Directional Next-Generation RNA Sequencing and Examination of Premature Termination Codon Mutations in Endoglin/Hereditary Haemorrhagic Telangiectasia

Hereditary haemorrhagic telangiectasia (HHT) is a disease characterised by abnormal vascular structures, and most commonly caused by mutations in ENG, ACVRL1 or SMAD4 encoding endothelial cell-expressed proteins involved in TGF-β superfamily signalling. The majority of mutations reported on the HHT mutation database are predicted to lead to stop codons, either due to frameshifts or direct nonsense substitutions. The proportion is higher for ENG (67%) and SMAD4 (65%) than for ACVRL1 (42%), p < 0.0001. Here, by focussing on ENG, we report why conventional views of these mutations may need to be revised. Of the 111 stop codon-generating ENG mutations, on ExPASy translation, all except one were premature termination codons (PTCs), sited at least 50-55 bp upstream of the final exon-exon boundary of the main endoglin isoform, L-endoglin. This strongly suggests that the mutated RNA species will undergo nonsense-mediated decay. We provide new in vitro expression data to support dominant negative activity of stable truncated endoglin proteins but suggest these will not generate HHT: the single natural stop codon mutation in L-endoglin (sited within 50-55 nucleotides of the final exon-exon boundary) is unlikely to generate functional protein since it replaces the entire transmembrane domain, as would 8 further natural stop codon mutations, if the minor S-endoglin isoform were implicated in HHT pathogenesis. Finally, next-generation RNA sequencing data of 7 different RNA libraries from primary human endothelial cells demonstrate that multiple intronic regions of ENG are transcribed. The potential consequences of heterozygous deletions or duplications of such regions are discussed. These data support the haploinsufficiency model for HHT pathogenesis, explain why final exon mutations have not been detected to date in HHT, emphasise the potential need for functional examination of non-PTC-generating mutations, and lead to proposals for an alternate stratification system of mutational types for HHT genotype-phenotype correlations

Patient, clinician and independent observer perspectives of shared decision making in adult orthodontics.

OBJECTIVES: To investigate and compare the extent of shared decision making (SDM) in orthodontics from the perspective of patients, clinicians and independent observers. DESIGN: A cross-sectional, observational study. SETTING: NHS teaching hospital. PARTICIPANTS: A total of 31 adult patients and their treating clinicians were included in the study. METHODS: The extent of SDM in new patient orthodontic consultations was measured using three versions of a validated instrument: the self-administered patient dyadic-OPTION scale; the self-administered clinician dyadic-OPTION scale; and an independent observer-rated OPTION12 scale. Patients and clinicians completed the 12-item dyadic-OPTION questionnaire independently at the end of the consultation to rate their perceived levels of SDM. The consultations were also audio-recorded and two calibrated raters independently rated the extent of SDM in these consultations using the OPTION12 scale. RESULTS: There was excellent inter-rater reliability between the two independent raters using the OPTION12 scale (intraclass correlation coefficient (ICC) = 0.909). The mean patient, clinician and independent observer OPTION scores for SDM were 90.4% (SD 9.1%, range 70.8% to 100%), 76.2% (SD 8.95%, range 62.5% to 95.8%) and 42.6% (SD 17.4%, range 13.5% to 68.8%), respectively. There was no significant correlation between the OPTION scores for the three groups (ICC = -0.323). CONCLUSIONS: The results showed that generally high levels of SDM were perceived by patients and clinicians but lower levels of SDM were scored by the independent observers. However, it could be argued that the patient's perception of SDM is the most important measure as it is their care that is affected by their involvement

Characterization of selected Nigerian biomass for combustion and pyrolysis applications

Biomass is the most utilized form of renewable energy, especially in developing nations, and is a possible replacement for fossil fuel in power generation. The most commonly used method for recovering energy from biomass is combustion. Many countries are exploring the utilization of energy crops and indigenous residues to deliver sustainable sources of biomass. For these bio-resources, detailed characterization of the fuel properties is essential in order to optimize the combustion processes. In this study, some potential energy crops and woods from Nigeria, namely Terminalia superba, Gmelina arborea, Lophira alata, Nauclea diderrichii, and also one abundant agricultural residue, palm kernel expellers (PKE), were characterized for their combustion properties. Standard characterization methods such as proximate and ultimate analyses, metals analysis, and ash fusion test were used for this purpose and the results were compared with some U.K. biomass. In addition, their thermal conversion was assessed by thermogravimetric analysis and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). Finally, combustion studies were conducted by suspending single biomass particles in a methane flame to obtain information on reactivities and combustion characteristics. Results indicate that the ash fractions in the Nigerian woods were low in K, Si, and Ca, resulting in low calculated alkali indices, hence these fuels are not predicted to cause severe fouling problems. Furthermore, the analysis of the evolved product during devolatilisation from Py-GC-MS suggests that the content of oil is high in Gmelina. Finally, the results from the single particle combustion experiments revealed a longer char burn out rate for Lophira and Nauclea when compared with those of Terminalia and Gmelina

Combined Inflammatory and Metabolic Defects Reflected by Reduced Serum Protein Levels in Patients with Buruli Ulcer Disease

Buruli ulcer is a skin disease caused by Mycobacterium ulcerans that is spreading in tropical countries, with major public health and economic implications in West Africa. Multi-analyte profiling of serum proteins in patients and endemic controls revealed that Buruli ulcer disease down-regulates the circulating levels of a large array of inflammatory mediators, without impacting on the leukocyte composition of peripheral blood. Notably, several proteins contributing to acute phase reaction, lipid metabolism, coagulation and tissue remodelling were also impacted. Their down-regulation was selective and persisted after the elimination of bacteria with antibiotic therapy. It involved proteins with various functions and origins, suggesting that M. ulcerans infection causes global and chronic defects in the host’s protein metabolism. Accordingly, patients had reduced levels of total serum proteins and blood urea, in the absence of signs of malnutrition, or functional failure of liver or kidney. Interestingly, slow healers had deeper metabolic and coagulation defects at the start of antibiotic therapy. In addition to providing novel insight into Buruli ulcer pathogenesis, our study therefore identifies a unique proteomic signature for this disease

The relationship between 2d knee valgus angle during single leg squat (sls), single leg landing (sll), and forward running

BACKGROUND: Two-dimensional (2D) analysis of knee valgus during common athletic screening tasks such as SLS has been purported to identify individuals who may be at a high-risk of ACL injury. There is limited literature exploring the relationships between joint motion during SLS and other athletic tasks associated with knee joint injuries, in order to evaluate the effectiveness of the SLS to identify athletes with hazardous knee motion in a range of athletic tasks. OBJECTIVE: To assess the relationship between the 2D knee valgus angle among three tasks (SLS, SLL, and Running). DESIGN: A correlational study. SETTING: Undertaken in the Human Performance Laboratory at the University of Salford. PARTICIPANTS: 15 recreational athletes, 7 males and 8 females, were recruited (age 25.25.1 years; height 1.67.38 m; and mass 67.610.93 kg). INTERVENTION: Participants performed a series of SLS, SLL from a 31-cm step, and forward running at percieved maximum speed whilst being videotaped with a 2D digital camera. MAIN OUTCOME MEASUREMENTS: Maximum right knee valgus angle was quantified by measuring the frontal plane projection angle (FPPA) using Quintic Biomechanics v21 software package. RESULTS: A moderate correlation was shown between FPPA during SLS and SLL when the whole participant group was analysed (r=0.35). When split by gender female statistics showed good correlations between the majority of tasks; SLS and SLL (r=0.87), SLS and Run (r=0.59) but weaker between SLL and Run (0.26). CONCLUSIONS: In females the FPPA during SLS correlates with FPPA during SLL and running. This indicates that if a female patient has increased FPPA during SLS they are likely to have increased FPPA across all the tasks. This could potentially reduce the time and tasks required for screening, as only one task would need to be assessed. In males there is little correlation between tasks so the same would not apply in male subjects

Mycolactone Diffuses into the Peripheral Blood of Buruli Ulcer Patients - Implications for Diagnosis and Disease Monitoring.

BACKGROUND: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established. METHODOLOGY/PRINCIPAL FINDING: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone. CONCLUSIONS/SIGNIFICANCE: Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies

Localization of tenascin in human skin wounds

A total of 56 surgically treated human skin wounds with a wound age between 8h and 7 months were investigated. Tenascin was visualized by immunohistochemistry and appeared first in the wound area pericellularly around fibroblastic cells approximately 2 days after wounding. A network-like interstitial positive staining pattern was first detectable in 3-day-old skin wounds. In all wounds with an age of 5 days or more, intensive reactivity for tenascin could be observed in the lesional area (dermal-epidermal junction, wound edge, areas of bleeding). In wounds with an age of more than approximately 1.5 months no positive staining occurred in the scar tissue. In conclusion, for forensic purposes, positive staining for tenascin restricted to the pericellular area of fibroblastic cells indicates a wound age of at least 2 days. Network-like structures appear after approximately 3 days or more. Since tenascin seems to be regularly detectable in skin wounds older than 5 days, the lack of a positive reaction in a sufficient number of specimens indicates a wound age of less than 5 days. The lack of a positive reaction in the granulation tissue of wounds with advanced wound age indicates a survival time of more than about 1.5 months, but a positive staining in older wounds cannot be excluded

Frequent and Persistent PLCG1 Mutations in Sezary Cells Directly Enhance PLC gamma 1 Activity and Stimulate NF kappa B, AP-1, and NFAT Signaling

Phospholipase C Gamma 1 (PLCG1) is frequently mutated in primary cutaneous T-cell lymphoma (CTCL). This study functionally interrogated nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDM1161V) identified in Sézary Syndrome, the leukemic variant of CTCL. The mutations were demonstrated in diagnostic samples and persisted in multiple tumor compartments over time, except in patients who achieved a complete clinical remission. In basal conditions, the majority of the mutations confer PLCγ1 gain-of-function activity through increased inositol phosphate production and the downstream activation of NFκB, AP-1, and NFAT transcriptional activity. Phosphorylation of the p.Y783 residue is essential for the proximal activity of wild-type PLCγ1, but we provide evidence that activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 transcriptional activity. Finally, the gain-of-function effects associated with the p.VYEEDM1161V indel suggest that the C2 domain may have a role in regulating PLCγ1 activity. These data provide compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1

Are there valid proxy measures of clinical behaviour?

Background: Accurate measures of health professionals' clinical practice are critically important to guide health policy decisions, as well as for professional self-evaluation and for research-based investigation of clinical practice and process of care. It is often not feasible or ethical to measure behaviour through direct observation, and rigorous behavioural measures are difficult and costly to use. The aim of this review was to identify the current evidence relating to the relationships between proxy measures and direct measures of clinical behaviour. In particular, the accuracy of medical record review, clinician self-reported and patient-reported behaviour was assessed relative to directly observed behaviour. Methods: We searched: PsycINFO; MEDLINE; EMBASE; CINAHL; Cochrane Central Register of Controlled Trials; science/social science citation index; Current contents (social & behavioural med/clinical med); ISI conference proceedings; and Index to Theses. Inclusion criteria: empirical, quantitative studies; and examining clinical behaviours. An independent, direct measure of behaviour (by standardised patient, other trained observer or by video/audio recording) was considered the 'gold standard' for comparison. Proxy measures of behaviour included: retrospective self-report; patient-report; or chart-review. All titles, abstracts, and full text articles retrieved by electronic searching were screened for inclusion and abstracted independently by two reviewers. Disagreements were resolved by discussion with a third reviewer where necessary. Results: Fifteen reports originating from 11 studies met the inclusion criteria. The method of direct measurement was by standardised patient in six reports, trained observer in three reports, and audio/video recording in six reports. Multiple proxy measures of behaviour were compared in five of 15 reports. Only four of 15 reports used appropriate statistical methods to compare measures. Some direct measures failed to meet our validity criteria. The accuracy of patient report and chart review as proxy measures varied considerably across a wide range of clinical actions. The evidence for clinician self-report was inconclusive. Conclusion: Valid measures of clinical behaviour are of fundamental importance to accurately identify gaps in care delivery, improve quality of care, and ultimately to improve patient care. However, the evidence base for three commonly used proxy measures of clinicians' behaviour is very limited. Further research is needed to better establish the methods of development, application, and analysis for a range of both direct and proxy measures of behaviour