Comparison of child morbidity in two contrasting medieval cemeteries from Denmark

This study compares associations between demographic profiles, long bone lengths, bone mineral content, and frequencies of stress indicators in the preadult populations of two medieval skeletal assemblages from Denmark. One is from a leprosarium, and thus probably represents a disadvantaged group (Naestved). The other comes from a normal, and in comparison rather privileged, medieval community (AEbelholt). Previous studies of the adult population indicated differences between the two skeletal collections with regard to mortality, dental size, and metabolic and specific infectious disease. The two samples were analyzed against the view known as the "osteological paradox" (Wood et al. [1992] Curr. Anthropol. 33:343-370), according to which skeletons displaying pathological modification are likely to represent the healthier individuals of a population, whereas those without lesions would have died without acquiring modifications as a result of a depressed immune response. Results reveal that older age groups among the preadults from Naestved are shorter and have less bone mineral content than their peers from AEbelholt. On average, the Naestved children have a higher prevalence of stress indicators, and in some cases display skeletal signs of leprosy. This is likely a result of the combination of compromised health and social disadvantage, thus supporting a more traditional interpretation. The study provides insights into the health of children from two different biocultural settings of medieval Danish society and illustrates the importance of comparing samples of single age groups.

Nuclear magnetic resonance structure shows that the severe acute respiratory syndrome coronavirus-unique domain contains a macrodomain fold

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for "middle of the SARS-unique domain") in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1 ''-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.

Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [ nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

Ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the N-terminal domain of N protein

Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 A (monoclinic) and at 1.85 A (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

Cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) spike (S) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. To investigate SCoV S protein, full-length and individual domains of S protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of SARS. S protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection. Reactivity was evident by both flow cytometry and Western blot assays, but the pattern of reactivity varied according to assay and sequence of the antigen. The antibody response to SCoV S protein involves antibodies to both linear and conformational epitopes, with linear epitopes associated with the carboxyl domain and conformational epitopes associated with the amino terminal domain. Recombinant SCoV S protein appears to be a suitable antigen for the development of an efficient and sensitive diagnostic test for SARS, but our data suggest that assay format and choice of S antigen are important considerations.

Cost-benefit evaluation of routine influenza immunisation in people 65-74 years of age

OBJECTIVES: To determine the cost-effectiveness of influenza vaccination in people aged 65-74 years in the absence of co-morbidity. DESIGN: Primary research: randomised controlled trial. SETTING: Primary care. PARTICIPANTS: People without risk factors for influenza or contraindications to vaccination were identified from 20 general practitioner (GP) practices in Liverpool in September 1999 and invited to participate in the study. There were 5875/9727 (60.4%) people aged 65-74 years identified as potentially eligible and, of these, 729 (12%) were randomised. INTERVENTION: Participants were randomised to receive either influenza vaccine or placebo (ratio 3:1), with all individuals receiving pneumococcal vaccine unless administered in the previous 10 years. Of the 729 people randomised, 552 received vaccine and 177 received placebo; 726 individuals were administered pneumococcal vaccine. MAIN OUTCOME MEASURES AND METHODOLOGY OF ECONOMIC EVALUATION: GP attendance with influenza-like illness (ILI) or pneumonia (primary outcome measure); or any respiratory symptoms; hospitalisation with a respiratory illness; death; participant self-reported ILI; quality of life (QoL) measures at 2, 4 and 6 months post-study vaccination; adverse reactions 3 days after vaccination. A cost-effectiveness analysis was undertaken to identify the incremental cost associated with the avoidance of episodes of influenza in the vaccination population and an impact model was used to extrapolate the cost-effectiveness results obtained from the trial to assess their generalisability throughout the NHS. RESULTS: In England and Wales, weekly consultations for influenza and ILI remained at baseline levels (less than 50 per 100,000 population) until week 50/1999 and then increased rapidly, peaking during week 2/2000 with a rate of 231/100,000. This rate fell within the range of 'higher than expected seasonal activity' of 200-400/100,000. Rates then quickly declined, returning to baseline levels by week 5/2000. The predominant circulating strain during this period was influenza A (H3N2). Five (0.9%) people in the vaccine group were diagnosed by their GP with an ILI compared to two (1.1%) in the placebo group [relative risk (RR), 0.8; 95% confidence interval (CI) = 0.16 to 4.1]. No participants were diagnosed with pneumonia by their GP and there were no hospitalisations for respiratory illness in either group. Significantly fewer vaccinated individuals self-reported a single ILI (4.6% vs 8.9%, RR, 0.51; 95% CI for RR, 0.28 to 0.96). There was no significant difference in any of the QoL measurements over time between the two groups. Reported systemic side-effects showed no significant differences between groups. Local side-effects occurred with a significantly increased incidence in the vaccine group (11.3% vs 5.1%, p = 0.02). Each GP consultation avoided by vaccination was estimated from trial data to generate a net NHS cost of 174 pounds. CONCLUSIONS: No difference was seen between groups for the primary outcome measure, although the trial was underpowered to demonstrate a true difference. Vaccination had no significant effect on any of the QoL measures used, although vaccinated individuals were less likely to self-report ILI. The analysis did not suggest that influenza vaccination in healthy people aged 65-74 years would lead to lower NHS costs. Future research should look at ways to maximise vaccine uptake in people at greatest risk from influenza and also the level of vaccine protection afforded to people from different age and socio-economic populations.

Preventing drug related morbidity: a process for facilitating changes in practice

Aim: To describe how quantitative data obtained from applying a series of indicators for preventable drug related morbidity (PDRM) in the electronic patient record in English general practice can be used to facilitate changes aimed at helping to improve medicines management. Design: A multidisciplinary discussion forum held at each practice facilitated by a clinical researcher. Subjects and setting: Eight English general practices. Outcome measures: Issues discussed at the multidisciplinary discussion forum and ideas generated by practices for tackling these issues. Progress made by practices after 1, 3, and 6 months. Results: A number of clinical issues were raised by the practices and ideas for moving them forward were discussed. The issues that were easiest and most straightforward to deal with (for example, reviewing specific patient groups) were quickly addressed in most instances. Practices were less likely to have taken steps towards addressing issues at a systems level. Conclusions: Data generated from applying PDRM indicators can be used to facilitate practice-wide discussion on medicines management. Different practices place different priority levels on the issues they wish to pursue. Individual practice "ownership'' of these, together with having a central committed figure at the practice, is key to the success of the process.

A family study of co-morbidity between generalized social phobia and generalized anxiety disorder in a non-clinic sample

Background: High rates of co-morbidity between Generalized Social Phobia (GSP) and Generalized Anxiety Disorder (GAD) have been documented. The reason for this is unclear. Family studies are one means of clarifying the nature of co-morbidity between two disorders. Methods: Six models of co-morbidity between GSP and GAD were investigated in a family aggregation study of 403 first-degree relatives of non-clinical probands: 37 with GSP, 22 with GAD, 15 with co-morbid GSP/GAD, and 41 controls with no history of GSP or GAD. Psychiatric data were collected for probands and relatives. Mixed methods (direct and family history interviews) were utilised. Results: Primary contrasts (against controls) found an increased rate of pure GSP in the relatives of both GSP probands and co-morbid GSP/GAD probands, and found relatives of co-morbid GSP/GAD probands to have an increased rate of both pure GAD and comorbid GSP/GAD. Secondary contrasts found (i) increased GSP in the relatives of GSP only probands compared to the relatives of GAD only probands; and (ii) increased GAD in the relatives of co-morbid GSP/GAD probands compared to the relatives of GSP only probands. Limitations: The study did not directly interview all relatives, although the reliability of family history data was assessed. The study was based on an all-female proband sample. The implications of both these limitations are discussed. Conclusions: The results were most consistent with a co-morbidity model indicating independent familial transmission of GSP and GAD. This has clinical implications for the treatment of patients with both disorders. (C) 2006 Elsevier B.V. All fights reserved.

A qualitative exploration of the underlying causes of preventable drug-related morbidity in primary care, resulting in hospitalisation

This study has explored the underlying causes of preventable drug-related admissions to hospital, from primary care through semi-structured interviews and review of patients’ medical records. Analysis of the data has revealed that communication failures between different groups of healthcare professionals and between healthcare professionals and patients contribute to preventable drug-related admissions, as do knowledge gaps about medication in both healthcare professionals and patients. In addition, working conditions for community pharmacists severely limit their ability to effectively act as a safety barrier to patients receiving inappropriate medication. Limitations include heavy workloads, lack of access to patients’ clinical information, poor relationships with general practitioners and time restrictions. The results of this study represent an important addition to our understanding of the contribution of human error as an underlying cause of preventable drug-related morbidity, and the factors which contribute to errors occurring in the primary healthcare setting.

Pseudomonas aeruginosa elastase disables proteinase-activated receptor 2 in respiratory epithelial cells

Pseudomonas aeruginosa, a major lung pathogen in cystic fibrosis (CF) patients, secretes an elastolytic metalloproteinase (EPa) contributing to bacterial pathogenicity. Proteinase-activated receptor 2 (PAR2), implicated in the pulmonary innate defense, is activated by the cleavage of its extracellular N-terminal domain, unmasking a new N-terminal sequence starting with SLIGKV, which binds intramolecularly and activates PAR2. We show that EPa cleaves the N-terminal domain of PAR2 from the cell surface without triggering receptor endocytosis as trypsin does. As evaluated by measurements of cytosolic calcium as well as prostaglandin E(2) and interleukin-8 production, this cleavage does not activate PAR2, but rather disarms the receptor for subsequent activation by trypsin, but not by the synthetic receptor-activating peptide, SLIGKV-NH(2). Proteolysis by EPa of synthetic peptides representing the N-terminal cleavage/activation sequences of either human or rat PAR2 indicates that cleavages resulting from EPa activity would not produce receptor-activating tethered ligands, but would disarm PAR2 in regard to any further activating proteolysis by activating proteinases. Our data indicate that a pathogen-derived proteinase like EPa can potentially silence the function of PAR2 in the respiratory tract, thereby altering the host innate defense mechanisms and respiratory functions, and thus contributing to pathogenesis in the setting of a disease like CF.

Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles

Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6. IMPORTANCE Although the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.

Microbiological insights into respiratory infections and the opportunities for inhaled therapy

Respiratory infections represent the fourth most common cause of all deaths across age groups and countries. Treating these infections appropriately is a clear clinical priority and here we outline the types of therapy that are in current use for some of these infections. It is important that treatments are further improved and the potential of inhaled delivery to fulfil this need is considered. We describe novel methodologies that are being applied for the identification and enumeration of microorganisms in the respiratory tract, and propose that ways of improving therapy may arise from understanding better the etiology of respiratory infection and the impact of inhaled drug therapies. The potential for translational benefits for patients are also discussed.