Yeast-based directed-evolution for high-throughput structural stabilization of G protein-coupled receptors (GPCRs).

The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we present a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The yeast detergent-resistant cell wall presents a unique opportunity for compartmentalization, to physically link the receptor's phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method reveals a surprising amenability of the GPCR scaffold to stabilization, and suggests an intriguing possibility of amending the stability properties of GPCR by varying the structural status of the C-terminus.

A descriptive analysis of an outbreak of measles and a multilevel mixed-effects analysis of factors associated with case isolation in healthcare settings, London (February-June 2016).

OBJECTIVES: We described the epidemiology and healthcare exposures during a measles outbreak in London and identified factors associated with isolation on arrival to healthcare premises. STUDY DESIGN: We conducted a cohort study including all confirmed measles cases in London residents from February 1, 2016, to June 30, 2016, and semistructured interviews with two infection prevention and control teams (IPCTs). METHODS: We described the outbreak and conducted a multilevel mixed-effects analysis to assess the relationship between sociodemographic and clinical factors and isolation on arrival to healthcare premises. We summarised the interviews. RESULTS: There were 182 cases, mostly aged 17-35 years (46%; 84). Excluding cases younger than one year, 76% (92/120) were unvaccinated, including two healthcare workers. The majority presented with rash (97%; 174), and 42% (70/166) required hospitalisation. Of the recorded cases, 93% of cases (164/178) had visited a healthcare setting during their infectious period (median number of visits = 2). In 33% (59/178) of the visits, the case was isolated on arrival; when not isolated, four healthcare exposures resulted in further transmission. Presenting to the hospital as opposed to a general practitioner (GP) was associated with higher odds of isolation (odds ratio = 2.23, 95% confidence interval = 1.1-4.4) when adjusted for age, gender and presenting with a cough. The IPCT identified measles training using standardised risk assessments by triage nurses in accident and emergency and intelligence regarding measles activity in the community as positive measures to prevent healthcare exposures. CONCLUSIONS: We recommend opportunistic immunisation of unvaccinated young adults by GPs and that occupational health departments ensure their staff are protected against measles. Raising measles awareness in healthcare settings via training or regular sharing of current measles surveillance activity from public health to the IPCT and GP may improve triage and isolation of cases on arrival to healthcare premises.

Tracking sickness through social networks - the practical use of social network mapping in supporting the management of an E. coli O157 outbreak in a primary school in London.

This paper describes the practical use of social network diagrams in the management of an outbreak of Escherichia coli O157 (VTEC) in a primary school in London. The diagrams were created during the outbreak to establish the extent and nature of person-to-person transmission in the cases and their contacts. The diagrams supported a tailored public health action, and hence aided in the control of the outbreak. We conclude that for selected infectious diseases, social network diagrams can provide a valuable tool in the management of an outbreak.

Carbon monoxide from neighbouring restaurants: the need for an integrated multi-agency response.

BACKGROUND: Carbon monoxide (CO) is a colourless, odourless toxic gas produced during incomplete combustion of carbon-based fuels. Most CO incidents reported to the UK Health Protection Agency (HPA) are due to faulty gas appliances, and legislation exists to ensure gas appliances are properly installed. METHODS: We present three CO poisoning incidents of unusual origin reported to the HPA. In each, residents living above restaurants were poisoned after workers left charcoal smouldering overnight in specialist or traditional ovens whilst ventilation systems were turned off. This led to production of CO, which travelled through floorboards and built up to dangerous concentrations in the flats. RESULTS: Working with local authorities, these incidents were investigated and resolved, and work was conducted to prevent further occurrences. CONCLUSIONS: The novel nature of these CO incidents led to delays in recognition and subsequent remedial action. Although previously undescribed, it is likely that due to the number of residences built above restaurants and the rising popularity of traditional cooking methods, similar incidents may be occurring and could increase in frequency. Multi-agency response and reporting mechanisms could be strengthened. Awareness raising in professional groups and the public on the importance of correct ventilation of such appliances is vital.

Investigating the effect of high spring incidence of pandemic influenza A(H1N1) on early autumn incidence.

A pandemic H1N1 infection wave in the USA occurred during spring 2009. Some hypothesized that for regions affected by the spring wave, an autumn outbreak would be less likely or delayed compared to unaffected regions because of herd immunity. We investigated this hypothesis using the Outpatient Influenza-like Illness (ILI) Network, a collaboration among the Centers for Disease Control and Prevention, health departments, and care providers. We evaluated the likelihood of high early autumn incidence given high spring incidence in core-based statistical areas (CBSAs). Using a surrogate incidence measure based on influenza-related illness ratios, we calculated the odds of high early autumn incidence given high spring incidence. CBSAs with high spring ILI ratios proved more likely than unaffected CBSAs to have high early autumn ratios, suggesting that elevated spring illness did not protect against early autumn increases. These novel methods are applicable to planning and studies involving other infectious diseases.

Genome scan of clot lysis time and its association with thrombosis in a protein C-deficient kindred.

BACKGROUND: Previously, we found increased clot-lysis time (CLT), as measured with a plasma-based assay, to increase the risk of venous thrombosis in two population-based case-control studies. The genes influencing CLT are as yet unknown. PATIENTS/METHODS: We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n = 346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore, we tested for quantitative trait loci (QTLs) for CLT, using variance component linkage analysis. RESULTS: Protein C-deficient family members had shorter CLTs than non-deficient members (median CLT 67 min vs. 75 min). One standard deviation increase in CLT increased the risk of venous thrombosis 2.4-fold in non-deficient family members. Protein C deficiency without elevated CLT increased the risk 6.9-fold. Combining both risk factors yielded a 27.8-fold increased risk. The heritability of CLT was 42-52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin-activatable fibrinolysis inhibitor genotypes did not explain the variation in CLT. CONCLUSION: Hypofibrinolysis appears to increase thrombosis risk in this family, especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTLs were found on chromosomes 11 and 13.

Molecular modeling of retinoschisin with functional analysis of pathogenic mutations from human X-linked retinoschisis.

Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. RS1 is an adhesive protein which is proposed to preserve the structural and functional integrity of the retina, but there is very little evidence of the mechanism by which protein changes are related to XLRS disease. Here, we report molecular modeling of the RS1 protein and consider perturbations caused by mutations found in human XLRS subjects. In 60 XLRS patients who share 27 missense mutations, we then evaluated possible correlations of the molecular modeling with retinal function as determined by the electroretinogram (ERG) a- and b-waves. The b/a-wave ratio reflects visual-signal transfer in retina. We sorted the ERG b/a-ratios by patient age and by the mutation impact on protein structure. The majority of RS1 mutations caused minimal structure perturbation and targeted the protein surface. These patients' b/a-ratios were similar across younger and older subjects. Maximum structural perturbations from either the removal or insertion of cysteine residues or changes in the hydrophobic core were associated with greater difference in the b/a-ratio with age, with a significantly smaller ratio at younger ages, analogous to the ERG changes with age observed in mice with no RS1-protein expression due to a recombinant RS1-knockout gene. The molecular modeling suggests an association between the predicted structural alteration and/or damage to retinoschisin and the severity of XLRS as measured by the ERG analogous to the RS1-knockout mouse.

Effectiveness of dog rabies vaccination programmes: comparison of owner-charged and free vaccination campaigns.

We investigated the percentage of dogs that could be vaccinated against rabies by conducting a pilot campaign in N'Djaména, Chad. Owners were charged US$4.13 per dog vaccinated, and 24% of all dogs in the three city districts covered by the campaign were vaccinated. Total campaign costs were US$7623, resulting in an average of US$19.40 per vaccinated dog. This is five times more expensive than the cost per animal vaccinated during a previous free vaccination campaign for dog-owners, conducted in the same districts. The free campaign, which vaccinated 2605 more dogs than this campaign, cost an additional US$1.45 per extra dog vaccinated. Campaigns in which owners are charged for vaccinations result in lower vaccination rates than in free campaigns. Public health officials can use these results when evaluating the costs and benefits of subsidizing dog rabies vaccination programmes.

Validity and reproducibility of resting metabolic rate measurements in rural Bangladeshi women: comparison of measurements obtained by Medgem and by Deltatrac device.

OBJECTIVE: To assess reproducibility and validity of resting metabolic rate (RMR) of Bangladeshi women as measured with the MedGem device and using the Deltatrac metabolic monitor as a reference; and (2) to evaluate the FAO/WHO/UNU basal metabolic rate (BMR)-prediction equations. DESIGN: In each of two sessions, resting oxygen consumption was measured in triplicate by MedGem and in triplicate by Deltatrac device. SETTING: Matlab area, the rural field research area of the Centre for Health and Population Research, Bangladesh (ICDDR,B). SUBJECTS: A total of 37 nonpregnant, nonlactating women, aged 27.6 +/- 4.5 y, BMI 20.8 +/- 3.1 kg/m(2) participated. RESULTS: The difference in oxygen consumption by MedGem and Deltatrac device was significantly level dependent. Within-subject within-session variations (expressed as CV) were 9.0 and 3.0% (P < 0.01) and within-subject between-session variations were 8.2 and 4.5% (P < 0.01) for MedGem and Deltatrac, respectively. Mean RMR measured by Deltatrac (5.17 +/- 0.51 MJ/day) was not significantly different from the BMR predicted by the FAO/WHO/UNU equations (5.16 +/- 0.42 MJ/day) in the second session and only 0.19 MJ/day higher than predicted in the first session (P < 0.05). CONCLUSION: Reproducibility and validity of the MedGem device was poor compared to the Deltatrac reference method. The FAO/WHO/UNU BMR-prediction equations give a good estimation of the BMR of rural, nonpregnant, nonlactating Bangladeshi women of 18-35 y. SPONSORSHIP: Wageningen University (The Netherlands) and ICDDR,B (Bangladesh).

The public health response to a case of Lassa fever in London in 2000.

OBJECTIVES: Evaluation of the Department of Health 1996 guidance, the Memorandum on the Management and Control of Viral Haemorrhagic Fevers. METHODS: Description of the public health management in 2000 of the fifth UK patient confirmed to have Lassa fever. RESULTS: Delayed risk categorisation of the patient occurred for a variety of reasons. DH Guidance was followed once infection control advice was sought. Active surveillance of 125 contacts was extremely resource intense, involving over 3000 communications. Self-monitoring by healthcare workers should be considered in future. Advice on use of ribavirin prophylaxis is not included in the Memorandum, nor advice or templates for information sheets for contacts. Information sheets are now available from the Health Protection Agency in the event of future cases. International aspects not adequately addressed include the need for reliable risk assessment to be carried out before patients are medically evacuated from the country of origin, and the steps required to repatriate UK nationals. Effective and efficient communication is required between national and international organisations involved in such incidents. CONCLUSIONS: If guidelines are unclear or impracticable they will not be followed. It is important that lessons are learned and documented and that national guidance be regularly reviewed.

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.

Specialist vaccination advice and pockets of resistance to MMR vaccination: lessons from an outbreak of measles.

An outbreak of measles was associated with a private nursery school in north west London. Sixteen cases were identified of whom 13 were laboratory confirmed. The majority of cases were aged three years or younger. Older cases were siblings of younger cases. None of the cases had been vaccinated against measles. In the nursery school 33% of the children had not received MMR vaccination. Based on specialist advice from the Health Protection Agency the outbreak control team recommended that children with no history of MMR vaccination should have a first MMR dose as soon as possible and that children with one MMR dose should receive the second dose as soon as possible (minimum of one month between doses). Some parents had strong negative views about MMR and represented 'pockets of resistance' to vaccination advice. The specialist vaccination advice, which was different to national immunisation guidelines, also caused some confusion amongst other health professionals. With a decrease in MMR vaccination uptake and resulting increased potential for future measles outbreaks, clinicians should be aware of and understand specialist vaccination advice intended to deal with outbreaks.

Modeling potential responses to smallpox as a bioterrorist weapon.

We constructed a mathematical model to describe the spread of smallpox after a deliberate release of the virus. Assuming 100 persons initially infected and 3 persons infected per infectious person, quarantine alone could stop disease transmission but would require a minimum daily removal rate of 50% of those with overt symptoms. Vaccination would stop the outbreak within 365 days after release only if disease transmission were reduced to <0.85 persons infected per infectious person. A combined vaccination and quarantine campaign could stop an outbreak if a daily quarantine rate of 25% were achieved and vaccination reduced smallpox transmission by > or = 33%. In such a scenario, approximately 4,200 cases would occur and 365 days would be needed to stop the outbreak. Historical data indicate that a median of 2,155 smallpox vaccine doses per case were given to stop outbreaks, implying that a stockpile of 40 million doses should be adequate.

Introduction to health economics for physicians.

Since the 1960s, expenditure on health care in developed countries has risen faster than the general rate of inflation, thus making economic assessment of interventions an integral part of decision making in health services. This paper is the first in a series whose goal is to provide some basic principles of health economics that will allow practising physicians to understand better the economic relations between their practice of medicine, the health-care sector, and the national economy. Some of the most important principles described in this paper include opportunity costs, identifying the appropriate perspective, correctly categorising costs, and discounting costs and non-monetary benefits (eg, lives saved) over time. Economic analyses of medical interventions must also take into consideration the difference between efficacy and effectiveness. Efficacy is the maximum possible benefit, often achieved with carefully controlled trials, and effectiveness is the actual decrease in disease achieved when the intervention is applied over a large, non-homogeneous population. This introduction ends with three methods of assessing the costs and benefits of an intervention-namely, cost-benefit, cost-effectiveness, and cost-utility analyses.

A simplified technique of ptosis repair using a single adjustable suture.

PURPOSE: To describe a simplified technique for ptosis repair using a single adjustable suture. DESIGN: Retrospective noncomparative series. PARTICIPANTS: Fifty-one cases of ptosis repair performed or supervised by the same surgeon between 1993 and 1995. INTERVENTION: The surgical approach consisted of using a single adjustable "hang-back" 5-0 silk suture for plication of the superior portion of the levator aponeurosis. Lid height was then reassessed within the first 4 postoperative days and permanently readjusted by fixating the adjustable suture. Excluded were patients with history of unstable ptosis as a result of systemic disease or congenital, mechanical, and traumatic ptosis resulting from a mass or trauma. The follow-up period ranged from 3 to 31 months. MAIN OUTCOME MEASURE: Lid position. RESULTS: All eyelids included in this study were corrected to 1 mm of the desired result. There were no incidents of peaking or recurrence on long-term follow-up. Complications included one case of hematoma and a case of slight tenting of the eyelid margin. CONCLUSIONS: This procedure combines the physiologic approach of levator aponeurosis surgery with the simplicity and flexibility of a single adjustable "hang-back" type suture. It may readily be combined with other procedures such as blepharoplasty. It is also useful when the "ideal" lid level may not be determined until the postoperative period, such as eyes subject to ptosis by Hering's law, levator dehiscence, or those at increased potential risk for corneal exposure.

The economics of vaccinating restaurant workers against hepatitis A.

The economics of vaccinating restaurant workers against hepatitis A were studied using Monte Carlo simulation models, one with a restaurant-owner perspective, and one with a societal perspective. The restaurant model allowed for a different size, number of employees and employee turnover rate. Benefits were the avoidance of loss of business (including the possibility of bankruptcy) after publicity linking the restaurant to an outbreak associated with a case of hepatitis A in a food handler. Additional benefits in the societal model included reductions in costs of food handler-associated cases of hepatitis A. The outcome used was Net Present Value (NPV), allowing comparison between models. Regardless of the cost of vaccination ($50-140/employee), for a restauranteur to ensure that all employees were vaccinated at all times substantial costs were involved (i.e. negative NPV). Even a 75% probability of bankruptcy still resulted in negative NPVs at the 95th percentiles. For society, vaccination was only cost-saving (i.e. positive NPV) if done only during epidemics and if it cost < $20/employee. Vaccinating restaurant employees is unlikely to be economical from either the restaurant owner or the societal perspective, even during hepatitis A epidemics.

Effectiveness and cost-benefit of influenza vaccination of healthy working adults: A randomized controlled trial.

CONTEXT: Although the cost-effectiveness and cost-benefit of influenza vaccination are well established for persons aged 65 years or older, the benefits for healthy adults younger than 65 years are less clear. OBJECTIVE: To evaluate the effectiveness and cost-benefit of influenza vaccine in preventing influenza-like illness (ILI) and reducing societal costs of ILI among healthy working adults. DESIGN: Double-blind, randomized, placebo-controlled trial conducted during 2 influenza seasons. SETTING AND PARTICIPANTS: Healthy adults aged 18 to 64 years and employed full-time by a US manufacturing company (for 1997-1998 season, n = 1184; for 1998-1999 season, n = 1191). INTERVENTIONS: For each season, participants were randomly assigned to receive either trivalent inactivated influenza vaccine (n = 595 in 1997-1998 and n = 587 in 1998-1999) or sterile saline injection (placebo; n = 589 in 1997-1998 and n = 604 in 1998-1999). Participants in 1997-1998 were rerandomized if they participated in 1998-1999. MAIN OUTCOME MEASURES: Influenza-like illnesses and associated physician visits and work absenteeism reported in biweekly questionnaires by all participants, and serologically confirmed influenza illness among 23% of participants in each year (n = 275 in 1997-1998; n = 278 in 1998-1999); societal cost of ILI per vaccinated vs unvaccinated person. RESULTS: For 1997-1998 and 1998-1999, respectively, 95% (1130/1184) and 99% (1178/1191) of participants had complete follow-up, and 23% in each year had serologic testing. In 1997-1998, when the vaccine virus differed from the predominant circulating viruses, vaccine efficacy against serologically confirmed influenza illness was 50% (P =.33). In this season, vaccination did not reduce ILI, physician visits, or lost workdays; the net societal cost was $65.59 per person compared with no vaccination. In 1998-1999, the vaccine and predominant circulating viruses were well matched. Vaccine efficacy was 86% (P =.001), and vaccination reduced ILI, physician visits, and lost workdays by 34%, 42%, and 32%, respectively. However, vaccination resulted in a net societal cost of $11.17 per person compared with no vaccination. CONCLUSION: Influenza vaccination of healthy working adults younger than 65 years can reduce the rates of ILI, lost workdays, and physician visits during years when the vaccine and circulating viruses are similar, but vaccination may not provide overall economic benefits in most years. JAMA. 2000;284:1655-1663.

Limited genetic susceptibility to severe Graves' ophthalmopathy: no role for CTLA-4 but evidence for an environmental etiology.

Graves' disease (GD) is an autoimmune thyroid disease (AITD) characterized by hyperthyroidism and by the occurrence of a distinctive ophthalmopathy (orbitopathy), which presents with varying degrees of severity. Graves' disease clusters in families but the importance of heredity in the pathogenesis of the associated ophthalmopathy is unclear. We have studied the family history of 114 consecutive, ethnically mixed patients with severe Graves' ophthalmopathy (GO). Patients were selected by unambiguous single ascertainment. Seventy-seven percent of patients were female and 59% smoked. The mean age at onset of their GD was 43 years (range 17-78 years). Forty-one patients (36%) had a family history of AITD, defined as a first- and/or a second-degree relative affected with either Graves' disease (GD) or Hashimoto's thyroiditis (HT). The segregation ratio for AITD in nuclear families in our ascertained Graves' ophthalmopathy families was 0.07 (0.12 in Caucasians only). Hence, the higher prevalence of AITD among relatives of Graves' ophthalmopathy patients agreed with the known genetic predisposition to AITD and this predisposition was stronger in women than in men. However, only 3 of the 114 patients had a family history of severe Graves' ophthalmopathy (all second-degree relatives) and the segregation ratio for GO was 0. These data did not support a major role for familial factors in the development of severe Graves' ophthalmopathy distinct from Graves' disease itself. In addition, we tested 4 candidate genes, human leukocyte antigen (HLA), tumor necrosis factor-beta (TNF-beta), CTLA-4 and the thyrotropin receptor (TSHR), for association with Graves' ophthalmopathy. These were negative except for the HLA and CTLA-4 genes, which were found to be weakly associated with GO giving similar relative risk (RR) as in GD patients without ophthalmopathy. These data suggested that environmental factors, rather than major genes, were likely to predispose certain individuals with AITD to severe Graves' ophthalmopathy. Smoking remains one example of such potential external insults.