Direct assessment of nitrative stress in lipid environments: Applications of a designer lipid-based biosensor for peroxynitrite.

Lipid membranes and lipid-rich organelles are targets of peroxynitrite (ONOO-), a highly reactive species generated under nitrative stress. We report a membrane-localized phospholipid (DPPC-TC-ONOO-) that allows the detection of ONOO- in diverse lipid environments: biomimetic vesicles, mammalian cell compartments, and within the lung lining. DPPC-TC-ONOO- and POPC self-assemble to membrane vesicles that fluorogenically and selectively respond to ONOO-. DPPC-TC-ONOO-, delivered through lipid nanoparticles, allowed for ONOO- detection in the endoplasmic reticulum upon cytokine-induced nitrative stress in live mammalian cells. It also responded to ONOO- within lung tissue murine models upon acute lung injury. We observed nitrative stress around bronchioles in precision cut lung slices exposed to nitrogen mustard and in pulmonary macrophages following intratracheal bleomycin challenge. Results showed that DPPC-TC-ONOO- functions specifically toward iNOS, a key enzyme modulating nitrative stress, and offers significant advantages over its hydrophilic analog in terms of localization and signal generation.

A Mycobacterial Systems Resource for the Research Community.

Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulceransIMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

Quantitative risk assessment of the likeihood of introducing porcine reproductive and respiratory syndrome virus into New Zealand through the importation of pig meat.

A quantitative model was developed to estimate the likelihood of an incursion of porcine reproductive and respiratory syndrome virus (PRRSV) into New Zealand through the importation of fresh consumer-ready cuts of pig meat. A sensitivity analysis of all the inputs used in this model illustrated the importance of correctly modelling the available 'dose-response' data, and a mechanistic Beta-Poisson model was shown to be the most appropriate method for this in the authors' assessment. The output of this model predicts an average of approximately 1,200 years between PRRSV introductions resulting in primary infections in New Zealand. Given the uncertainties in the model, there is 95% confidence that this time period ranges from 52 to 6,200 years. The values chosen in this model are considered to provide a conservative estimate of the likelihood of introducing PRRSV into New Zealand via the importation of fresh pork.

Acknowledging and allocating responsibility for clinical inertia in the management of Type 2 diabetes in primary care: a qualitative study.

AIMS: Failure to intensify treatment in patients with Type 2 diabetes with suboptimal blood glucose control has been termed clinical inertia and has been shown to contribute to poorer patient outcomes. We aimed to identify and explore perceptions about clinical inertia from the perspective of primary healthcare providers. METHODS: A qualitative study was conducted in Leicestershire and Northamptonshire, UK. Purposive sampling was based on healthcare providers working in primary care settings with 'higher' and 'lower' target achievement based on routine data. Twenty semi-structured interviews were conducted, face-to-face or by telephone. Thematic analysis was informed by the constant comparative approach. RESULTS: An important broad theme that emerged during the analysis was related to attribution and explanation of responsibility for clinical inertia. This included general willingness to accept a degree of responsibility for clinical inertia. In some cases, however, participants had inaccurate perceptions about levels of target achievement in their primary care centres, as indicated by routine data. Participants sought to lessen their own sense of accountability by highlighting patient-level barriers such as comorbidities and human fallibility, and also system-level barriers, particularly time constraints. Perceptions about ways of addressing the problem of clinical inertia were not seen as straightforward, further emphasizing a complex and cumulative pattern of barriers. CONCLUSIONS: In order to understand and address the problem of clinical inertia, provider, patient- and system-level barriers should be considered together rather than as separate issues. Acknowledgement of responsibility should be regarded positively as a motivator for change.

Preclinical assessment of a new magnetic resonance-based technique for determining bone quality by characterization of trabecular microarchitecture.

The utility of HR-CT to study longitudinal changes in bone microarchitecture is limited by subject radiation exposure. Although MR is not subject to this limitation, it is limited both by patient movement that occurs during prolonged scanning at distal sites, and by the signal-to-noise ratio that is achievable for high-resolution images in a reasonable scan time at proximal sites. Recently, a novel MR-based technique, fine structure analysis (FSA) (Chase et al. Localised one-dimensional magnetic resonance spatial frequency spectroscopy. PCT/US2012/068284 2012, James and Chase Magnetic field gradient structure characteristic assessment using one-dimensional (1D) spatial frequency distribution analysis. 7932720 B2, 2011) has been developed which provides both high-resolution and fast scan times, but which generates at a designated set of spatial positions (voxels) a one-dimensional signal of spatial frequencies. Appendix 1 provides a brief introduction to FSA. This article describes an initial exploration of FSA for the rapid, non-invasive characterization of trabecular microarchitecture in a preclinical setting. For L4 vertebrae of sham and ovariectomized (OVX) rats, we compared FSA-generated metrics with those from CT datasets and from CT-derived histomorphometry parameters, trabecular number (Tb.N), bone volume density (BV/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). OVX caused a reduction of the higher frequency structures that correspond to a denser trabecular lattice, while increasing the preponderance of lower frequency structures, which correspond to a more open lattice. As one example measure, the centroid of the FSA spectrum (which we refer to as fSAcB) showed strong correlation in the same region with CT-derived histomorphometry values: Tb.Sp: r -0.63, p < 0.001; Tb.N: r 0.71, p < 0.001; BV/TV: r 0.64, p < 0.001, Tb.Th: r 0.44, p < 0.05. Furthermore, we found a 17.5% reduction in fSAcB in OVX rats (p < 0.0001). In a longitudinal study, FSA showed that the age-related increase in higher frequency structures was abolished in OVX rats, being replaced with a 78-194% increase in lower frequency structures (2.4-2.8 objects/mm range), indicating a more sparse trabecular lattice (p < 0.05). The MR-based fine structure analysis enables high-resolution, radiation-free, rapid quantification of bone structures in one dimension (the specific point and direction being chosen by the clinician) of the spine.

Diabetes self-management education: acceptability of using trained lay educators.

BACKGROUND: The use of lay people to deliver education programmes for people with chronic conditions is a potential method of addressing healthcare staff capacity and increasing the cost efficiency of delivering education. This qualitative substudy is embedded within an equivalence trial (2008-2011 including development stage). OBJECTIVES: In the qualitative substudy, we aimed to elicit the views of key stakeholders (patients, educators) about using lay people to deliver education to people recently diagnosed with type 2 diabetes, alongside a healthcare professional educator with an equal role. In this way, we sought to explore perceptions about acceptability and also contribute to understanding the reasons underlying positive or negative quantitative findings from main trial. METHODS: We conducted 27 telephone interviews with a purposive sample of patients, lay educators and healthcare professional educators involved in the main trial. Thematic analysis of transcribed data was underpinned by the constant comparative approach and structured using Framework methodology. RESULTS: Overall, the data suggested that the use of lay educators was acceptable to educators and patients. Perceived difference in knowledge levels between lay and healthcare professional educators did not appear to have an impact on perceived acceptability or the effectiveness of the education received. Additional themes explored were related to peer status of educators and feasibility. Some concerns were raised about lay educators with diabetes, transferring personal issues and about the impact of healthcare professional time taken up by mentoring and supporting lay educators. CONCLUSIONS: Positive perceptions about the use of lay educators support the positive quantitative findings from the main trial. Acceptability is an important consideration in relation to implementation of the model of delivery studied. Concerns raised within the interviews should be considered in the design of training for lay educators. TRIAL REGISTRATION NUMBER: ISRCTN 99350009.

Litigation after hip and knee replacement in the National Health Service.

The results of hip and knee replacement surgery are generally regarded as positive for patients. Nonetheless, they are both major operations and have recognised complications. We present a review of relevant claims made to the National Health Service Litigation Authority. Between 1995 and 2010 there were 1004 claims to a value of £41.5 million following hip replacement surgery and 523 claims to a value of £21 million for knee replacement. The most common complaint after hip surgery was related to residual neurological deficit, whereas after knee replacement it was related to infection. Vascular complications resulted in the highest costs per case in each group.Although there has been a large increase in the number of operations performed, there has not been a corresponding relative increase in litigation. The reasons for litigation have remained largely unchanged over time after hip replacement. In the case of knee replacement, although there has been a reduction in claims for infection, there has been an increase in claims for technical errors. There has also been a rise in claims for non-specified dissatisfaction. This information is of value to surgeons and can be used to minimise the potential mismatch between patient expectation, informed consent and outcome.

February 2010: a dramatic shift away from private fee-for-service plans in rural Medicare Advantage enrollment.

In a reversal of recent trends, private fee-for-service (PFFS) enrollment fell dramatically in rural areas in early 2010. As a result, Medicare Advantage (MA) enrollment in rural areas (excluding other prepaid plans) fell slightly in early 2010 for the first time in years. The dramatic drop in PFFS enrollment was offset by increases in enrollment in preferred provider organization (PPO) and health maintenance organization (HMO) plans. Although PFFS enrollment has driven the rapid growth of the MA program in recent years, particularly in rural areas, the MA market share held by PFFS plans in rural areas fell significantly from 52% in December 2009 to 38% in February 2010. The decline was driven by some plans leaving the market and by underutilized and duplicative plans consolidating; factors contributing to this trend include slow growth in payment rates, legislative changes requiring PFFS plans to form provider networks by 2011, and significant increases in beneficiary premiums. Nationwide, enrollment in PPO and HMO plans grew, offsetting the decline in PFFS enrollment and contributing to a slight growth in total MA enrollment in 2010.

Assessment of the impact of flares in ankylosing spondylitis disease activity using the Flare Illustration.

OBJECTIVES: Many AS patients report periods of perceived higher disease activity (flares). This pilot study aims to document disease activity patterns reported by AS patients and examine associations with disease-specific health status measures. METHODS: Consecutive AS patients (n = 114) were asked whether they experience flares, and if they experience symptoms of AS between flares. They were shown the Flare Illustration of disease patterns over time and asked to select the pattern that best described their disease (i) since symptom onset and (ii) in the past year. Associations between reported disease pattern and disease activity (Bath AS Disease Activity Index, BASDAI); functional impairment (Bath AS Functional Index, BASFI); AS Quality of Life (ASQoL); Back Pain (Nocturnal and Overall) and demographic features were assessed in a subsample (n = 83) (statistical significance defined at P 70% of patients) and patterns with constant symptoms since onset (vs intermittent symptoms) were associated with worse health status (ASQoL: P = 0.007; BASDAI: P = 0.029; BASFI: P = 0.013, overall back pain: P = 0.025). CONCLUSIONS: Almost all AS patients report flares in disease activity: 70-80% report constant symptoms with single/repeated flares, while 20-30% report flares with no intermittent symptoms. The former is associated with a significantly poorer health status. These findings will be validated in a prospective study.

Cost-effectiveness of a disease management programme for secondary prevention of coronary heart disease and heart failure in primary care.

OBJECTIVE: To determine if a disease management programme for patients with coronary heart disease and heart failure represents an efficient use of health services resources. METHODS: We carried out an economic evaluation alongside a cluster randomised control trial of 1163 patients with coronary heart disease and chronic heart failure in 20 primary care practices in the United Kingdom. Practices were randomised to either a control group, where patients received standard general practice care, or an intervention group where patients had access to a specialist nurse-led disease management programme. We estimated costs in both groups for coronary heart disease-related resource use. The main outcome measure used in the economic evaluation was quality adjusted life years (QALY) measured using the EuroQol. RESULTS: The disease management programme was associated with an increase in the QALY measured of 0.03 per year and an increase in the total NHS costs of 425 pounds (540 euros), of this only 83 pounds was directly associated with the provision of the nurse clinics. The clinics generated additional QALY at an incremental cost of 13 pounds 158 per QALY compared to the control group. CONCLUSIONS: The use of a nurse-led disease management programme is associated with increased costs in other coronary heart disease-related services as well as for the costs of the clinics. They are also associated with improvements in health. Even in the short term these disease management programmes may represent a cost-effective service, as additional QALY are generated at an acceptable extra cost.

In vitro simulation and quantification of wear within the patellofemoral joint replacement.

Quantification of the wear rate in vitro is now considered an essential step in the development of a new joint replacement prior to clinical trials. However, little research exists around in vitro simulation of wear in the patellofemoral joint (PFJ) despite over 200,000 being implanted annually within the European Union. A method to simulate wear in the laboratory using four input degrees of freedom within the PFJ of total knee replacement (TKR) has been developed. Wear simulation was validated through comparison of functional kinematics and patellar surface damage modes produced in vitro to clinical outcomes. The technique has been shown to replicate the prescribed in vivo kinematics in a reproducible and repeatable manner. The wear scar areas were similar to those found in vivo. However, geometrical measurements of wear were not reliable due to creep and geometry changes. As has been found previously with tibial inserts, geometrical determination of wear volume was not found to be an effective method of comparing wear from simulators and retrievals. Change in volume calculated gravimetrically was seen to be the most repeatable measure of patellar wear in vitro.

Cost effectiveness analysis of using quantitative ultrasound as a selective pre-screen for bone densitometry.

It has been suggested that quantitative ultrasound (QUS) could be used as a selective population pre-screen, to maximise the cost effectiveness of referral for dual energy X-ray absorptiometry (DXA) assessment of bone mineral density (BMD). We set out to examine how such an approach might perform in the assessment of women who were referred by general practitioners for DXA via the open access service in Cardiff. In 115 women aged 40-80 (mean 69) years we used DXA to measure BMD at lumbar spine and hip, and QUS to measure broadband ultrasound attenuation (BUA) in the heel. A bottom-up approach was used to estimate the costs of DXA and QUS. We examined the cost effectiveness of using QUS as a pre-screen, only referring subjects for the more expensive DXA assessment if BUA were less than a pre-determined threshold. The unit costs of pencil-beam DXA and QUS were approximately 44 UK pounds and 16 UK pounds respectively. We identified a BUA threshold of 60 dB/MHz as the most cost effective, and calculated a sensitivity of 81% and specificity of 89% in identifying those subjects whom DXA assessment subsequently identified as having osteoporosis. At the BUA threshold of 60 dB/MHz, pre-screening saved 969 UK pounds at the expense of missing ten women with osteoporosis as diagnosed by DXA. Therefore the cost per additional woman with osteoporosis identified using DXA alone was only 97 UK pounds. QUS assessment does not appear to have a significant cost effective benefit as a pre-screen for DXA in the studied population. A QUS pre-screen would be cost effective only if this investigation could be performed at a substantially lower cost.

Power Doppler ultrasound assessment of rheumatoid hand synovitis.

OBJECTIVE: To evaluate power Doppler ultrasound (PD) as a technique in assessing response to treatment with steroids in rheumatoid hand synovitis. METHODS: Twelve patients with rheumatoid hand synovitis were assessed before and after treatment with steroids. Variables used to assess synovitis activity in each patient included patient visual analog scale (VAS) score for pain, physician assessment score (PAS), erythrocyte sedimentation rate (ESR), and PD of the metacarpophalangeal joints. RESULTS: Nine female and 3 male patients were studied; mean age was 53.3+/-6.5 yrs and mean disease duration 6.5+/-4.5 yrs. All patients had a good clinical response to steroid treatment and there was a significant improvement in the synovitis activity assessments. Wilcoxon signed-rank test using the exact method was applied to the change in disease activity variables. For PD signal, p < 0.002; VAS, p < 0.0016; ESR, p < 0.031; PAS, p < 0.008. CONCLUSION: PD quantifies synovitis and may be a useful adjunct to disease assessment and the response to treatment in RA.

Physician prescribing of anorexigenics for weight loss in Salt Lake County, Utah.

The objective of this survey of primary care physicians in Salt Lake County, Utah, was to determine how many of these providers were prescribing anorexigenics (antiobesity drugs) and to evaluate their knowledge of side effects and drug interactions. A confidential survey was sent to 377 family practice, internal medicine, and obstetrics-gynecology physicians, of whom 236 (62.5%) responded. Of those who answered the survey completely, 146 (65.2%) prescribed anorexigenics to 5,107 patients. Family practice physicians, male physicians less than 40 years old, and internal medicine physicians less than 40 years old prescribed anorexigenics more frequently than their counterparts. Female physicians (vs male) and female family practice physicians (vs male family practice physicians) reported a higher proportion of psychiatric comorbidity among patients for whom they prescribed the drugs. Respondents had limited knowledge of side effects and drug interactions of anorexigenics. Better understanding of physicians' prescribing patterns of these agents is relevant for current and future optimal treatment of obesity.

Cost effectiveness analysis of BMD referral for DXA using ultrasound as a selective pre-screen in a group of women with low trauma Colles' fractures.

Measurements of bone density (BMD) are central to the World Health Organisation (WHO) approach to the definition of osteoporosis. Dual energy X- ray absorptiometry (DXA) remains the gold standard technique for measuring the bone mineral density (BMD) but Quantitative Ultrasound (QUS) is an attractive alternative method of bone assessment because it is easy to use and relatively inexpensive. It has been suggested that QUS could be used as a selective population pre-screen, to maximise the cost effectiveness of referral for DXA assessment of BMD. We set out to examine how such an approach might perform in the assessment of women with low trauma Colles' fracture. In 46 women aged 50-80 (mean 67) years we used DXA to measure BMD at lumbar spine and hip, and heel bone ultrasound to measure Broad Band Attenuation (BUA) and Velocity of Sound (VOS). We calculated local costs of pounds sterling 45 for DXA and pounds sterling 15 for QUS. We identified a BUA threshold of 60 dB/MHz as most cost effective as pre-screen, and calculated a sensitivity of 93% and specificity of 84% in identifying those subjects who were subsequently identified as having osteoporosis by DXA. DXA assessment of all patients had a cost of pounds sterling 77 per osteoporotic subject identified. We examined the cost-effectiveness of using QUS as a pre-screen, only referring subjects for more expensive DXA assessment if BUA was less than 60 dB/MHz. However this approach had no advantage, still costing pounds sterling 78 per osteoporotic subject identified. QUS assessment does not appear cost-effective as a pre-screen for DXA, even in this high risk group of women with low trauma Colles' fracture. A QUS pre-screen would only be cost-effective if the scan could be performed at a substantially lower cost.

Bone assessment in elderly women: what does a low bone ultrasound result tell us about bone mineral density?

Access to dual energy X-ray absorptiometry (DXA) can prove difficult for frail or elderly patients, and bone ultrasound may offer a practical alternative. Even after adjustment for bone mineral density (BMD), ultrasound readings are able to predict hip fracture in elderly women. We consider how bone ultrasound might contribute to bone assessment in a clinical setting. DXA remains the gold standard for bone assessment, with osteoporosis defined as a BMD result more than 2.5 S.D. below the young adult mean. Using an equivalent approach we defined an osteoporotic ultrasound result as broadband ultrasound attenuation (BUA)<54 dB/MHz. In 73 women aged 29-86 (mean 65) years DXA was used to measure BMD at lumbar spine and hip, and ultrasound to measure BUA at the heel. Correlation of BUA with BMD at femoral neck (r=0.64, P<0.001), and lumbar spine (r=0.55, P<0.001) was consistent with previously reported figures for this ultrasound system. All subjects with BUA below the 54 dB/MHz threshold value were shown to have low femoral neck BMD. Women (42%) aged over 65, but only 18% of younger women had low BUA results. In women over 65 years of age measurements of BUA achieved a sensitivity of 61% and specificity of 100% in prediction of low femoral neck BMD. Although a normal BUA did not exclude an osteoporotic BMD result at hip or lumbar spine, a low BUA appeared a highly specific predictor of low BMD at these sites. Since all those women identified as having a low BUA at the heel also had low BMD results, ultrasound appeared to identify a subgroup of elderly patients at a very high risk of fracture.

In vitro and in vivo assessment of adenovirus 41 as a vector for gene delivery to the intestine.

In order to identify suitable adenoviral vectors for efficient delivery of transgenic proteins and peptides to the intestine, the ability of adenovirus types 5 and 41 (an enterotropic serotype) to bind to and enter undifferentiated and differentiated enterocytes was assessed. FACS analysis showed no significant difference between the virions in their ability to bind to undifferentiated Caco-2 cells as 81.6% of the cellular population bound adenovirus 5 (Ad 5) and 79.8% bound Ad 41. Both virions were also efficiently internalized in this cell type as 99.6% of the cells took up Ad 5, while 95.9% took up Ad 41. In studies with differentiated enterocytes, probable targets for oral gene delivery but rather resistant to adenovirus-mediated gene transfer, 28.4% of the population internalized the Ad 5 vector and less than 10% bound the virus. Adenovirus 41 was efficiently internalized in differentiated enterocytes as 89.6% of the cellular population took up the virus while 37.4% bound the virus. These results were consistent with those observed in vivo in rat jejunum. Thus, molecularly engineered Ad 41-based recombinants could be highly efficient vectors for delivery of transgenic proteins to differentiated enterocytes.