In a study of a standardized fracture of the tibia in dogs, we examined the relationship between extraction of strontium-85, as measured by a multiple indicator-dilution technique, and blood flow, as determined by an iodoantipyrine-washout technique. Although the blood flow at the fracture site increased from a control value of 1.5 milliliters per 100 grams of bone per minute to a value of 6.65 milliliters per 100 grams of bone per minute at two weeks, the maximum instantaneous extraction of 85Sr by the fractured tibiae did not change from a value of about 0.77 of the amount injected. These results suggest that the increase in blood flow is accomplished by recruitment of capillaries, that the permeabilities of the capillary beds in the fractured tibia and in the normal tibia are similar, and that the pattern of flow in the fractured tibia remains non-uniform. The increased capillary surface area available for exchange is suggested as the reason that strontium extraction was unchanged despite the increased flow.
This study has shown that calcium is only moderately rapidly exchangeable across the capillary membrane and made available for uptake by osteoblasts and deposition in callus at the fracture site. Because calcium delivery via capillary is essential, and because capillary surface area is ordinarily increased in the region of a healing fracture, we can now see clearly that clinical treatment, in particular manipulative intervention, should be carried out in a fashion that minimizes damage to the capillary bed. The exchange processes described in this study are the same as those involved in the deposition of agents used for bone-scanning, and thus this information provides a basis for the timing of scanning studies and for their interpretation.
Maternal mortality in much of sub-Saharan Africa is very high whereas there has been a steady decline in over the past 60 years in Europe. Perinatal mortality is 12 times higher than maternal mortality accounting for about 7 million neonatal deaths; many of these in sub-Saharan countries. Many of these deaths are preventable. Countries, like Malawi, do not have the resources nor highly trained medical specialists using complex technologies within their healthcare system. Much of the burden falls on healthcare staff other than doctors including non-physician clinicians (NPCs) such as clinical officers, midwives and community health-workers. The aim of this trial is to evaluate a project which is training NPCs as advanced leaders by providing them with skills and knowledge in advanced neonatal and obstetric care. Training that will hopefully be cascaded to their colleagues (other NPCs, midwives, nurses).
This is a cluster randomised controlled trial with the unit of randomisation being the 14 districts of central and northern Malawi (one large district was divided into two giving an overall total of 15). Eight districts will be randomly allocated the intervention. Within these eight districts 50 NPCs will be selected and will be enrolled on the training programme (the intervention). Primary outcome will be maternal and perinatal (defined as until discharge from health facility) mortality. Data will be harvested from all facilities in both intervention and control districts for the lifetime of the project (3–4 years) and comparisons made. In addition a process evaluation using both quantitative and qualitative (e.g. interviews) will be undertaken to evaluate the intervention implementation.
Education and training of NPCs is a key to improving healthcare for mothers and babies in countries like Malawi. Some of the challenges faced are discussed as are the potential limitations. It is hoped that the findings from this trial will lead to a sustainable improvement in healthcare and workforce development and training.
Virtual Patients (VPs) are web-based representations of realistic clinical cases. They are proposed as being an optimal method for teaching clinical reasoning skills. International standards exist which define precisely what constitutes a VP. There are multiple design possibilities for VPs, however there is little formal evidence to support individual design features. The purpose of this trial is to explore the effect of two different potentially important design features on clinical reasoning skills and the student experience. These are the branching case pathways (present or absent) and structured clinical reasoning feedback (present or absent).
This is a multi-centre randomised 2x2 factorial design study evaluating two independent variables of VP design, branching (present or absent), and structured clinical reasoning feedback (present or absent).The study will be carried out in medical student volunteers in one year group from three university medical schools in the United Kingdom, Warwick, Keele and Birmingham. There are four core musculoskeletal topics. Each case can be designed in four different ways, equating to 16 VPs required for the research. Students will be randomised to four groups, completing the four VP topics in the same order, but with each group exposed to a different VP design sequentially. All students will be exposed to the four designs. Primary outcomes are performance for each case design in a standardized fifteen item clinical reasoning assessment, integrated into each VP, which is identical for each topic. Additionally a 15-item self-reported evaluation is completed for each VP, based on a widely used EViP tool. Student patterns of use of the VPs will be recorded.
In one centre, formative clinical and examination performance will be recorded, along with a self reported pre and post-intervention reasoning score, the DTI. Our power calculations indicate a sample size of 112 is required for both primary outcomes.
This trial will provide robust evidence to support the effectiveness of different designs of virtual patients, based on student performance and evaluation. The cases and all learning materials will be open access and available on a Creative Commons Attribution-Share-Alike license.
Virtual patients; Clinical reasoning; Elearning; Education; Undergraduate; Musculoskeletal; Rheumatology
Tuberous sclerosis complex (TSC) is a genetic condition characterized by the growth of benign tumours in multiple organs, including the brain and kidneys, alongside intellectual disability and seizures. Identification of a causative mutation in TSC1 or TSC2 is important for accurate genetic counselling in affected families, but it is not always clear from genetic data whether a sequence variant is pathogenic or not. In vitro functional analysis could provide support for determining whether an unclassified TSC1 or TSC2 variant is disease-causing. We have performed a detailed functional analysis of four patient-derived TSC2 mutations, E92V, R505Q, H597R and L1624P. One mutant, E92V, functioned similarly to wild-type TSC2, whereas H597R and L1624P had abnormal function in all assays, consistent with available clinical and segregation information. One TSC2 mutation, R505Q, was identified in a patient with intellectual disability, seizures and autistic spectrum disorder but who did not fulfil the diagnostic criteria for TSC. The R505Q mutation was also found in two relatives, one with mild learning difficulties and one without apparent phenotypic abnormality. R505Q TSC2 exhibited partially disrupted function in our assays. These data highlight the difficulties of assessing pathogenicity of a mutation and suggest that multiple lines of evidence, both genetic and functional, are required to assess the pathogenicity of some mutations.
TSC; mutation; function; pathogenicity
The membrane-bound Toll-like receptors (TLRs) trigger innate immune responses following recognition of a wide variety of pathogen-derived compounds. Despite the wide range of ligands recognized by TLRs, the receptors share a common structural framework in their extracellular, ligand-binding domains. These domains all adopt horseshoe-shaped structures built from leucine-rich repeat motifs. Typically, upon ligand binding, two extracellular domains form an “m”-shaped dimer sandwiching the ligand molecule bringing the transmembrane and cytoplasmic domains in close proximity and triggering a downstream signalling cascade. Although the ligand-induced dimerization of these receptors has many common features, the nature of the interactions of the TLR extracellular domains with their ligands varies markedly between TLR paralogs.
Toll-like receptor; innate immunity; inflammation; leucine-rich repeat; dsRNA; Pattern recognition receptor
Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z+ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28z+ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.
Toll-like receptors (TLRs) recognize conserved molecular patterns in invading pathogens and trigger innate immune responses. TLR3 recognizes dsRNA, a molecular signature of most viruses via its ectodomain (ECD). The TLR3-ECD structure consists of a 23 turn coil bent into the shape of a horseshoe with specialized domains capping the N and C terminal ends of the coil. TLR3-ECDs bind as dimeric units to dsRNA oligonucleotides of at least 45 bp in length, the minimal length required for signal transduction. X-ray analysis has shown that each TLR3-ECD of a dimer binds dsRNA at two sites located at opposite ends of the TLR3 “horseshoe” on the one lateral face that lacks N-linked glycans. Intermolecular contacts between the C-terminal domains of two TLR3-ECDs stabilizes the dimer and positions the C-terminal residues within 20–25Å of each other, which is thought to be essential for transducing a signal across the plasma membrane in intact TLR3 molecules. Interestingly, in TLRs 1, 2 and 4, which bind lipid ligands using very different interactions from TLR3, the ligands nevertheless promote the formation of a dimer in which the same two lateral surfaces as in the TLR3-ECD:dsRNA complex face each other, bringing their C-termini in close proximity. Thus, a pattern is emerging in which pathogen-derived substances bind to TLR-ECDs, thereby promoting the formation of a dimer in which the glycan-free ligand-binding surfaces face each other and the two C-termini are brought in close proximity for signal transduction.
Toll-like receptor; innate immunity; inflammation; leucine-rich repeat; dsRNA; Pattern recognition receptor
The extractions of 85Sr2+, 18F−, sucrose-14C, EDTA-51Cr, and antipyrine-14C in bone were determined by the multiple indicator-dilution method. Fluoride and strontium extractions were 18 to 70% during a single transcapillary passage, and those of EDTA and sucrose were from 11 to 59%, whereas extraction of antipyrine was 87%. Injections of 85Sr2+ and 18F− made when perfusion was done alternately with blood and plasma resulted in similar fractional extractions. When flow and extraction were measured simultaneously, extraction was related inversely to flow.
Bone blood flow; Capillary permeability; Tracer exchange
Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the β-propeller and α/β-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.
Diabetes; Protein Cross-linking; Protein Drug Interactions; Serine Protease; X-ray Crystallography; Conformational Change; Induced Fit; Neurological Disease; Prolyl Endopeptidase
Toll-like receptor 3 (TLR3) recognizes dsRNA, a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECD) dimerize on oligonucleotides of at least 40–50 bp in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 Å resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 “horseshoe”, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll IL-1 Receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.
Lofexidine is an alpha-2-A noradrenergic receptor agonist that is approved in the United Kingdom for the treatment of opioid withdrawal symptoms. Lofexidine has been reported to have more significant effects on decreasing opioid withdrawal symptoms with less hypotension than clonidine.
To demonstrate that lofexidine is well tolerated and effective in the alleviation of observationally-defined opioid withdrawal symptoms in opioid dependent individuals undergoing medically supervised opioid detoxification as compared to placebo.
An inpatient, Phase 3, placebo-controlled, double blind, randomized multi-site trial with three phases: (1) Opioid Agonist Stabilization Phase (days 1–3), (2) Detoxification/Medication or Placebo Phase (days 4–8), and (3) Post Detoxification/Medication Phase (days 9–11).
Sixty-eight opioid dependent subjects were enrolled at three sites with 35 randomized to lofexidine and 33 to placebo.
Main Outcome Measure
Modified Himmelsbach Opiate Withdrawal Scale (MHOWS) on study day 5 (2nd opioid detoxification treatment day).
Due to significant findings, the study was terminated early. On the study day 5 MHOWS, subjects treated with lofexidine had significantly lower scores (equating to fewer/less severe withdrawal symptoms) than placebo subjects (Least squares means 19.5 ± 2.1 versus 30.9 ± 2.7; p=0.0019). Lofexidine subjects had significantly better retention in treatment than placebo subjects (38.2% versus 15.2%; Log rank test p=0.01).
Lofexidine is well tolerated and more efficacious than placebo for reducing opioid withdrawal symptoms in inpatients undergoing medically supervised opioid detoxification.
trial registry name A Phase 3 Placebo-Controlled, Double-Blind Multi-Site Trial of Lofexidine for Opiate Withdrawal, registration number NCT00032942, URL for the registry http://clinicaltrials.gov/ct/show/NCT00032942?order=4.
Lofexidine; Alpha 2 Agonist; Opioid Withdrawal Treatment; Phase 3; Placebo-Controlled; Double-Blind; Multi-Site Trail
It is well established that in nature, bacteria are found primarily as residents of surface-associated communities called biofilms. These structures form in a sequential process initiated by attachment of cells to a surface, followed by the formation of matrix-enmeshed microcolonies, and culminating in dispersion of the bacteria from the mature biofilm. In the present study, we have demonstrated that, during growth, Pseudomonas aeruginosa produces an organic compound we have identified as cis-2-decenoic acid, which is capable of inducing the dispersion of established biofilms and of inhibiting biofilm development. When added exogenously to P. aeruginosa PAO1 biofilms at a native concentration of 2.5 nM, cis-2-decenoic acid was shown to induce the dispersion of biofilm microcolonies. This molecule was also shown to induce dispersion of biofilms, formed by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Bacillus subtilis, Staphylococcus aureus, and the yeast Candida albicans. Active at nanomolar concentrations, cis-2-decenoic acid appears to be functionally and structurally related to the class of short-chain fatty acid signaling molecules such as diffusible signal factor, which act as cell-to-cell communication molecules in bacteria and fungi.
At postgraduate level evidence based medicine (EBM) is currently taught through tutor based lectures. Computer based sessions fit around doctors' workloads, and standardise the quality of educational provision. There have been no randomized controlled trials comparing computer based sessions with traditional lectures at postgraduate level within medicine.
This was a randomised controlled trial involving six postgraduate education centres in the West Midlands, U.K. Fifty five newly qualified foundation year one doctors (U.S internship equivalent) were randomised to either computer based sessions or an equivalent lecture in EBM and systematic reviews. The change from pre to post-intervention score was measured using a validated questionnaire assessing knowledge (primary outcome) and attitudes (secondary outcome).
Both groups were similar at baseline. Participants' improvement in knowledge in the computer based group was equivalent to the lecture based group (gain in score: 2.1 [S.D = 2.0] versus 1.9 [S.D = 2.4]; ANCOVA p = 0.078). Attitudinal gains were similar in both groups.
On the basis of our findings we feel computer based teaching and learning is as effective as typical lecture based teaching sessions for educating postgraduates in EBM and systematic reviews.
The crystal structure of 2,2-trimethylenedioxy-4,4,6,6-tetrachlorocyclo triphosphazene has been determined at 120, 274 and 293 K. The result at 293 K confirms the room temperature Cmc21 structure, but at the lower temperatures the space group is Pna21. Nevertheless the basic structure remains the same, with only small displacements of the atoms, amounting to an average of 25 pm between 120 and 293 K.
X-ray diffraction and DSC results indicate that the phase transition takes place in two steps between 274 – 293 K and provides an understanding of previous NQR results. In the intermediate temperature range the molecules are displaced from their room temperature positions in such a way as to give an average structure with Cmc21 symmetry.
The overall phase transition is consistent with the occurrence of a soft lattice mode at room temperature in which a large displacement of the molecule in the x-direction is coupled with a flexing motion about an axis defined by the nitrogen atoms in the N1 and N3 positions.
Phenotypic and genetic evidence supporting the notion of biofilm formation as a developmental process is growing. In the present work, we provide additional support for this hypothesis by identifying the onset of accumulation of biofilm-stage specific proteins during Pseudomonas aeruginosa biofilm maturation and by tracking the abundance of these proteins in planktonic and three biofilm developmental stages. The onset of protein production was found to correlate with the progression of biofilms in developmental stages. Protein identification revealed that proteins with similar function grouped within similar protein abundance patterns. Metabolic and housekeeping proteins were found to group within a pattern separate from virulence, antibiotic resistance, and quorum-sensing-related proteins. The latter were produced in a progressive manner, indicating that attendant features that are characteristic of biofilms such as antibiotic resistance and virulence may be part of the biofilm developmental process. Mutations in genes for selected proteins from several protein production patterns were made, and the impact of these mutations on biofilm development was evaluated. The proteins cytochrome c oxidase, a probable chemotaxis transducer, a two-component response regulator, and MexH were produced only in mature and late-stage biofilms. Mutations in the genes encoding these proteins did not confer defects in growth, initial attachment, early biofilm formation, or twitching motility but were observed to arrest biofilm development at the stage of cell cluster formation we call the maturation-1 stage. The results indicated that expression of theses genes was required for the progression of biofilms into three-dimensional structures on abiotic surfaces and the completion of the biofilm developmental cycle. Reverse transcription-PCR analysis confirmed the detectable change in expression of the respective genes ccoO, PA4101, and PA4208. We propose a possible mechanism for the role of these biofilm-specific proteins in biofilm formation.
Several studies have reported health effects of concentrated ambient particles (CAP) in rodents and humans; however, toxicity end points in rodents have provided inconsistent results. In 2000 we conducted six 1-day exposure studies where spontaneously hypertensive (SH) rats were exposed to filtered air or CAPs (≤ 2.5 μm, 1,138–1,765 μg/m3) for 4 hr (analyzed 1–3 hr afterward). In seven 2-day exposure studies in 2001, SH and Wistar Kyoto (WKY) rats were exposed to filtered air or CAP (≤ 2.5 μm, 144–2,758 μg/m3) for 4 hr/day × 2 days (analyzed 1 day afterward). Despite consistent and high CAP concentrations in the 1-day exposure studies, no biologic effects were noted. The exposure concentrations varied among the seven 2-day exposure studies. Except in the first study when CAP concentration was highest, lavageable total cells and macrophages decreased and neutrophils increased in WKY rats. SH rats demonstrated a consistent increase of lavage fluid γ -glutamyltransferase activity and plasma fibrinogen. Inspiratory and expiratory times increased in SH but not in WKY rats. Significant correlations were found between CAP mass (microgram per cubic meter) and sulfate, organic carbon, or zinc. No biologic effects correlated with CAP mass. Despite low chamber mass in the last six of seven 2-day exposure studies, the levels of zinc, copper, and aluminum were enriched severalfold, and organic carbon was increased to some extent when expressed per milligram of CAP. Biologic effects were evident in those six studies. These studies demonstrate a pattern of rat strain–specific pulmonary and systemic effects that are not linked to high mass but appear to be dependent on CAP chemical composition.
concentrated ambient particles; fibrinogen; γ-glutamyltransferase; hypertensive rats; lung inflammation; macrophages; neutrophils; Wistar Kyoto rats
Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.
Virtual patients (VPs) are online representations of clinical cases used in medical education. Widely adopted, they are well placed to teach clinical reasoning skills. International technology standards mean VPs can be created, shared and repurposed between institutions. A systematic review has highlighted the lack of evidence to support which of the numerous VP designs may be effective, and why. We set out to research the influence of VP design on medical undergraduates.
This is a grounded theory study into the influence of VP design on undergraduate medical students. Following a review of the literature and publicly available VP cases, we identified important design properties. We integrated them into two substantial VPs produced for this research. Using purposeful iterative sampling, 46 medical undergraduates were recruited to participate in six focus groups. Participants completed both VPs, an evaluation and a 1-hour focus group discussion. These were digitally recorded, transcribed and analysed using grounded theory, supported by computer-assisted analysis. Following open, axial and selective coding, we produced a theoretical model describing how students learn from VPs.
We identified a central core phenomenon designated ‘learning from the VP’. This had four categories: VP Construction; External Preconditions; Student–VP Interaction, and Consequences. From these, we constructed a three-layer model describing the interactions of students with VPs. The inner layer consists of the student's cognitive and behavioural preconditions prior to sitting a case. The middle layer considers the VP as an ‘encoded object’, an e-learning artefact and as a ‘constructed activity’, with associated pedagogic and organisational elements. The outer layer describes cognitive and behavioural change.
This is the first grounded theory study to explore VP design. This original research has produced a model which enhances understanding of how and why the delivery and design of VPs influence learning. The model may be of practical use to authors, institutions and researchers.