceptible to cultural differences between North American and Japanese contexts. Our findings clearly demonstrated the scoring capacity, generalizability, and extrapolability of the J-MES.

Mathermycin, an anti-cancer molecule that targets cell surface phospholipids.

Mathermycin, a lantipeptide isolated from marine actinomycete Marinactinospora thermotolerans, is an antibiotic that has been shown to disrupt bacterial plasma membrane. We now provide evidences that mathermycin can also disrupt cancer, but not normal, cell plasma membranes through targeting phosphatidylethanolamine (PE), which is located only in the inner leaflet of the plasma membrane in normal cells but in both the inner and outer leaflets of the membrane in tumor cells. Our data shows that mathermycin inhibits the metabolic activity and induces mainly necrotic death of all cancer cell lines with EC50 between 4.2 and 16.9 µM, while normal cell lines have EC50 between 113 and 129 µM. The cytotoxicity of mathermycin could be inhibited by exogenous PE, but not phosphoserine and phosphocholine. The formation of mathermycin-PE complexes was confirmed by in silico analysis, HPLC and MS spectrometer. Furthermore, mathermycin exhibited similar cytotoxicity toward cancer and multidrug resistant cancer cells, which could be due to its ability to inhibit mitochondrial function, as shown by our data from the Seahorse™ metabolic analyzer. This study demonstrates that mathermycin is a potentially effective class of anti-tumor chemotherapeutics that do not easily develop resistance due to a mechanism of action targeting PE.

Oligomycin-induced proton uncoupling.

Oligomycin is a classical mitochondrial reagent that binds to the proton channel on the Fo component of ATP synthase. As a result, oligomycin blocks mitochondrial ATP synthesis, proton translocation, and O2 uptake. Here we show that oligomycin induces proton uncoupling subsequent to inhibition of ATP synthesis, as evidenced by recovery of O2 uptake to near baseline levels. Uncoupling is uniquely rapid and readily observed in HepG2 cells but is also observed at longer times in the unrelated H1299 cell line. Proton fluxes plateau at oligomycin concentrations in the region 0.25-5 µM. At the plateau, fluxes are lower than expected for the classical mitochondrial permeability transition pore, although in H1229 cells, fluxes increase to levels consistent with pore opening at higher oligomycin concentrations. Uncoupling is observed in cells metabolizing either pyruvate or lactate and reversed by addition of glucose to restore ATP synthesis. Uncoupling is not sensitive to cyclosporin A and is not reversed by the ANT inhibitor bongkrekic acid. However, bongkrekic acid inhibits uncoupling if added before oligomycin, which we interpret in terms of maintenance of mitochondrial ATP levels.

A case of acute kidney injury secondary to black cherry concentrate in a patient with chronic kidney disease secondary to type 2 diabetes mellitus.

There are many herbal products which are accessible to patients, and they may provide with many health benefits. Nevertheless, some of these supplements can lead to significant morbidity as they can also have important side effects and impact patient's organ systems. In this case report, we present a patient with chronic kidney disease secondary to type II diabetes mellitus who develops acute kidney injury and metabolic disturbances secondary to consuming black cherry concentrate as a mean to self-manage his gout flare. The most likely mechanism of injury was cyclooxygenase inhibition by anthocyanins, molecular compounds found in cherries that have a similar mechanism of action to nonsteroidal anti-inflammatory medications. Patient's kidney injury and metabolic disturbances improved after the discontinuation of black cherry concentrate. This is the second case report that presents a correlation between consumption of cherry concentrate in a patient with chronic kidney disease and acute kidney injury.

Reducing Glucokinase Activity Restores Endogenous Pulsatility and Enhances Insulin Secretion in Islets From db/db Mice.

An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and ß-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.

Internal Medicine Point-of-Care Ultrasound Curriculum: Consensus Recommendations from the Canadian Internal Medicine Ultrasound (CIMUS) Group.

Bedside point-of-care ultrasound (POCUS) is increasingly used to assess medical patients. At present, no consensus exists for what POCUS curriculum is appropriate for internal medicine residency training programs. This document details the consensus-based recommendations by the Canadian Internal Medicine Ultrasound (CIMUS) group, comprising 39 members, representing 14 institutions across Canada. Guiding principles for selecting curricular content were determined a priori. Consensus was defined as agreement by at least 80% of the members on POCUS applications deemed appropriate for teaching and assessment of trainees in the core (internal medicine postgraduate years [PGY] 1-3) and expanded (general internal medicine PGY 4-5) training programs. We recommend four POCUS applications for the core PGY 1-3 curriculum (inferior vena cava, lung B lines, pleural effusion, and abdominal free fluid) and three ultrasound-guided procedures (central venous catheterization, thoracentesis, and paracentesis). For the expanded PGY 4-5 curriculum, we recommend an additional seven applications (internal jugular vein, lung consolidation, pneumothorax, knee effusion, gross left ventricular systolic function, pericardial effusion, and right ventricular strain) and four ultrasound-guided procedures (knee arthrocentesis, arterial line insertion, arterial blood gas sampling, and peripheral venous catheterization). These recommendations will provide a framework for training programs at a national level.

What adult electrocardiogram (ECG) diagnoses and/or findings do residents in emergency medicine need to know?

OBJECTIVE: There is no evidence-based description of electrocardiogram (ECG) interpretation competencies for emergency medicine (EM) trainees. The first step in defining these competencies is to develop a prioritized list of adult ECG findings relevant to EM contexts. The purpose of this study was to categorize the importance of various adult ECG diagnoses and/or findings for the EM trainee. METHODS: We developed a list of potentially important adult ECG diagnoses/findings and conducted a Delphi opinion-soliciting process. Participants used a 4-point Likert scale to rate the importance of each diagnosis for EM trainees. Consensus was defined as a minimum of 75% agreement at the second round or later. In the absence of consensus, stability was defined as a shift of 20% or less after successive rounds. RESULTS: A purposive sampling of 22 emergency physicians participated in the Delphi process, and 16 (72%) completed the process. Of those, 15 were from 11 different EM training programs across Canada and one was an expert in EM electrocardiography. Overall, 78 diagnoses reached consensus, 42 achieved stability and one diagnosis achieved neither consensus nor stability. Out of 121 potentially important adult ECG diagnoses, 53 (44%) were considered "must know" diagnoses, 61 (50%) "should know" diagnoses, and 7 (6%) "nice to know" diagnoses. CONCLUSION: We have categorized adult ECG diagnoses within an EM training context, knowledge of which may allow clinical EM teachers to establish educational priorities. This categorization will also facilitate the development of an educational framework to establish EM trainee competency in ECG interpretation.

SEURAT-1 liver gold reference compounds: a mechanism-based review.

There is an urgent need for the development of alternative methods to replace animal testing for the prediction of repeat dose chemical toxicity. To address this need, the European Commission and Cosmetics Europe have jointly funded a research program for 'Safety Evaluation Ultimately Replacing Animal Testing.' The goal of this program was the development of in vitro cellular systems and associated computational capabilities for the prediction of hepatic, cardiac, renal, neuronal, muscle, and skin toxicities. An essential component of this effort is the choice of appropriate reference compounds that can be used in the development and validation of assays. In this review, we focus on the selection of reference compounds for liver pathologies in the broad categories of cytotoxicity and lipid disorders. Mitochondrial impairment, oxidative stress, and apoptosis are considered under the category of cytotoxicity, while steatosis, cholestasis, and phospholipidosis are considered under the category of lipid dysregulation. We focused on four compound classes capable of initiating such events, i.e., chemically reactive compounds, compounds with specific cellular targets, compounds that modulate lipid regulatory networks, and compounds that disrupt the plasma membrane. We describe the molecular mechanisms of these compounds and the cellular response networks which they elicit. This information will be helpful to both improve our understanding of mode of action and help in the selection of appropriate mechanistic biomarkers, allowing us to progress the development of animal-free models with improved predictivity to the human situation.

Patient-centred education: what do students think?

CONTEXT: Medical educators endeavour to foster patient-centred learning. Although studies of patient-educators report general increases in patient-centredness, no formal review of students' reflections on the role of patients in their education has yet been undertaken. Our research questions were: (i) What themes might be identified through a qualitative analysis of students' reflective writing on patient-centred education? (ii) What are common students' perceptions regarding patients as educators? METHODS: For two academic years, Year 2 pre-clinical students (189 and 167 students, respectively, in each academic year) submitted a 250-word writing assignment in response to one of four questions meant to promote reflection on the role of patients in their education. Using a grounded theory approach, we performed a qualitative analysis of these written reflections for emerging themes. A synthesis of these themes was prepared and was presented for validation and discussion by two focus groups of six and three students, respectively. We analysed the transcripts of the focus group discussions and compared them with results from the analysis of written reflections and used them to further inform and refine our initial thematic framework. RESULTS: A total of 356 reflective writing assignments were analysed. The major themes were: (i) students seeing the condition within the context of patients' lives; (ii) patients supporting students' learning; (iii) students recognising patients' needs; (iv) students seeing the patient as a capable part of the team, and (v) students recognising the complexity of practising medicine. The two focus group discussions confirmed these main themes, but placed greater emphasis on the first and second themes. These themes mapped closely to the conceptualisation of patient-centred care defined by the International Alliance of Patients' Organizations. CONCLUSIONS: Students' reflections on their experiences of patient-educators cover an important and broad range of key concepts in patient-centred care that are well aligned with patient-generated conceptualisations of patient-centred care.

Pharmacologic stimulation of cytochrome P450 46A1 and cerebral cholesterol turnover in mice.

Cytochrome P450 46A1 (CYP46A1) is a brain-specific cholesterol 24-hydroxylase responsible for the majority of cholesterol elimination from the brain. Genetically increased CYP46A1 expression in mice leads to improved cognition and decreases manifestations of Alzheimer disease. We found that four pharmaceuticals (efavirenz (EFV), acetaminophen, mirtazapine, and galantamine) prescribed for indications unrelated to cholesterol maintenance increased CYP46A1 activity in vitro. We then evaluated the anti-HIV medication EFV for the mode of interaction with CYP46A1 and the effect on mice. We propose a model for CYP46A1 activation by EFV and show that EFV enhanced CYP46A1 activity and cerebral cholesterol turnover in animals with no effect on the levels of brain cholesterol. The doses of EFV administered to mice and required for the stimulation of their cerebral cholesterol turnover are a hundred times lower than those prescribed to HIV patients. At such small doses, EFV may be devoid of adverse effects elicited by high drug concentrations. CYP46A1 could be a novel therapeutic target and a tool to further investigate the physiological and medical significance of cerebral cholesterol turnover.

In silico and intuitive predictions of CYP46A1 inhibition by marketed drugs with subsequent enzyme crystallization in complex with fluvoxamine.

Cytochrome P450 46A1 (cholesterol 24-hydroxylase) is an important brain enzyme that may be inhibited by structurally distinct pharmaceutical agents both in vitro and in vivo. To identify additional inhibitors of CYP46A1 among U.S. Food and Drug Administration-approved therapeutic agents, we used in silico and intuitive predictions and evaluated some of the predicted binders in the enzyme and spectral binding assays. We tested a total of 298 marketed drugs for the inhibition of CYP46A1-mediated cholesterol hydroxylation in vitro and found that 13 of them reduce CYP46A1 activity by >50%. Of these 13 inhibitors, 7 elicited a spectral response in CYP46A1 with apparent spectral K(d) values in a low micromolar range. One of the identified tight binders, the widely used antidepressant fluvoxamine, was cocrystallized with CYP46A1. The structure of this complex was determined at a 2.5 Å resolution and revealed the details of drug binding to the CYP46A1 active site. The NH(2)-containing arm of the Y-shaped fluvoxamine coordinates the CYP46A1 heme iron, whereas the methoxy-containing arm points away from the heme group and has multiple hydrophobic interactions with aliphatic amino acid residues. The CF(3)-phenyl ring faces the entrance to the substrate access channel and has contacts with the aromatic side chains. The crystal structure suggests that only certain drug conformers can enter the P450 substrate access channel and reach the active site. Once inside the active site, the conformer probably further adjusts its configuration and elicits the movement of the protein side chains.

The Postoperative Pain Assessment Skills pilot trial.

UNLABELLED: BACKGROUND/ OBJECTIVES: Pain-related misbeliefs among health care professionals (HCPs) are common and contribute to ineffective postoperative pain assessment. While standardized patients (SPs) have been effectively used to improve HCPs' assessment skills, not all centres have SP programs. The present equivalence randomized controlled pilot trial examined the efficacy of an alternative simulation method - deteriorating patient-based simulation (DPS) - versus SPs for improving HCPs' pain knowledge and assessment skills. METHODS: Seventy-two HCPs were randomly assigned to a 3 h SP or DPS simulation intervention. Measures were recorded at baseline, immediate postintervention and two months postintervention. The primary outcome was HCPs' pain assessment performance as measured by the postoperative Pain Assessment Skills Tool (PAST). Secondary outcomes included HCPs knowledge of pain-related misbeliefs, and perceived satisfaction and quality of the simulation. These outcomes were measured by the Pain Beliefs Scale (PBS), the Satisfaction with Simulated Learning Scale (SSLS) and the Simulation Design Scale (SDS), respectively. Student's t tests were used to test for overall group differences in postintervention PAST, SSLS and SDS scores. One-way analysis of covariance tested for overall group differences in PBS scores. RESULTS: DPS and SP groups did not differ on post-test PAST, SSLS or SDS scores. Knowledge of pain-related misbeliefs was also similar between groups. CONCLUSIONS: These pilot data suggest that DPS is an effective simulation alternative for HCPs' education on postoperative pain assessment, with improvements in performance and knowledge comparable with SP-based simulation. An equivalence trial to examine the effectiveness of deteriorating patient-based simulation versus standardized patients is warranted.

The impact of trained patient educators on musculoskeletal clinical skills attainment in pre-clerkship medical students.

BACKGROUND: Despite the high burden of musculoskeletal (MSK) diseases, few generalists are comfortable teaching MSK physical examination (PE) skills. Patient Partners® in Arthritis (PP®IA) is a standardized patient educator program that could potentially supplement current MSK PE teaching. This study aims to determine if differences exist in MSK PE skills between non-MSK specialist physician and PP®IA taught students. METHODS: Pre-clerkship medical students attended 2-hour small group MSK PE teaching by either non-MSK specialist physician tutors or by PP®IA. All students underwent an MSK OSCE and completed retrospective pre-post questionnaires regarding comfort with MSK PE and interest in MSK. RESULTS: 83 students completed the OSCE (42 PP®IA, 41 physician taught) and 82 completed the questionnaire (42 PP®IA, 40 physician taught). There were no significant differences between groups in OSCE scores. For all questionnaire items, post-session ratings were significantly higher than pre-session ratings for both groups. In exploratory analysis PP®IA students showed significantly greater improvement in 12 of 22 questions including three of five patient-centred learning questions. CONCLUSIONS: PP®IA MSK PE teaching is as good as non-MSK specialist physician tutor teaching when measured by a five station OSCE and provide an excellent complementary resource to address current deficits in MSK PE teaching.

Use of the one-minute preceptor as a teaching tool in the gross anatomy laboratory.

The one-minute preceptor (OMP) is a time-efficient technique used for teaching in busy clinical settings. It consists of five microskills: (1) get a commitment from the student, (2) probe for supporting evidence, (3) reinforce what was done right, (4) correct errors and fill in omissions, and (5) teach a general rule. It can also be used to address structure-identification questions in gross anatomy laboratory small-group settings. The OMP is an active learner-centered teaching approach that prepares students for a style of learning that they need to master in clinical settings, provides novice anatomy teachers with an efficient and effective teaching strategy, and moves anatomy learning beyond mere name tagging to active knowledge construction.

Musculoskeletal examination teaching by patients versus physicians: how are they different? Neither better nor worse, but complementary.

BACKGROUND: Musculoskeletal (MSK) complaints comprise 12-20% of primary healthcare; however, practicing physicians' MSK physical examination (PE) skills are weak. Further, there is a shortage of specialists able to effectively teach this subject. Previous evaluations of patient educators have yielded mixed results. AIMS: The aim of this study is to document how teaching by patient educators and physician tutors in MSK PE skills differs. METHODS: A qualitative researcher observed, video-recorded, and took notes during preclerkship MSK PE teaching sessions given by patient educators or physician tutors. The researcher identified themes which were evaluated by collective case study methods. RESULTS: Two patient educator and four physician groups were evaluated. The patient educators were more consistent regarding content and style than the physicians. There appeared to be a continuum in teaching organization from patient educator to novice physician tutors to experienced physician tutors. The patient educators consistently covered all major joints (physicians did not); physicians were more likely to request verbalization of actions, relate findings to history, receive questions, and use opportunistic teaching moments. CONCLUSIONS: Understanding preclerkship MSK teaching by patient educators compared to physician tutors is necessary for appropriate targeting of the existing Patient Partners® in Arthritis patient educator program and to guide the development of future MSK teaching initiatives.

Transplant-insert-constrain-relax-assemble (TICRA): protein-ligand complex structure modeling and application to kinases.

We introduce TICRA (transplant-insert-constrain-relax-assemble), a method for modeling the structure of unknown protein-ligand complexes using the X-ray crystal structures of homologous proteins and ligands with known activity. We present results from modeling the structures of protein kinase-inhibitor complexes using p38 and Lck as examples. These examples show that the TICRA method may be used prospectively to create and refine models for protein kinase-inhibitor complexes with an overall backbone rmsd of less than 0.75 Å for the kinase domain, when compared to published X-ray crystal structures. Further refinement of the models of the kinase domains of p38 and Lck in complex with their cognate ligands from the published crystal structures was able to improve the rmsd's of the model complexes to below 0.5 Å. Our results show that TICRA is a useful approach to the problem of structure-based drug design in cases where little structural information is available for the target proteins and the binding mode of active compounds is unknown.