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"Clinical features of culture-proven Mycoplasma pneumoniae infections at King Abdulaziz University Hospital, Jeddah, Saudi Arabia"
"OBJECTIVE: This retrospective chart review describes the epidemiology and clinical features of 40 patients with culture-proven Mycoplasma pneumoniae infections at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. METHODS: Patients with positive M. pneumoniae cultures from respiratory specimens from January 1997 through December 1998 were identified through the Microbiology records. Charts of patients were reviewed. RESULTS: 40 patients were identified, 33 (82.5%) of whom required admission. Most infections (92.5%) were community-acquired. The infection affected all age groups but was most common in infants (32.5%) and pre-school children (22.5%). It occurred year-round but was most common in the fall (35%) and spring (30%). More than three-quarters of patients (77.5%) had comorbidities. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common symptoms, and crepitations (60%), and wheezes (40%) were the most common signs. Most patients with pneumonia had crepitations (79.2%) but only 25% had bronchial breathing. Immunocompromised patients were more likely than non-immunocompromised patients to present with pneumonia (8/9 versus 16/31, P = 0.05). Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications, 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities. CONCLUSION: our results were similar to published data except for the finding that infections were more common in infants and preschool children and that the mortality rate of pneumonia in patients with comorbidities was high."
"Mycoplasma pneumoniae is a common cause of upper and lower respiratory tract infections. It remains one of the most frequent causes of atypical pneumonia particu-larly among young adults. [1, 2, 3, 4, 5] Although it is highly transmissible, most infections caused by this organism are relatively minor and include pharyngitis, tracheobronchitis, bronchiolitis, and croup with one fifth of in-fections being asymptomatic. [6, 7] Only 3 -10% of infected subjects develop symptoms consistent with bronchopneumonia and mortality from infection is rare. [6, 7] The organism is fastidious and difficult to grow on cultures. Therefore, diagnosis of infections caused by this organism is usually confirmed with serological tests or polymerase chain reaction-gene amplification techniques. At King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, the facility to perform Mycoplasma culture has been available since January 1997. As published information concerning M. pneumoniae infections in Saudi Arabia is scarce, [8, 9, 10] we wished to study the epidemiology and clinical features of cultureproven infections caused by this organism at this hospital. KAUH is a tertiary care teaching hospital with a bed capacity of 265 beds and annual admissions of 18000 to 19000 patients. Patients with M. pneumoniae positive cultures from respiratory specimens were identified over a 24-months" period from January, 1997 through December, 1998 for this review. During the study period, respiratory specimens (sputum, nasopharyngeal aspiration, endotracheal secretion, and bronchoalveolar lavage) for M. pneumoniae culture were obtained from patients with upper or lower respiratory tract infections seen as inpatients or in the outpatient or emergency departments. Respiratory specimens were aslo Gram-stained and cultured for bacteria and viruses. M. pneumoniae serological tests for IgG or IgM were not available at KAUH during the study period. All positive culture results were obtained from the Microbiology laboratory records. Charts of patients were reviewed with standardized data collection. Information collected included patients' demographics, comorbidities, clinical manifestations, complications, and outcome. M. pneumoniae was cultured using the classic M. pneumoniae agar medium (M.P. agar) and the Pneumofast tray (Pneumofast ® , International Microbio, Signes, France). Specimens were processed according to the instructions of the manufacturer. The M.P. agars and Pneumofast trays were incubated anaerobically at 37°C and inspected daily for 4 weeks. The organism was identified based on typical colonial morphology (granular colonies, rarely fried-egg-like, 10-150 ∝ in diameter) on the M.P. agar medium and the change in the Pneumofast broth color from red to orange then to yellow (glucose fermentation) in the absence of turbidity of the broth. Antibiotic sensitivity profile on the Pneumofast tray was also used for identification according to the instructions of the manufacturer. Bacterial and viral cultures were performed using standard methods. M. pneumoniae isolates were considered community-acquired if they were recovered from unhospitalized patients or within 72 hours of admission to the hospital, and nosocomial if they were recovered beyond that period. Pneumonia was diagnosed based on clinical symptoms and signs, along with radiographic evidence of pneumonia when possible. Severe pneumonia was defined as pneumonia associated with tachycardia (>140 /minute), tachypnoea (>30/minute), hypotension (Systolic blood pressure <90 mmHg), hypoxemia (arterial oxygen partial pressure <8 kPa or oxygen saturation <90%), and/or more than 2 areas of consolidation. Outcome of patients with M. pneumoniae infection was classified into 4 categories; uneventful recovery, recovery following complications, death due to M. pneumoniae infection, or death unrelated to M. pneumoniae infection. The Statistical Package for Social Sciences (SPSS) program was used for data analysis. Comparison of categorical data was by Chi-square statistic or Fisher's exact test for small expected values. A total of 40 respiratory specimens from 40 patients were positive for M. pneumoniae over the 24-months study period. The demographic and epidemiological characteristics of the patients are summarized in Table 1 . Of all isolates, 37 (92.5%) were community-acquired and 3 (7.5%) were nosocomial. Thirty-three (82.5%) patients required admission to the hospital and the remaining 7 (17.5%) were treated as outpatients. Twenty-four isolates (60%) were associated with pneumonia, 14 (35%) with upper respiratory tract infections, and 2 (5%) with bronchiolitis. Of the 24 cases of pneumonia, 21 were confirmed radiologically and the remaining 3 were diagnosed clinically. The two cases of bronchiolitis occurred in 2 children, one and three years old. Thirty-one patients (77.5%) had comorbidities. Eleven patients (27.5%) had cardiopulmonary comorbidities (asthma, 8, lung fibrosis, 1, congestive heart failure, 1, congenial heart disease, 1), 9 patients (22.5%) were immunocompromised (malignancy, 7, steroid therapy, 3, Human immunodeficiency virus infection, 1), and 11 patients (27.5%) had other comorbidities (premature newborns, 2, and one each of myelodysplastic syndrome, myelopro-liferative disorder, sickle cell anemia, Evan's syndrome, Down syndrome, sarcoidosis, demyelinating disease, cerebral palsy, and spinal muscle atrophy). Organisms concomitantly isolated with M. pneumoniae from the respiratory tract included herpes simplex virus type 1 (2 occasions), adenovirus (2 occasions), cytomegalo virus (1 occasion), respiratory syncytial virus (1 occasion), and bacterial isolates (2 occasions: Acinetobacter species, 1, and Enter obacter cloacae, 1). Clinical manifestations associated with M. pneumoniae infections are summarized in Table 2 . Pneumonia was more common than upper respiratory tract infections (57.5 % versus 27.5%, respectively). Immunocompromised patients were more likely to present with pneumonia as opposed to upper respiratory tract infection or bronchiolitis than non-immunocompromised patients (8/9 versus 16/31, P = 0.05). Similarly, there was a tendency for patients 60 years of age or older to present with pneumonia more frequently than those below 60 (4/4 versus 20/36, P = 0.1). Of the 24 patients with clinically or radiologically confirmed pneumonia, 19 (79.2%) had crepitations and only 6 (25%) had bronchial breath sounds on physical examination. Of the 16 patients in whom wheezes were detected, 9 (56.3%) were not known to have asthma or other obstructive airway disease. Table 3 . Of the 24 patients with pneumonia, 21 (87.5%) were admitted to the hospital, and 20 (83.3%) had comorbidities. All patients with upper respiratory tract infections (11 patients) or bronchiolitis (2 patients) had uneventful recovery. Of the 24 patients with pneumonia, 14 (58.3%) had uneventful recovery, 4 (16.7%) recovered following some complications (acute respiratory distress syndrome, 2, respiratory failure, 1, septic shock, 1), 3 (12.5%) died because of M pneumoniae infection, and 3 (12.5%) died due to underlying comorbidities. The 3 patients who died of M pneumoniae pneumonia had other comorbidities; one had congestive heart failure, the second had congenital heart disease, and the third was a 3months old infant born prematurely at 32 weeks of gestation who previously had 3 episodes of pneumonia due to other pathogens. Mycoplasma pneumoniae is one of the most common causes of atypical pneumonia accounting for 5-23% of community-acquired pneumonia, [1, 2, 3, 4, 5] In a study of 511 children with acute respiratory tract infection in Riyadh, Saudi Arabia, Mycoplasma pneumoniae was found to be the second most common causative agent after Respiratory syncytial virus (RSV) accounting for 9% of all cases, [8] In a study of 112 adult patients with community acquired pneumonia admitted to a military hospital in Riyadh, Saudi Arabia, this organism accounted for 6% of all cases, [9] In another retrospective study of 567 pneumonic episodes in adult patients from Al-Qassim area, the organism accounted for 23% of all episodes, [10] The organism also causes other relatively minor infections such as pharyngitis, tracheobronchitis, bronchiolitis, and croup. It is transmitted from person-to-person by infected respiratory droplets during close contact. It is most common in school-aged children, military recruits, and college students. [11] Most cases occur singly or as family outbreaks. Larger outbreaks can also occur in closed populations such as military recruit camps or boarding schools, [12] Infection occurs most frequently during the fall and winter in temperate climates but may develop year-round, [13] The average incubation period is 3 weeks following exposure, [6] Although rare, complications are protean and may involve virtually any organ system such as the respiratory system (e.g.: pleurisy, pneumothorax, acute respiratory distress syndrome, lung abscess), the hematologic system (e.g.: hemolytic anemia, intravascular coagulation, thrombocytopenia), the dermatologic system (e.g.: maculopapular or urticarial rashes, erythema multiforme, erythema nodosum), the musculoskeletal system (e.g.: myalgias, arthralgias, arthritis), the cardiovascular system (e.g.: pericarditis, myocarditis), the nervous system (e.g.: meningoencephalitis, Guillain-Barre syndrome, neuropathies, acute psychosis), or the eye (optic disc edema, optic nerve atrophy, retinal exudation and hemorrhages). [6, 7, 14, 15, 16, 17, 18] Immunity following infection is not long lasting. [11] In our study, the infection affected all age groups but was most common in infants (32.5%) and preschool children (22.5%), and least common in adults aged 15 to 30 years (2.5%) and elderly above 70 years of age (5%). This contrasts with data from temperate countries where the infection is most common in school-aged children, and young adults. [11] One possible explanation for this difference is that infants and preschool children perhaps had more severe infections than did school-aged children, and young adults which prompted presentation of the former group to the hospital. The infection occurred year-round but was most common in the fall (35%), and spring (30%), and least common in the summer (10%). Most infections were community-acquired (92.5%). More than one half of patients (57.5%) presented with pneumonia, and about a third (27.5%) presented with upper respiratory tract infection, Immunocompromised patients and patients 60 years of age or older were more likely to present with pneumonia as opposed to upper respiratory tract infection than non-immunocompromised patients or those below 60 years of age. Cough (82.5%), fever (75%), and malaise (58.8%) were the most common presenting symptoms. Cough was usually dry or slightly productive of white sputum and mild to moderate in severity. Most febrile patients had mild to mod- erate fever of 39°C or less; high-grade fever of more than 39°C was rare. Crepitations (60%), and wheezes (40%) were the most common signs. Wheezes were as common in patients with no history of obstructive airway disease (9 patients) as it was in those with such a history (7 patients). Bronchial breathing as a sign of consolidation was detected in only one fourth of patients with pneumonia, which is consistent with the known disparity between clinical and radiological signs of M pneumoniae pneumonia. Crepitations, however, were detected in the majority (79.2%) of patients. Pleuritic chest pain and pleural effusion were rare. More than half (56.5%) of the patients with pneumonia had uneventful recovery. Mortality from M. pneumoniae pneumonia was high (12.5%) and occurred only in patients with underlying comorbidities. None of the 9 patients with no underlying comorbidities died of M pneumoniae pneumonia. The relatively high complications rate (16.7%) and mortality (12.5%) related to M. pneumoniae pneumonia are likely due to selection bias as most patients with pneumonia were sick enough to require admission to the hospital (21/24 or 87.5%) and most of them had comorbidities (20/24 or 83.3%). In conclusion, our data shed some light on the epidemiology and clinical features of M pneumoniae infections in one of the Saudi tertiary care centers. The data are comparable to those of other countries except for the finding that infections were more common in infants and preschool children than in school children and young adults. Additionally, mortality attributable to M. pneumoniae pneumonia was relatively high in patients with comorbidities. It is hoped this information will assist clinicians in their approach and management of respiratory tract infections."
1
"Nitric oxide: a pro-inflammatory mediator in lung disease?"
"Inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO•) and increased indices of NO• -dependent oxidative stress. Although NO• is known to have anti-microbial, anti-inflammatory and anti-oxidant properties, various lines of evidence support the contribution of NO• to lung injury in several disease models. On the basis of biochemical evidence, it is often presumed that such NO• -dependent oxidations are due to the formation of the oxidant peroxynitrite, although alternative mechanisms involving the phagocyte-derived heme proteins myeloperoxidase and eosinophil peroxidase might be operative during conditions of inflammation. Because of the overwhelming literature on NO• generation and activities in the respiratory tract, it would be beyond the scope of this commentary to review this area comprehensively. Instead, it focuses on recent evidence and concepts of the presumed contribution of NO• to inflammatory diseases of the lung."
"Since its discovery as a biological messenger molecule more than 10 years ago, the gaseous molecule nitric oxide (NO • ) is now well recognized for its involvement in diverse biological processes, including vasodilation, bronchodilation, neurotransmission, tumor surveillance, antimicrobial defense and regulation of inflammatory-immune processes [1] [2] [3] . In the respiratory tract, NO • is generated enzymically by three distinct isoforms of NO • synthase (NOS-1, NOS-2 and NOS-3) that are present to different extents in numerous cell types, including airway and alveolar epithelial cells, neuronal cells, macrophages, neutrophils, mast cells, and endothelial and smoothmuscle cells. In contrast with the other two NOS isoforms (NOS-1 and NOS-3), which are expressed constitutively and activated by mediator-induced or stress-induced cell activation, NOS-2 activity is primarily regulated transcriptionally and is commonly induced by bacterial products and pro-inflammatory cytokines. As such, inflammatory diseases of the respiratory tract, such as asthma, acute respiratory distress syndrome (ARDS) and bronchiectasis, are commonly characterized by an increased expression of NOS-2 within respiratory epithelial and inflammatory-immune cells, and a markedly elevated local production of NO • , presumably as an additional host defense mechanism against bacterial or viral infections. The drawback of such excessive NO • production is its accelerated metabolism to a family of potentially harmful reactive nitrogen species (RNS), including peroxynitrite (ONOO -) and nitrogen dioxide (NO 2 • ), especially in the presence of phagocyte-generated oxidants. The formation of such RNS is thought to be the prime reason why NO • can in many cases contribute to the etiology of inflammatory lung disease [4] [5] [6] . Despite extensive research into both pro-inflammatory and anti-inflammatory actions of NO • , the overall contribution of NO • to inflammatory conditions of the lung is not easily predicted and seems to depend on many factors, such as the site, time and degree of NO • production in relation to the local redox status, and the acute or chronic nature of the immune response. In addition, our current understanding of the pro-inflammatory or pro-injurious mechanisms of NO • or related RNS is incomplete; this commentary will focus primarily on these latter aspects. To explore a role for NO • (or NOS) in infectious or inflammatory diseases, two general research approaches have been taken: the use of pharmacological inhibitors of NOS isoenzymes, and the targeted deletion of individual NOS enzymes in mice. Both approaches suffer from the shortcoming that animal models of respiratory tract diseases generally do not faithfully reflect human disease. The use of NOS inhibitors to determine the contribution of individual NOS isoenzymes is also hindered by problems related to specificity and pharmacokinetic concerns. However, the unconditional gene disruption of one or more NOS isoforms, leading to lifelong deficiency, can have a markedly different outcome from pharmacological inhibition at a certain stage of disease, as the involvement of individual NOS isoenzymes can be different depending on disease stage and severity. Despite these inherent limitations, studies with the targeted deletion of NOS isoforms have led to some insights, indicating a role for NO • and NOS-2 in the etiology of some inflammatory lung diseases. For instance, mice deficient in NOS-2 are less susceptible to lethality after intranasal inoculation with influenza A virus, suffer less lung injury after administration of endotoxin, and display reduced allergic eosinophilia in airways and lung injury in a model of asthma, than their wild-type counterparts [7] [8] [9] . However, although the contribution of NOS-2 is expected in inflammatory conditions, recent studies have determined that NOS-1, rather than NOS-2, seems to be primarily involved in the development of airway hyper-reactivity in a similar asthma model [10] . The linkage of NOS-1 to the etiology of asthma was more recently supported in asthmatic humans by an association of a NOS-1 gene polymorphism with this disease, although the physiological basis for this association remains unclear [11] . Despite the potential contribution of NOS-2-derived NO • to lung injury after endotoxemia, the sequestration of neutrophils in the lung and their adhesion to postcapillary and postsinusoidal venules after administration of endotoxin were found to be markedly increased in NOS-2-deficient mice, and NOS-2 deficiency did not alleviate endotoxininduced mortality. It therefore seems that the 'harmful' and 'protective' effects of NOS-2 might contend with each other within the same model, which makes the assessment of the potential role of NOS in human disease even more difficult. In this context, it is interesting to note that humans or animals with cystic fibrosis have subnormal levels of NOS-2 in their respiratory epithelium, related to a gene mutation in the cystic fibrosis transmembrane conductance regulator [12] . This relative absence of epithelial NOS-2 might be one of the contributing factors behind the excessively exuberant respiratory tract inflammatory response in patients with cystic fibrosis, even in the absence of detectable respiratory infections. Overall, the apparently contrasting findings associated with NOS deficiency, together with concerns about animal disease models used, make interpretations and conclusions with regard to human lung disease all the more difficult. Pharmacological inhibitors of NOS have also been found to reduce oxidative injury in several animal models of lung injury, such as ischemia/reperfusion, radiation, paraquat toxicity, and endotoxemia (see, for example, [13] [14] [15] ). However, results are again not always consistent, and in some cases NOS inhibition has been found to worsen lung injury, indicating anti-inflammatory or protective roles for NO • . All in all, despite these inconsistencies, there is ample evidence from such studies to suggest a contributing role of NO • in various respiratory disease conditions, which continues to stimulate research into mechanistic aspects underlying such pro-inflammatory roles and modulation of NO • generation as a potential therapeutic target. Although the pro-inflammatory and injurious effects of NO • might be mediated by a number of diverse mechanisms, it is commonly assumed that such actions are largely due to the generation of reactive by-products generated during the oxidative metabolism of NO • ; these are collectively termed RNS. One of the prime suspects commonly implicated in the adverse or injurious properties of NO • is ONOO -, a potent oxidative species formed by its almost diffusion-limited reaction with superoxide (O 2 •-), which is a product of activated phagocytes and of endothelial or epithelial cells [4, 5, 13] . The formation of ONOOseems highly feasible under conditions of elevated production of both NO • and O 2 •in vivo, and its oxidative and cytotoxic potential is well documented [5, 6] . However, because the direct detection of ONOOunder inflammatory conditions is virtually impossible because of its instability and high reactivity, the formation of ONOOin vivo can be demonstrated only by indirect methods. Thus, many investigators have relied on the analysis of characteristic oxidation products in biological molecules, such as proteins and DNA, most notably free or protein-associated 3-nitrotyrosine, a product of tyrosine oxidation that can be formed by ONOO -(and several other RNS) but not by NO • itself (see, for commentary review reports primary research http://respiratory-research.com/content/1/2/067 example, [5] ). Indeed, elevated levels of 3-nitrotyrosine have been observed in many different inflammatory conditions of the respiratory tract [16] , which illustrates the endogenous formation of ONOOor related RNS in these cases. However, without known evidence for functional consequences of (protein) tyrosine nitration, the detection of 3-nitrotyrosine should not be regarded as direct proof of a pro-inflammatory role of NO • . Moreover, although the detection of 3-nitrotyrosine has in most cases been interpreted as conclusive evidence for the formation of ONOOin vivo (see, for example, [17] ), it should be realized that other RNS formed by alternative mechanisms might also contribute to endogenous tyrosine nitration. Indeed, it has recently become clear that the presence of inflammatory-immune cells, and specifically their heme peroxidases myeloperoxidase (MPO) and eosinophil peroxidase (EPO), can catalyze the oxidization of NO • and/or its metabolite NO 2 to more reactive RNS and thereby contribute to protein nitration [16, 18, 19] . This notion is further supported by the fact that 3-nitrotyrosine is commonly detected in tissues affected by active inflammation, mostly in and around these phagocytic cells and macrophages, which can also contain active peroxidases originating from apoptotic neutrophils or eosinophils. Hence, the detection of 3-nitrotyrosine in vivo cannot be used as direct proof of the formation of ONOO -, but merely indicates the formation of RNS by multiple oxidative pathways, possibly including ONOObut more probably involving the activity of phagocyte peroxidases [16, 20] . In this regard, a preliminary study with EPO-deficient mice has recently demonstrated the critical importance of EPO in the formation of 3-nitrotyrosine in a mouse model of asthma [21] . Future studies with animals deficient in MPO and/or EPO will undoubtedly help to clarify this issue. Given the considerable interest in 3-nitrotyrosine as a collective marker of the endogenous formation of NO •derived RNS, the crucial question remains of whether the detection of 3-nitrotyrosine adequately reflects the toxic or injurious properties of NO • . The formation of ONOO -(or of other RNS that can induce tyrosine nitration) might in fact represent a mechanism of decreasing excessive levels of NO • that might exert pro-inflammatory actions by other mechanisms. For instance, NO • can promote the expression of pro-inflammatory cytokines or cyclo-oxygenase (responsible for the formation of inflammatory prostanoids) by mechanisms independent of ONOO - [22, 23] , and the removal of NO • would minimize these responses. Furthermore, although ONOOor related NO •derived oxidants can be cytotoxic or induce apoptosis, these effects might not necessarily relate to their ability to cause protein nitration (see, for example, [16]). For instance, the bactericidal and cytotoxic properties of ONOOare minimized by the presence of CO 2 , even though aromatic nitration and other radical-induced modifications are enhanced [5] . Similarly, the presence of NO 2 in the incubation medium decreases the cytotoxicity of MPO-derived hypochlorous acid (HOCl) toward epithelial cells or bacteria, despite increased tyrosine nitration of cellular proteins (A van der Vliet and M Syvanen, unpublished data). Thus, it would seem that the cytotoxic properties of NO • and/or its metabolites might instead be mediated through preferred reactions with other biological targets, and these might not necessarily be correlated with the degree of tyrosine nitration. The extent of nitrotyrosine immunoreactivity in bronchial biopsies of asthmatic patients was correlated directly with measured levels of exhaled NO • and inversely with the provocation concentration for methacholine (PC 20 ) and forced expiratory volume in 1 s [24] . However, an immunohistochemical analysis of nitrotyrosine and apoptosis in pulmonary tissue samples from lung transplant recipients did not identify patients with an imminent risk of developing obliterative bronchiolitis [25] . It is therefore still unclear to what degree tyrosine nitration relates to disease progression. Several studies with purified enzymes have suggested that nitration of critical tyrosine residues adversely affects enzyme activity, but there is as yet no conclusive evidence in vivo for biological or cellular changes as a direct result of tyrosine nitration [16, 20] . For instance, tyrosine nitration was suggested to have an effect on cellular pathways by affecting cytoskeletal proteins or tyrosine phosphorylation, thereby affecting processes involved in, for example, cell proliferation or differentiation [16, 26] . Recent studies have provided support for selective tyrosine nitration within certain proteins [27, 28] and of selective cellular targets for nitration by RNS (see, for example, [29, 30] ), and such specificity might indicate a potential physiological role for this protein modification. However, in none of these cases could tyrosine nitration be linked directly to changes in enzyme function. Chemical studies have indicated that tyrosine nitration by RNS accounts for only a minor fraction of oxidant involved, and reactions with other biological targets (thiols, selenoproteins, or transition metal ions) are much more prominent [5, 6] . Indeed, the extent of tyrosine nitration in vivo is very low (1-1000 per 10 6 tyrosine residues according to best estimates [16]), although different analytical methods used to detect 3-nitrotyrosine in biological systems have often given inconsistent results. It is important to note that recent rigorous studies have unveiled substantial sources of artifact during sample preparation, which might frequently have led to an overestimation of tyrosine nitration in vivo in previous studies [31] . On the basis of current knowledge, the formation of 3-nitrotyrosine seems to be merely a marker of NO •derived oxidants, with as yet questionable pathophysiological significance. In view of the low efficiency of tyrosine nitration by biological RNS, and the endogenous presence of variable factors that influence protein nitration (antioxidants or other RNS scavengers), it seems unlikely that tyrosine nitration is a reliable mechanism of, for example, enzyme regulation. Nevertheless, the recent discovery of enzymic 'denitration' mechanisms that can reverse tyrosine nitration [32] merits further investigation of the possibility that tyrosine nitration might reflect a signaling pathway, for example analogous to tyrosine phosphorylation or sulfation. The biological effects of NO • are mediated by various actions, either by NO • itself or by secondary RNS, and the overall biochemistry of NO • is deceptively complex. Moreover, the metabolism and chemistry of NO • depend importantly on local concentrations and pH; the recently described acidification of the airway surface in asthmatics [33] might significantly affect NO • metabolism in these patients. It is well known that interactions with the ion centers of iron or other transition metals are responsible for many of the signaling properties of NO • ; the activation of the heme enzyme guanylyl cyclase and the consequent formation of cGMP is involved not only in smooth-muscle relaxation but also in the activation of certain transcription factors, the expression of several pro-inflammatory and anti-inflammatory genes (including cytokines and cyclo-oxygenase), and the production of respiratory mucus [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . In addition to such direct signaling properties, many actions of NO • might be due largely to secondary RNS that can react with multiple additional targets, in some cases forming nitroso or nitro adducts as potentially unique NO • -mediated signaling mechanisms. As discussed, the formation of protein nitrotyrosine has been postulated as a potential RNSspecific signaling pathway. Even more interest has been given to the reversible S-nitros(yl)ation of protein cysteine residues, which has been proposed to affect a number of redox-sensitive signaling pathways, for example by the activation of p21 ras or the inhibition of protein tyrosine phosphatases [35, 36] . Similar modifications of reactive cysteine residues in transcription factors such as nuclear factor-κB or of caspases contribute to the regulation of gene expression and apoptosis [37] [38] [39] . The precise mechanisms leading to protein S-nitrosylation in vivo are still not clarified, but might involve dinitrogen trioxide (formed during the autoxidation of NO • ), iron-nitrosyl complexes, and perhaps ONOO - [16] ; changes in NO • metabolism during inflammatory lung diseases undoubtedly affect such NO • -dependent signaling pathways. In addition, S-nitrosylation can be reversed by either enzymic (thioredoxin or glutaredoxin) or chemical (metals or oxidants) mechanisms, and evidence is increasing that this reversible modification is complementary to more widely accepted oxidant-dependent redox signaling pathways [40] . The reported alterations in S-nitrosothiol levels in tracheal secretions of patients with asthma or cystic fibrosis further point to altered NO • metabolism in these cases, and might provide new clues to the role of S-nitrosylation in controlling such disease processes [41, 42] . Unfortunately, technical limitations to detect S-nitrosylation in specific protein targets in vivo have limited a full understanding of this potential signaling pathway; further research in these areas can be expected to establish more clearly its significance in the pathophysiological properties of NO • . Despite the by now overwhelming evidence for the increased formation of NO • and NO • -derived oxidants in many different lung diseases, the exact contribution of NO • or its metabolites to inflammatory lung disease is still unclear. Indeed, NO • might have distinctly different roles in different stages of respiratory tract inflammatory diseases, being pro-inflammatory or pro-injurious in acute and severe stages but perhaps being protective and antiinflammatory in more stable conditions; it is uncertain whether NOS is a suitable therapeutic target in the management of inflammatory lung disease. Caution is clearly needed when interpreting observations of tyrosine nitration in animal models of disease or in human tissues, which does not automatically implicate ONOO -(as often thought), but rather indicates the formation of RNS by various mechanisms. Furthermore, animal models of chronic lung disease that usually reflect short-term or acute inflammation might not always be applicable to chronic airway diseases in humans. For instance, phagocyte degranulation, a common feature observed in association with human airway inflammatory diseases such as asthma, does not seem to occur in mouse models of asthma [43] . Therefore the importance of granule proteins, such as heme peroxidases, in the pathology of human airway diseases might not be adequately reflected in such animal models. More work with animal models more characteristic of human diseases or with biopsy materials from human subjects will be required to unravel the precise role of NO • in inflammatory lung disease, and might establish more clearly whether the pharmacological inhibition of NOS isoenzymes can be beneficial. This brings up the interesting paradox that, despite presumed adverse roles of NO • in such inflammatory lung diseases as septic shock and ARDS, NO • inhalation has been suggested as a potential therapeutic strategy to improve overall gas exchange [44] . Intriguingly, in a rat model of endotoxemia, inhalation of NO • was found to reduce neutrophilic inflammation and protein nitration [45] , again supporting the crucial involvement of inflammatory-immune cells in this protein modification. For a better assessment of the role of NO • in respiratory tract diseases in humans, the production of RNS and/or characteristic markers would need to be more carefully monitored during various disease stages. Care should be given to analytical techniques, their quantitative capacity and the possibility of artifacts. The monitoring of exhaled NO • , although convenient and non-invasive, does not reflect the actual production or fate of NO • in the respiratory tract and is not well correlated with NOS activity in the lung [46] . We therefore need to continue research into the local biochemistry of NO • in the lung, taking into account the presence of secreted or phagocyte peroxidases and possible changes in local pH, as in asthmatic airways [33] , that might modulate NO • activity and metabolism. This might result in a better understanding of relationships between the various metabolic endproducts of NO • (NO 2 -, NO 3 -, or nitroso and nitro adducts) and its pro-inflammatory or injurious properties."
2
"Surfactant protein-D and pulmonary host defense"
"Surfactant protein-D (SP-D) participates in the innate response to inhaled microorganisms and organic antigens, and contributes to immune and inflammatory regulation within the lung. SP-D is synthesized and secreted by alveolar and bronchiolar epithelial cells, but is also expressed by epithelial cells lining various exocrine ducts and the mucosa of the gastrointestinal and genitourinary tracts. SP-D, a collagenous calcium-dependent lectin (or collectin), binds to surface glycoconjugates expressed by a wide variety of microorganisms, and to oligosaccharides associated with the surface of various complex organic antigens. SP-D also specifically interacts with glycoconjugates and other molecules expressed on the surface of macrophages, neutrophils, and lymphocytes. In addition, SP-D binds to specific surfactant-associated lipids and can influence the organization of lipid mixtures containing phosphatidylinositol in vitro. Consistent with these diverse in vitro activities is the observation that SP-D-deficient transgenic mice show abnormal accumulations of surfactant lipids, and respond abnormally to challenge with respiratory viruses and bacterial lipopolysaccharides. The phenotype of macrophages isolated from the lungs of SP-D-deficient mice is altered, and there is circumstantial evidence that abnormal oxidant metabolism and/or increased metalloproteinase expression contributes to the development of emphysema. The expression of SP-D is increased in response to many forms of lung injury, and deficient accumulation of appropriately oligomerized SP-D might contribute to the pathogenesis of a variety of human lung diseases."
"Surfactant protein-D (SP-D) is a member of the collagenous subfamily of calcium-dependent lectins (collectins) that includes pulmonary surfactant protein A (SP-A) and the serum mannose-binding lectin [1] [2] [3] . Collectins inter-act with a wide variety of microorganisms, lipids, and organic particulate antigens, and can modulate the function of immune effector cells and their responses to these ligands. This article reviews what is currently known about the sites of production, structure, function, and regulated expression of SP-D. Emphasis will be placed on functional attributes, known ligand interactions, and structure-function relationships believed to be important for host defense. For additional information on SP-A and other members of the collectin family, the reader is referred to other recent reviews [4] [5] [6] . SP-D is synthesized and secreted into the airspaces of the lung by the respiratory epithelium [1] . At the alveolar level, SP-D is constitutively synthesized and secreted by alveolar type II cells. More proximally in the lung, SP-D is secreted by a subset of bronchiolar epithelial cells, the non-ciliated Clara cells. Because SP-D is stored within the secretory granules of Clara cells [7, 8] , it seems likely that SP-D is subject to regulated secretion via granule exocy-tosis at this level of the respiratory tract. In some species, SP-D is also synthesized by epithelial cells and/or submucosal glands associated with the bronchi and trachea [9] . Although many alveolar macrophages show strong cytoplasmic and/or membrane labeling with antibody against SP-D, they do not contain detectable SP-D message. The lung seems to be the major site of SP-D production. However, there is increasing evidence for extrapulmonary sites of expression as assessed with monoclonal or affinity-purified antibodies, reverse-transcriptase-mediated PCR (RT-PCR), and/or hybridization assays of tissues from humans and other large mammals [10 • ,11-14] (summarized in Table 1 ). It is difficult to entirely exclude crossreactions or amplification of related sequences; however, localization to many of these sites in human tissues was confirmed by using monoclonal antibodies in combination with RT-PCR with sequencing of the amplified products [10 • ]. Non-pulmonary expression seems to be largely restricted to cells lining epithelial surfaces or ducts and certain glandular epithelial cells that are in direct or indirect continuity with the environment. Notable exceptions to this generalization might include heart, brain, pancreatic islets, and testicular Leydig cells. SP-D has also been identified in amnionic epithelial cells by immunohistochemistry [15] ; however, it is unclear whether this is synthesized locally or derived from the lung by way of the amniotic fluid. Interestingly, in many of these sites SP-D microscopically co-localizes with gp-340, an SP-D binding protein and putative SP-D receptor [10 • ]. Sites of extrapulmonary expression have also been described in small mammals. In the rat, SP-D message was identified in RNA extracted from skin and blood vessel [16] , and both protein and message were identified in gastric mucosa [17] and mesentery [13] . Using RT-PCR, SP-D message has also been identified in mouse stomach, heart, and kidney [14] . SP-D (43 kDa, reduced) consists of at least four discrete structural domains: a short, N-terminal domain; a relatively long collagenous domain, a short amphipathic connecting peptide or coiled-coil neck domain, and a C-terminal, Ctype lectin carbohydrate recognition domain (CRD). Each molecule consists of trimeric subunits (3 × 43 kDa), which associate at their N-termini (Fig. 1) . Although most preparations of SP-D contain a predominance of dodecamers (that is, four trimeric subunits), the proportions of various oligomers vary between species. For example, rat lavage and recombinant rat SP-D are almost exclusively assembled as dodecamers (four trimers), whereas recombinant human SP-D is secreted as trimers, dodecamers and higher-order multimers [18] . SP-D isolated from the lavage of some patients with alveolar proteinosis consists predominantly of higher-order multimers, which can contain up to 32 (or more) trimeric subunits (Fig. 1 ). Recent crystallographic and mutagenesis studies suggest that the structural determinants of saccharide binding are similar to those originally described for mannose-binding lectin [19,20,21 • ,22 • ]. At least two bound calcium ions and two intrachain disulfide crosslinks stabilize the required tertiary structure, and Glu321 and Asn323 within the CRD participate in glucose/mannose type recognition. Interactions with at least one glycolipid ligand, phosphatidylinositol (PI), require the participation of the C-terminal end of the protein [23, 24] . A trimeric cluster of CRDs is necessary for high-affinity binding to carbohydrate ligands [21 • ,25]. The crystal structure of human SP-D suggests that the spatial distribution of CRDs within a trimeric subunit permits simultaneous and cooperative interactions with two or three glycoconjugates displayed on the surface of a particulate ligand [21 • ]. Furthermore, solid-phase binding studies have shown that monomeric CRDs have an approximately 10-fold lower binding affinity for multivalent ligands than trimeric CRDs. Crystallographic studies of human SP-D further suggest that the spatial organization of CRDs within a trimer is stabilized by interactions of the C-terminal sequence with the trimeric neck domain [21 • ,26]. Interestingly, the three CRDs show a deviation from threefold asymmetry, suggesting some flexibility of the CRDs in relation to the neck. Thus, the dependence of the binding of PI on the C-terminal sequence could reflect conformational effects, rather than the direct participation of this sequence in ligand interactions. The collagen domain length of SP-D is highly conserved and lacks interruptions in the repeating Gly-X-Y sequence (in which X and Y are different amino acids). As for other collagenous proteins, this domain is enriched in imino acids and contains hydroxyproline. Unlike SP-A, SP-D also contains hydroxylysine. Although the collagen domain of rat, human, bovine, and mouse SP-D lacks cysteine residues, cDNA sequencing has identified a codon for cysteine within the collagen domain of pig SP-D [27 • ]; this suggests the possibility of alternative patterns of chain association and oligomeric assembly for pig SP-D. The first translated exon of SP-D contains a highly conserved and unusually hydrophilic Gly-X-Y sequence that shows little homology with the remainder of the collagen sequence. The functional significance of this region is unknown. However, it has been suggested that this region contributes to oligomer assembly or mediates interactions with cellular receptors. The collagen domain determines the maximal spatial separation of trimeric, C-terminal lectin domains within SP-D molecules, but might also contribute to normal oligomeric assembly and secretion. For example, deletion of the entire collagen domain of rat SP-D results in the secretion of trimers rather than dodecamers [28] . In addition, 2,2-dipyridyl, an inhibitor of prolyl hydroxylation that interferes with the formation of a stable collagen helix, causes the intracellular accumulation of 43 kDa monomers and dimers [29] . In any case, the complete conservation of the number of Gly-X-Y triplets suggests that the spatial separation of trimeric CRDs is critical for normal SP-D function. The N-terminal peptide of the mature protein contains two conserved cysteine residues at positions 15 and 20. These residues participate in interchain disulfide crosslinks that stabilize the trimer, as well as the N-terminal association of four or more trimeric subunits. Stable oligomerization of trimeric subunits permits cooperative or bridging interactions between spatially separated binding sites on the same surface or on different particles. The process of forming interchain disulfide bonds is complex, and appropriate crosslinking of the N-terminal domains might be rate limiting for secretion [30] . Subcellular fractionation studies suggest that interchain bonds form initially between the three chains of a trimeric subunit. Subsequent rearrangements within the rough endoplasmic reticulum might allow the covalent crosslinking of a single chain from one subunit and two crosslinked chains of another, with the associated elimination of free thiol groups. Mutant proteins that contain unpaired N-terminal cysteine residues are not secreted. However, it is unclear whether this results from abnormalities in disulfide bonding itself, or the failure to stabilize the required N-terminal conformation. The collagen domain contains hydroxylysyl-derived glycosides and a single N-linked oligosaccharide. In most species (human, rat, mouse, and cow) the site of N-linked glycosylation is located near the N-terminal end of the collagenous domain. Recently, it was shown that pig SP-D has an additional potential site of N-linked glycosylation within the CRD [27 • ]. Although rat and human lung lavage SP-D seem to be sialylated, as suggested by charge heterogeneity and cleavage with highly purified neuraminidase, preparations of human amniotic fluid and bovine lavage SP-D recovered from amniotic fluid showed predominantly complex type biantennary structures and no sialic acid [31] . A variant form of SP-D (50 kDa) has been identified in lavage from a subset of human lavage samples; this protein shows O-linked glycosylation of threonyl residues within the N-terminal peptide domain [32 • ]. At present, the functional significance of these sugars is not known. The presence of O-linked glycosylation within the N-terminal domain might be predicted to interfere with normal dodecamer assembly. In this regard, the O-glycosylated 50 kDa form of human SP-D is recovered as trimeric subunits or smaller species. As for many glycoproteins, the functional role of the attached carbohydrate is unknown. Mutational analysis has shown that the N-linked sugar on rat SP-D is not required for secretion, for dodecamer formation, or for interactions with a variety of microorganisms [29,33]. Consistent with its designation as a 'mannose-type' C-type lectin, SP-D preferentially binds to simple and complex saccharides containing mannose, glucose, or inositol [34, 35] . SP-D also interacts with specific constituents of pulmonary surfactant including PI [36-38] and glucosylceramide [39] . Binding to glucosylceramide involves interactions of the carbohydrate-binding sequences of the CRD with the glucosyl moiety. However, the interaction of SP-D with PI involves interactions with the lipid, as well as CRD-dependent interactions with the inositol moiety [24, 40] . Microorganisms are surfaced with a diverse and complex array of polysaccharides and glycoconjugates, and most classes of microorganism contain one or more sugars recognized by SP-D. However, the outcome of this interaction depends on the specific organism and can be modified by the conditions of microbial growth. The potential consequences of this interaction include the following: varying degrees of lectin-dependent aggregation (namely, microbial agglutination), enhanced binding of microorganisms or microbial aggregates to their 'receptors' on host cells, phagocyte activation, and opsonic enhancement of phagocytosis and killing, potentially involving one or more cellular receptors for SP-D. Binding to organisms in suspension is often -but not always -accompanied by some degree of aggregation. SP-D binds to purified lipopolysaccharide (LPS) isolated from a variety of Gram-negative organisms [35, 41] . In addition, LPS is the major cell wall component that is labeled on lectin blotting of outer membranes isolated from Escherichia coli [41] . The latter interactions involve the recognition of the core oligosaccharide domain, which contains glucose and heptose [41] . SP-D interacts preferentially with purified LPS molecules characterized by short or absent O-antigens and preferentially agglutinates bacterial strains expressing a predominance of rough (O-antigen-deficient) LPS [41, 44] . Although the core oligosaccharide domain of LPS constitutes the major ligand for SP-D on at least some Gram-negative bacteria, the mechanism of interaction with this group of microorganisms is probably heterogeneous. SP-D binds to some smooth, unencapsulated strains of Gram-negative bacteria by immunofluorescence. The mechanism is uncertain; the quantity or quality of binding differs from that observed for rough strains and does not necessarily result in agglutination. LPS molecules on the surfaces of bacteria show heterogeneity in the extent of maturation, so it is possible that this interaction is mediated by a subpopulation of LPS with deficient O-antigens and that the density of binding sites is too low for high-affinity binding. The recognition of the surface glycoconjugates on Gramnegative bacteria by SP-D depends not only on the expression of lectin-specific residues by a given strain or species, but also on the accessibility of these residues [1, 45] . For example, SP-D binds inefficiently to the core region of LPS of encapsulated Klebsiella, but efficiently agglutinates the corresponding unencapsulated phase variants. Interactions of SP-D with the core oligosaccharides of Gram-negative organisms are also influenced by the number of repeating saccharide units associated with the terminal O-antigen of the LPS [41,44]. Other potential ligands include the O-antigen domain of LPS, certain capsular polysaccharides, and membraneassociated glycoproteins. In this regard, SP-D can bind to di-mannose containing O-antigens expressed by a subset of Klebsiella serotypes (I Ofek, H Sahly and EC Crouch, unpublished data). Although other C-type lectins, specifically SP-A and the mannose receptor, can interact with specific capsular polysaccharides [46], a specific interaction of SP-D with capsular glycoconjugates or exopolysaccharides has not been described. The mechanism of interaction with Gram-positive organisms has not been elucidated. Lipoteichoic acids, which are the major glycolipids associated with the Gram-positive cell wall, do not detectably compete with LPS for binding to SP-D (I Ofek, A Mesika, M Kalina, Y Keisari, D Chang, D McGregor and EC Crouch, manuscript submitted). In preliminary studies we observed that binding was competed only partly with maltose and/or EDTA, raising the possibility that binding might be more complex than for some Gram-negative organisms. . However, similar effects were observed when the neutrophils were preincubated with SP-D, and there was only a slight enhancement of uptake when bacteria were incubated with human SP-D and washed before their addition to neutrophils. Notably, the extent of binding and internalization was dependent on the extent of multimerization, with human SP-D multimers demonstrating the highest potency. Differences in cell type, the extent of SP-D multimerization, or differences in size or organization of bacterial aggregates could account for some of the apparent inconsistencies. Although LPS mediates the binding of SP-D to at least some Gram-negative bacteria, SP-D can also bind to spe- In the latter study the authors suggested that fungal aggregation inhibits phagocytosis. Interestingly, SP-D binding directly inhibited fungal growth and decreased the outgrowth of pseudohyphae, the invasive form of the fungus, in the absence of phagocytic cells [57] . It is possible that these effects are also secondary to agglutination, possibly as result of nutrient deprivation. Purified rat and human SP-D inhibit the infectivity and hemagglutination activity of influenza SP-D can interact with host cells, both directly and indirectly. As indicated above, SP-D can enhance the phagocytosis and killing of certain microorganisms and enhance the oxidant response to microbial binding. However, at present there is only one study that suggests that the enhancement of phagocytosis by SP-D might involve the participation of an opsonic receptor. Furthermore, the enhanced uptake of IAV seems to be mediated by viral aggregation, with enhanced interactions of the virus with its natural receptors on the host cell. In any case, SP-D can interact directly with host cells, and in some cases can influence their behavior. SP-D is chemotactic and haptotactic for neutrophils and certain mononuclear phagocytes [59 • ,67-69] and can elicit directional actin polymerization in alveolar macrophages [69] . In this regard, SP-D is considerably more potent than SP-A. Early studies with natural proteins isolated from silicotic animals reported directed effects on the oxidant metabolism of isolated alveolar macrophages [70] . However, such effects can probably be attributed to endotoxin contamination and/or aggregation. Purified dodecamers do not significantly increase the production of nitric oxide [71] or of proinflammatory cytokines such as tumor necrosis factor-α (Y Kesari, H Wang, A Mesika, E Crouch and I Ofek, unpublished data). Interestingly, purified SP-D has been reported to increase the production of several metalloproteinases in the absence of a significant effect on proinflammatory cytokine production [72] . Despite the ability of SP-D to modulate a variety of cellular functions, little is currently known about potential cellular receptors for this protein. compartments [73] , but it is unclear whether the uptake is receptor dependent and whether SP-D is being internalized in association with specific ligands. There are at least two classes of binding to host cells: CRD-dependent and CRD-independent. Some studies have demonstrated CRD-dependent binding to phagocytes that can be inhibited with EDTA or competing saccharides, both in vitro and in vivo. As indicated above, the ability of SP-D to elicit the chemotaxis of neutrophilic and monocytic cells depends on the lectin activity of SP-D [68] . In addition, Kuan and coworkers reported that extracting formaldehyde-fixed alveolar macrophages with detergents largely eliminates the binding of purified SP-D, suggesting a membrane-associated ligand or glycolipid receptor [73] . Dong and Wright have extended these findings and suggest that PI can contribute to SP-D binding by alveolar macrophages [74] . It is of interest that SP-D can bind to recombinant sCD14 through interactions with N-linked oligosaccharides [51 • ]. Given that the membrane-associated form of CD14 is widely expressed on host cells, it is possible that CD14 can serve as a binding site on macrophages and other cell types. The phagocytic uptake of certain bacteria by neutrophils is also inhibited by calcium chelation or competing sugars [42]; however, this could result from the inhibition of microbial agglutination rather than lectin-dependent interactions with the phagocyte. Wang et al suggested that SP-D can bind to lymphocytic cells by a lectin-dependent mechanism [75 •• ] . In this regard, it is interesting to note that glucosylceramide, a ligand for SP-D in vitro, is one of the most abundant neutral glycolipids expressed by lymphoid cells. Reid and co-workers were the first to present evidence for lectin-independent binding [76] . These and other studies suggested that binding does not involve known C1q or collectin receptors. The only putative receptor protein, gp-340, is a widely expressed member of the scavenger receptor superfamily [77,78 • ]. It binds to the CRD of SP-D in a calcium-dependent manner that does not require the lectin activity of SP-D. Although the protein has been immunolocalized to alveolar macrophage membranes and distributes together with SP-D in many different human tissues [10 • ,77], it has not yet been shown to mediate the binding of SP-D to these cells or to participate in signal transduction events. The cDNAs isolated from lung have not shown a membrane-spanning region [77] , and the protein is abundant as a soluble component in BAL. Given that gp-340 is a highly multimerized protein that contains numerous potential ligand binding domains (Fig. 1b) , it is possible that the protein cooperates with SP-D in the neu-tralization or clearance of certain ligands rather than specifically mediating the interactions of SP-D with host cells. Wright and co-workers have demonstrated the binding of SP-D to isolated type II pneumocytes. The mechanism seems distinct from the binding to macrophages [79 • ]. The binding was dependent on concentration, time, and temperature and required calcium; it was not sensitive to protease treatment or to PI-phospholipase C. Although the internalized SP-D was degraded or recycled to lamellar bodies, SP-D binding did not alter the uptake of surfactant lipids. SP-D has demonstrated comparatively few direct effects on the metabolism of host cells, at least in situations where self-aggregation and endotoxin contamination have been excluded. One possible explanation is that modulation of cellular function requires the prior interaction of SP-D with a ligand. This would have numerous potential physiological advantages, because the presence of 'active' protein might be restricted to sites of microbial or antigenic deposition. The binding of complex, multivalent, particulate antigens to two or more CRDs could markedly alter the conformation of SP-D molecules, with respect to the spatial orientation of the arms in relation to the N-terminal crosslinking domain and/or with respect to the spatial orientation of the CRDs within a given trimeric subunit. Thus, the 'charging' of SP-D with a particulate ligand could lead to local or distant conformational changes that expose 'cryptic' binding sites for cellular receptors. There is some preliminary evidence consistent with the notion that the interaction of SP-D with a ligand alters its capacity to activate host cells. Table 3 and discussed below. SP-D can be isolated in different multimeric forms from proteinosis lavage [32 • ] and are produced by Chinese hamster ovary K1 cells transfected with human SP-D cDNA [18] . As described previously, the effects of SP-D on the neutrophil response to influenza virus are highly dependent on the ability of SP-D to agglutinate the viral particles, and the agglutination activity is directly correlated with the extent of multimerization. Trimers can bind to the virus but have little capacity to modulate neutrophil interactions. By contrast, highly multimerized proteins show greater activity than dodecamers [81] . Given these observations, factors that favor enhanced oligomerization or lead to the accumulation of trimeric subunits promote might influence SP-D function. For example, the liberation of active trimers by a hypothetical microbial protease could lead to the accumulation of molecules that might inhibit the aggregation-dependent activities of SP-D. In contrast, recombinant trimeric CRDs can stimulate chemotaxis [67] and decrease viral infectivity [65 • ]. Although higher-order oligomers of SP-D can self-aggregate and precipitate in the presence of calcium in vitro, the functional consequences are not known. The lectin activity of SP-A is decreased after the nitric oxide-dependent nitration of tyrosine residues [82] , and nitration decreases the ability of SP-A to enhance the adherence of Pneumocystis to alveolar macrophages [83] . However, similar findings have not yet been reported for SP-D. Conditions of mildly acidic pH, as might be found in endocytic compartments, are predicted to disrupt the lectin-dependent activities of SP-D [34]. Proteolytic degradation remains an important possibility. However, SP-D is highly resistant to degradation by a wide variety of neutral proteases in vitro, and degradation products have not yet been shown to accumulate under pathological conditions in vivo. Glucose concentrations at levels encountered in diabetes can interfere with SP-D's ability to interact with specific strains of IAV or other microorganisms in vitro [84 • ]. Many microorganisms release cell wall polysaccharides or glycoconjugates, which might interfere with the binding of collectins to the same or other organisms. In this regard, SP-D recovered from rats after the instillation of LPS into the airway shows decreased lectin activity, which is attributed to occupancy of the CRD with LPS [49 • ]. It seems reasonable to speculate that some organisms might compete with other organisms for binding to SP-D. Such a situation could conceivably predispose to secondary infections. Lastly, the potential inhibitory effects of competing saccharide ligands presents important methodological considerations for experiments using carbohydrate-containing cell culture medium or buffers. Non C-type lectins (such as ficolins) It is difficult to predict the functions of SP-D within the airspace. Other lectins with overlapping specificity are also present. Although the levels of mannose-binding lectin are probably low in the absence of increased vascular permeability, SP-A and the macrophage mannose receptor could conceivably interact with the same ligands in the distal airways and alveoli. Such interactions could lead to antagonistic or cooperative effects. Furthermore, we have little knowledge regarding the microanatomic distribution of these molecules in specific circumstances in vivo. Although most SP-A is probably associated with the insoluble phase of the alveolar lining material, and the macrophage mannose receptor is membrane-associated, the distribution might be altered in the setting of lung injury. Models of SP-D deficiency show no detectable anatomical or physiological abnormalities at birth. However, the animals gradually develop a patchy, subpleural alveolar lipidosis with associated type II cell hypertrophy, the accumulation of enlarged and foamy macrophages, and an apparent expansion of peribronchial lymphoid tissue [85 • ,86 • ]. Interestingly, the mice eventually develop distal-acinar emphysema and areas of subpleural fibrosis, which could reflect a continuing inflammatory reaction associated with abnormal oxidant metabolism and metalloproteinase activity [87 • ]. By contrast, SP-A-deficient mice (-/-) show essentially normal respiratory function and surfactant lipid metabolism [88, 89] but numerous apparent host defense abnormalities [90] . The capacity of SP-D to bind to specific strains of influenza A in vitro is highly correlated with the capacity of the virus to proliferate in mice in vivo [62] . Specifically, strains with more oligosaccharide attachments on the HA are preferentially neutralized by SP-D in vitro and show decreased proliferation in mice. Because the administration of mannan together with the virus increased the replication of IAV in the lung, the involvement of a mannose-type, C-type lectin was implicated. SP-D-sensitive IAV strains also replicate to higher titers in the lungs of diabetic mice than in nondiabetic controls [84 • ]. Replication of the virus is positively correlated with blood glucose level, and decreases in response to insulin treatment. Significantly, blood glucose levels comparable to those measured in the diabetic mice were sufficient to inhibit the interaction of SP-D with these viral strains in vitro. PR-8, a strain that does not interact with SP-D but does interact with SP-A, replicated to the same extent in diabetic and control mice. SP-D levels increase in association with certain infections. For example, SP-D levels, but not the levels of serum mannose-binding lectin, increase markedly after IAV infection [62] . Impressive increases in SP-D have also been observed in murine models of Pneumocystis carinii [91] and P. aeruginosa infection [92] . SP-D-deficient mice have not yet been extensively characterized with respect to host defense function. However, they show decreased viral clearance and enhanced inflammation after challenge with respiratory syncytial virus [93] and IAV (AM Levine, personal communication). In addition, they show increased inflammation, increased oxidant production, and decreased macrophage phagocytosis in response to intratracheally instilled group B streptococcus and Haemophilus influenzae (AM Levine, personal communication). Although the overexpression of wild-type SP-D in type II pneumocytes with the SP-D-deficient mice can prevent the lipidosis and inflammatory changes [94] , the ability of overexpressed wild-type SP-D or exogenous SP-D to ameliorate these abnormalities has not yet been described. The coexisting pulmonary abnormalities also complicate the interpretation of challenge models. For example, macrophage activation might enhance killing and offset any decrease that results more directly from SP-D deficiency. SP-D deficiency modifies the host response to instilled LPS with decreased lung injury and inflammatory cell recruitment [50]. Molecules that can bind to potential antigens and deliver them to macrophages and other antigen-presenting cells might contribute to the development of acquired immunity. In this regard, a few published observations suggest possible roles in the development of humoral and/or cellular immunity in response to microorganisms or complex organic antigens. For example, SP-D can decrease interleukin-2dependent T-lymphocyte proliferation [95 • ]. Interestingly, single-arm mutants were at least as potent as intact dodecamers in mediating this effect. SP-D also binds to oligosaccharides associated with dust mite allergen [96 • ], and can inhibit the binding of specific IgE to these allergens, possibly through direct, CRD-dependent binding to lymphocytes [96 • ]. Thus, alterations in the level of SP-D (or the state of oligomerization) might influence the development of immunological responses and contribute to the pathogenesis of asthma and other hypersensitivity disorders. There are other potential interplays between humoral immunity and collectins with regard to antimicrobial host defense. For example, increased glycosylation of IAV coat proteins, an adaptation that is believed to help the virus to evade antibody-mediated neutralization, is associated with increased reactivity with SP-D and other collectins [62]. Thus, the relative potential importance of antibody and collectin-mediated host defenses might be influenced by subtle variations in the structure of the microbial surface. There is little recent information on the developmental regulation of SP-D expression. In general, SP-D increases rapidly late in gestation [97] [98] [99] [100] . The production of SP-D increases during the culture of fetal lung explants, and expression can be increased with glucocorticoids [98, 100, 101] . The exposure of fetal rats to glucocorticoids in vivo leads to precocious expression with increased numbers of SP-D-expressing cells and increased cellular levels of SP-D message [98, 101, 102] . Although SP-D is produced constitutively within the lung, protein accumulation and gene expression are inducible and increases in SP-D expression have been observed in a number of disease states or models (Tables 4 and 5 ). In general, the synthesis and secretion of SP-D increase in association with lung injury and activation of the respiratory epithelium [1] . For example, levels of SP-D mRNA and SP-D accumulation are increased within 24-72 h after intratracheal instillation of LPS [103 • ], and SP-D expression by alveolar and bronchiolar epithelial cells increases after exposure of rats to 95% O 2 for 12 h [104] . Keratinocyte growth factor (KGF) increases SP-D expression and protein production in association with pneumocyte hyperplasia and after injury caused by bleomycin [105] . In addition, the levels of SP-D can increase markedly in response to the overexpression of certain cytokines, such as interleukin-4, or in response to microbial challenge [91, 92] . Studies of the upstream regulatory region of the SP-D gene have demonstrated increased promoter activity in the presence of glucocorticoids, which is consistent with the findings in vivo and in lung organ culture [106] . However, no functional glucocorticoid response elements have been identified, and the effects of dexamethasone seem to be secondary and involve the effects of other transregulatory molecules. The activity of the human SP-D promoter is dependent on a conserved activator protein-1 (AP-1) element (-109) that binds to members of the fos and jun families of transcriptional factors [107] . In addition, the promoter contains multiple functional binding sites for CCAAT-enhancer-binding protein (C/EBP) transcription factors. Mutagenesis experiments suggest that these are required for basal and stimulated promoter activity, and promoter activity is markedly increased in H441 cells after co-transfection with C/EBPβ cDNA (YC He and E crouch, unpublished data). The importance of the conserved AP-1 element and the presence of multiple binding sites for C/EBP transcription factors is consistent with the observed modulation of SP-D expression in the setting of tissue injury. SP-D promoter activity is not dependent on the binding of thyroid transcription factor 1 (TTF-1) [107] . However, promoter activity is dependent on two interacting forkhead binding sites, upstream and downstream of the AP-1 element; these sites bind to hepatic nuclear factor-3α and apparently other forkhead box proteins in H441 lung adenocarcinoma nuclear extracts [107] . Initial comparison of genomic and cDNA sequence suggested the existence of genetic polymorphisms in the SP-D coding sequence, including one in the N-terminal propeptide domain (Thr11 compared with Met11 in the mature protein) and three additional differences within the collagen domain at positions 102, 160, and 186 [108] . The latter substitutions are conservative to the extent that they are not expected to disrupt the collagen helix. Floros Table 5 Increased SP-D accumulation or expression in animal models Silicosis Rat [118] Hyperoxia Rat [104] Endotoxin (LPS) Rat [103] Challenge with P. aeruginosa Mouse [92] Challenge with IAV Mouse [62] Challenge with Pneumocystis carinii SCID mouse [91] Rat [119] Overexpression of interleukin-4 Mouse [120] SCID, severe combined immunodeficiency. and co-workers have recently confirmed the existence of polymorphisms at positions 11 and 160 of the mature protein [109] . The potential biological significance, if any, is not known. Interestingly, the 50 kDa variant of SP-D showed O-linked glycosylation of Thr11 [32 • ], suggesting that this polymorphism might be associated with altered glycosylation. Interestingly, the 50 kDa variant was recovered as trimeric subunits, raising the possibility that differences in the glycosylation of residue 11, which is immediately N-terminal to Cys15, could influence multimerization and the capacity of SP-D to participate in bridging interactions. There is increasing evidence that SP-D interacts specifically with a wide variety of respiratory pathogens, modulates the leukocyte response to these organisms, and participates in aspects of pulmonary immune and inflammatory regulation (Table 6) . SP-D can influence the activity of phagocytes through CRD-dependent and CRD-independent interactions. At least some of the effects of SP-D result from aggregation with enhanced binding of the agglutinated ligand to their natural 'receptors'. Although the lung is the major site of SP-D expression, it is likely that the protein has more generalized roles in host defense and the acute response to infection and tissue injury. 16 "
3
"Role of endothelin-1 in lung disease"
"Endothelin-1 (ET-1) is a 21 amino acid peptide with diverse biological activity that has been implicated in numerous diseases. ET-1 is a potent mitogen regulator of smooth muscle tone, and inflammatory mediator that may play a key role in diseases of the airways, pulmonary circulation, and inflammatory lung diseases, both acute and chronic. This review will focus on the biology of ET-1 and its role in lung disease."
"from Xenopus laevis [16] . ETA receptors in normal lung are found in greatest abundance on vascular and airway smooth muscle, whereas ETB receptors are most often found on the endothelium. Clearance of ET-1 from the circulation is mediated by the ETB receptor primarily in the lung, but also in the kidney and liver [17] . Activation of both ETA and ETB receptors on smooth muscle cells leads to vasoconstriction whereas ETB receptor activation leads to bronchoconstriction. Activation of ETB receptors located on endothelial cells leads to vasodilation by increasing nitric oxide (NO) production. The mitogenic and inflammatory modulator functions of ET-1 are primarily mediated by ETA receptor activity. Binding of the ligand to its receptor results in coupling of cell-specific G proteins that activate or inhibit adenylate cyclase, stimulate phosphatidyl-inositol-specific phosholipase, open voltage gated calcium and potassium channels, and so on. The varied effects of ET-1 receptor activation thus depend on the G protein and signal transduction pathways active in the cell of interest [18] . A growing number of receptor antagonists exist with variable selectivity for one or both receptor subtypes. Regulation of ET-1 is at the level of transcription, with stimuli including shear stress, hypoxia, cytokines (IL-2, IL-1β, tumor necrosis factor α, IFN-β, etc), lipopolysaccharides, and many growth factors (transforming growth factor-β, platelet-derived growth factor, epidermal growth factor, etc) inducing transcription of ET-1 mRNA and secretion of protein [18] . ET-1 acting in an autocrine fashion may also increase ET-1 expression [19] . ET-1 expression is decreased by NO [20] . Some stimuli may additionally enhance preproET-1 mRNA stability, leading to increased and sustained ET-1 expression. The number of ETA and ETB receptors is also cell specific and regulated by a variety of growth factors [18] . Because ET-1 and receptor expression is influenced by many diverse physical and biochemical mechanisms, the role of ET-1 in pathologic states has been difficult to define, and these are addressed in subsequent parts of this article. In the airway, ET-1 is localized primarily to the bronchial smooth muscle with low expression in the epithelium. Cellular subsets of the epithelium that secrete ET-1 include mucous cells, serous cells, and Clara cells [21] . ET binding sites are found on bronchial smooth muscle, alveolar septae, endothelial cells, and parasympathetic ganglia [22, 23] . ET-1 expression in the airways, as previously noted, is regulated by inflammatory mediators. Eosinophilic airway inflammation, as may be seen in severe asthma, is associated with increased ET-1 levels in the lung [24] . ET-1 secretion may also act in an autocrine or paracrine fashion, via the ETA receptor, leading to increased transepithelial potential difference and ciliary beat frequency, and to exerting mitogenic effects on airway epithelium and smooth muscle cells [25] [26] [27] [28] . All three endothelins cause bronchoconstriction in intact airways, with ET-1 being the most potent. Denuded bronchi constrict equally to all three endothelins, suggesting considerable modulation of ET-1 effects by the epithelium [29] . The vast majority of ET-1 binding sites on bronchial smooth muscle are ETB receptors, and bronchoconstriction in human bronchi is not inhibited by ETA antagonists but augmented by ETB receptor agonists [30] [31] [32] . Since cultured airway epithelium secretes equal amounts of ET-1 and ET-3, which have equivalent affinity for the ETB receptor, bronchoconstriction could be mediated by both endothelins [33] . While ET-1 stimulates release of multiple cytokines important in airway inflammation, it does not enhance secretion of histamine or leukotrienes. ET-1 does increase prostaglandin release [32] . Inhibition of cyclo-oxygenase, however, has no effect on bronchoconstriction suggesting that, despite the release of multiple mediators, ET-1 mediated bronchoconstriction is a direct effect of activation of the ETB receptor [32] . ETA mediated bronchoconstriction may also be important following ETB receptor desensitization or denudation of the airway epithelium, as may occur during airway inflammation and during the late, sustained airway response to inhaled antigens [31, 34, 35] . Interestingly, heterozygous ET-1 knockout mice, with a 50% reduction in ET-1 peptide, have airway hyperresponsiveness but not remodeling, suggesting the decrease in ET-1 modulates bronchoconstriction activity by a functional mechanism, possibly by decreasing basal NO production [36, 37] . Asthma is also an inflammatory airway disease characterized by bronchoconstriction and hyperreactivity with influx of inflammatory cells, mucus production, edema, and airway thickening. ET-1 may have important roles in each of these processes. While ET-1 causes immediate bronchoconstriction [38] , it also increases bronchial reactivity to inhaled antigens [35] as well as influx of inflammatory cells [39, 40] , increased cytokine production [40] , airway edema [41] , and airway remodeling [28, 42, 43] . Airway inflammation also leads to increased ET-1 synthesis, possibly perpetuating the inflammation and bronchoconstriction [44] . ET-1 release from cultured peripheral mononuclear and bronchial epithelial cells from asthmatics is also increased [45, 46] . Inhibition of ETA or combined ETA and ETB receptors additionally leads to decreased airway inflammation in antigen-challenged animals, suggesting that the proinflammatory effects of ET-1 in the airway are mediated by ETA receptors [39, 47] . Children with asthma have increased circulating levels of ET-1 [48] . Adult asthmatics have normal levels between attacks but, during acute attacks, have elevated serum ET-1 levels that correlate inversely with airflow measurements and decrease with treatment [49] . Bronchoalveolar lavage (BAL) ET-1 in asthmatics is similarly increased to concentrations that cause bronchoconstriction and inversely correlates with forced expiratory volume in 1 s (FEV 1 ) [29, 50, 51] . As in cultured epithelial cells, ET-1 and ET-3 are found in equal amounts in BAL fluid from asthmatics [33, 52] . There is also a relative increase in ETB versus ETA receptor expression in asthmatic patients, which may contribute to increased bronchoconstriction [53] . Not all asthmatics, however, have increased ET-1 as patients with nocturnal asthma have decreased BAL ET-1 levels [54] . Treatment of acute asthma exacerbations with steroids, beta-adrenergic agonists or phosphodiesterase inhibitors resulted in decreased BAL ET-1 [52, 55] . Immunostaining and in situ hybridization for ET-1 in biopsy specimens from asthmatics have shown an increase in ET-1 in the bronchial epithelium that correlates with asthma symptoms [46, 56] . Cigarette smoking leads to increased circulating ET-1 [57] but patients with chronic obstructive pulmonary disease, in the absence of pulmonary hypertension and hypoxemia, do not have increased plasma ET-1 [58] [59] [60] . Increases in urinary ET-1 instead correlate with decreases in oxygenation, possibly through hypoxic release of ET-1 from the kidney [61, 62] . Smokers also have impaired ET-1 mediated vasodilation that correlates with bronchial hyperresponsiveness and may contribute to pulmonary hypertension [63, 64] . ET-1 has been implicated in the pathogenesis of bronchiectasis by its ability to promote neutrophil chemotaxis, adherence, and activation [65] [66] [67] [68] [69] . Sputum ET-1 levels are increased in patients with cystic fibrosis [59] , and sputum ET-1 correlated with Pseduomonas infection in noncystic fibrosis related bronchiectasis [70] . ET-1 has also been implicated in the pathogenesis of bronchiolitis obliterans (BO), which is characterized by injury to small conducting airways resulting in formation of proliferative, collagen rich tissue obliterating airway architecture. BO is the leading cause of late mortality from lung transplantation, and ET-1 is increased in lung allografts [71] . The pro-inflammatory and mitogenic properties of ET-1 in the airways has led to speculation that ET-1 may be involved in formation of the lesion [28] . This is further supported by the increase in BAL ET-1 in lung allografts [72, 73] . The in vivo gene transfer of ET-1 to the airway epithelium using the hemagglutinating virus of Japan in rats recently resulted in pathologic changes in the distal airways identical to those seen in human BO specimens [74] . These changes were not due to nonspecific effects of the hemagglutinating virus of Japan itself, but could be attributed to the presence of the ET-1 gene, which was localized to the airway epithelium, hyperplastic lesions, and alveolar cells. Pulmonary hypertension is a rare and progressive disease characterized by increases in normally low pulmonary vascular tone, pulmonary vascular remodeling, and progressive right heart failure. ET-1 has been implicated as a mediator in the changes seen in pulmonary hypertension. In the pulmonary vasculature, ET-1 is found primarily in endothelial cells and to a lesser extent in the vascular smooth muscle cells. The endothelium secretes ET-1 primarily to the basolateral surface of the cell. ET-1 secretion may be increased by a variety of stimuli including cytokines, catecholamines, and physical forces such as shear stress, and decreased by NO, prostaglandins, and oxidant stress [20, [75] [76] [77] [78] . Hypoxia has been reported to increase, have no effect, or decrease ET-1 release from endothelial cells [79] [80] [81] [82] [83] . Activation of the receptors for ET-1 in the pulmonary vasculature leads to both vasodilation and vasoconstriction, and depends on both cell type and receptor. In the whole lung, ETA receptors are the most abundant and are localized to the medial layer of the arteries, decreasing in intensity in the peripheral circulation [84, 85] . ETB receptors are also found in the media of the pulmonary vessels, increasing in intensity in the distal circulation, while intimal ETB receptors are localized in the larger elastic arteries [85] . This distribution of receptors has important implications in understanding ET-1 regulation of vascular tone. Vascular ET-1 receptors may be increased by several factors including angiotensin and hypoxia [80, [85] [86] [87] . ET-1 can act as both a vasodilator and vasoconstrictor in the pulmonary circulation. Generation of NO or opening of ATP-sensitive potassium channels leading to hyperpolarization results in vasodilation mediated by ETB receptors on pulmonary endothelium [88, 89] . In hypertensive, chronically hypoxic lungs with increased ETB receptor expression, augmented vasodilation is due to increased ETB mediated NO release that is inhibited by hypoxic ventilation, while inhibition of NO synthesis leads to increased ET-1 mediated vasoconstriction [85, [90] [91] [92] . Both ETA and ETB receptors, conversely, acting on vascular smooth muscle, mediate ET-1 induced vasoconstriction. In the normal lung, ET-1 causes vasoconstriction primarily by activation of the ETA receptors in the large, conducting vessels of the lung [93, 94] . In the smaller, resistance vessels of the lung, ETB receptors in the media predominate and are responsible for the ET-1 induced vasoconstriction [93] . Interestingly, preconstriction of the pulmonary circulation resulted in a shift from primarily ETA mediated to ETB mediated vasoconstriction [94] . The overall effect of ET-1 on vascular tone depends on both the dose and on the pre-existing tone in the lung. ET-1 administration during acute hypoxic vasoconstriction will result in transient pulmonary vasodilation [89] . This effect is dose dependent, with lower doses leading to vasodilation while higher or repetitive doses cause vasoconstriction following an initial, brief vasodilation [89] . The role of ET-1 in the acute hypoxic vasoconstriction in the lung is not certain. ETA receptor antagonism attenuates hypoxic pulmonary vasoconstriction in several species [95] , and ET-1 may be implicated in the mechanism of acute hypoxic response by inhibition of K-ATP channels [96] . Several lines of evidence have suggested the importance of ET-1 in chronic hypoxic pulmonary hypertension. ET-1 is increased in plasma and lungs of rats following exposure to hypoxia [80, 97] . Treatment with either ETA or combined ETA and ETB receptor antagonists additionally attenuates the development of hypoxic pulmonary hypertension [98, 99] . ET-1 has also been implicated in the vascular remodeling associated with chronic hypoxia through its mitogenic effects on vascular smooth muscle cells [98, 100] . ET-1 has also been implicated in other animal models of pulmonary hypertension. ET-1 is increased in fawn hooded rats that develop severe pulmonary hypertension when raised under conditions of mild hypoxia and in monocrotaline treated rats [101, 102] . The increase in ET-1 in both of these forms of pulmonary hypertension may be contributing to increases in vascular tone as well as in vascular remodeling [103] [104] [105] [106] 114] . Interestingly, transgenic mice overexpressing the human preproET-1 gene, with modestly increased lung ET-1 levels (35-50%), do not develop pulmonary hypertension under normoxic conditions or an exaggerated response to chronic hypoxia [107] . Human pulmonary hypertension is classified as primary, or unexplained, or secondary to other cardiopulmonary diseases or connective tissue diseases (ie scleroderma). Hallmarks of the disease include progressive increases in pulmonary vascular resistance and pulmonary vascular remodeling, with thickening of the medial layer small pulmonary arterioles and formation of the complex plexiform lesion [108] . Circulating ET-1 is increased in humans with pulmonary hypertension, either primary or due to other cardiopulmonary disease [109] . Levels are highest in patients with primary pulmonary hypertension. Since the lung is the major source for clearance of ET-1 from the circulation, increased arterio-venous ratios as seen in primary pulmonary hypertension suggest either decreased clearance or increased production in the lung [17, 109] . ET-1 is also increased in lungs of patients with pulmonary hypertension, with the greatest increase seen in the small resistance arteries and the plexiform lesions [110] , and may correlate with pulmonary vascular resistance [111] . Interestingly, treatment with continuous infusion of prostacyclin resulted in clinical improvement and a decrease in the arterio-venous ratio of ET-1 [112] , possibly by decreasing ET-1 synthesis from endothelial cells [76] . Studies using ET-1 receptor antagonists in the treatment of primary pulmonary hypertension are underway and may offer hope to patients with this disease by inhibiting this pluripotent peptide's effects on vascular tone and remodeling. Several lines of evidence suggest the importance of ET-1 in lung allograft survival and rejection. The peptide has been implicated as an important factor in ischemia-reperfusion injury at the time of transplant as well as in acute and chronic rejection of the allograft. Circulating ET-1 is increased in humans undergoing lung transplant immediately following perfusion of the allograft. Plasma ET-1 increased threefold within minutes, remained high for 12 hours following transplantation, and declined to near normal levels within 24 hours [113] . This increase in ET-1 correlated with the increase in pulmonary vascular resistance occurring about 6 hours post-transplantation, suggesting that the release of ET-1 in the circulation may have mediated this event. ET-1 in BAL fluid from recipients of lung allografts is similarly increased several fold and remains elevated up to 2 years post-transplant [72, 73] . In recipients of single lung transplants, ET-1 was increased 10-fold in BAL fluid from the transplanted lung compared with the native lung, suggesting that the increase in ET-1 was due to the graft and not the underlying disease requiring transplant [72] . ET-1 in BAL fluid did not, however, correlate with episodes of infection or rejection. The cellular source of ET-1 in lung allografts is unknown. The expression of ET-1 in nontransplanted human lungs is low and found primarily in the vascular endothelium [114] . Transbronchial biopsy specimens obtained either for surveillance or for clinical suspicion of infection or rejection following transplantation revealed the presence of ET-1 in the airway epithelium and in alveolar macrophages [115] . ET-1 was occasionally seen in lymphocytes but not in the endothelium or pneumocytes. ET-1 localization was no different in surveillance specimens compared with infected or rejecting lungs, or changed over time from transplantation. This study suggests that the source of the increased BAL ET-1 in transplanted lungs is due to the increased number of alveolar inflammatory cells and de novo expression in the airway epithelium. The biologic importance of the ET-1 from inflammatory cells is supported by the observation that peripheral mononuclear cells from dogs with mild to moderate lung allograft rejection cause vasoconstriction in pulmonary arterial rings, which is attenuated by the ETA blocker BQ123 [116] . Analysis of ET-1 binding activity in failed transplanted human lungs suggested that ET-1 binding activity was not different compared with normal lung in the lung parenchyma, bronchial smooth muscle, or perivascular infiltrates. ET-1 binding was, however, decreased in small muscular arteries (pulmonary arteries and bronchial arteries) in the failed transplants, suggesting a role for ET-1 in impaired vasoregulation of transplanted lungs [117] . Ischemia-reperfusion injury is the leading cause of early post-operative graft failure and death. In its severest manifestation, increased pulmonary vascular resistance, hypoxia, and pulmonary edema lead to cor pulmonale and death [118] . ET-1 has been implicated as a mediator of these events. The increase in pulmonary vascular resistance observed in human recipients of lung allografts follows an increase in circulating ET-1 and falls with decreases in circulating ET-1 [113] . A similar pattern is seen in dogs subjected to allotransplantation [119] . Conscious dogs with left pulmonary allografts demonstrate an increase in both resting pulmonary perfusion pressure and acute pulmonary vasoconstrictor response to hypoxia [120] . Administration of ETA selective or combined ETA and ETB receptor blockers did not change the resting tone. ETB receptor mediated hypoxic pulmonary vasoconstriction appeared, however, to be increased in allograft recipients. In another study, administration of a mixed ETA and ETB receptor antagonist (SB209670) to dogs before reperfusion of the allograft resulted in a marked increase in oxygenation, decreases in pulmonary arterial pressures and improved survival compared with control animals [121] . In a model of ischemia reperfusion, inhibitors of ECE additionally attenuated the increase in circulating ET-1 and the severity of lung injury [122] . ET-1 receptor antagonists did not, however, completely eliminate the ischemia-reperfusion injury, suggesting that changes in other vasoactive mediators, such as an increase in thromboxane, a decrease in prostaglandins, or a decrease in NO, may also contribute to the increased pulmonary vascular resistance. Administration of NO donor (FK409) to both donor and recipient dogs before lung transplantation reduced pulmonary arterial pressure, lung edema, and inflammation, and improved survival. This suggests that reductions in NO following transplantation may be partly responsible for early graft failure [123] . Treatment with NO donor was also associated with a decrease in plasma ET-1 levels. Acute rejection is manifested by diffuse infiltrates, hypoxia, and airflow limitation, and may lead to respiratory insufficiency and death. BAL ET-1 was increased in dogs during episodes of acute rejection that decreased with immunosuppressive treatment [124] . Acute episodes of rejection in humans, however, are not associated with further increases in BAL ET-1 [72] . Chronic rejection of allografts, manifested as BO, is the major cause of morbidity and mortality in long-term lung transplant survivors [71] . The etiology of BO following transplant is unclear but may be related to repeated episodes of acute rejection, chronic low-grade rejection, or organizing pneumonia [125] . As discussed earlier, a chronic increase in ET-1, as seen in lung allografts, may contribute to bronchospasm and proliferative bronchiolitis obliterans due to the bronchoconstrictor and smooth muscle mitogenic effects of ET-1 [28, 126] . This is further supported by the increase of BAL ET-1 in the transplanted lung, which is susceptible to BO, but not the native lung in recipients of single lung transplants [72] . The mitogenic effects of ET-1 may play a role in the development of pulmonary malignancy as well as metastasis to the lung. Many human tumor cell lines, including prostate, breast, gastric, ovary, colon, etc, produce ET-1. The importance of the ET-1 may lie in its mitogenic effects on tumor growth and survival. This has been suggested by blockade of ETA receptors resulting in a decrease in mitogenic effects of ET-1 in a prostate cancer and colorectal cell lines [127, 128] . ET-1 receptors in tumor cells may also be altered with increases in the ETA receptor and downregulation of ETB receptors [129] . Other tumors may have an increase in ETB receptors, however, and blockade of ETB results in a decrease in tumor growth [130, 131] . Tumor cells may, as a result of this altered balance, lose the ability to respond to regulatory signals from their environment. ET-1 may additionally protect against Fas-ligand mediated apoptosis [132] . ET-1 has been detected using immunohistochemistry and in situ hybridization in pulmonary adenocarcinomas and squamous cell tumors and, to a lesser extent, small cell and carcinoid tumors [133] . In situ hybridization also demonstrated a similar pattern of ET-1 mRNA expression in non-neuroendocrine tumors. ET-1 receptors have also been found in a variety of pulmonary tumor cell lines. ETA receptors were found in small cell tumors, adenocarcinomas and large cell tumors, while ETB receptors were expressed primarily in adenocarcinomas and small cell tumors [134] . ECE, which converts big ET-1 to ET-1, the committed step in ET-1 biosynthesis, was also found in human lung tumors but not in adjacent normal lung [135] . These findings, combined with the presence of ET-1 in lung tumors, suggest a possible autocrine loop that sustains and supports the growth of lung tumors. A recent study, however, suggested that, while ETA and ECE-1 were detectable in lung tumors, these genes were downregulated compared with normal bronchial epithelial cell lines [136] . It was proposed that the role of ET-1 in lung tumors is not that of an autocrine factor, but that of a paracrine growth factor to the stroma and vasculature surrounding the tumor allowing angiogenesis. Tumor angiogenesis is necessary for continued growth of the tumor beyond the limits of oxygen diffusion. The growth of vessels into the tumor is also important to metastatic potential of the tumor. ET-1 may play an important role in angiogenesis and tumor growth and survival Available online http://respiratory-research.com/content/2/2/090 commentary review reports primary research through induction of vascular endothelial growth factor expression and sprouting of new vessels into the tumor and surrounding tissue [137, 138] . ET-1 binding activity was found in blood vessels and vascular stroma surrounding lung tumors at the time of resection, most markedly surrounding squamous cell tumors [139] . ET-1 production may be further augmented by the hypoxic environment found within large solid tumors [140] . Since metastasis is dependent on neo-vascularization, ET-1 may also be an important mediator of this phenomenon. ET-1 receptor antagonists may have a useful role in the treatment of neoplastic disease by inhibiting growth as well as metastatic potential of human tumors. Experimental lung injury of many different types results in increased circulating ET-1, BAL ET-1, and lung tissue ET-1 [18] . ET-1 levels in humans are also increased in sepsis, burns, disseminated intravascular coagulation, acute lung injury, and acute respiratory distress syndrome (ARDS) [141] [142] [143] [144] [145] [146] [147] . ET-1 increases also correlate with a poorer outcome with multiple organ failure, increased pulmonary arterial pressure, increased airway pressure and decreased PiO 2 /FiO 2 , while clinical improvement correlates with decreased ET-1 levels [144, 145, 147] . The arterio-venous ratio for ET-1 is increased in patients with ARDS but it is not clear whether this is due to increased secretion of ET-1 in the lungs or decreased clearance [142, 144] . In patients who succumbed to ARDS, there was also a marked increase in tissue ET-1 immunostaining in vascular endothelium, alveolar macrophages, smooth muscle, and airway epithelium compared with lungs of patients who died without ARDS. Interestingly, these same patients also had a decrease in immunostaining for both endothelial nitric oxide synthase and inducible nitric oxide synthase in the lung [148] . ARDS is also characterized by the presence of inflammatory cells in the lung. Since ET-1 may act as an immune modulator, an increase in ET-1 may contribute to lung injury by inducing expression of cytokines including tumor necrosis factor and IL-6 and IL-8 [149] . These cytokines may in turn stimulate the production of many inflammatory mediators, leading to lung injury. ET-1 additionally activated neutrophils, and increased neutrophil migration and trapping in the lung [65] [66] [67] [68] [69] . Another hallmark of ARDS is disruption and dysfunction of the pulmonary vascular endothelium leading to accumulation of lung water. The role of endothelin in formation of pulmonary edema is uncertain. Infusion of ET-1 raises pulmonary vascular pressure, but it is uncertain whether ET-1 by itself increased pulmonary protein or fluid transport in the lung [150] [151] [152] . ET-1 may rather be acting synergistically with other mediators to lead to pulmonary edema [153, 154] . Pulmonary fibrosis is the final outcome for a variety of injurious processes involving the lung parenchyma. The final common pathway in response to injury to the alveolar wall involves recruitment of inflammatory cells, release of inflammatory mediators, and resolution. The reparative phase occasionally becomes disordered, resulting in progressive fibrosis. ET-1 in the lung may be important in the initial events in lung injury by activating neutrophils to aggregate and release elastase and oxygen radicals, increasing neutrophil adherence, activating mast cells, and inducing cytokine production from monocytes [65] [66] [67] [68] [69] 149, 155] . Among the many cytokines induced by ET-1 that are important in mediating pulmonary fibrosis are transforming growth factor-β and tumor necrosis factor α [156, 157] . ET-1 is also profibrotic by stimulating fibroblast replication, migration, contraction, and collagen synthesis and secretion while decreasing collagen degradation [158] [159] [160] [161] [162] . ET-1 additionally enhances the conversion of fibroblasts into contractile myelofibroblasts [43, 163] . ET-1 also increases fibronectin production by bronchial epithelial cells [164] . Finally, ET-1 has mitogenic effects on vascular and airway smooth muscle [126, 28] . ET-1 may thus play an important role in the initial injury and eventual fibrotic reparative process of many inflammatory events in the lung. Several lines of evidence regarding the importance of ET-1 in pulmonary fibrosis are available. Plasma and BAL ET-1 levels are increased in idiopathic pulmonary fibrosis [50, 165] . Lung biopsies from patients with idiopathic pulmonary fibrosis have additionally increased ET-1 immunostaining in airway epithelial cells and type II pneumocytes, which correlates with disease activity [166] . Scleroderma is commonly associated with pulmonary hypertension and pulmonary fibrosis. Plasma and BAL ET-1 is increased in these patients [160, 167, 168] , but it is unclear whether the presence of either pulmonary hypertension or pulmonary fibrosis increases these levels further [167] . BAL fluid from patients with scleroderma increased proliferation of cultured lung fibroblasts, which was inhibited by ETA receptor antagonist. This suggests that the ET-1 in the airspace may be contributing significantly to the fibrotic response [160] . An increase in ET-1 binding has also been reported in lung tissue from patients with scleroderma associated pulmonary fibrosis [169] . Pulmonary inflammatory cells also appear to be primed for ET-1 production because cultured alveolar macrophages from patients with scleroderma and lung involvement secrete increased amounts of ET-1 in response to stimulation with lipopolysaccharide [170] . These observations collectively suggest that augmented ET-1 release may contribute to and perpetuate the inflammatory process. Bleomycin-induced pulmonary fibrosis in animals is associated with increased ET-1 expression in alveolar macrophages and epithelium [171] . The increase in ET-1 proceeds the development of pulmonary fibrosis. The use of ET-1 receptor antagonists has produced mixed results in limiting the development of bleomycin-induced fibrosis. A decrease in fibroblast replication and secretion of extracellular matrix proteins in vitro but not a decrease in lung collagen content in vivo has been shown using ETA or combined ETA and ETB receptor antagonists after bleomycin [172] . Another group did, however, observe a decrease in fibrotic area in lungs of rats following bleomycin that were treated with a mixed ETA and ETB receptor antagonist [173] . While ET-1 seems to correlate with pulmonary fibrosis, it remains uncertain whether the increase in ET-1 is a cause or consequence of the lung disease. Pulmonary fibrosis was recently reported in mice that constitutively overexpress human ET-1 [107] . These mice were known to develop progressive nephrosclerosis in the absence of systemic hypertension [174] . The transgene was localized throughout the lung, with the strongest expression in the bronchial wall. In the lung, the mice developed age-dependent accumulation of collagen and accumulation of CD4+ lymphocytes in the perivascular space. This observation suggests that an increase in lung ET-1 alone may play a causative role in the development of pulmonary fibrosis [107, 175] . Since its discovery 12 years ago, much evidence has accumulated regarding the biologic activity and potential role of ET-1 in a variety of diseases of the respiratory track. As compelling as much of this evidence is, the causal relationship between ET-1 activity and disease is not complete. The increasing use of ECE and endothelin receptor antagonists in experimental and human respiratory disorders will help to clarify the role of this pluripotent peptide in health and disease."
4
"Gene expression in epithelial cells in response to pneumovirus infection"
"Respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM) are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner."
"RSV and PVM are viruses of the family Paramyxoviridae, subfamily pneumovirus; they are enveloped, singlestranded, nonsegmented RNA viruses that can cause intense viral bronchiolitis in humans and mice, respectively. In its most severe form, the lower respiratory tract infection caused by pneumoviruses is associated with the development of peribronchiolar infiltrates that are accompanied by submucosal edema and bronchorrhea, and ultimately leads to bronchiolar obstruction and compromised oxygen transfer. As the infection is confined to the respiratory epithelium, the responses of these cells are clearly of primary importance in determining the nature and extent of the resulting inflammatory process. Most of our understanding of responses to pneumovirus infection has emerged from studies of RSV infection of human epithelial target cells in vitro; a list of genes and/or gene products produced by epithelial cells in response to RSV infection in vitro is provided in Table 1 . At the cellular level, epithelial cells initially respond to RSV infection by reducing their ciliary beat frequency. Production and release of chemoattractant cytokines (chemokines) can be observed as early as 12 h after infection, leading to the recruitment of specific leukocyte subsets to the lung tissue. RSV-infected epithelial cells become resistant to tumor necrosis factor (TNF)-α-induced apoptosis, but later fuse to form giant-cell syncytia and die by cellular necrosis. We review the molecular bases (to the extent that they are understood) of these specific responses, and discuss several novel strategies that may permit us to study the responses to RSV and PVM infection in a more coherent and systematic manner. Tristram et al [1] observed that explanted respiratory epithelial cells slow their ciliary beat frequency almost immediately after exposure to RSV, with complete ciliostasis seen as early as 6 h after the initial infection. The molecular basis of ciliostasis remains completely unknown. The chemokines and cytokines with production and release that has been associated with RSV infection of human epithelial cells are listed in Table 1 . Much of this work was also recently reviewed elsewhere [2, 3] . We focus here on the three chemokines whose molecular mechanisms and physiologic implications are best understood. The earliest reports on this subject described production of the neutrophil chemoattractant IL-8 from tissue culture supernatants from RSV-infected cells [4] [5] [6] and in nasal secretions from patients with viral rhinitis [7] . IL-8 has since been detected in lower airway secretions from patients with severe RSV bronchiolitis [8] , and the neutrophil influx observed in response to this infection is probably due, at least in part, to the activity of this chemokine. At the cellular level IL-8 production can be observed in response to inactivated RSV virions, whereas IL-8 production in response to active infection was inhibited by ribavarin, amiloride, and antioxidants [9, 10] . Several groups have demonstrated activation of the transcription factor nuclear factor-κB (NF-κB) in response to RSV infection, and NF-κB is recognized for its central role in eliciting the production of IL-8 [9, 11, 12] . The transcription factor NF-IL-6 is also produced in response to RSV infection [13] , and participates in a co-operative manner with NF-κB in the regulation of IL-8 gene expression [11] , although later studies suggest that activator protein-1 may function preferentially in this role [14] . Interestingly, the NF-κB regulator IκBα, which functions by inhibiting NF-κB activation in response to TNF-α, was produced with different kinetics and does not promote a reversal of NF-κB activation in response to RSV infection as it does in response to TNF-α [15] . Most recently, Casola et al [16] demonstrated that the IL-8 promoter contains independent response elements, with nucleotides -162 to -132 representing a unique RSV response element that is distinct from elements necessary for IL-8 production in response to TNF-α. This concept of a stimulus-specific response will probably make an important contribution toward our understanding of how pneumoviruses promote transcription of unique and specific sets of independent gene products. The pleiotropic chemokine regulated upon activation, normal T-cell expressed and secreted (RANTES) has also been detected in supernatants from RSV-infected epithelial cells in culture [17, 18] , as well as in upper and lower airway secretions from patients infected with this virus [7, 8] . RANTES acts as a chemoattractant for eosinophils and monocytes in vitro, although its role in vivo is somewhat less clear. Similar to IL-8, RANTES can be produced in vitro in response to inactivated virions [8] , and involves NF-κB activation, binding, and nuclear translocation [19] . However, Koga et al [20] demonstrated that stabilization of RANTES mRNA, a response to RSV infection mediated in part by nucleotides 11-389 of the RANTES gene, is probably the primary mechanism underlying increased production and secretion of RANTES protein. Further studies will determine whether a similar mechanism is also in place for IL-8 and other RSV-mediated responses. Several groups have recently shown that macrophage inflammatory protein (MIP)-1α is released from RSVinfected cells in culture [7, 21] ; MIP-1α was also detected in upper and lower airway secretions from RSV-infected patients [7, 8] . Interestingly, of the three aforementioned chemokines, MIP-1α is the one that is most closely correlated with the presence of eosinophil degranulation products; this, together with data from our PVM model of pneumovirus infection [22] , has suggested to us that MIP-1α plays a pivotal role in eosinophil recruitment in response to primary pneumovirus infection. Interestingly, production of MIP-1α in cell culture requires active viral replication [8] , which suggests that this response may proceed by a mechanism that is completely distinct from that which elicits production of RANTES and IL-8. However, no reports to date have addressed the molecular mechanism that underlies the RSV-mediated MIP-1α response. A list of cell-surface molecules that have been reported as expressed in response to RSV infection is shown in Table 1 . We focus here on the expression of intercellular adhesion molecule (ICAM)-1 (CD54) and the leukocyte integrin CD18. Increased expression of this cell-surface adhesion protein was observed in both respiratory epithelial cell lines [23, 24] and in human nasal epithelial cells [25] in response to infection with RSV in vitro. Chini et al [26] demonstrated that the expression of ICAM-1 mRNA, similar to IL-8 and RANTES, was dependent on an intact NF-κB site in the gene promoter, and demonstrated a role for the consensus binding site for the factor CCAAT/ enhancer-binding protein. Stark et al [27] demonstrated that ICAM-1 and CD18 expressed in response to RSV serve to enhance neutrophil and eosinophil binding to epithelial cells. CD18 is a polypeptide of the integrin family that functions in mediating cell-cell interactions. Several groups have observed expression of CD18 on epithelial cells in response to RSV infection [27, 28] , with CD18 shown to enhance the degranulation of eosinophils in this specific setting [28] . Of particular interest are the recent findings relating expression of CD18 (along with CD14) to earlier literature on bacterial superinfections in the setting of viral infections. Earlier studies [29, 30] reported enhanced binding of bacteria to respiratory epithelial cells that were infected with RSV, findings that had clinical implications relating to acute bacterial otitis media in infants. Two more recent studies addressed the question of binding sites. Saadi et al [31] determined that two strains of the pathogen Bordetella pertussis bound more efficiently to RSV-infected cells, and that the binding was reduced upon pretreatment of the cells with anti-CD14 or anti-CD18 antibodies. Similarly, Raza et al [32] reported that both CD14 and CD18 on RSV-infected epithelial cells contributed to the binding of nonpilate Neisseria meningitidis. In vivo testing is required before the clinical significance of these intriguing findings can be appreciated. RSV-infected epithelial cells in culture do not show features that are suggestive of apoptosis (ie no evidence of membrane blebbing, fragmentation of chromosomal DNA, or characteristic changes in nuclear morphology). Takeuchi et al [33] showed that, although RSV-infected epithelial cells express a number of apoptosis-associated genes, including interferon regulatory factor-1, IL-1β-converting enzyme and caspase 3, they do not undergo formal apoptosis. As part of our attempts to understand mucosal responses in a more systematic manner (see below), we discovered that RSV-infected epithelial cells express the recently described antiapoptosis gene IEX-1L [34] . In our studies, we found that expression of IEX-1L is a response to active virus; no gene expression was observed in response to irradiated, replication-incompetent virus. Moreover, expression of IEX-1L is not observed in response to adenoviral infection, suggesting that expression of this gene is not a universal response to cellular perturbation, or indeed to all viral infections. Functionally, we also demonstrated that RSV infection protects epithelial cells from TNF-α-induced apoptosis, an effect that is temporally associated with the expression of IEX-1L. Apoptosis is generally considered to be a highly efficient self-defense mechanism employed by host target cells, because it permits the infected host to dispose of viral proteins and nucleic acids on a single-cell basis without inducing an inflammatory response. It is thus not surprising that many viruses have evolved strategies to circumvent this response. Of interest, Krilov et al [35] demonstrated that monocytes and cord blood mononuclear cells are similarly protected from apoptosis when infected with RSV. Although virus-induced protection from apoptosis appears advantageous to the virus alone, another interpretation may be considered. Because respiratory epithelial cells are now recognized as a major source of leukocyte chemoattractants, and because leukocyte recruitment to the lung has been associated with enhanced viral clearance and prolonged survival in pneumovirus infection [22] , the ability to maintain chemoattractant production from viable cells may ultimately benefit the host organism as well. Available online http://respiratory-research.com/content/2/4/225 In tissue culture, RSV-infection is characterized by the formation of giant-cell syncytia. The mechanisms for the formation of these fused masses of cells depend in part on the expression of the RSV-specific fusion (F) protein on the surface of infected host cells, and in part on virusmediated changes in cytoskeletal architecture. It is important to note that RSV-induced changes in cytoskeletal architecture are not restricted to cell lines grown in vitro, as giant-cell syncytia have also been found in pathologic lung specimens obtained from both humans and animals that were infected with RSV. Again, as part of our systematic study of gene expression in response to pneumovirus infection, we found that human respiratory epithelial cells respond to RSV infection with increased expression of the cytoskeletal protein cytokeratin-17 [36] . Cytokeratin-17 is a 46-kDa cytoskeletal protein that belongs to the class I acidic cytokeratin family. In the lung, expression of cytokeratin-17 is normally restricted to basal epithelial cells of the larynx, trachea, and bronchi. In RSV-infected cells, we found expression of Ck-17 predominantly at sites of syncytia formation, and thus provided the first description of a unique component of these pathognomonic structures at the molecular level. Similar to what has been reported for the production of IL-8, expression of Ck-17 is dependent on the activity of the transcription factor NF-κB, and future studies will determine the role of the NF-κB consensus site (-200 to -208 of the cytokeratin-17 promoter) in mediating this response. To date, efforts to study pneumovirus-induced alterations in gene expression have relied heavily on in vitro models of virus-infected cells and cell lines. The intrinsic value of characterizing the genes identified in this artificial system is by definition limited, and the clinical and physiologic sig-nificance of any findings must ultimately be tested in vivo. To some extent, the study of gene products in clinical specimens is possible, but this approach is limited, cumbersome, and dictated by sample availability. It is clear that an appropriate animal model of inflammatory bronchiolitis is required to characterize the alterations in gene expression discovered using the available in vitro models. Although RSV has been used for the study of specific allergic reactions to viral antigens, it is not a natural pathogen of mice, and intranasal inoculation of virus at high titer results in, at best, a minimal primary infection with a correspondingly minimal inflammatory response. In order to study gene expression in response to primary pneumovirus infection in vivo, we developed a novel mouse model of inflammatory bronchiolitis using the natural rodent pneumovirus pathogen and closest phylogenetic relative of RSV [37] -PVM. We presented our initial findings on PVM infection in mice in three recent publications [22, 38, 39] . A summary of these findings is presented in Table 2 and Fig. 1 . To begin, we described the cellular and biochemical pathology observed in response to PVM infection in mice [38] . We found that infection could be established with as few as 30 plaque-forming units (pfu) of PVM in the inoculum, with infection resulting in significant morbidity and mortality, and viral recoveries in the order of 10 8 pfu/g lung tissue. We also noted inflammatory bronchiolitis as among the immediate responses to this infection, with bronchoalveolar lavage fluid containing virtually 100% neutrophils and eosinophils obtained as early as 3 days after inoculation. Furthermore, we found that infection was accompanied by the production of the proinflammatory chemokine MIP-1α, which was previously shown by Cook et al [40] to be an important component of the inflammatory response to the orthomyxovirus influenza virus. We also described the role played by MIP-1α in the pathogenesis of PVM-induced bronchiolitis [22] . Specifi- cally, we explored the responses of gene-deleted mice to infection with PVM, and found no inflammatory response in mice deficient in MIP-1α expression (MIP-1α -/-) and only minimal virus-induced inflammation in mice that lacked the major MIP-1α receptor on granulocytes chemokine receptor (CCR)1 (CCR1 -/-). Although the inflammatory response is often considered to be unnecessary and indeed detrimental, we demonstrated that the absence of granulocytic inflammation was associated with enhanced recovery of infectious virions, as well as with accelerated mortality. These results suggest that the MIP-1α/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of viral infection. Our most recent report on this subject [39] presents a direct comparison between the responses of mice to challenge with PVM and RSV. Although RSV is not a natural pathogen of mice, it has been used extensively in mouse models of human infection because a limited, or 'semipermissive' infection can be established via intranasal inocula-tion of virus at very high titers. In this regard, we found (as have others) that RSV infection did not result in any measurable degree of morbidity, and that viral recovery was significantly lower than that found in the inoculum; these results suggested that there was no significant viral replication in mouse lung tissue. We further demonstrated that the inflammatory response to RSV challenge was minimal, as few leukocytes were recruited to the lungs (Fig. 1) . Taken together, our results suggest that infection of mice with PVM provides a superior model for the study of acute inflammatory bronchiolitis in response to pneumovirus infection in vivo. The advantages of this model include the following: clinical parameters -morbidity and mortalitythat can be measured clearly and specifically; clear evidence of viral replication in lung tissue, with incremental recoveries that, at peak, are in excess of 10 8 pfu/g in response to as few as 30 pfu in the inoculum; and a dramatic granulocytic response that is modulated at least in part by the proinflammatory chemokine MIP-1α and its receptor CCR1. Traditionally, analysis of gene expression through measurement of steady-state levels of individual mRNAs could be conducted only one gene at a time using northern blotting, dot blots, or quantitative reverse transcription-PCR. Differential display, serial analysis of gene expression, and total gene analysis offer great promise, because they are multiplex technologies that provide simultaneous analysis of multiple mRNAs isolated under conditions of interest via PCR amplification techniques. DNA hybridization arrays are theoretically the most efficient of the gene expression analysis techniques. Although many skeptics have described these genome-based approaches as expensive, nonhypothesis-driven 'fishing expeditions', we view them as broad-based screening techniques that will enable us to identify patterns of gene expression that can then be subjected to careful characterization and analysis. Differential display is a semiquantitative, reverse transcription-PCR-based technique that is used to compare mRNAs from two or more conditions of interest. Both increased and decreased expression of specific amplicons will be evident -an obvious advantage to this approach. Total RNA can be isolated from virus infected versus uninfected cells or mouse lungs both before and during infection, and differential display is performed using degenerate T11(XY) anchoring primers and random upstream oligomers, as described elsewhere [34, 36] . The resulting PCR products are separated by electrophoresis, and the gel is dried and exposed to film. An example of our results comparing cDNA amplicons from RNA extracted from RSV-infected epithelial cells daily for 4 days is shown in Fig. 2 . Differentially expressed bands are cut from the gel, eluted and reamplified using the same primers that generated the original signal, and northern blots generated from RNA extracted from pneumovirus-infected cells or tissue over time and probed with the differentially expressed amplicons serve to confirm differential expression of the identified sequence. The DNA sequences of the newly identified differentially expressed amplicons are compared with sequences present in the GenBank database. Viral sequences are expected to be upregulated over time and can be identified immediately, because the entire genomes of both PVM and RSV are present in GenBank. In cases in which the amplicon represents a newly discovered gene, potential openreading frames are compared with sequences that are present in the Swiss protein database; motifs that share homologies with known proteins represent important clues to the identity of the differentially expressed gene. With the help of differential display, we have identified and characterized several genes that are upregulated in RSV-infected respiratory epithelial cells. Two specific examples of genes that were found to be induced during RSV infection, and later characterized as playing independent roles in the pathophysiology of RSV infection, are the antiapoptosis gene IEX-1L [34] and the gene that encodes the cytoskeletal protein cytokeratin-17 [36] . Unlike DNA viruses, which are known to encode virus-specific antiapoptosis genes, RSV -an RNA virus with a small (approximately 15.2 kb) viral genome -was shown to alter host cell expression of the apoptosis inhibitor IEX-1L. After demonstrating that IEX-1L mRNA was present at sevenfold higher concentrations in RSV-infected respiratory epithelial cells when compared with uninfected cells, we concluded that this cellular response protected against TNF-α-induced programmed cell death during viral infection. Further efforts to determine which of the 11 RSV proteins participate in the trans-activation of the IEX-1L gene (either directly or indirectly) are ongoing. A second example of a gene that is specifically upregulated in RSV-infected respiratory epithelium, as identified by differential display, is that which encodes cytokeratin-17 [36] . Upon characterizing the molecular events that are important for cytokeratin-17 induction, we demonstrated a link to an NF-κB signaling pathway. Above, we discussed the importance of this transcription factor in the regulation of proinflammatory cytokine gene expression, and because of this involvement we were not surprised to discover its role in virus-induced cytokeratin-17 gene regulation. Perhaps the most interesting observation made during these experiments was the in situ localization of cytokeratin-17 protein to areas of cytopathic syncytia formation, suggesting a role for this cytoskeletal protein in their formation. Of note, we observed a dramatic decrease in RSV replication and in syncytia formation when we blocked cytokeratin-17 expression, suggesting that blocking syncytia formation, at least in part, impairs the direct cell-cell spread and productive replication of virus. Although there are several companies that market these systems and components, the cytokine gene macroarray systems recently developed by R&D Systems (Sigma Genosys ® ; Minneapolis, MN, USA) and Clontech (Atlas ® ; Palo Alto, CA, USA) represent some of the newer opportunities available that have a focus on gene products that are known to be involved in inflammation. These arrays consist of different cDNAs printed as PCR products onto charged nylon membranes. An example of our experience with the Sigma Genosys array is shown in Fig. 3 . For this example, total RNA was extracted from RSV-infected HEp-2 cells and uninfected controls at day 3 after infection. Three micrograms of total RNA was used in a cDNA synthesis reaction, using a proprietary mixture of 378 primer pairs and trace amounts of 32 P-radiolabeled dCTP. The resulting radiolabeled products were hybridized to the macroarrays overnight at 65°C, and then washed and exposed to film. The arrow highlights one of the most obviously upregulated sequences from this experiment, which was identified as the gene encoding human MIP-1α. The physiologic importance of MIP-1α upregulation during human RSV infection and during rodent PVM bronchiolitis has already been described. Microarrays can be differentiated from macroarrays in several ways. Among these differences, the microarray matrix is a glass or plastic slide, probes are labeled with fluorescent dye rather than via radioisotopes, and, most significantly, microarrays generally include a larger number and a higher density of imbedded sequences than do macroarrays. Although this may seem to be highly appealing at first, the massive amounts of data generated by microarray technology poses new challenges with respect to data normalization, management, and development of mathematical models to assist in data interpretation. The pneumoviruses RSV and PVM enter respiratory epithelial cells via a receptor-mediated event. During hostcell attachment and internalization, the target cell begins to alter its gene expression, which, among other events, involves the transcriptional upregulation of cytokine and chemokine genes. As RSV replication progresses, additional changes in cellular gene expression can be observed, including induction of the potent antiapoptosis gene IEX-1L and increased expression of the otherwise quiescent gene that encodes cytokeratin-17. What we know regarding the physiologic importance of these genes and their gene products has been described, but there is more to be learned. As the available technologies evolve, we can continue to capitalize on the use of Display of amplicons generated from RNA extracted from RSV-infected cells at daily intervals following infection (days 0-4) using a single anchoring primer, T11GC (downstream primer 8) and (A-H) eight random 10mers. Two differentially expressed sequences are highlighted by arrows (the black arrow shows an upregulated amplicon, and the white arrow highlights a downregulated amplicon). Several other differentially expressed signals are also seen. genomic approaches as large-scale screening tools to identify genes that play important roles in the pathophysiology of pneumovirus infection. These elegant and simple tools will provide us with the means for thorough and systematic exploration of gene expression, both in the estab- Cytokine macroarray probed with radiolabelled cDNA generated from total RNA extracted from epithelial cells 48 h after RSV infection (upper panel) or 48 h after exposure to conditioned medium (lower panel). Signal intensity of each gene under each condition is compared. The arrow highlights the signal for human MIP-1α present at 12-fold higher concentration in infected epithelial cells compared with the uninfected controls."
5
"Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis"
"Nidovirus subgenomic mRNAs contain a leader sequence derived from the 5′ end of the genome fused to different sequences (‘bodies’) derived from the 3′ end. Their generation involves a unique mechanism of discontinuous subgenomic RNA synthesis that resembles copy-choice RNA recombination. During this process, the nascent RNA strand is transferred from one site in the template to another, during either plus or minus strand synthesis, to yield subgenomic RNA molecules. Central to this process are transcription-regulating sequences (TRSs), which are present at both template sites and ensure the fidelity of strand transfer. Here we present results of a comprehensive co-variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. While the leader TRS merely plays a targeting role for strand transfer, the body TRS fulfils multiple functions. The sequences of mRNA leader–body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis."
"The genetic information of RNA viruses is organized very ef®ciently. Practically every nucleotide of their genome is utilized, either as protein-coding sequence or as cis-acting signals for translation, RNA synthesis or RNA encapsidation. As part of their genome expression strategy, several groups of positive-strand RNA (+RNA) viruses produce subgenomic (sg) mRNAs (reviewed by Miller and Koev, 2000) . The replication of their genomic RNA, which is also the mRNA for the viral replicase, is supplemented with the generation of sg transcripts to express structural and auxiliary proteins, which are encoded downstream of the replicase gene in the genome. Sg mRNAs of +RNA viruses are always 3¢-co-terminal with the genomic RNA, but different mechanisms are used for their synthesis. Some viruses, such as brome mosaic virus, initiate sg mRNA synthesis internally on the full-length minus strand RNA template (Miller et al., 1985) . Others, exempli®ed by red clover necrotic mosaic virus (RCNMV), may rely on premature termination of minus strand synthesis from the genomic RNA template, followed by the synthesis of sg plus strands from the truncated minus strand template (Sit et al., 1998) . Members of the order Nidovirales, which includes coronaviruses and arteriviruses, have evolved a third and unique mechanism, which employs discontinuous RNA synthesis for the generation of an extensive set of sg RNAs (reviewed by Brian and Spaan, 1997; Lai and Cavanagh, 1997; Snijder and Meulenberg, 1998) . Nidovirus sg mRNAs differ fundamentally from other viral sg RNAs in that they are not only 3¢-coterminal, but also 5¢-co-terminal with the genome ( Figure 1A) . A 5¢ common leader sequence of 65±221 nucleotides, derived from the 5¢ end of the genomic RNA, is attached to the 3¢ part of each sg RNA (thè mRNA body'). Various models have been put forward to explain the cotranscriptional fusion of non-contiguous parts of the nidovirus genome during sg RNA synthesis ( Figure 1B and C). Central to each of these models are short transcription-regulating sequences (TRSs), which are present both at the 3¢ end of the leader and at the 5¢ end of the sg RNA body regions in the genomic RNA. The TRS is copied into the mRNA and connects its leader and body part (Spaan et al., 1983; Lai et al., 1984) . Synthesis of sg mRNAs initially was proposed to be primed by free leader transcripts, which would base-pair to the complementary TRS regions in the full-length minus strand, and would be extended subsequently to make sg plus strands ( Figure 1B ; Baric et al., 1983 Baric et al., , 1985 . This model, however, was based on the report that sg minus strands were not present in coronavirus-infected cells (Lai et al., 1982) . The subsequent discovery of such molecules (Sethna et al., 1989) resulted in reconsideration of the initial`leader-primed transcription' model. Sawicki and Sawicki (1995) have proposed an alternative model ( Figure 1C ), in which the discontinuous step occurs during minus instead of plus strand RNA synthesis. In this model, minus strand synthesis would be attenuated after copying a body TRS from the plus strand template. Next, the nascent minus strand, with the TRS complement at its 3¢ end, would be transferred to the leader TRS and attach by means of TRS±TRS base pairing. RNA synthesis would be reinitiated to complete the sg minus strand by adding the complement of the genomic leader sequence. Subsequently, the sg minus strand would be used as template for sg mRNA synthesis, and the presence of the leader complement at its 3¢ end might allow the use of the same RNA signals that direct genome synthesis from the fulllength minus strand. Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis The EMBO Journal Vol. 20 No. 24 pp. 7220±7228, 2001 Using site-directed mutagenesis of TRSs of the arterivirus equine arteritis virus (EAV), we have shown previously that base pairing between the sense leader TRS and antisense body TRSs is crucial for sg mRNA synthesis (van Marle et al., 1999a) . However, base pairing is only one step of the nascent strand transfer process and is essential in both models outlined in Figure 1 . The EAV genomic RNA contains several sequences that match the leader TRS precisely, but nevertheless are not used for sg RNA synthesis (den Boon et al., 1996; Pasternak et al., 2000) . This suggests that leader±body TRS similarity alone is, though necessary, not suf®cient for the strand transfer to occur. To gain further insight into the cis-acting signals regulating sg RNA synthesis, we performed a comprehensive site-directed mutagenesis study of the EAV leader and body TRSs. Every nucleotide of the TRS (5¢-UCAACU-3¢) was substituted with each of the three alternative nucleotides. Our analysis revealed a number of striking similarities with the process of copy-choice RNA recombination, as it occurs in RNA viruses. Whereas the leader TRS plays only a targeting role in translocation of the nascent strand, body TRS nucleotides appear to ful®l diverse position-speci®c and base-speci®c functions. In addition, the sequence of the leader±body junctions of the sg mRNAs produced by these mutants provided strong evidence for the discontinuous minus strand extension model. EAV genome replication is not signi®cantly affected by leader TRS and body TRS mutations To dissect EAV RNA synthesis, we routinely use a fulllength cDNA clone (van Dinten et al., 1997) , from which infectious EAV RNA is in vitro transcribed. Following transfection of the RNA into baby hamster kidney (BHK-21) cells, intracellular RNA is isolated and analysed by northern blot hybridization and RT±PCR (van Marle et al., 1999a) . Due to differences in transfection ef®ciency, the total amount of virus-speci®c RNA (genomic RNA and sg mRNA) isolated from transfected cell cultures is somewhat variable. Thus, the accurate quantitation of sg mRNA synthesis by TRS mutants requires an internal standard for transfection ef®ciency. The amount of viral genomic RNA can be this standard, but only if its ampli®cation is not dramatically affected by the TRS mutations. To prove that this is the case, we used the previously described mutants L4, B4 and LB4 (van Marle et al., 1999a) , in which ®ve nucleotides of the TRS (5¢-UCAAC-3¢) were replaced by the sequence 5¢-AGUUG-3¢, either in the leader TRS (L4), RNA7 body TRS (B4) or both TRSs (LB4). The three mutants were tested in three independent experiments. Intracellular RNA was isolated at 14 h posttransfection, early enough to prevent spread of the wildtype control virus to non-transfected cells (®rst cycle analysis). Transfection ef®ciencies were determined by immuno¯uorescence assays (see Materials and methods) and varied between 10 and 23% (data not shown). Prior to RNA analysis, the amount of isolated intracellular RNA was corrected for the transfection ef®ciency of the sample, so that each lane in Figure 2 represents EAV-speci®c RNA from an approximately equal number of EAV-positive cells. Phosphoimager quantitation revealed that genomic RNA replication of mutants L4, B4 and LB4 varied by not more than 30% (Table I) . These differences could re¯ect, for example, a slight in¯uence of RNA secondary structure changes in the TRS regions on genomic RNA synthesis. Remarkably, however, the genomic RNA level of the leader±body TRS double mutant LB4 was not affected by more than 10%. In view of the results obtained with these pentanucleotide TRS mutants, we assumed that the amount of genomic RNA could indeed be used as an internal standard during the analysis of mutants containing only single nucleotide replacements in leader TRS and/or RNA7 body TRS. The regions of the genome specifying the leader (L) sequence, the replicase gene (ORFs 1a and 1b) and the structural genes are indicated. The nested set of EAV mRNAs (genome and sg mRNAs 2±7) is depicted below. The black boxes in the genomic RNA indicate the position of leader and major body TRSs. (B and C) Alternative models for nidovirus discontinuous sg RNA synthesis. The discontinuous step may occur during either plus strand (B) or minus strand (C) RNA synthesis. In the latter case, sg mRNAs would be synthesized from an sg minus strand template. For details see text. Northern analysis of EAV-speci®c RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from TRS pentanucleotide mutants (UCAAC to AGUUG). The results of two independent experiments are shown. The RNA±RNA interaction between the leader and body TRSs is not the only factor that regulates EAV sg RNA synthesis There are numerous examples of regulatory RNA±RNA interactions in both eukaryotic and prokaryotic cells, as well as in RNA viruses. Essential processes such as translation, replication and encapsidation of RNA virus genomes frequently depend on RNA±RNA interactions and higher order RNA structures. Regulation of sg RNA synthesis of +RNA viruses by RNA±RNA interactions is also not without precedent. In tomato bushy stunt virus, an RNA element located 1000 nucleotide upstream of the sg RNA2 promoter base-pairs with the promoter and is necessary for sg RNA production (Zhang et al., 1999) . Similarly, base pairing interactions between complementary sequences in the 5¢ end of the potato virus X genomic RNA and sequences upstream of two major sg RNA promoters are required for ef®cient sg RNA synthesis (Kim and Hemenway, 1999) . In RCNMV, an intermolecular RNA±RNA interaction is required for sg RNA synthesis (Sit et al., 1998) . Recently, we have established the pivotal role of an interaction between sense and antisense RNA sequences in the life cycle of EAV (van Marle et al., 1999a) . In that study, the role of TRS nucleotides C 2 and C 5 was tested by substituting them with G. It was concluded that base pairing between the sense leader TRS and the antisense body TRS plays a crucial role in nidovirus sg RNA synthesis. We now took a more systematic approach and performed an extensive site-directed co-variation mutagenesis study of the entire leader TRS and RNA7 body TRS, which directs the synthesis of the most abundant EAV sg RNA. Every nucleotide of the TRS (5¢-UCA-ACU-3¢) was replaced with each of the other possible nucleotides. As in the study of van Marle et al. (1999a) , every mutation was introduced into leader TRS, RNA7 body TRS and both TRSs, resulting in 54 mutant constructs. Each mutant was given a unique name: e.g. BU 1 A refers to a mutant in which a U has been changed to A at position 1 of the body TRS; LU 1 A refers to the same substitution in the leader TRS; and DU 1 A means that these two substitutions were combined in one double mutant construct. The amount of sg RNA7 was quantitated by phosphoimager scanning of hybridized gels and was corrected for the amount of genomic RNA in the same lane (as outlined above). Figure 3 shows the relative sg RNA7 level of the 54 mutants, compared with the RNA7 level of the wild-type control. For a selection of 11 interesting mutants (see below), the analysis was repeated three times (Figure 4 ), without observing signi®cant variations in sg RNA synthesis. The comprehensive analysis of the effects of TRS mutations considerably expanded our understanding of van Dinten et al., 1997) was taken along as a positive control. For every mutant, the level of sg RNA7 synthesis was calculated as [(sg/g)/(sg/g) wt ] 3 100%: it was corrected for the level of genomic RNA (used as an internal standard; see text) and subsequently was related to the level of sg RNA7 produced by the wild-type control in the same experiment, which was also corrected for the corresponding genomic RNA level. The relative sg RNA7 level of the wild-type control was set at 100%. A.O. Pasternak et al. discontinuous sg RNA synthesis. Remarkably, the effects of single (leader or body) TRS mutations were mostly base speci®c, i.e. different nucleotide substitutions at the same position affected sg RNA7 synthesis to different extents. For example, at position 1, the BU 1 A mutant retained 44% of the wild-type RNA7 synthesis level, whereas both the BU 1 C and BU 1 G mutants lost RNA7 synthesis almost completely. Conversely, when U 1 of the leader TRS was changed to A or G, RNA7 synthesis was completely abolished, whereas 13% of the wild-type level was still maintained by LU 1 C. For position 2, only the BC 2 U mutant retained 30% of the wild-type RNA7 synthesis level, while all the other position 2 single mutants have lost 90% or more of wild-type RNA7 synthesis. Another example is position 6: BU 6 C left only 5% of wild-type RNA7 synthesis, whereas BU 6 A produced much higher RNA7 levels. This implied that for some positions (1, 2 and 6), certain mismatches in the duplex between plus leader TRS and minus body TRS, such as U±U (BU 1 A and BU 6 A) or C±A (LU 1 C and BC 2 U), are allowed to a limited extent. In contrast, no mismatches were allowed for position 5, where all single nucleotide substitutions abolished RNA7 synthesis almost completely. Surprisingly, both body TRS U to C substitutions at positions 1 and 6 (BU 1 C and BU 6 C) resulted in low levels of RNA7, despite the fact that these mutations allow the formation of a G±U base pair between the plus leader TRS, providing the U nucleotide, and the minus body TRS, providing the G. On the other hand, for positions 3 and 4, G±U base pairing was shown to be functional, because mutants LA 3 G and LA 4 G, which can form G±U base pairs between the G in the plus leader TRS and U in the minus body TRS, were the only position 3 and 4 single mutants that produced reasonable levels of RNA7. Taken together, these ®ndings suggest that other factors, besides leader± body base pairing, also play a role in sg RNA synthesis and that the primary sequence (or secondary structure) of TRSs may dictate strong base preferences at certain positions. Our analysis of the degree of complementation by the double mutants provided strong support for this assumption. Differentiating between effects at the level of primary TRS sequence and the level of leader±body duplex formation For some TRS nucleotides (2, 5 and 6, except in the case of DU 6 C), the RNA7 level of double mutants was clearly higher than that of the corresponding single mutants. This means that base pairing between these leader and body TRS nucleotides is involved in sg RNA synthesis. However, none of these double mutants reached the wild-type sg RNA7 level. In the other double mutants (all position 1, 3 and 4 mutants, and DU 6 C), in clear contradiction to the predictions of the`base pairing model', RNA7 synthesis was not signi®cantly restored. Moreover, a comparison of the values for the B and D mutants in Figure 3 showed that, for almost all of these mutants (e.g. the position 1 mutants), the amount of sg RNA7 produced by the double mutant appeared to be limited by the level allowed by the body TRS mutation. Sometimes the RNA7 level of the double mutant was even less than that of the leader mutant (DU 1 C, DA 3 G, DA 4 G or DU 6 C). Clearly, for these substitutions, restoration of the possibilities for leader±body duplex formation did not restore sg RNA synthesis. Apparently this is because the effect of body TRS mutations at the level of primary sequence or secondary structure can be`dominant' over the duplex-restoring effects of the double mutations. Body TRS mutants thus fell into two distinct types, determined by the position and chemistry of the substitution. In mutants of the ®rst type, sg RNA synthesis was impaired mainly because of the disruption of the leader± body TRS duplex. This effect could be compensated for by introduction of the corresponding mutation in the leader TRS and, in the double mutant, sg RNA synthesis was restored compared with the corresponding single mutants. In mutants of the second type, sg RNA synthesis was down-regulated as a consequence of both TRS duplex disruption and disruption of the primary sequence (or secondary structure) of the body TRS. Obviously, the latter effect could not be compensated for by mutating the leader TRS, and the corresponding double mutants did not show restoration of sg RNA synthesis. In contrast to our ®ndings with the body TRS mutants, we did not obtain leader TRS mutations that appeared to determine the level of sg RNA7 synthesis of the corresponding double mutant (Figure 3) . Thus, effects of mutations in the leader TRS were not`dominant' over the duplex-restoring effects of the double mutations, suggesting that they only affected duplex formation. This indicated that the leader TRS probably does not have an additional, sequence-speci®c function in sg RNA synthesis in addition to its participation in TRS±TRS base pairing. The fact that single leader TRS mutations at all six Nidovirus discontinuous subgenomic RNA synthesis positions severely repressed RNA7 synthesis indicated that base pairing of every TRS nucleotide contributes to sg RNA production. In this respect, it was signi®cant that the two leader TRS mutants with the highest RNA7 levels, LA 3 G and LA 4 G, can form G±U base pairs to maintain the duplex. The observation that leader TRS mutations could bè rescued' by introducing complementary mutations in the body TRS, but that many body TRS mutations could not bè rescued' by corresponding changes in the leader TRS, is clearly illustrated by the U 1 A mutants. Due to the restoration of TRS base pairing possibilities, the RNA7 synthesis of double mutant DU 1 A was signi®cantly increased compared with that of LU 1 A, but not above the level of BU 1 A. Thus, restoration of the leader±body duplex in DU 1 A exerted a clear effect on sg RNA7 production compared with LU 1 A, but had no effect on sg RNA synthesis compared with BU 1 A. This exempli®ed the dominant nature of a mutation in the primary sequence of a body TRS. In contrast, for instance, the BC 2 U mutation probably affected duplex formation only, because RNA7 synthesis was restored almost to wildtype levels in the DC 2 U double mutant. These results indicate that there are strong base preference constraints for some body TRS positions. To interpret these base preferences accurately, it is necessary to limit the analysis to the double mutants only, because in these mutants the down-regulation of sg RNA synthesis was only due to the sequence changes in the body TRS, and not to the disruption of the leader±body TRS duplex. There were strict preferences for positions 1, 3 and 4 of the body TRS: at position 1, only the U to A substitution allowed for a signi®cant RNA7 level (~40% of wild-type); and at positions 3 and 4, only the A to U mutants retained 15±20% of the wild-type level. For positions 2 and 5, the sequence constraints were less stringent (all substitutions allowed for >20% of wild-type level), but still only DC 2 A and DC 2 U reached >50%. At position 6 of the body TRS, only U to C was not allowed, whereas the other two double mutants still produced 50% or more of RNA7. In other words, the functional EAV RNA7 body TRS (based on the analysis of our single nucleotide substitutions) can be described as U 1 (C/u/a) 2 A 3 A 4 C 5 (U/a/g) 6 , with wild-type nucleotides shown in upper case and nucleotides that allowed for at least 50% of the wild-type RNA7 level shown in lower case. Remarkably, TRS nucleotides A 3 , A 4 and C 5 are conserved in the TRSs of all other arteriviruses (Snijder and Meulenberg, 1998) . Also the fact that DC 2 U retained 80% of RNA7 synthesis corresponded nicely to the presence of a U at this position in other arteriviruses. Until recently (Almazan et al., 2000; Thiel et al., 2001) , infectious cDNA clones were lacking for coronaviruses. Consequently, most studies on coronavirus sg RNA synthesis were carried out using defective interfering (DI) RNAs. These replicons carried body TRSs from which moderate levels of sg mRNAs could be produced in the presence of helper virus. Using this system, Joo and Makino (1992) and van der Most et al. (1994) performed body TRS mutagenesis studies for the murine coronavirus (MHV). Joo and Makino systematically mutagenized the core of the MHV body TRS. In contrast to our results, they found that in only two of 21 body TRS mutants was sg RNA synthesis from the DI RNA genome abolished, whereas all others supported normal levels of sg RNA production. Thus, it is possible that the MHV TRS which was used in that study is more tolerant to single-nucleotide mismatches than the EAV sg RNA7 TRS. In a similar study, van der Most et al. (1994) observed that U to C substitutions at positions 1 and 3 of the MHV body TRS, which maintained the duplex by changing a U±A base pair into a U±G base pair, reduced sg RNA levels more strongly than substitutions that disrupted the duplex (van der Most et al., 1994) . This implies that, as in the case of EAV, leader±body TRS duplex formation is not the only factor that determines coronavirus sg RNA synthesis. However, because of the limitations of the DI RNA system, the leader TRS could not be mutagenized in these studies, and body TRS-speci®c effects could not be distinguished from effects at the level of leader±body duplex formation. The discontinuous step in nidovirus sg RNA synthesis occurs during minus strand RNA synthesis Due to recent studies of arterivirus and coronavirus sg RNA synthesis (van Marle et al., 1999a; Baric and Yount, 2000; Sawicki et al., 2001) , the discontinuous minus strand extension model ( Figure 1C ) has been gaining more and more ground. This model predicts that the TRSderived sequence that forms the leader±body junction in the sg mRNA is a copy of the body TRS, and not of the leader TRS. The leader-primed transcription model predicts the opposite ( Figure 1B) . Therefore, determining the origin of the leader±body junction of sg mRNAs would help to distinguish between the two models. However, in the wild-type situation, EAV leader and body TRSs are identical and consequently one cannot determine the origin of the sg mRNA leader±body junction. This problem could be overcome by tracing the mutations introduced in leader or RNA7 body TRS mutants, most of which retained part of their ability to produce mRNA7. In a previous study (van Marle et al., 1999a) , we found that nucleotides 2 and 5 of the mRNA7 leader±body junction sequence were derived exclusively from the body TRS, and not from the leader TRS. This was shown by direct sequencing of RT±PCR products obtained from the residual mRNA7 produced by mutants BC 2 G, LC 2 G, BC 5 G and LC 5 G ( van Marle et al., 1999a) . Using the same approach, we analysed mRNA7 from mutants BC 2 A and BC 2 U, and these transcripts also contained the mutated nucleotide derived from the body TRS (data not shown). Assuming that only one crossover event occurs during leader±body joining, we could thus map this crossover between positions ±1 and +2 of the sg RNA junction sequence. This left the intriguing question of whether the crossover site could be mapped even more precisely. In other words, was nucleotide +1 of the junction sequence derived from the body TRS or the leader TRS? Using the position 1 mutants described above, we could answer this question ( Figure 5) . The most striking result was that mRNA7 of mutants BU 1 A, BU 1 G and LU 1 C contained exclusively the body TRS-derived nucleotide at position +1. Thus, for these mutants, the crossover site could be mapped precisely between TRS nucleotide positions ±1 and +1, meaning that the complete leader± body junction sequence in an EAV sg mRNA can be body TRS derived. On the other hand, sg RNAs from mutants LU 1 A, BU 1 C and LU 1 G contained mixed populations of leader TRS-and body TRS-derived nucleotides at position +1 ( Figure 5 ): A and U for LU 1 A, C and U for BU 1 C, and G and U for LU 1 G. Remarkably, this pattern correlated with the relative amounts of sg mRNA7 produced by these mutants (Figure 3 ). Mutants that produced populations of sg RNAs that were mixed with respect to the origin of the nucleotide at position +1 of the leader±body junction had lost RNA7 synthesis almost completely. On the other hand, mutants that contained exclusively the body nucleotide at position +1 retained higher levels of RNA7 synthesis. This observation may be explained as follows: in the wild-type situation, the large majority of the crossovers probably occur between positions ±1 and +1, leading to a body TRS-derived nucleotide at position +1 in the sg RNA; however, a low number of crossovers take place between nucleotides +1 and +2, resulting in a leader TRS-derived nucleotide at position +1. Mutants in which almost all sg RNA synthesis is blocked by a substitution at position +1 may somehow be de®cient in the crossover between ±1 and +1, but may have retained the ability for crossovers between +1 and +2, which were detected by sequence analysis. Conversely, in position +1 mutants that retain reasonable sg RNA synthesis, most crossovers occur between positions ±1 and +1, and they obscure the minority of crossovers between +1 and +2 in the sequencing electropherogram. Alternatively, position +1 TRS mutations that strongly interfere with sg RNA synthesis may force a shift of the crossover site in the remaining molecules. We believe that our present ®ndings strongly support the discontinuous minus strand extension model. Indeed, the fact that a complete body TRS can be copied into the sg RNA is very dif®cult to reconcile with the alternative model, in which sg RNA synthesis from the genomic minus strand template is primed by free plus strand leader transcripts that contain the leader TRS at their 3¢ end ( Figure 1B) . To explain the presence of a complete copy of the body TRS in the sg mRNA in this model, one would have to assume that a 3¢±5¢ exonuclease activity trims back the free leader transcript prior to its extension into an sg mRNA (Baker and Lai, 1990) . Note that there would not be a single base pair left to hold these`trimmed' leader molecules on the template. Such an enzymatic activity, which is unprecedented in +RNA viruses, exists in yeast retrotransposon Ty5 (Ke et al., 1999) , in which reverse transcription is primed by an internal region in a tRNA. However, in this system, it is not a part of the duplex that is removed, but the single-stranded 3¢ tail of the tRNA, which cannot base-pair with the Ty5 RNA. Removal of the TRS at the 3¢ end of the nidovirus leader, which has already base paired with the template, would be very energetically unfavourable for the RdRp. Instead of starting elongation using the intact and properly positioned leader as a primer, it would have to disrupt the newly formed duplex, degrade part of the leader RNA and then reinitiate polymerization, without any base pairing between primer and template. It has been shown that in¯uenza virus transcription does not require a sequence match between the (cellular) RNA primer and the (viral) template (Plotch et al., 1981) . However, if in the nidovirus system the`trimmed' leader RNA could also be ®xed on the template solely by RNA±protein interactions, the targeting of the nascent strand by TRS base pairing would be extremely puzzling. Sequence data of sg RNA leader±body junctions from other arteriviruses are also dif®cult to reconcile with the leader-primed transcription model. For the porcine and simian arteriviruses (Meulenberg et al., 1993; Godeny et al., 1998) , the leader±body junctions of some sg RNAs mapped two nucleotides upstream of the body TRS, which again would not leave a single nucleotide to hold the putative free leader on the template after the hypothetical back trimming'. On the other hand, these ®ndings and our data can be explained readily by the discontinuous minus strand extension model ( Figure 1C ). The six-nucleotide Fig. 5 . Sequence analysis of mRNA7 leader±body junctions from position 1 TRS mutants. Sequences were determined directly from sg mRNA7-speci®c RT±PCR products. For the U 1 A and U 1 C mutants, the sequence shown corresponds to the plus strand of sg RNA7. For sequencing-related technical reasons, the minus strand sequence was determined for the U 1 G mutants; a mirror image of the electropherogram is shown with the corresponding plus strand sequence listed at the top of the panel. For every mutant, a sequence alignment of the leader (red) and body (blue) TRSs and surrounding sequences is shown (TRSs are boxed). The mRNA7 leader±body junctions detected by our sequence analysis are shown in yellow. duplex formed between the body TRS complement at the 3¢ end of the leaderless sg minus strand and the leader TRS in the genomic RNA template should suf®ce to position the nascent minus strand properly for subsequent elongation to add the complement of the leader sequence. In most cases, the nascent minus strand contains the entire body TRS complement at its 3¢ end at the moment of strand transfer, leading to a body TRS-derived leader±body junction sequence in the sg mRNA molecule. In a small number of transcripts, however, minus strand synthesis appears to be interrupted before nucleotide +1 of the body TRS is copied and, after strand transfer, resumes by incorporating the complement of the +1 nucleotide of the leader TRS. As stated above, we postulate that the detection of this phenomenon is determined by the level of crossovers between the ±1 and +1 position that is allowed by the mutations introduced at the +1 position of body TRS or leader TRS. We cannot, however, formally exclude that a`back trimming' activity degrades the 3¢-terminal nucleotide of the minus strand before or after strand transfer. However, note that in the discontinuous minus strand extension model ( Figure 1C ), such an activity would not disturb the proper positioning of the nascent minus strand on the leader template, because the TRS± TRS duplex would be shortened by one nucleotide only. Nidovirus discontinuous minus strand extension resembles similarity-assisted, copy-choice RNA recombination Due to their discontinuous sg RNA synthesis, nidoviruses occupy a special`niche' in the +RNA virus world. Their mode of sg RNA production is clearly different from that of other +RNA viruses and resembles another welldocumented +RNA virus feature: RNA recombination (for recent reviews see Nagy and Simon, 1997; Aaziz and Tepfer, 1999; Worobey and Holmes, 1999) . Most of the experimental evidence supports an RdRp template switch (Kirkegaard and Baltimore, 1986) as the main mechanism of RNA recombination. Mechanistically, such a template switch involves the transfer of a nascent strand from one RNA template (donor) to the other (acceptor). Also, nidovirus discontinuous sg RNA synthesis involves transfer of a nascent RNA strand, the sg RNA, but now from one site to another in the same template. Based on the data currently available, we refer to the discontinuous minus strand extension model as our working model for nidovirus sg RNA synthesis. If one applies the`recombination terms' to this model (Chang et al., 1996; Brian and Spaan, 1997; van Marle et al., 1999a) , the donor strand would be the body part of the genomic RNA template, the acceptor strand would be the leader part of the genomic RNA template and the nascent strand would be the discontinuously synthesized minus strand. Nagy and Simon (1997) have de®ned three main classes of RNA recombination: similarity-essential, similarity-non-essential and similarity-assisted recombination. The latter is de®ned as a mechanism in which strand transfer is determined by both sequence similarity between the parental RNAs and additional RNA determinants, present in only one of the parental RNAs. The results of our present study strongly suggest that nidovirus discontinuous sg RNA synthesis can be considered a special case of high-frequency similarity-assisted RNA recombination. While the only obvious function of the leader TRS is to ensure the ®delity of the strand transfer by base pairing with the 3¢ end of the nascent strand, the body TRS in the donor template indeed has additional, sequence-speci®c functions. One of these functions apparently is to pause (or terminate) nascent strand synthesis and thereby provide the opportunity for strand transfer. In addition, body TRS-derived nucleotides may play a role in the reinitiation of nascent strand synthesis on the acceptor template. Given the compact nature of the EAV TRS, it is quite possible that some nucleotides ful®l multiple tasks. RNA secondary structure of the body TRS may regulate sg RNA synthesis The sequence-speci®c function of the body TRS, revealed in this study, may be exerted at the level of either primary sequence or secondary structure. For a number of +RNA viruses, RNA secondary structure motifs located in the (proximity of) sg RNA promoters are vital for sg RNA synthesis. In alfalfa mosaic virus (Haasnoot et al., 2000) , turnip crinkle virus (TCV) (Wang et al., 1999) and barley yellow dwarf virus (Koev et al., 1999) , stem±loop structures in sg RNA promoter regions of the template strand are required for sg RNA synthesis. The sg RNA1 promoter of the latter virus is especially interesting, since it contains two stem±loop domains. For one of them, secondary structure, but not the primary sequence, is important for sg RNA synthesis, whereas the other domain acts through primary sequence, and not secondary structure (Koev et al., 1999) . Similarly, RNA secondary structure may play only a minor role in the sequence-speci®c recognition of the BMV sg RNA promoter by the RdRp Siegel et al., 1997) . We have suggested previously that RNA secondary structure of body TRS regions contributes to their attenuating potential and thereby determines the relative portion of the nascent minus strands that is transferred to the leader TRS in the template (Pasternak et al., 2000) . At present, it is unknown whether EAV body TRSs are part of an RNA structural motif that is essential for body TRS function, or whether they are recognized by a protein factor in a sequence-speci®c manner. However, the latter seems less likely than the former, since even LB4 (Figure 2 ), in which ®ve TRS nucleotides were substituted, still produced some sg RNA7, although~30-fold less than the wild-type control. The fact that some sequences in the EAV genome match the leader TRS perfectly, but are not used for sg mRNA synthesis, also argues against the recognition of a speci®c sequence (Pasternak et al., 2000) . More probably, mutagenesis of the RNA7 body TRS disturbed an RNA structure that is necessary for its function. This could, for example, explain the fact that the BU 6 C substitution reduced the amount of RNA7 by 20-fold (and could not be rescued by the same mutation in the leader TRS), whereas the wild-type RNA6 body TRS contains a C at the same position. If a protein factor were involved in sequence-speci®c TRS recognition, then one would expect it to recognize all TRSs similarly. If RNA structure is important for recognition by such a protein, then the BU 6 C substitution probably disturbs a structural motif of the RNA7 TRS, which is not present in the RNA6 TRS. On the other hand, conservation of part of the TRS in other arteriviruses suggests a sequence-speci®c recognition. Further studies are required to distinguish between these possibilities. In the TCV satellite RNA recombination system, the hairpin structure in the acceptor strand, as well as the donor±acceptor homology region, are necessary for the template switch . The hairpin has been postulated to bind the RdRp, whereas the homology region targets the nascent strand to the crossover site. The TCV RdRp probably recognizes the secondary and/or tertiary structure of the hairpin, while individual nucleotides play a less important role . In EAV, the leader TRS in the acceptor template is predicted to reside in the loop of an extensive hairpin, and its base pairing interaction with the body TRS complement at the 3¢ end of the nascent minus strand would resemble certain antisense RNA-regulated control mechanisms that are based on interactions between single-stranded tails and hairpin loops (van Marle et al., 1999a, and references therein) . It is possible that the EAV RdRp, or its accessory proteins, also binds to the stem of the long hairpin that presents the leader TRS. In any case, the leader TRS itself does not seem to be recognized by a protein in a sequence-speci®c manner. The body TRS is a better candidate to serve as a protein recognition site. This protein would then mediate the pausing of the nascent strand synthesis and/or nascent strand transfer. This would resemble the DNA-dependent RNA polymerase I termination system, in which speci®c DNA-binding terminator proteins bind to termination sequences (Reeder and Lang, 1997) , or a function of the HIV nucleocapsid protein, which promotes the minus strand strong-stop DNA transfer (Guo et al., 1997) . The EAV replicase component nsp1, which recently was shown to possess an sg RNA synthesis-speci®c activity (Tijms et al., 2001) , may be a good candidate for such a regulatory role. Residues predicted to form a zinc ®nger structure in nsp1 were shown to be necessary for sg RNA synthesis. Interestingly, zinc ®nger structures in the HIV nucleocapsid protein facilitate strand transfer (Guo et al., 2000) . Finally, it should be noted that the RNA structure of the nascent strand may also in¯uence pausing, strand transfer or reinitiation, as illustrated by the fact that stable hairpin structures in the nascent strand promote termination of transcription by Escherichia coli RNA polymerase (Wilson and von Hippel, 1995) . Site-directed mutagenesis, RNA transfections and immuno¯uorescence analysis Site-directed mutagenesis of EAV leader and body TRSs was carried out as described by van Marle et al. (1999a) , and all mutant constructs were sequenced. Following in vitro transcription from infectious cDNA clones, full-length EAV RNA was introduced into BHK-21 cells by electroporation, as described by van Dinten et al. (1997) . Immuno¯uorescence assays with EAV-speci®c antisera were performed at 14 h posttransfection as described by van der Meer et al. (1998) . To visualize the nuclei for cell counting, nuclear DNA was stained with 5 mg/ml Hoechst B2883 (Sigma). Cells were counted using the Scion Image software (Scion Corporation) and the percentage of transfected cells was calculated on the basis of the number of cells positive for the EAV replicase component nsp3 (Pedersen et al., 1999) . For RNA analyses, cells were lysed at 14 h post-transfection. Intracellular RNA isolation was performed using the acidic phenol method as described by Pasternak et al. (2000) . Total intracellular RNA was resolved in denaturing agarose±formaldehyde gels. Hybridization of dried gels with the radioactively labelled oligonucleotide probe E154, which is complementary to the 3¢ end of the EAV genome and recognizes all viral mRNA molecules (genomic and subgenomic), and phosphoimager quantitation of individual bands were performed as described by Pasternak et al. (2000) . To determine the leader±body junction sequence of sg mRNA7, mRNA7-speci®c RT±PCRs were carried out as described by van Marle et al. (1999b) using an antisense (RT and PCR) primer from the RNA7 body region and a sense PCR primer matching a part of the leader sequence. RT±PCR products were sequenced directly as described by Pasternak et al. (2000) using the leader-derived primer, an ABI PRISMÔ sequencing kit (Perkin Elmer) and an ABI PRISMÔ 310 Genetic Analyser (Perkin Elmer)."
6
"Debate: Transfusing to normal haemoglobin levels will not improve outcome"
"Recent evidence suggests that critically ill patients are able to tolerate lower levels of haemoglobin than was previously believed. It is our goal to show that transfusing to a level of 100 g/l does not improve mortality and other clinically important outcomes in a critical care setting. Although many questions remain, many laboratory and clinical studies, including a recent randomized controlled trial (RCT), have established that transfusing to normal haemoglobin concentrations does not improve organ failure and mortality in the critically ill patient. In addition, a restrictive transfusion strategy will reduce exposure to allogeneic transfusions, result in more efficient use of red blood cells (RBCs), save blood overall, and decrease health care costs."
"Anaemia is a common condition in critically ill patients, and RBC transfusions are often used in the treatment and management of this patient population. In fact, one study [1] reported that 25% of all critically ill patients received RBC transfusions. Many laboratory studies [2] [3] [4] [5] [6] [7] [8] have examined the physiological responses (ie compensatory mechanisms) of the body to anaemia, which include the following [9] : increased cardiac output, decreased blood viscosity, capillary changes, increased oxygen extraction, and other tissue adaptations to meet oxygen requirements. Although critically ill patients are affected by a number of factors that predispose them to the adverse consequences of anaemia, persistence of this condition is of particular concern because it may cause the compensatory mechanisms in these patients to become impaired, risking oxygen deprivation in vital organs [9] . However, critically ill patients may also be at increased risk from the adverse effects of RBC transfusions, such as pulmonary oedema from volume overload, immune suppression resulting in increased risk of infection, and microcirculatory injury from poorly deformable RBCs. It is the aim of the present commentary to justify the statement 'Transfusing to normal haemoglobin concentration will not improve outcome.' If we define normal haemoglobin as being greater than 115 g/l for women and greater than 125 g/l for men, then there is no evidence in the literature to justify maintaining such high concentrations by the use of RBC transfusions in any anaemic patient. There may, however, be some debate about adopting a transfusion threshold of 100 g/l, which is well below 'normal'. transfusion threshold would, obviously, reduce the number of allogeneic RBCs transfused. It is our goal to impress upon the reader that transfusing to a level equal to or greater than 100 g/l does not improve mortality and other clinically important outcomes in a critical care setting. We first explore the reasons why a reduction in the total number of allogeneic blood transfusions would be beneficial. Second, we examine the current evidence for using a lower transfusion strategy, specifically that employed in the Transfusion Requirements In Critical Care (TRICC) trial. RBC transfusions have inherent risks that may be categorized as follows [11] [12] [13] [14] [15] : transfusion-transmitted infections; immune-related reactions (acute or delayed haemolytic reactions, febrile, allergic, anaphylactic reactions and graft-versus-host disease); and nonimmunerelated reactions (fluid overload, hypothermia, electrolyte toxicity and iron overload). Major improvements in donor screening procedures and laboratory testing have dramatically improved the safety of the blood supply [16] . Currently, the risk of transmitting an infectious agent through blood transfusion ranges from 1:100,000 for hepatitis B virus to 1:1,000,000 for HIV (Canadian Blood Services, personal communication, 2000). The most important threats to the blood supply remain new and unknown pathogens. More recently, concern has focused on the potential transmission of prions through RBCs. Also, infectious agents with long latency periods pose particular risks to young individuals who require RBCs, such as multiple trauma victims. The risk : benefit ratio for a 24-year-old trauma victim with a 50-year life expectancy differs markedly from that for a person aged 80 years who is undergoing coronary artery bypass surgery. In summary, because there is a risk of transmitting diseases through the blood supply, we should always strive to use RBCs according to the best available evidence in order to ensure that we do more good than harm to our patients. It is a long-standing observation [17] [18] [19] [20] [21] that blood transfusions are associated with immune suppression. This clinical phenomenon was first observed in renal transplant patients who had received blood transfusions while on dialysis before the transplant [22] , and has been observed repeatedly in transplant centres around the world [23, 24] . Recently, Opelz et al [25] reported a multicentre clinical trial in which all renal allograft recipients received modern immunosuppressive regimens. Those patients who were allocated to receive three allogeneic RBC units before renal transplant had a 1-year graft survival rate of 90%, as compared with 82% for patients who were not transfused (P = 0.02). These data suggest that there are long-term immunosuppressive effects following transfusion of nonleukocyte-reduced allogeneic RBCs. A large number of studies [26] [27] [28] [29] [30] [31] [32] [33] [34] have also suggested that allogeneic transfusions accelerate cancer growth, perhaps due to altered immune surveillance. These altered immune responses after allogeneic RBC transfusions may also predispose critically ill transfusion recipients to nosocomial infections [35] [36] [37] [38] [39] [40] and increased rates of multiplesystem organ failure [41] , which may ultimately result in higher mortality rates. However, most studies that examined the association between cancer recurrence and postoperative infection after transfusion [42, 43] only provided weak causal inferences because of poor study design and the lack of independence between allogeneic RBC transfusions and the potential complication. A recent meta-analysis [44] combined the results from seven RCTs, and was unable to detect clinically important decreases in mortality and postoperative infections. We added the results of a new RCT by van de Watering et al [45] to the above meta-analysis. The relative risk for allcause mortality was 1.05 (95% confidence interval 0.88-1.25), and was 1.10 (95% confidence interval 0.85-1.43) for postoperative infections. However, this meta-analysis excluded two positive RCTs [40, 46] because of the significant statistical heterogeneity introduced by these two studies. If all available RCTs are combined, ignoring heterogeneity, then the relative risk difference for postoperative infections across all studies is 1.60 (95% confidence interval 1.00-2.56; P = 0.05). Thus, the available evidence suggests that universal prestorage leukoreduction could have clinical effects that range from none to decreasing rates of infection by as much as 50% in high-risk patients. In summary, despite convincing laboratory evidence and some supportive clinical studies, the clinical significance of the immunosuppressive effects of allogeneic RBC transfusions have not been clearly established [47] . More importantly, the impact of a leukoreduction programme has not been studied in a large population of patients who are expected to have significant exposure to allogeneic RBCs. The majority of complications from allogeneic RBC transfusion, however, are no more frequent in the intensive care setting than in other patient populations, with the possible exception of pulmonary oedema, hypothermia and electrolyte disturbance. Hypothermia and electrolyte disturbances occur most frequently with massive transfusions. In critically ill patients, the optimal effective circulatory volume may be difficult to determine, and as a consequence pulmonary oedema may be a much more frequent complication of RBC transfusion than in other patient populations. This may explain the significantly higher rate of pulmonary oedema in patients transfused using a threshold of 100 g/l (5.3% in the restrictive transfusion group versus 10.7% in the liberal transfusion group; P < 0.01), as reported in the TRICC trial [10] . As an alternative explanation, the more frequent use of RBCs might have resulted in more frequent episodes of transfusion-related acute lung injury in the liberal strategy group (7.7% in the restrictive strategy versus 11.4% in the liberal strategy; P = 0.06), as reported in the TRICC trial. Clinical evidence is also insufficient to definitively establish a correlation between the age of RBCs being transfused and patient mortality; however, laboratory evidence has shown many storage-related changes that may result in impairment of blood flow and oxygen delivery at the microcirculatory level. Marik et al [48] demonstrated an association between a fall in gastric intramucosal pH and transfusion of RBCs stored for longer than 15 days. In addition, there is ample laboratory evidence that prolonged RBC storage adversely affects RBCs, potentially results in the generation of cytokines, and alters host immune function. In another study, Fitzgerald et al [49] , using an animal model of transfusion, consistently observed a lack of efficacy of transfused, stored rat blood to improve tissue oxygen consumption as compared with fresh cells or other blood substitutes. Three retrospective clinical studies tested the association between the age of transfused blood and duration of stay in the intensive care unit (ICU) [50] and mortality [51, 52] . Martin et al [50] observed a statistically significant association between the transfusion of aged blood (>14 days old) and increased duration of ICU stay (P = 0.003) in 698 critically ill patients. In patients who received a transfusion, aged RBCs was the only predictor of duration of stay (P < 0.0001). In survivors, only median age of blood was predictive of duration of stay (P < 0.0001). Purdy et al [51] demonstrated a negative correlation (r = -0.73) between the proportion of RBC units of a given age transfused to survivors and increasing age of RBCs in patients admitted to the ICU with a diagnosis of severe sepsis (n = 31). Those investigators also noted that these latter units were more likely to be older. A recently reported study by Vamvakas and Carven [52] evaluated the effect of duration of RBC storage on postoperative pneumonia in 416 consecutive patients undergoing coronary artery bypass grafting. Those investigators noted an adjusted increase of 1% in the risk of postoperative pneumonia per day of average increase in the duration of RBC storage (P < 0.005) in transfused patients. Each of these three studies also noted that patients who received a large number of RBC units had a higher mortality. Although these risks are relatively small when viewed collectively, they become significant when one considers that 25% of all critically ill patients in Canada are transfused during their ICU stay [1] . Until recently, physicians have depended on clinical judgement when deciding at what haemoglobin level to transfuse a critically ill patient. As a result, significant variation has been shown to exist in transfusion practice among Canadian critical care physicians [53] , which is due largely to a lack of published data on the subject. An arbitrary haemoglobin level of 100 g/l has historically been used as a threshold to transfuse critically ill patients. Six observational studies investigated the importance of anaemia on transfusion practices in various settings. Of these, three large cohort studies, which were performed in different patient populations (intensive care [1] , coronary artery bypass surgery [54] and hip fractures [55]), reached different conclusions. RBC transfusions in particular improved outcome in critically ill patients with cardiovascular disease, but increased the risk of myocardial infarction in coronary artery bypass surgery patients. Transfusion had no impact on short-term or long-term mortality in hip-fracture patients. Three smaller studies [56] [57] [58] evaluated the relationship between anaemia and adverse outcomes in vascular disease patients. Although increased numbers of ischaemic events were observed in anaemic patients, the validity of these studies is uncertain, given that the decision to transfuse a patient was often correlated with illness burden of the patient. It is also possible that comorbidity was not adequately adjusted for in those studies. Transfusion thresholds were compared in 10 randomized clinical trials [10, [59] [60] [61] [62] [63] [64] [65] [66] [67] . Although the clinical settings varied, each trial randomized patients to be transfused on the basis of a 'conservative' or a 'liberal' strategy. The definitions of conservative and liberal strategies varied, and actually overlapped between studies. Of these 10 trials, only three included more than 100 patients and only one trial evaluated the impact of transfusion on symptoms. In the first trial of patients undergoing elective coronary artery bypass surgery [65] , the differences between perioperative haemoglobin levels were small, event rates were very low, and there were no differences in any outcome. In the second trial [67] , patients undergoing knee arthroplasty were randomly assigned to receive autologous blood transfusion immediately after surgery or to receive autologous blood if haemoglobin level fell below 9 g/dl [67] . Again, no differences in outcome were observed. The third trial of hip fracture patients undergoing surgical repair [64] found no differences in outcomes; however, five deaths were recorded at 60 days after surgery in the symptomatic group, and two deaths occurred in the 10 g/dl group. The numbers of patients in these trials were too small to evaluate the effect of lower transfusion triggers on clinically important outcomes such as mortality, morbidity and functional status. In 1999, Hebert et al [10] reported the results of the TRICC trial. Patients (n = 838) were randomized either to a restrictive strategy (haemoglobin concentration maintained between 70 and 90 g/l, with a trigger set at 70 g/l) or to a liberal strategy (haemoglobin concentration maintained between 100 and 120 g/l, with a trigger at 100 g/l). To date, the TRICC trial is the only large study that has investigated these parameters. The groups were comparable at baseline. The average daily haemoglobin concentration ranged from 85 ± 7.2 g/l in the restrictive group to 107 ± 7.3 g/l in the liberal group (P < 0.01). The average number of transfusions was reduced by 52% in the restrictive group (2.6 ± 4.1 versus 5.6 ± 5.3 RBCs/patient; P < 0.01). Cardiac events, primarily pulmonary oedema and myocardial infarction, were more frequent in the liberal strategy (P < 0.01; Table 1 ). On examination of composite outcomes, the number of patients with multiorgan failure was found to be substantially increased in both groups, with the results being marginally better in the restrictive strategy group (20.6% versus 26.0%; P = 0.07; Table 2 ). Overall, the restrictive transfusion group showed a lower 30-day mortality (18.7% versus 23.3%; P = 0.11; Fig. 1 ). Kaplan-Meier survival curves, however, were significantly different in the subgroup of patients with an Acute Physiology and Chronic Health Evaluation II score of 20 or less (P = 0.02; Fig. 2 ). In addition, although 60-day mortality (22.8% versus 26.5%; P = 0.23) and ICU mortality (13.9% versus 16.2%; P = 0.29) were not statistically significant, they did show a consistent trend in terms of absolute values that favoured the restrictive strategy. The key observation from the TRICC trial is not that the restrictive strategy is better, but rather that it is at worst equivalent to the liberal strategy and at best superior to the liberal strategy. At this juncture, preclinical and clinical evidence support the adoption of a more restrictive transfusion strategy in most critically ill patients. However, there remain divergent views regarding the risks and benefits of treating anaemia in patients with cardiovascular disease. Laboratory-based studies [68, 69] suggest that patients with cardiovascular disease may require higher haemoglobin concentrations to maintain oxygen delivery in partially occluded or diseased coronary arteries. Studies to demonstrate how anaemia affects contractile function of the left ventricle have rarely shown important effects above haemoglobin concentrations of 70 g/l. Indeed, it is more important to address the underlying pathophysiological causes of the acute coronary syndrome with proven therapy such as aspirin and β-blockers, rather than treating mild-to-moderate anaemia as an initial step. If the effects of RBC transfusion were either limited or increased then there would be no debate; however, the use of allogeneic RBCs has been shown to be associated with immunomodulation [12, 47] and/or alteration in the delivery of oxygen in the microcirculation [70, 71] , resulting in increased rates of infections and organ failure. Few clinical studies have attempted to elucidate the risk : benefit ratio of anaemia and transfusion in cardiac patients. Two small RCTs [62, 72] examined transfusion practice in patients undergoing coronary artery bypass grafting, and concluded that a conservative approach to the administration of RBCs may be safe. However, two recent cohort studies suggested that anaemia may increase the risk of mortality in critical illness [73] and following surgery in patients with cardiovascular disease [74] . There were 418 and 420 patients in the restrictive and liberal transfusion groups, respectively. *Difference calculated by subtracting mean values of restrictive group from those of liberal group. † Three patients were lost to 60-day mortality rate; therefore n = 835. ‡ Nonsurvivors are considered to have all organs failing on date of death. § Changes in MOD score from baseline, while also incorporating adjustment for death. Data from Hébert et al [10] . In a study of Jehovah's Witnesses (a group that refuses RBC transfusion on religious grounds) undergoing surgical procedures [74] , it was noted that mortality was significantly increased in patients with cardiac disease after a decrease in haemoglobin levels from 100-110 g/l to 60-69 g/l. In that study, patients with no cardiac disease and similar changes in haemoglobin levels showed no increase in mortality, which is in accordance with the results of the TRICC trial [10] . In the study by Hébert et al. [73] of 4470 critically ill patients, a correlation between Critical Care Vol 5 No 2 Alvarez et al [10] . Kaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (all patients). Data from Hébert et al [10] . Kaplan-Meier estimates of survival in the 30 days after admission to the ICU in the restrictive and liberal transfusion strategy groups (patients with APACHE II score ≤20). Data from Hébert et al [10] . anaemia and mortality rates was observed. Those investigators also found that the risk of anaemia appeared to decrease with RBC transfusion in patients with cardiac disease. In patients with cardiac disease, mortality increased when haemoglobin concentrations were below 95 g/l, as compared with anaemic patients with other diagnoses (55% versus 42%; P = 0.09). In the subgroup of patients with cardiac disease, increasing haemoglobin values in anaemic patients was associated with improved survival (odds ratio 0.80 for each 10 g/l increase; P = 0.012). One possible explanation for the discrepancy between the TRICC trial and this observational study may be that the attending physicians who recruited patients into the study did not enter those patients who were considered to have severe cardiac disease. Hébert et al. [73] sought to examine further whether a restrictive transfusion strategy was at least as effective as a liberal strategy in critically ill patients with cardiac disease. In the subgroup of patients with cardiovascular disease from the TRICC trial, those investigators suggested that most haemodynamically stable critically ill patients with cardiovascular disease may be transfused when haemoglobin concentrations fall below 70 g/l, and that the hemoglobin concentration should be maintained between 70 and 90 g/l. Average daily haemoglobin concentrations were 85 ± 6.2 g/l in the restrictive transfusion group and 103 ± 6.7 g/l in the liberal transfusion group (P < 0.01). In the 357 patients with cardiovascular disease, the 30-day mortality rate was 23% in the restrictive transfusion group versus 23% in the liberal group (95% confidence interval of the difference -8.4% to 9.1%; P = 1.00). Other mortality rates, including 60-day (26% versus 27%; P = 0.90), ICU (19% versus 16%; P = 0.49) and hospital mortality (27% versus 28%; P = 0.81), were not significantly different between groups. Kaplan-Meier survival curves comparing time to death demonstrated similar trends in the two groups ( Fig. 3 ; P = 0.98). The multiple organ dysfunction (MOD) scores, during the entire study period, were also not significantly different between groups (8.6 ± 4.9 versus 9.0 ± 4.4; P = 0.40), but the change in MOD score from baseline values was significantly lower in the restrictive group than in the liberal group (0.2 ± 4.2 versus 1.3 ± 4.4; P = 0.02). Combined measures of morbidity and mortality, or composite outcomes, were also examined. When all patients who died were given a score of 24, the total MOD score between groups was not different (P = 0.39), or were the changes in MOD scores significantly different from baseline (2.7 ± 6.9 versus 4.0 ± 7.3; P = 0.08). Among the specific subset of cardiac patients with ischaemic heart disease (n = 257), there were no discernible differences in 30-day and 60-day as well as ICU mortality rates. However, a nonsignificant (P = 0.3) decrease in overall survival rate in the restrictive group was noted in those patients with confirmed ischaemic heart disease, severe peripheral vascular disease or severe comorbid cardiac disease (Fig. 4) . In conclusion, a restrictive RBC transfusion strategy generally appears to be safe in most critically ill patients with cardiovascular disease, with the possible exception of patients experiencing acute myocardial infarction or unstable angina. Survival over 30 days in patients with ischemic heart disease in the restrictive and liberal allogeneic RBC transfusion strategy groups. This graph illustrates Kaplan-Meier survival curves for all patients with ischemic heart disease in both study groups. There is no difference in mortality in patients in the restrictive group (dashed line) as compared to the liberal group (solid line) (P = 0.30). The need to reduce the amount of allogeneic blood transfusions in order to reduce the associated risks has been firmly established. RBCs are associated with clinically important immune suppression, and stored RBCs have been shown to cause adverse microcirculatory effects that result in increased organ failure. The question for some time has been whether critically ill patients are able to tolerate lower levels of haemoglobin without deleterious effects, thus reducing the amount of exposure to allogeneic transfusions. In the only large RCT, Hébert et al [10] established that there was no difference in mortality rates between restrictive and liberal transfusion strategies in noncardiac, critically ill patients. Although those investigators were able to show convincing trends that the liberal strategy may in fact be deleterious in terms of absolute values, statistical significance was not achieved. However, the fact that no difference between the two strategies was achieved is of great importance, because this means that the total number of transfusions can be reduced by approximately half without any impact on mortality. In addition, these findings are easily put into clinical practice. Although many questions remain, the TRICC trial and many laboratory and clinical studies have established that transfusing to normal haemoglobin concentrations does not improve organ failure and mortality in the critically ill patient. As such, a restrictive transfusion strategy will reduce exposure to allogeneic transfusions, result in more efficient use of RBCs, save blood overall, and decrease health care costs."
7
"The 21st International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, 20-23 March 2001"
"The 21st International Symposium on Intensive Care and Emergency Medicine was dominated by the results of recent clinical trials in sepsis and acute respiratory distress syndrome (ARDS). The promise of extracorporeal liver replacement therapy and noninvasive ventilation were other areas of interest. Ethical issues also received attention. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of a high standard. The meeting was marked by a sense of renewed enthusiasm that positive progress is occurring in intensive care medicine."
"This year's symposium was dominated by the results of recent clinical trials. After 10 years of 'magic bullet' trials in sepsis, a number of successful therapeutic options are now emerging. In addition, recent advances in our understanding of the soup of mediators observed in sepsis offer yet more tantalizing targets for new therapies. In contrast, the eagerly awaited results from Italy of the prone positioning trial in ARDS were disheartening. The epidemiology of both sepsis and ARDS, and their impact on clinical studies and the future provision of critical care were also hot topics. The era of extracorporeal liver replacement therapy is upon us, with considerable early promise and the probability of wide availability. Finally, as always, ethics remained an area of interest. This report summarizes and discusses the presentations on the above topics. Angus (Pittsburgh, PA, USA) presented his group's work on the epidemiology of sepsis in the USA (accepted for publication in Critical Care Medicine). They developed a method for identifying hospitalized patients with sepsis based on ICD9 criteria, the most widely recorded coding system used in US hospitals. Prospective testing of the method found it to be both sensitive and reliable. They then applied it to a representative selection of US hospitals. Their results indicated that about 50% of intensive care unit (ICU) patients have systemic inflammatory response syndrome, and that approximately 20% of these progress to severe sepsis. Mortality for severe sepsis was greater than 30%. Demographically, those at the extremes of age represent the most at-risk groups, in whom the mortality is also the highest. These data provides yet another reminder that the increasing demands on health care resources caused by the ageing population is predicted to exceed intensive care provision within the next The 21st International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, 20-23 March 2001 10-20 years. Finally, those investigators found a striking demographic peak in patients aged 20-30 years, which they attributed largely to human immunodeficiency virus. The long-standing debate between the two schools of sepsis theory -microcirculatory dys-autoregulation versus cellular dysfunction -shows signs of resolution. New techniques for studying tissue oxygen tension, presented by Ince (Amsterdam, The Netherlands), provide more evidence that microcirculatory dys-autoregulation results in significant shunting. This occurs predominantly in the submucosal and serosal portions of organs, and is an early event. These studies show that the macroscopic restoration of global oxygen delivery fails to improve oxygen consumption as the mucosa becomes hyperoxic, whereas the submucosa and serosa remain hypoxic. Somewhat counterintuitively, this can be reversed in the face of resistant hypotension with vasodilators, at least in animal models. The cellular dysfunction camp, although still somewhat doubtful as to the importance of these microcirculatory findings, have now clearly established that their championed mechanism of mitochondrial failure is a late but crucial event in the evolution of sepsis. Fink (Pittsburgh, PA, USA) presented evidence that mitochondrial failure in septic cells is triggered by the activation of the enzyme poly-adenosine diphosphate ribose polymerase [1] . This enzyme represents a significant target for novel therapies, which are apparently already in development. The debate regarding the toxicity of oxygen and the formation of free radicals continues despite the absence of demonstrated effectiveness of scavenging therapies, and is a testament to the incomplete understanding of this area. The round-table conference preceding this year's symposium concentrated on distilling current knowledge on the microscopic events in critically ill patients into an explanation of the macroscopic multiorgan failure that is so commonly encountered. The conclusions of the conference appeared to relate mostly to future directions for research, in particular the study of organ-organ interactions. Marshall (Toronto, Canada) proposed the development of an alternative to the much-maligned physiological scoring systems, based on the staging systems widely used in the field of oncology. He proposed that mediator levels, in addition to physiological variables, will soon be used usefully to characterize septic patients. He also suggested that, in the light of the recent successful mediator trials in sepsis, future therapies will be directed in a manner analogous to the control of glucose in diabetic patients. The natural anticoagulants antithrombin III (AT III), tissue factor pathway inhibitor (TFPI) and activated protein C (APC), and the cytokine tumour necrosis factor (TNF)-α are the latest inflammatory mediators to be targeted in large multicentre clinical trials in an attempt to improve the current dismal outcome for patients with severe sepsis. The KyberSept AT III study recruited over 2300 patients from 200 centres, with high Simplified Acute Physiology Scale scores (median 50), and a mortality of nearly 40% [2] . Unfortunately, no overall benefit was shown between AT III and placebo, although results were more encouraging in an analysis of the subgroup of patients who received AT III but no heparin, which is known to inhibit AT III. Interestingly, improvements in quality of life scores were seen in survivors who received AT III in comparison to those who received placebo, suggesting that morbidity may be reduced, although again this was an analysis of a subgroup. Patients in the AT III group who received concomitant heparin had a significantly higher incidence of bleeding events, and outcome worsened as the dose of heparin increased. Explanations for the failure of this study included the inhibitory effects of heparin and the failure to achieve AT III activity levels of greater that 200% from baseline in the treatment population, a level established as required for therapeutic benefit in phase II trials. Phase II clinical trial results using TFPI (TFPI n = 141, placebo n = 69; unpublished data) show a mortality benefit in the sicker sepsis patients who already have coagulation problems. Results of the phase III multicentre study are expected to be presented at the 22nd International Symposium on Intensive Care and Emergency Medicine, in Brussels in 2002. Human trials of various anti-TNF-α formulations have been variable to date, and include North American sepsis trial (NORASEPT) I [3] , International sepsis trial (INTERSEPT) [4] and NORASEPT II [5] . Possible reasons have included a lack of biological activity of the anti-TNF-α formulation studied, inappropriate timing of therapy, redundancy of proinflammatory mediators and hetereogeneity of patient populations. The Monoclonal Anti-TNF, A Randomized controlled Sepsis Trial (MONARCS) study used a different anti-TNF-α formulation (F[ab′]2 fragment of a murine monoclonal antibody to human TNF-α), and stratified patients based on demonstrable abnormalities in immunological pathways (highly elevated interleukin-6 levels -a circulating cytokine that is induced by TNF-α). Unpublished results revealed 28-day mortality rates of 44 and 48% in the anti-TNF-α and placebo groups, respectively, in those patients who had high interleukin-6 levels on recruitment to the study (n = 488 anti-TNF-α, n = 510 placebo). This represented a relative mortality reduction of 14%. Relative mortality reduction in all patients (n = 1305 anti-TNF-α, n = 1329 placebo), independent of baseline interleukin-6 levels, was only 10%. The Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study is hot off the press [6] , and presentation of the results at the congress allowed those of us who still carry the unopened New England Journal of Medicine issue in our briefcases to catch up! A total of 164 sites from 11 countries recruited 1690 patients with severe sepsis, before the trial was prematurely stopped following the second safety analysis. Twenty-eight-day all-cause mortality rates for placebo and APC were 31 and 25% respectively, with a relative risk reduction of 19%. Resolution of cardiovascular and respiratory function was more rapid in survivors who received APC, although ICU and hospital stay did not differ. There was a trend towards an increase in serious bleeding events in the APC group (3% APC versus 2% placebo), but these events were primarily due to trauma or instrumentation. Although this is an exciting breakthrough, we all recognize that when APC reaches the market place it will seriously stretch ICU finances, especially because there appear to be other mediators on the horizon that we will be encouraged to use, in combination, to fight the inflammatory 'soup'. Two opposing epidemiological views of ARDS were presented by Lemaire (Créteil, France) and Evans (London, UK). Broad agreement does seem to exist as to the incidence of this condition, which is in the order of 10/100,000, although there is significant variation between countries. It was argued that this variation results from the availability of ventilated beds, with higher incidences apparent in countries with greater provision, emphasizing that this condition can be considered the result of intensive care intervention or, as one speaker put it, 'physician-induced lung injury'. Early results from the Acute Lung Injury Verification of European Epidemiology (ALIVE) study (unpublished data), sponsored by the European Society of Intensive Care Medicine, are at odds with recent trial findings. The ALIVE study, which included over 6000 patients surveyed in 1998, found a 50-60% 28-day mortality, which compares to only 20-30% in the control groups of recent trials. Pneumonia was the commonest cause, responsible for 50% of cases, with sepsis identified as the cause in a further 20-30%. Astonishingly, this study found the ratio of arterial oxygen tension to fractional inspired oxygen at ICU admission was highly predictive of mortality, despite continuing controversy regarding this measurement. A diverse range of views were presented from the Third International Consensus Conference on ARDS (unpublished data), held in Barcelona late last year. The decision as to how to change the defining criteria for this condition remains unresolved. The debates surrounding chest X-ray criteria, the use of the ratio of arterial oxygen tension to fractional inspired oxygen, and the level/utility of pulmonary artery wedge pressure measurements continue. In addition, a debate has arisen as to whether ARDS can be a unilateral process, and whether it can coexist with cardiac failure. There appears to be increasing recognition that ARDS represents only a small subset of patients with acute lung failure (approximately 30%). Surprisingly little is known about the remainder of this larger group. In contrast to the ALIVE study, several centres have reported their 28-day mortality at 40%, which represents an improvement from the 60% of 10 years ago. However, it was argued that a 28-day follow-up period is too short for clinical trials, as the long-term quality of life for patients with ARDS is poor compared with that of critically ill patients without this condition. Results suggest that the recovery of lung function is good overall, but is dependent on severity. Treatment recommendations include the universal adoption of the US National Institutes of Health protective lung ventilation strategy [7] . There was general agreement that recruitment manoeuvres are beneficial, but how and when to employ them remains controversial. Rouby (Paris, France) put forward a new classification for ARDS based on computed tomography findings. He observed that patients can be split into three groups, depending on the appearance of the upper lobes. In group 1 the upper lobes are normal, and positive end-expiratory pressure (PEEP) is of little benefit and results in significant over-distension. Survival in this group is approximately 60%. In group 2 the upper lobes are abnormal, PEEP is of dramatic benefit, but survival is only approximately 25%. In group 3 there are mixed/patchy abnormalities, the effects of PEEP are less predictable, but, as in group 1, survival is approximately 60%. Gattinoni (Milan, Italy) presented the results of the longawaited Italian multicentre randomized controlled trial of prone positioning in ARDS (unpublished data). This trial was terminated after 3 years despite having only recruited 304 patients; enrollment of 750 patients was originally planned, in order to achieve sufficient statistical power. At trial outset, recruitment was encumbered by the lack of familiarity with and scepticism regarding this procedure in many of the centres. However, by the end of the study many participants were unwilling to enter patients into the trial, because they felt it unethical to deny them this intervention. The trial protocol resulted in patients in the treatment group being prone for an average of only 7 out of 24 h for a 10-day period. Overall there was no difference in mortality between the control and treatment groups at day 10, time of ICU discharge, or at 6 months. Interestingly, analysis of subgroups revealed a significant difference in the outcome at 10 days for patients with the most severe disease, although this disappeared by ICU discharge. In retrospect, the design of this ambitious trial was flawed by its failure to establish the optimal utilization of this manoeuvre. The opening session reported that we are making progress in supporting the failing liver (Wendon, London, UK). Current optimism should probably be limited to extracorporeal methods, because the molecular adsorbent recirculating system (essentially extracorporeal albumin dialysis) has been shown to have beneficial clinical effects as well as improved survival in two small randomized controlled trials [8, 9] . The equipment is familiar to all those who use dialytic therapies, and we will undoubtedly hear more about this system in the next few years. The slide of a patient reading the newspaper through a transparent helmet, while receiving noninvasive ventilation (NIV) resembled pictures of a NASA astronaut! However, it was reported to be well tolerated for prolonged periods, and significantly reduces the complications associated with NIV (pressure areas, tolerance of mask). The recent Consensus Conference [10] examined weaning aspects of NIV and emphasized the reduced weaning time and avoidance of reintubation, but called for more randomized controlled trials. Finally, although continuous positive airway pressure has been shown to be beneficial in pulmonary oedema, caution is still advised with the use of bilevel positive airway pressure because of the reporting of myocardial infarction in several studies. However, the groups studied were unmatched and starting points were different, so conclusions should not be drawn until randomized controlled trial results are available in this area. This was a well-attended session, which, according to Levy (Providence, RI, USA), was in complete contrast to the interest shown in the USA for the subject. Although there were few new data in the session, the emphasis on a strategy for lawsuits was welcome. Suggestions included statements from scientific societies at a national and international level, open reporting in medical files of decisions to withdraw or withhold treatment, and family involvement in decision making that will ultimately involve better media education. The last day of this year's symposium was sadly abandoned by many due to the Belgian rail strike. Despite this, the usual convivial atmosphere, both in and around the congress, was as abundant as ever. Overall, the 'state of the art' lectures, pro/con debates, seminars and tutorials were of the usual high standard, although, yet again, access to many of the symposium's venues was limited by the lack of capacity of the secondary rooms. The 21st International Symposium was marked by a sense of renewed enthusiasm that real positive progress is occurring at the coal face of intensive care."
8
"Heme oxygenase-1 and carbon monoxide in pulmonary medicine"
"Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states."
"The heme oxygenase-1/carbon monoxide (HO-1/CO) system has recently seen an explosion of research interest due to its newly discovered physiological effects. This metabolic pathway, first characterized by Tenhunen et al. [1, 2] , has only recently revealed its surprising cytoprotective properties [3, 4] . Research in HO-1/CO now embraces the entire field of medicine where reactive oxygen/nitrogen species, inflammation, growth control, and apoptosis represent important pathophysiological mechanisms [3] [4] [5] [6] . Indeed, the number of publications in recent years concerning HO-1 has increased exponentially, while the list of diseases and physiological responses associated with changes in HO-1 continues to expand [5] . Until now, relatively few studies have addressed the role of HO-1/CO in pulmonary medicine. Several investigators have focused on the diagnostic application of the HO-1/CO system, by measuring exhaled CO (E-CO) in various pathological pulmonary conditions, such as asthma or chronic obstructive pulmonary disease (COPD) [7] . In another experimental approach, investigators have examined the expression of HO-1 in lung tissue from healthy or diseased subjects [8, 9] . This review will highlight the actions of HO-1/CO in the context of heme degradation have antioxidant properties [18, 19] . The liberated heme iron undergoes detoxification either by extracellular efflux or by sequestration into ferritin, an intracellular iron-storage molecule with potential cytoprotective function [20] [21] [22] [23] . Of the three known isoforms of HO (HO-1, HO-2, and HO-3), only HO-1 responds to xenobiotic induction [24] [25] [26] [27] . Constitutively expressed in many tissues, HO-2 occurs at high levels in nervous and vascular tissues, and may respond to regulation by glucocorticoids [25, 28, 29] . HO-1 and HO-2 differ in genetic origin, in primary structure, in molecular weight, and in their substrate and kinetic parameters [25, 26] . HO-3 displays a high sequence homology with HO-2 but has little enzymatic activity [27] . This review will focus on the inducible, HO-1, form. In addition to the physiological substrate heme, HO-1 responds to induction by a wide variety of stimuli associated with oxidative stress. Such inducing agents include hypoxia, hyperoxia, cytokines, nitric oxide (NO), heavy metals, ultraviolet-A (320-380 nm) radiation, heat shock, shear stress, hydrogen peroxide, and thiol (-SH)-reactive substances [3] . The multiplicity of toxic inducers suggest that HO-1 may function as a critical cytoprotective molecule [3, 4] . Many studies have suggested that HO-1 acts as an inducible defense against oxidative stress, in models of inflammation, ischemia-reperfusion, hypoxia, and hyperoxia-mediated injury (reviewed in [3] ). The mechanisms by which HO-1 can mediate cytoprotection are still poorly understood. All three products of the HO reaction potentially participate in cellular defense, of which the gaseous molecule CO has recently received the most attention [30, 31] . The administration of CO at low concentrations can compensate for the protective effects of HO-1 in the presence of competitive inhibitors of HO-1 activity [32] [33] [34] . While HO-1 gene transfer confers protection against oxidative stress in a number of systems, clearly not all studies support a beneficial role for HO-1 expression. Cell-culture studies have suggested that the protective effects of HO-1 overexpression fall within a critical range, such that the excess production of HO-1 or HO-2 may be counterprotective due to a transient excess of reactive iron generated during active heme metabolism [35, 36] . Thus, an important caveat of comparative studies on the therapeutic effects of CO administration versus HO-1 gene delivery arises from the fact that the latter approach, in addition to producing CO, may have profound effects on intracellular iron metabolism. HO-1 expression is primarily regulated at the transcriptional level. Genetic analyses have revealed two enhancer sequences (E1, E2) in the murine HO-1 gene located at -4 kb (E1) and -10 kbp (E2) of the transcriptional start site [37, 38] . These enhancers mediate the induction of HO-1 by many agents, including heavy metals, phorbol esters, endotoxin, oxidants, and heme. E1 and E2 contain repeated stress-responsive elements, which consist of overlapping binding sites for transcription factors including activator protein-1 (AP-1), v-Maf oncoprotein, and the cap'n'collar/basic-leucine zipper family of proteins (CNC-bZIP), of which Nrf2 (NF-E2-related factor) may play a critical role in HO-1 transcription [39] . The promoter region of HO-1 also contains potential binding sites for nuclear factor κB (NF-κB), though the functional significance of these are not clear [40] . Both NF-κB and AP-1 have been identified as regulatory elements responsive to oxidative cellular stress [40, 41] . In response to hyperoxic stress, AP-1 factors mediated the induction of HO-1 in cooperation with signal-transducer and activator of transcription (STAT) proteins [41] . Furthermore, a distinct hypoxia-response element (HRE), which mediates the HO-1 response to hypoxia, represents a binding site for the hypoxia-inducible factor-1 (HIF-1) [42] . The toxic properties of CO are well known in the field of pulmonary medicine. This invisible, odorless gas still claims many victims each year by accidental exposure. CO evolves from the combustion of organic materials and is present in smoke and automobile exhaust. The toxic actions of CO relate to its high affinity for hemoglobin (240-fold greater than that of O 2 ). CO replaces O 2 rapidly from hemoglobin, causing tissue hypoxia [43] [44] [45] . At high concentrations, other mechanisms of CO-induced toxicity may include apoptosis, lipid peroxidation, and inhibition of drug metabolism and respiratory enzyme functions [44] . Only recently has it become known that, at very low concentrations, CO participates in many physiological reactions. Where a CO exposure of 10,000 parts per million (ppm) (1% by volume CO in air) is toxic, 100-250 ppm (one hundredth to one fortieth as much) will stimulate the physiological effects without apparent toxicity [4] . The majority of endogenous CO production originates from active heme metabolism (>86%), though a portion may be produced in lipid peroxidation and drug metabolism reactions [46] . Cigarette smoking, still practiced by many lung patients, represents a major source of chronic lowlevel exposure to CO. Inhaled CO initially targets alveolar macrophages and respiratory epithelial cells. The exact mechanisms by which CO acts at the molecular level remain incompletely understood. CO potentially exerts its physiological effects by influencing at least three known pathways (Fig. 2 ). By complexation with the heme moiety of the enzyme, CO activates soluble guanylate cyclase (sGC), stimulating the production of cyclic 3':5'guanosine monophosphate (cGMP) [47] . The sGC/cGMP pathway mediates the effects of CO on vascular relaxation, smooth muscle cell relaxation, bronchodilation, neurotransmission, and the inhibition of platelet aggregation, coagulation, and smooth muscle proliferation [48] [49] [50] [51] . Furthermore, CO may cause vascular relaxation by directly activating calcium-dependent potassium channels [52] [53] [54] . CO potentially influences other intracellular signal transduction pathways. The mitogen-activated protein kinase (MAPK) pathways, which transduce oxidative stress and inflammatory signaling (i.e. response to lipopolysaccharide), may represent an important target Possible mechanism(s) of carbon monoxide action Figure 2 Possible mechanism(s) of carbon monoxide action. Endogenous carbon monoxide (CO) arises principally as a product of heme metabolism, from the action of heme oxygenase enzymes, although a portion may arise from environmental sources such as pharmacological administration or accidental exposure, or other endogenous processes such as drug and lipid metabolism. The vasoregulatory properties of CO, including its effects on cellular proliferation, platelet aggregation, and vasodilation, have been largely ascribed to the stimulation of guanylate cyclase by direct heme binding, leading to the generation of cyclic GMP. The anti-inflammatory properties of CO are associated with the downregulation of proinflammatory cytokine production, dependent on the selective modulation of mitogen-activated protein kinase (MAPK), such as the 38 kilodalton protein (p38MAPK). In addition to these two mechanisms, CO may potentially interact with any hemoprotein target, though the functional consequences of these interactions with respect to cellular signaling remain poorly understood. Anti-Platelet Aggregation Anti-Proliferation ? Inhibition of pro-inflammatory cytokine production Modulation of hemoprotein function of CO action [32, 34, 55, 56 ]. An anti-apoptotic effect of CO and its relation to MAPK has recently been described. The overexpression of HO-1 or the exogenous administration of CO prevented tumor necrosis factor α (TNF-α)induced apoptosis in murine fibroblasts [57] . In endothelial cells, the anti-apoptotic effect of CO depended on the modulation of the p38 (38 kilodalton protein) MAPK pathway [34] . The role of the remaining heme metabolites, (i.e. Fe and biliverdin IXα) in the modulation of apoptosis is currently being investigated and is beyond the scope of this review. Recent studies have reported a potent anti-inflammatory effect of CO, involving the inhibition of proinflammatory cytokine production after endotoxin stimulation, dependent on the modulation of p38 MAPK [32] . The clinical relevance of p38 MAPK lies in the possibility of modulating this pathway in various clinical conditions to downregulate the inflammatory response [58] . Oxidative stress arising from an imbalance between oxidants and antioxidants plays a central role in the pathogenesis of airway disease [59] . In lung tissue, HO-1 expression may occur in respiratory epithelial cells, fibroblasts, endothelial cells, and to a large extent in alveolar macrophages [41, 60, 61] . HO-1 induction in these tissues, in vitro and in vivo, responds to common causes of oxidative stress to the airways, including hyperoxia, hypoxia, endotoxemia, heavy metal exposure, bleomycin, diesel exhaust particles, and allergen exposure [4, 41, 61] . Induction of HO-1 or administration of CO can protect cells from these stressful stimuli [10, 41] . In one of the experiments that best illustrate the protective role of CO in vivo, rats were exposed to hyperoxia (>98% O 2 ) in the absence or presence of CO at low concentration (250 ppm). The CO-treated rats showed increased survival and a diminished inflammatory response to the hyperoxia [11] . As demonstrated in a model of endotoxin-induced inflammation, the protection afforded by CO most likely resulted from the downregulated synthesis of proinflammatory cytokines (i.e. TNF-α, IL-1β) and the upregulation of the anti-inflammatory cytokine interleukin-10 (IL-10) [32] . Furthermore, increases in exhaled CO (E-CO) have been reported in a number of pathological pulmonary conditions, such as unstable asthma, COPD, and infectious lung disease; these increases may reflect increased endogenous HO-1 activity [7] . Elevated carboxyhemoglobin (Hb-CO) levels have also been reported in these same diseases in nonsmoking subjects, where both the E-CO and Hb-CO levels decrease to normal levels in response to therapy [62] . E-CO in humans originates primarily from both systemic heme metabolism, which produces CO in various tissues, and localized (lung) heme metabolism, as a result of the combined action of inducible HO-1 and constitutive HO-2 enzymatic activity. Endogenously produced or inspired CO is eliminated exclusively by respiration [63] . Elevation of E-CO may also reflect an increase in exogenous sources such as smoking or air pollution. In addition to changes in environmental factors, elevations of E-CO in lung diseases may reflect an increase in blood Hb-CO levels in response to systemic inflammation, as well as an increase in pulmonary HO-1 expression in response to local inflammation [9, 62, 64] . The diagnostic value of measuring E-CO remains controversial due to many conflicting reports (i.e. some reports indicate differences in E-CO measurements between disease activity and controls, and some reports do not). The possible explanations for these discrepancies include large differences in patient populations and in the methods used for measuring E-CO, and undefined corrections for background levels of CO. Furthermore, remarkable differences arise between studies in the magnitude of the E-CO levels in the control groups as well as in treated or untreated asthma patients. When active or passive smoking occurs, or in the presence of high background levels of CO, the measurement of E-CO is not particularly useful for monitoring airway inflammation. In patients who smoke, E-CO can be used only to confirm the smoking habit [65, 66] . Comparable to the beginning era of measurements of exhaled NO, a standardization in techniques and agreement on background correction should be reached for E-CO measurements, to allow proper conclusions to be drawn in this area of investigation. Asthma, a form of allergic lung disease, features an accumulation of inflammatory cells and mucus in the airways, associated with bronchoconstriction and a generalized airflow limitation. Inflammation, a key component of asthma, involves multiple cells and mediators where an imbalance in oxidants/antioxidants contributes to cell damage. Several pathways associated with oxidative stress may participate in asthma. For example, the redox-sensitive transcription factors NF-κB and AP-1 control the expression of proinflammatory mediators [59, [67] [68] [69] . In light of the potential protective effects of HO-1/CO on inflammatory processes, the study of HO-1 in asthma has gained popularity. In a mouse model of asthma, HO-1 expression increased in lung tissue in response to ovalbumin aerosol challenge, indicating a role for HO-1 in asthma [70] . In a similar model of aeroallergen-induced asthma in ovalbumin-sensitized mice, exposure to a CO atmosphere resulted in a marked attenuation of eosinophil content in bronchoalveolar lavage fluid (BALF) and downregulation of the proinflammatory cytokine IL-5 [10] . This experiment showed that exogenous CO can inhibit asthmatic responses to allergens in mice. Recent human studies have revealed higher HO-1 expression in the alveolar macrophages and higher E-CO in untreated asthmatic patients than in healthy nonsmoking controls [71, 72] . Patients with exacerbations of asthma and patients who were withdrawn from inhaled steroids showed higher E-CO levels than steroid-treated asthmatics or healthy controls [73] . Higher levels of E-CO may also occur in children with persistent asthma than in healthy controls [74] . E-CO levels may correlate with functional parameters such as peak expiratory flow rate. A low rate in asthma exacerbations correlated with high E-CO, whereas normalization of the rate with oral glucocorticoid treatment resulted in a reduction of E-CO [75] . Furthermore, increased E-CO was associated with greater expression of HO-1 in airway alveolar macrophages obtained by induced sputum in untreated asthmatic patients than in controls. These asthma patients also showed higher bilirubin levels in the induced sputum, indicating higher HO activity [71] . Furthermore, patients with asthma show an increased Hb-CO level at the time of exacerbation, with values decreasing to control levels after oral glucocorticoid treatment [62] . In human asthmatics, E-CO and airway eosinophil counts decreased in response to a one-month treatment with inhaled corticosteroids [73] . In direct contrast to such studies promoting E-CO as a useful noninvasive tool for monitoring airway inflammation, other studies reported no difference in E-CO levels of asthma patients versus healthy controls, or between patients with stable and unstable asthma. In one such report, no further change in E-CO occurred in asthma patients after a one-month treatment of inhaled corticosteroids, despite observed decreases in airway eosinophil content and bronchial responsiveness to metacholine [76] . A recent study accentuates this finding in asthma excerbations, where no decrease in E-CO of children with asthma could be detected after oral prednisolone treatment [77] . In human allergic responses, results on elevation of E-CO are also inconclusive. A clear elevation of E-CO after allergen exposure occurred in patients with asthma during the late response, and during the early response immediately after the inhalation [78] . However, another report showed that no elevation of E-CO occurred in allergen-induced asthma within 48 hours after allergen challenge [79] . Finally, increases in E-CO were measured in allergic rhinitis, correlating with seasonal changes in exposure to allergen (pollen) [80] . Airway inflammation plays an important role in the development of COPD, characterized by the presence of macrophages, neutrophils, and inflammatory mediators such as proteinases, oxidants, and cytokines. Further-more, the inflammatory consequences of chronic microbiological infections may contribute to the progression of the disease. The current paradigm for the pathogenesis of COPD involves imbalances in protease/antiprotease activities and antioxidant/pro-oxidant status. Proteases with tissue-degrading capacity, (i.e. elastases and matrix metalloproteinases), when insufficiently inhibited by antiproteases, can induce tissue damage leading to emphysema. Oxidants that supersede cellular antioxidant defenses can furthermore inactivate antiproteases, cause direct injury to lung tissue, and interfere with the repair of the extracellular matrix. Smoking plays an important role in both hypotheses. Cigarette smoke will act primarily on alveolar macrophages and epithelial cells, which react to this oxidative stress by producing proinflammatory cytokines and chemokines and releasing growth factors. Nevertheless, smoking cannot be the only factor in the development of COPD, since only 15-20% of smokers develop the disease [81, 82] . Exposure to reactive oxygen species (from cigarette smoke or chronic infections) and an imbalance in oxidant/antioxidant status are the main risk factors for the development of COPD. To defend against oxidative stress, cells and tissues contain endogenous antioxidant defense systems, which include millimolar concentrations of the tripeptide glutathione (GSH). A close relation exists between GSH concentration and HO-1, whereby depletion of GSH augments the transcriptional regulation of HO-1 by oxidants, suggesting that the HO-1/CO system acts as a secondary defense against oxidative stress [83] [84] [85] [86] . Accumulating clinical evidence suggests that HO-1/ CO may also play an important part in COPD. Alveolar macrophages, which produce a strong HO-1 response to stimuli, may represent the main source of CO production in the airways [60, 64] . Patients with COPD have displayed higher E-CO than healthy nonsmoking controls [87] . Furthermore, much higher levels of HO-1 have been observed in the airways of smokers than in nonsmokers [64] . Among subjects who formerly smoked, patients with COPD have lower HO-1 expression in alveolar macrophages than healthy subjects [88] . A microsatellite polymorphism that is linked with the development of COPD may occur in the promoter region of HO-1, resulting in a lower production of HO-1 in people who have the polymorphism. Thus, a genetically dependent downregulation of HO-1 expression may arise in subpopulations, possibly linked to increased susceptibility to oxidative stress [89] [90] [91] . Future studies on both genetic predisposition and possible therapeutic modalities will reveal the involvement of the HO-1/CO system in COPD. Cystic fibrosis (CF) involves a deposition of hyperviscous mucus in the airways associated with pulmonary dysfunc-tion and pancreatic insufficiency, which may be accompanied by chronic microbiological infections. E-CO readings were higher in untreated versus oral-steroidtreated CF patients [92] . Furthermore, E-CO increased in patients during exacerbations of CF, correlating to deterioration of the forced expiratory volume in one second (FEV 1 ), with normalization of the E-CO levels after treatment [93] . E-CO levels may correlate with exhaled ethane, a product of lipid peroxidation that serves as an indirect marker of oxidative stress. Both E-CO and exhaled ethane were higher in steroid-treated and untreated CF patients than in healthy controls [94] . E-CO was higher in children with CF than in control patients. In addition to the inflammatory and oxidative stress responses to continuous infectious pressure in these patients, E-CO may possibly respond to hypoxia. E-CO increased further in CF children following an exercise test, and correlated with the degree of oxyhemoglobin desaturation, a finding suggestive of an increased HO-1 expression in CF patients during hypoxic states induced by exercise [95] . In patients with pneumonia, higher Hb-CO levels can be measured at the onset of illness, with values decreasing to control levels after antibiotic treatment [62] . E-CO levels were reported to be higher in lower-respiratory-tract infections and bronchiectasis, with normalization after antibiotic treatment [96, 97] . Furthermore, E-CO levels in upper-respiratory-tract infections were higher than in healthy controls [74, 80] . The relationship between higher measured E-CO in these infectious states and higher Hb-CO levels cannot be concluded from these studies. The role of HO-1 in the development of interstitial lung disease remains undetermined. Comparative immunohistochemical analysis has revealed that lung tissue of control subjects, patients with sarcoidosis, usual interstitial pneumonia, and desquamative interstitial pneumonia, all showed a high expression of HO-1 in the alveolar macrophages but a weak expression in the fibrotic areas [98] . The antiproliferative properties of HO-1 suggest a possible beneficial role in limiting fibrosis; however, this hypothesis is complicated by a newly discovered relation between IL-10 and HO-1. IL-10 produced by bronchial epithelial cells promotes the growth and proliferation of lung fibroblasts [99] . HO-1 expression and CO treatment have been shown to increase the production of IL-10 in macrophages following proinflammatory stimuli [32] . Conversely, IL-10 induces HO-1 production, which is apparently required for the anti-inflammatory action of IL-10 [100] . A recent report clearly shows the suppression of bleomycin-induced pulmonary fibrosis by adenovirus-mediated HO-1 gene transfer and overexpression in C57BL/6 mice, involving the inhibition of apoptotic cell death [101] . Overall, more research is needed to elucidate the mechanisms of HO-1 in interstitial lung disease and its possible therapeutic implications. HO-1 action may be of great importance in solid tumors, an environment that fosters hypoxia, oxidative stress, and neovascularization. HO-1 may have both pro-and antagonistic effects on tumor growth and survival. HO-1 and CO cause growth arrest in cell-culture systems and thus may represent a potential therapeutic modality in modulating tumor growth [16] . The overexpression of HO-1 or administration of CO in mesothelioma and adenocarcinoma mouse models resulted in improved survival (>90%) as well as reduction in tumor size (>50%) [17] . Furthermore, HO-1 expression in oral squamous cell carcinomas can be useful in identifying patients at low risk of lymph node metastasis. High expression of HO-1 was detected in groups without lymph node metastasis in this report [102] . In contrast to growth arrest, HO-1 may protect solid tumors from oxidative stress and hypoxia, possibly by promoting neovascularization. In one study, zinc protoporphyrin, a competitive inhibitor of HO-1 enzyme activity, suppressed tumor growth [103] . CO may represent a critical mediator of the body's adaptive response to hypoxia, a common feature in pulmonary vascular disease [104] . Since CO can modulate vascular tone by inducing cGMP and large, calcium-dependent potassium channels, HO-1 and CO probably play important roles in pulmonary vascular diseases [54] . A NOmediated HO-1 induction occurred in the hepatopulmonary syndrome during cirrhosis, associated with enhancement of vascular relaxation [105] . In portopulmonary hypertension, elevated levels of cGMP and inducible nitric oxide synthase (iNOS) expression in the vascular endothelium, and HO-1 expression in macrophages and bronchial epithelium have been described [106] . In transgenic mice models, ho-1 -/and ho-1 +/+ mice did not differ in their development of pulmonary hypertension following chronic hypoxia treatment, despite the development of right ventricular dilation and right myocardial infarction in ho-1 -/mice [107] . The preinduction of HO-1 protein with chemical inducers, however, prevented the development of pulmonary hypertension in the rat lung as a consequence of chronic hypoxia treatment [108] . Transgenic mice overexpressing HO-1 in the lung were resistant to hypoxia-induced inflammation and hypertension [109] . Further research is needed to elucidate the potential role of HO-1 and CO in primary human lung vascular diseases such as primary pulmonary hypertension. Supplemental oxygen therapy is often used clinically in the treatment of respiratory failure. Exposure to high oxygen tension (hyperoxia) may cause acute and chronic lung injury, by inducing an extensive inflammatory response in the lung that degrades the alveolar-capillary barrier, leading to impaired gas exchange and pulmonary edema [110, 111] . Hyperoxia-induced lung injury causes symptoms in rodents that resemble human acute respiratory distress syndrome [112] . Hyperoxia induced HO-1 expression in adult rats but apparently not in neonatal rats, in which the expression and activities of HO-1 and HO-2 are developmentally upregulated during the prenatal and early postnatal period [113] . Both HO-1 and HO-2 potentially influence pulmonary adaptation to high O 2 levels. In one example, the adenoviral-mediated gene transfer of HO-1 into rat lungs protected against the development of lung apoptosis and inflammation during hyperoxia [114] . In vitro studies showed that the overexpression of HO-1 in lung epithelial cells or rat fetal lung cells caused growth arrest and conferred resistance against hyperoxia-induced cell death [15, 16] . An oxygen-tolerant variant of hamster fibroblasts that moderately overexpressed HO-1 in comparison with the parent line resisted oxygen toxicity in vitro. The treatment of this oxygen-tolerant strain with HO-1 antisense oligonucleotides reduced the resistance to hyperoxia. In contrast, additional, vector-mediated, HO-1 expression did not further increase oxygen tolerance in this model [115] . In vivo studies with gene-deleted mouse strains have provided much information on the roles of HO-1 and HO-2 in oxygen tolerance. Dennery et al. demonstrated that heme oxygenase-2 knockout mice (ho-2 -/-) were more sensitive to the lethal effects of hyperoxia than wild-type mice [116] . In addition to the absence of HO-2 expression, however, the mice displayed a compensatory increase in HO-1 protein expression, and higher total lung HO activity. Thus, in this model, the combination of HO-2 deletion and HO-1 overexpression resulted in a hyperoxiasensitive phenotype. Recent studies of Dennery et al. have shown that HO-1-deleted (ho-1 -/-) mice were more resistant to the lethal effects of hyperoxia than the corresponding wild type [117] . The hyperoxia resistance observed in the ho-1 -/strain could be reversed by the reintroduction of HO-1 by adenoviral-mediated gene transfer [117] . In contrast, mouse embryo fibroblasts derived from ho-1 -/mice showed increased sensitivity to the toxic effects of hemin and H 2 O 2 and generated more intracellular reactive oxygen species in response to these agents [118] . Both ho-1 -/-and ho-2 -/strains were anemic, yet displayed abnormal accumulations of tissue iron. Specifically, ho-1 -/accumulated nonheme iron in the kidney and liver and had decreased total iron content in the lung, while ho-2 -/mice accumulated total lung iron in the absence of a compensatory increase in ferritin levels [116, 119] . The mechanism(s) by which HO-1 or HO-2 deletions result in accumulation of tissue iron remain unclear. These studies, taken together, have indicated that animals deficient in either HO-1 and HO-2 display altered sensitivity to oxidative stress conditions. Aberrations in the distribution of intra-and extra-cellular iron, may underlie in part, the differential sensitivity observed [116, 117] . Otterbein et al. have shown that exogenous CO, through anti-inflammatory action, may protect the lung in a rat model of hyperoxia-induced lung injury. The presence of CO (250 ppm) prolonged the survival of rats in a hyperoxic (>95% O 2 ) environment, and inhibited the appearance of markers of hyperoxia-induced lung injury (i.e. hemorrhage, fibrin deposition, edema, airway protein accumulation, and BALF neutrophil influx) [11] . Furthermore, in a mouse model, CO inhibited the expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in mice induced by the hyperoxia treatment. Using genedeleted mice, Otterbein and colleagues also observed that the protection afforded by CO in this model, similar to a lipopolysaccharide-induced model of lung injury, depended on the p38 MAPK pathway (Otterbein et al., unpublished observation, as reviewed in [3] ). In direct contrast to these studies, the group of Piantadosi and colleagues reported no significant difference in the hyperoxia tolerance of rats at CO doses between 50 and 500 ppm [120] . In their model, CO did not alter the accumulation of fluid in the airway. Furthermore, CO, when applied in combination with hyperoxia, increased the activity of myeloperoxidase, a marker of airway neutrophil influx. This study also suggested that inhalation of CO (50-500 ppm) did not alter the expression of HO-1 or other antioxidant enzymes such as Manganese superoxide dismutase (MnSOD) in vivo [120] . Furthermore, Piantadosi and colleagues were able to induce oxygen tolerance in rats and HO-1 expression with hemoglobin treatment, but this tolerance also occurred in the presence of HO inhibitors, thereby not supporting a role for HO activity in oxygen tolerance [121] . Although no consensus has been reached as to the protective role of CO inhalation and/or HO-1 induction in hyperoxic lung injury, human studies will be required to show if CO will supersede NO in providing a significant therapeutic benefit in the context of severe lung diseases [122] . While antioxidant therapies have been examined, until now no human studies exist on the role of HO-1 and CO in acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia [123] . Lung transplantation is the ultimate and often last therapeutic option for several end-stage lung diseases. After lung transplantation, there remains an ongoing hazardous situation in which both acute and chronic graft failure, as well as complications of the toxic immunosuppressive regimen used (i.e. severe bacterial, fungal, and viral infections; renal failure; and Epstein-Barr-virus-related lymphomas), determine the outcome [124] . The development of chronic graft failure, obliterative bronchiolitis (OB), determines the overall outcome after lung transplantation. OB, which may develop during the first months after transplantation, is the main cause of morbidity and death following the first half-year after transplantation, despite therapeutic intervention. Once OB has developed, retransplantation remains the only therapeutic option available [124, 125] . Little is known about the pathophysiological background of OB. The possible determinants of developing OB include ongoing immunological allograft response, HLADR mismatch, cytomegalovirus infection, acute rejection episodes, organ-ischemia time, and recipient age [125] . OB patients displayed elevated neutrophil counts in the BALF, and evidence of increased oxidant activity, such as increased methionine oxidation in BALF protein and decreases in the ratio of GSH to oxidized glutathione (GSSG) in epithelial lining fluid. [126, 127] . So far, only very limited research data are available on the possible role for HO-1 in allograft rejection after lung transplantation. Higher HO-1 expression has been detected in alveolar macrophages from lung tissue in lung transplant recipients with either acute or chronic graft failure than in stable recipients [128] . The protective role of HO-1 against allograft rejection has been shown in other transplantation models, in which solid organ transplantation typically benefits from HO-1 modulation. A higher expression of protective genes such as HO-1 has been observed in episodes of acute renal allograft rejection [129] . Furthermore, the induction of HO-1 alleviates graft-versus-host disease [130] . Adenoviral-HO-1 gene therapy resulted in remarkable protection against rejection in rat liver transplants [131] . The upregulation of HO-1 protected pancreatic islet cells from Fas-mediated apoptosis in a dose-dependent fashion, supporting an anti-apoptotic function of HO-1 [132, 133] . HO-1 may confer protection in the early phase after transplantation by inducing Th2-dependent cytokines such as IL-4 and IL-10, while suppressing interferon-γ and IL-2 production, as demonstrated in a rat liver allograft model [134] . Beneficial effects of HO-1 modulation have also been described in xenotransplantation models, in which HO-1 gene expression appears functionally associated with xenograft survival [135] . In a mouse-to-rat heart trans-plant model, the effects of HO-1 upregulation could be mimicked by CO administration, suggesting that HOderived CO suppressed the graft rejection [136] . The authors proposed that CO suppressed graft rejection by inhibition of platelet aggregation, a process that facilitates vascular thrombosis and myocardial infarction. HO-1 may also contribute to ischemic preconditioning, a process of acquired cellular protection against ischemia/ reperfusion injury, as observed in guinea pig transplanted lungs [137] . HO-1 overexpression provided potent protection against cold ischemia/reperfusion injury in a rat model through an anti-apoptotic pathway [138, 139] . The induction of HO-1 in rats undergoing liver transplantation with cobalt-protoporphyrin or adenoviral-HO-1 gene therapy resulted in protection against ischemia/ reperfusion injury and improved survival after transplantation, possibly by suppression of Th1-cytokine production and decreased apoptosis after reperfusion [140, 141] . Until now, no reports have addressed E-CO measurements in lung transplantation, where it is possible that differences in E-CO will be found in patients with acute and chronic allograft rejection. The evolution of CO in exhaled breath may serve as a general marker and diagnostic indicator of inflammatory disease states of the lung, though more research will be required to verify its reliability. Increases in exhaled CO presumably reflect changes in systemic and airway heme metabolic activity from the action of HO enzymes. Evidence from numerous in vitro and animal studies indicates that HO-1 provides a protective function in many, if not all, diseases that involve inflammation and oxidative stress. Thus, the exploitation of HO-1 for therapeutic gain could be achieved through the modulation of HO-1 enzyme activity or its up-and downstream regulatory factors, either by gene transfer, pharmacological inducers, or direct application of CO by gas administration or chemical delivery [142] [143] [144] [145] . The CO-releasing molecules (transition metal carbonyls) developed by Motterlini et al. [144] show promise in the pharmacological delivery of CO for therapeutic applications in vascular and immune regulation. The CO-releasing molecules have been shown to limit hypertension in vivo and promote vasorelaxation in isolated heart and aortic rings [144] . Ultimately, the challenge remains in applying the therapeutic potentials of HO-1 to the treatment of human diseases. In vivo models of transplantation have shown that HO-1 gene therapy protects against allograft rejection [129, 134] . Given the toxic therapy that every transplant patient receives, especially after lung transplantation, the field of transplantation medicine may bring the first frontier for human applications of HO-1 gene therapy or exogenous CO administration. The potential use of inhalation CO as a clinical therapeutic in inflammatory lung diseases has also appeared on the horizon. In one promising study, an inhalation dose of 1500 ppm CO at the rate of 20 times per day for a week produced no cardiovascular side effects [146] . Cigarette smoking and CO inhalation at identical intervals produced comparable Hb-CO levels of approximately 5%. The question of whether or not CO can be used as an inhalation therapy will soon be replaced by questions of "how much, how long, and how often?" The fear of administering CO must be weighed against the severe toxicity of the immunosuppressive agents in current use, and the often negative outcome of solid organ transplantation. "
9
"Technical Description of RODS: A Real-time Public Health Surveillance System"
"This report describes the design and implementation of the Real-time Outbreak and Disease Surveillance (RODS) system, a computer-based public health surveillance system for early detection of disease outbreaks. Hospitals send RODS data from clinical encounters over virtual private networks and leased lines using the Health Level 7 (HL7) message protocol. The data are sent in real time. RODS automatically classifies the registration chief complaint from the visit into one of seven syndrome categories using Bayesian classifiers. It stores the data in a relational database, aggregates the data for analysis using data warehousing techniques, applies univariate and multivariate statistical detection algorithms to the data, and alerts users of when the algorithms identify anomalous patterns in the syndrome counts. RODS also has a Web-based user interface that supports temporal and spatial analyses. RODS processes sales of over-the-counter health care products in a similar manner but receives such data in batch mode on a daily basis. RODS was used during the 2002 Winter Olympics and currently operates in two states—Pennsylvania and Utah. It has been and continues to be a resource for implementing, evaluating, and applying new methods of public health surveillance."
"Unfortunately, conventional public health disease surveillance-which relies on physician and laboratory reporting and manual analysis of surveillance data-is ill equipped for timely detection of such threats. 3 The reportable disease system relies on health care professionals to recognize, diagnose, and report cases and suspected outbreaks to public health officials 4, 5 ; however, it is unlikely that without an event or alert to raise his or her index of suspicion, a physician will attribute the early symptoms and signs of disease in a bioattack victim appropriately and report the case. 6 A key limitation of the current system is that the lone physician is blind to the cases his or her colleagues in a nearby hospital are seeing-knowledge that might lead the physician to consider uncommon diseases more strongly in his or her diagnostic reasoning. Mandatory laboratory reporting 4 is also illequipped for early detection, because it takes time before tests are ordered and specimens are obtained, transported, processed, and resulted. Sufficiently early detection of a biological attack may be accomplished through surveillance schemes that can detect infected individuals earlier in the disease process. For completeness, we note that biosensors are being developed (and deployed) that detect organisms in the air and that this type of detection, if feasible, occurs fundamentally much earlier, because the delay introduced by the incubation period of the disease is eliminated from the surveillance system. 7 However, such approaches face unsolved technical problems in the analysis of contaminated specimens (the norm in air sampling). Biosensors also need to be in the right place-on every person's lapel or every street corner and hallway-to provide complete surveillance coverage. Surveillance methods that can detect disease at an earlier stage are an important research direction for public health surveillance. These methods are generally referred to as syndromic surveillance because they have the goal of recognition of outbreaks based on the symptoms and signs of infection and even its effects on human behavior prior to first contact with the health care system. 8 Because the data used by syndromic surveillance systems cannot be used to establish a specific diagnosis in any particular individual, syndromic surveillance systems must be designed to detect signature patterns of disease in a population to achieve sufficient specificity. For example, it would be absurd to use only the symptom of fever to attempt to establish a working diagnosis of inhalational anthrax in an individual, but it would be very reasonable to establish a working diagnosis of anthrax release in a community if we were to observe a pattern of 1,000 individuals with fever distributed in a linear streak across an urban region consistent with the prevailing wind direction two days earlier. It would be beyond reasonable and, in fact, imperative to establish a working diagnosis of public health emergency if presented with such information. One recent example of a form of syndromic surveillance is drop-in surveillance-the stationing of public health workers in emergency departments (EDs) and special clinics during high-profile events such as the Super Bowl to capture data on patients presenting with symptoms potentially indicative of bioterrorism. The major disadvantage of this approach is the cost of round-the-clock staffing for manual data collection. A less expensive approach-and the one taken in the Realtime Outbreak and Disease Surveillance (RODS) system-is detection based on data collected routinely for other purposes. Examples of such data include absenteeism data, sales of over-the-counter (OTC) health care products, and chief complaints from EDs. 9 The expenses of manual data collection are avoided; however, the data obtained typically are noisy approximations of what could be obtained by direct interviewing of the patient (in the case of individual level data). Both approaches may play complementary roles with current methods of public health surveillance 10-12 by assisting the physician and public health official with a continuously updated picture of the ''health status'' of a population. 13, 14 A focus of our research has been syndromic surveillance from free-text chief complaints routinely collected by triage nurses in EDs and acute care clinics during patient registration. We have deployed this type of surveillance at the 2002 Winter Olympics and in the States of Pennsylvania and Utah. We described a previous version of the RODS system, 12 but the system has undergone considerable subsequent development both architecturally and functionally. This report provides a detailed description of the current version of RODS, an example of a computer-based public health surveillance system that adheres to the National Electronic Disease Surveillance System (NEDSS) specifications of the Centers for Disease Control and Prevention (CDC). 15, 16 Background The role of public health surveillance is to collect, analyze, and interpret data about biological agents, diseases, risk factors, and other health events and to provide timely dissemination of collected information to decision makers. 17 Conventionally, public health surveillance relies on manual operations and off-line analysis. Existing syndromic surveillance systems include the CDC's drop-in surveillance systems, 8 Early Notification of Community-based Epidemics (ESSENCE), 10,18 the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS), 19 the Rapid Syndrome Validation Project (RSVP), 20 and the eight systems discussed by Lober et al. 11 Lober et al. summarized desirable characteristics of syndromic surveillance systems and analyzed the extent to which systems that were in existence in 2001 had those characteristics. 11 A limitation of most systems (e.g., ESSENCE, 10 Children's Hospital in Boston, 11 University of Washington 11 ) was batch transfer of data, which may delay detection by as long as the time interval (periodicity) between batch transfers. For example, a surveillance system with daily batch transfer may delay by one day the detection of an outbreak. Some systems required manual data input (e.g., CDC's dropin surveillance systems, RSVP, 20 and LEADERS 19 ), which is labor-intensive and, in the worst case, requires round-theclock staffing. Manual data input is not a feasible mid-or long-term solution even if the approach is to add items to existing encounter forms (where the items still may be ignored by busy clinicians). A third limitation for existing surveillance systems is that the systems may not exploit existing standards or communication protocols like Heath Level 7 (HL7) even when they are available. The data type most commonly used among surveillance systems is symptoms or diagnoses of patients from ED and/or physician office visits. Other types of data identified in that study include emergency call center and nurse advice lines. Other types of data being used include sales of over-thecounter health care products, prescriptions, telephone call volumes to health care providers and drug stores, and absenteeism. We have conducted studies demonstrating that the free-text chief complaint data that we use correlate with outbreaks. 21, 22 Design Objectives The overall design objective for RODS is similar to that of an early warning system for missile defense; namely, to collect whatever data are required to achieve early detection from as wide an area as necessary and to analyze the data in a way that they can be used effectively by decision makers. It is required that this analysis be done in close to real time. This design objective is complex and difficult to operationalize because of the large number of organisms and the even larger number of possible routes of dissemination all requiring potentially different types of data for their detection, different algorithms, and different time urgencies. For this reason, our focus since beginning the project in 1999 has been on the specific problem of detecting a large-scale outbreak due to an outdoor (outside buildings) aerosol release of anthrax. Additional design objectives were adherence to NEDSS standards to ensure future interoperability with other types of public health surveillance systems, scalability, and that the system could not rely on manual data entry, except when it was done in a focused way in response to the system's own analysis of passively collected data. This report describes RODS 1.5, which was completely rewritten as a Java 2 Enterprise Edition (J2EE) application since the previous publication describing it. RODS 1.5 is multidata type enabled, which means that any time series data can be incorporated into the databases and user interfaces. The deployed RODS system currently displays and analyzes health care delivery site registrations and separately monitors sales of OTC health care products. Overview RODS uses clinical data that are already being collected by health care providers and systems during the registration process. When a patient arrives at an ED (or an InstaCare in Utah), the registration clerk or triage nurse elicits the patient's reason for visit (i.e., the chief complaint), age, gender, home zip code, and other data and enter the data in a registration computer. The registration computer then generates an HL7 ADT (admission, discharge, and transfer) message and transmits it to the health system's HL7 message router (also called an integration engine). There usually is only one message router per health system even if there are many hospitals and facilities. These processes are all routine existing business activities and do not need to be created de novo for public health surveillance. Figure 1 shows the flow of clinical data to and within RODS. The hospital's HL7 message router, upon receipt of an HL7 message from a registration computer, deletes identifiable information from the message and then transmits it to RODS over a secure virtual private network (VPN), or a leased line, or both (during the 2002 Winter Olympics we utilized both types of connections to each facility for fault tolerance). The RODS HL7 listener maintains the connection with the health system's message router and parses the HL7 message as described in more detail below. It then passes the chief complaint portion of the message to a Bayesian text classifier that assigns each free-text chief complaint to one of seven syndromic categories (or to an eighth category, other). The database stores the category data, which then are used by applications such as detection algorithms and user interfaces. Data about sales of OTC health care products are processed separately by the National Retail Data Monitor, which is discussed in detail in another article in this issue of JAMIA. 23 The processing was kept separate intentionally because, in the future, the servers for the National Retail Data Monitor may operate in different physical locations than RODS. The RODS user interfaces can and do display sales of OTC health care products as will be discussed, but other user interfaces can be connected to the National Retail Data Monitor as well. Prior to September 2001, RODS received data only from hospitals associated with the UPMC Health System, and efforts to recruit other hospitals met with resistance. After the terrorist attacks (including anthrax) in the Fall of 2001, other hospitals agreed to participate. Although data in this project are de-identified, certain information such as the number of ED visits by zip code were considered proprietary information by some health systems. Health Insurance Portability and Accountability Act (HIPAA) concerns also were very prominent in the discussions. Data-sharing agreements were executed with every participating health system that addressed these concerns. As an additional precaution, all RODS project members meet annually with University of Pittsburgh council to review obligations and are required to sign an agreement every year stating that they understand the terms of the data-sharing agreements and agree to abide by the terms. RODS began as a research project at the University of Pittsburgh in 1999 and has functioned with IRB approvals since that time. Health care facilities send admission, discharge, and transfer (ADT) HL7 messages to RODS for patient visits in EDs and walk-in clinics. A minimal data set is sent, as shown in Figure 2 , which qualifies as a HIPAA Limited Data Set. 24 Currently the data elements are age (without date of birth), gender, home zip code, and free-text chief complaint. The HL7 listener receives HL7 messages from the message routers located in each health system. The HL7 listener then passes the received HL7 message to the HL7 parser bean, an Enterprise JavaBean (EJB) in the RODS business logic tier. The HL7 parser bean uses regular expressions to parse the fields in an HL7 message. The HL7 parser bean then stores the parsed elements into a database through a managed database connection pool. Although nearly all health systems utilize the HL7 messaging standard, the location of individual data elements in an HL7 message may differ from health system to health system. For example, some care providers' systems record free-text chief complaint in the DG1 segment instead of the PV2 segment of an HL7 message. To resolve this mapping problem, a configuration file written in eXtensible Markup Language (XML), a standard protocol often used to define hierarchical data elements, defines where each of the data elements can be found in the HL7 message. When an HL7 listener starts up, it reads the hospital-dependent configuration file and passes the configuration information to the parser bean. We also use this configuration file to define the database table and field in which the HL7 parser bean should store each data element. This approach is useful because it allows the HL7 data to be stored to an external database. We anticipate that health departments with existing NEDSS or other public health surveillance databases may wish to use just this component of RODS for real-time collection of clinical data. For hospitals that do not have HL7 message routers (two of approximately 60 in our experience to date), RODS accepts ED registration data files through either a secure Web-based data upload interface or a secure file transfer protocol. In general, these types of data transfers are technically trivial and for that reason are used by many groups but do not have the reliability of a HL7 connection (and have very undesirable time latencies). RODS checks the integrity of the data in the HL7 messages that it receives. This processing is necessary because hospital data flows may have undesirable characteristics such as duplicates. RODS identifies and deletes duplicates by using a database trigger that creates a composite primary key before inserting the data. RODS also filters out scheduling messages, which are identified by the fact that they have future admitted date and time. RODS monitors all data feeds to ensure continuous connections with health systems. If RODS does not receive data for six hours, it sends an alert to the RODS administrator and the sending health system's administrator. Because the commercial message routers that hospitals use queue up HL7 messages when encountering networking or system problems, data integrity is preserved. RODS uses an Oracle8i database to store ED registration data. (Oracle, Redwood Shores, CA). To ensure fast response for an online query (e.g., the daily counts of respiratory syndrome in a county for the past six months), we developed a cache For connectivity with the HL7 message routers, we utilize hardware-based routers. The VPN router is a Cisco PIX 501 and the leased-line routers are a pair of Cisco 2600s (Cisco Systems, Inc., San Jose, CA). All of the RODS processes can be run on a single computer, but in our current implementation-serving Pennsylvania F i g u r e 2. Sample HL7 admission, discharge, and transfer (ADT) message from an emergency department. The circled fields are age, gender, home zip code, admitted date and time, and free-text chief complaint, respectively. and Utah as an application service provider-we use five dedicated servers: firewall, database, Web server, a geographic information system (GIS) server, and computation. The processes are written in Java code and can run on most platforms, but here we describe the specific platforms we use to indicate approximate sizing and processing requirements. We developed RODS applications using the Java 2 Enterprise Edition Software Toolkit (J2EE SDK) from Sun Microsystems for cross-platform Java application development and deployment. 26 We followed contemporary application programming practices-a multitiered application consisting of a client tier (custom applications such as HL7 listeners and detection algorithms), business logic tier, database tier, and Web tier. Business logic such as the HL7 parser bean was implemented as Enterprise JavaBeans (EJBs). NEDSS specifies EJB as the standard for application logic. RODS uses Jboss, an opensource J2EE application server, to run all EJBs. 10 The Web tier comprises the graphical user-interface to RODS and uses Java Server Pages (JSP), Java Servlets, and ArcIMS. The database tier was implemented in Oracle 8i. RODS uses a naive Bayesian classifier called Complaint Coder (CoCo) to classify free-text chief complaints into one of the following syndromic categories: constitutional, respiratory, gastrointestinal, neurological, botulinic, rash, hemorrhagic, and other. CoCo computes the probability of each category, conditioned on each word in a free-text chief complaint and assigns a patient to the category with the highest probability. 27 The probability distributions used by CoCo are learned from a manually created training set. CoCo can be retrained with local data, and it can be trained to detect a different set of syndromes than we currently use. CoCo runs as a local process on the RODS database server. CoCo was developed at the University of Pittsburgh and is available for free download at <http://health.pitt.edu/rods/sw>. Over the course of the project, RODS has used two detection algorithms. These algorithms have not been formally field tested because the emphasis of the project to date has been on developing the data collection infrastructure more than field testing of algorithms. The Recursive-Least-Square (RLS) adaptive filter 28 currently runs every four hours, and alerts are sent to public health officials in Utah and Pennsylvania. RLS, a dynamic autoregressive linear model, computes an expected count for each syndrome category for seven counties in Utah and 16 counties in Pennsylvania as well as for the combined counts for each state. We use RLS because it has a minimal reliance on historical data for setting model parameters and a high sensitivity to rapid increases in a time series e.g., a sudden increase in daily counts. RLS triggers an alert when the current actual count exceeds the 95% confidence interval for the predicted count. During the 2002 Olympics we also used the What's Strange About Recent Events (WSARE 1.0) algorithm. 29 WSARE performs a heuristic search over combinations of temporal and spatial features to detect anomalous densities of cases in space and time. Such features include all aspects of recent patient records, including syndromal categories, age, gender, and geographical information about patients. The criteria used in the past for sending a WSARE 1.0 alert was that there has been an increase in the number of patients with specific characteristics relative to the counts on the same day of the week during recent weeks and the p-value after careful adjustment for multiple testing for the increase was #0.05. Version 3.0 of WSARE, which will incorporate a Bayesian model for computing expected counts rather than using unadjusted historical counts currently, is under development. When an algorithm triggers an alert based on the above criteria, RODS sends e-mail and/or page alerts to its users. RODS uses an XML-based configuration file to define users' e-mail and pager addresses. The e-mail version of the alert includes a URL link to a graph of the time series that triggered the alarm with two comparison time series: total visits for the same time period and normalized counts. RODS has a password-protected, encrypted Web site at which users can review health care registration and sales of OTC health care products on epidemic plots and maps. When a user logs in, RODS will check the user's profile and will display data only for his or her health department's jurisdiction. The interface comprises three screens-Main, Epiplot, and Mapplot. The main screen alternates views automatically among each of the available data sources (currently health care registrations and OTC products in Pennsylvania and Utah and OTC sales only for other states). The view alternates every two minutes as shown in Figure 3 . The clinic visits view shows daily total visits and seven daily syndromes for the past week. The OTC data view shows daily sales for five product categories and the total, also for the past week. Users also can set the view to a specific county in a state. If the normalize control box is checked, the counts in the time series being displayed will be divided by (normalized by) the total daily sales of OTC health care products or ED visits for the region. The Epiplot screen provides a general epidemic plotting capability. The user can simultaneously view a mixture of different syndromes and OTC product categories for any geographic region (state, county, or zip code), and for any time interval. The user also can retrieve case details as shown in Figure 4 . The Get Cases button queries the database for the admission date, age, zip code, and chief complaint (verbatim, not classified into syndrome category) of all patients in the time interval and typically is used to examine an anomalous density (spike) of cases. The Download Data button will download data as a compressed comma separated file for further analyses. The Mapplot screen is an interface to ArcIMS, an Internetenabled GIS product developed by Environmental Systems Research Institute, Inc. Mapplot colors zip code regions to indicate the proportion of patients presenting with a particular syndrome. The GIS server also can overlay state boundaries, county boundaries, water bodies, hospital locations, landmarks, streets, and highways on the public health data as shown in Figure 5 . Similar to Epiplot, Mapplot also can display case details for a user-selected zip code. RODS has been in operation for four years and, like most production systems, has acquired many fault-tolerant features. For example, at the software level, HL7 listeners continue to receive messages and temporarily store the messages when the database is off-line. A data manager program runs every ten minutes and, on finding such a cache, it loads the unstored messages to the database when the database is back on-line. In addition, the data manager program monitors and restarts HL7 listeners as necessary. The database uses ''archive log'' mode to log every transaction to ensure that the database can recover from a system failure. The hardware architecture also is fault tolerant. All servers have dual power supplies and dual network cards. All hard drives use Redundant Arrays of Inexpensive Disk configurations. In addition to dual power supplies, all machines are connected to an uninterrupted power supply that is capable of sending an e-mail alert to the RODS administrator when the main power is down. An important component of RODS that currently is used only at the UPMC Health System in Pittsburgh is the Health System Resident Component (HSRC). The HSRC is located within the firewall of a health system and connects directly to the HL7 message router. The HSRC currently receives a diverse set of clinical data from the HL7 message router including culture results, radiology reports, and dictated F i g u r e 3. Health care registrations view in the Main screen of RODS. The Main screen alternates views every 2 minutes among data types available in the public health jurisdiction. The figure shows eight plots of health care registration data-total visits, botulinic, constitutional, gastrointestinal (GI), hemorrhagic, neurological, rash, and respiratory. After 2 minutes, over-the-counter data will be displayed. The Main screen can be used as a ''situation room'' display. emergency room notes. Its purpose is to provide additional public health surveillance functions that would not be possible if it were located outside of the firewall due to restrictions on the release of identifiable clinical data. The HSRC uses patient identifiers to link laboratory and radiology information to perform case detection. In the past, we have used HSRC to monitor for patients with both a gram-positive rod in a preliminary microbiology culture report and ''mediastinal widening'' in a radiology report. The HSRC is a case detector in a distributed outbreak detection system that is capable of achieving much higher specificity of patient diagnostic categorization through access to more information. HSRC also removes identifiable information before transmitting data to the RODS system, a function provided by the health system's message router in other hospitals that connect to RODS. The HSRC at UPMC Health System functions as an electronic laboratory reporting system, although the state and local health departments are not yet ready to receive real-time messaging from the system. Currently, it sends email alerts to the director of the laboratory and hospital infection control group about positive cultures for organisms that are required to be reported to public health in the state of Pennsylvania. 30 It also sends messages to hospital infection control when it detects organisms that cause nosocomial infections. These organisms include Clostridium difficile, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus. We have been able in HSRC to prototype one additional feature, which is a ''look-back'' function that facilitates very rapid outbreak investigations by providing access to electronic medical records to public health investigators as shown in Figure 6 . This feature requires a token that can be passed to a hospital information system that can uniquely identify a patient, and the reason we have prototyped this feature in the HSRC and not in RODS is simply that HSRC runs within the firewall so an unencrypted token can be used. The lookback is accomplished as follows: when a public health user identifies an anonymous patient record of interest (e.g., one of 20 patients with diarrhea today from one zip code), HSRC calls the UPMC Health System Web-based electronic medical record system and passes it the patient identifier. UPMC Health System then requests the user to log in using the UPMC-issued password before providing access to the record directly from its own secure Web site. This approach is not intended to be implemented in HSRC, but rather in the RODS system outside of the firewall of a health system. It is intended to use encrypted identifiers that the health system would decrypt to retrieve the correct record. The HSRC could provide the encryption-decryption service or it could be provided by another data system in the hospital. We estimate that the prevalence of health systems that have Web-based results review in the United States is 30% to 50% and growing so that this approach could very quickly improve the efficiency of outbreak investigations. For these reasons, we have moved to an application service provider model for dissemination in which we encourage state and local health departments to form coalitions to support shared services. We also have been fortunate to have sufficient grant funding from the Commonwealth of Pennsylvania to be able to support these services on an interim basis while sustainable funding models evolve. Our original design objectives for RODS were real-time collection of data with sufficient geographic coverage and sampling density to provide early syndromic warning of a large-scale aerosol release of anthrax. Although we have not achieved all of our initial design objectives, progress has been substantial. The research identified two types of data-freetext chief complaints and sales of OTC health care prod- ucts-that can be obtained in real time or near real time at sampling levels of 70% or higher for most of the United States. These results were obtained through large-scale deployments of RODS in Pennsylvania and Utah and through building the National Retail Data Monitor described in the accompanying article in this issue of JAMIA. The deployments also provided insights about organizational and technical success factors that would inform an effort to scale the project nationally. The project established the importance of HL7 message routers (also known as integration engines) for public health surveillance. HL7 message routers are a mature, highly prevalent technology in health care. We demonstrated that free-text triage chief complaints can be obtained in real time from most U.S. hospitals through message routers and that these data represent early syndromal information about disease. Many other clinical data of value to public health are transmitted using the HL7 standard (e.g., orders for diagnostic tests, especially microbiology tests, reports of chest radiographs, medications, and test results) and can be integrated into RODS or other surveillance systems capable of receiving HL7 messages. As a result of our efforts to disseminate this technology by giving it away, we have learned that most health departments do not have the technical resources to build and maintain real-time electronic disease surveillance systems. Our application service provider model has been much more success-ful, and we now recommend that states form coalitions to share the costs of such services. The project very early identified the need for a computing component to reside within the firewall of a health system, connected to the hospital's HL7 message router. This component would function as a case detector in a distributed public health surveillance scheme linking laboratory and radiology data to increase the specificity of case detection. It has proven very difficult to disseminate this technology, perhaps due to the complexity of the idea. Nevertheless, the threat of bioterrorism has created a need for such technology, and this approach, or something with equivalent function, must be deployed. Adherence to NEDSS architectural standards was an early design objective that we have met. RODS 1.5 closely follows NEDSS architectural, software, messaging, and data specifications. Our success is a strong validation of those standards. We will gain further understanding of the standards as we attempt to use RODS components including HL7 listeners, natural language parsers, message parsers, databases, user interfaces, notification subsystems, and detection algorithms with other NEDSS compliant systems. An ongoing project will use RODS to collect chief complaints and integrate them into the Utah Department of Health's planned NEDSS system. We have demonstrated the ability to rapidly deploy RODS in a special event with the added advantage that the system F i g u r e 6. Look-back function of RODS. The user has selected one patient to investigate using the screen that is in the background and partly hidden by overlap. RODS has logged the user into the results-review function of an electronic medical record and requested that patient's chart, which is shown on the screen in the foreground. persisted after the event. This experience suggests strongly that RODS or similar systems be considered an alternative to drop-in surveillance. Our future plans are to meet our initial design objective to develop early-warning capability for a large, outdoor release of anthrax, especially ensuring that the data and analysis produced by RODS are reviewed by public health. This goal will require improvements in the interfaces and the detection algorithms to reduce false alarms and to vastly improve the efficiency with which anomalies are evaluated by use of multiple types of data, better interfaces, and implementation of the look-back function. We would like to enlarge as quickly as possible the application service provider to include more states and more types of clinical data so that states will be in a position to prospectively evaluate the detection performance from different types of data on naturally occurring outbreaks. Our long-term goals are to add additional disease scenarios to the design objectives such as detection of in-building anthrax release, vector-borne disease, food-borne disease, and a communicable disease such as severe acute respiratory syndrome (SARS). RODS is a NEDSS-compliant public health surveillance system that focuses on real-time collection and analysis of data routinely collected for other purposes. RODS is deployed in two states and was installed quickly in seven weeks for the 2002 Olympics. Our experience demonstrates the feasibility of such a surveillance system and the challenges involved. Outbreaks, emerging infections, and bioterrorism have become serious threats. It is our hope that the front-line of public health workers, astute citizens, and health care workers will detect outbreaks early enough so that systems such as RODS are not needed. However, timely outbreak detection is too important to be left to human detection alone. The notion that public health can operate optimally without timely electronic information is as unwise as having commercial airline pilots taking off without weather forecasts and radar."
10
"Conservation of polyamine regulation by translational frameshifting from yeast to mammals"
"Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals."
"The ef®ciency of +1 ribosomal frameshifting at a speci®c codon is used as a sensor to regulate polyamine levels in mammalian cells. The frameshifting occurs in decoding the gene antizyme 1, which has two partially overlapping open reading frames (ORFs). Protein sequencing showed that the reading-frame shift occurs at the last codon of ORF1, causing a proportion of ribosomes to enter ORF2 to synthesize a transframe protein (Matsufuji et al., 1995) . ORF2 encodes the main functional domains (Matsufuji et al., 1990; Miyazaki et al., 1992) of antizyme but has no ribosome initiation site of its own. The antizyme 1 protein binds to ornithine decarboxylase (ODC) (Murakami et al., 1992a; Cof®no, 1993, 1994) , inhibits it (Heller et al., 1976) and targets it for degradation by the 26S proteosome without ubiquitylation (Murakami et al., 1992b (Murakami et al., , 1999 . ODC catalyzes the ®rst and usually ratelimiting step in the synthesis of polyamines, conversion of ornithine to putrescine. Putrescine is a substrate for the synthesis of spermidine and spermine. Because of its inhibition of ODC, antizyme 1 is a negative regulator of the synthesis of polyamines. In addition, antizyme 1 is a negative regulator of the polyamine transporter (Mitchell et al., 1994; Suzuki et al., 1994; Sakata et al., 1997) . As discovered by Matsufuji and colleagues (Gesteland et al., 1992) and Rom and Kahana (1994) , increasing polyamine levels elevate frameshifting in decoding antizyme 1 mRNA and so increase the level of antizyme 1. Since antizyme 1 negatively regulates the synthesis and uptake of polyamines, the frameshifting is the sensor for an autoregulatory circuit. A second mammalian paralog of antizyme, antizyme 2, has very similar properties to antizyme 1, including the regulatory frameshifting, but does not stimulate degradation of ODC under certain conditions where antizyme 1 is active (Ivanov et al., 1998a; Zhu et al., 1999; Y.Murakami, S.Matsufuji, I.P.Ivanov, R.F.Gesteland and J.F.Atkins, in preparation) . Just like antizyme 1, antizyme 2 mRNA is ubiquitously expressed in the body but is 16 times less abundant than mRNA of antizyme 1 (Ivanov et al., 1998a) . In addition to antizyme 1 and 2, mammals have a third paralog of the gene, antizyme 3 (also encoded by two ORFs), which is expressed only during spermatogenesis (Ivanov et al., 2000) . Zebra®sh also have multiple antizyme genes, which differ in their expression patterns and activities (Saito et al., 2000) . Numerous studies have addressed the regulation of fungal ODC in response to exogenously added polyamines. In the cases examined, Physarum polycephalum (Mitchell and Wilson, 1983) , Saccharomyces cerevisiae (Fonzi, 1989; Toth and Cof®no, 1999) and Neurospora crassa (Barnett et al., 1988; Williams et al., 1992) , added polyamines, especially spermidine, result in signi®cant repression of ODC activity. The mechanisms of repression seem to vary from fungus to fungus and are apparently different from the mechanism of polyamine-dependent regulation of ODC in higher eukaryotes. In some cases, the existence of an antizyme-like protein has been suggested but has either been disproved, as in the case of N.crassa (Barnett et al., 1988) , or has never been substantiated, as is the case with S.cerevisiae. As expected from their small cationic nature and ability to neutralize negative charges locally, polyamines play key roles in processes ranging from the functioning of certain ion channels (Williams, 1997) , nucleic acid packaging, DNA replication, apoptosis, transcription and translation. The role of polyamines can be complex as illustrated by the transfer of the butylamine moiety of spermidine to a lysine residue to form hypusine in mammalian translation initiation factor eIF-5A, the only known substrate for this reaction (Tome et al., 1997; Lee et al., 1999) . Spermine negatively regulates the growth of prostatic carcinoma cells at their primary site (Smith et al., 1995) , but at later stages of tumor progression it fails to induce antizyme, which correlates with cells becoming refractory to spermine (Koike et al., 1999) . Lack of antizyme function is also important in the early deregulation of cellular proliferation in oral tumors (Tsuji Conservation of polyamine regulation by translational frameshifting from yeast to mammals The EMBO Journal Vol. 19 No. 8 pp. 1907±1917, 2000 ã European Molecular Biology Organization et al., 1998) and probably others. The levels of polyamines are altered in many tumors, and inhibitors of polyamine synthesis are being tested for antiproliferative and cell death effects. The synthesis of ODC varies during the cell cycle in normal cells (Linden et al., 1985; Fredlund et al., 1995) . It is induced by many growth stimuli and is constitutively elevated in transformed cells (Pegg, 1988; Auvinen et al., 1992) with some phosphorylated ODC being translocated to the surface membrane where it is important for mitotic cytoskeleton rearrangement events (Heiskala et al., 1999) . Antizyme is one example of certain mRNA-contained signals that can elevate speci®c frameshifting >1000-fold above the background level of normal translational errors. In addition to antizyme, frameshifting is also involved in the decoding of some bacterial and yeast genes and especially in many mammalian Retroviruses and Coronaviruses, plant viruses and bacterial insertion sequences (Atkins et al., 1999) . The site of frameshifting in both mammalian antizyme 1 and 2 mRNAs is UCC UGA, where quadruplet translocation occurs at UCCU (underlined) to shift reading to the +1 frame, immediately before the UGA stop codon of the initiating frame (Matsufuji et al., 1995; Ivanov et al., 1998a) . For the frameshifting to occur with an ef®ciency of 20% or more, it is important that the 3¢ base of the quadruplet is the ®rst base of a stop codon. Other important features are a pseudoknot just 3¢ of the shift site and a speci®c sequence 5¢ of the shift site (Matsufuji et al., 1995; Ivanov et al., 1998a) . A pseudoknot 3¢ of the shift site is a common stimulator for eukaryotic ±1 frameshifting, but the synthesis of antizyme is the only known case utilizing +1 frameshifting. Comparative analysis of RNA sequences from different organisms is informative about important features and the different options selected by evolution. Since most of the known examples of programmed frameshifting are in viruses or chromosomal mobile elements, the opportunity for comparison of frameshift cassettes in divergent organisms where the time of divergence can be approximated is limited. A start has been made with the frameshifting required for bacterial release factor 2 expression (Persson and Atkins, 1998) , but antizyme provides the ®rst opportunity for such a comparison in eukaryotes. Antizyme genes in genetically tractable lower eukaryotes would be helpful for understanding the functionally important interactions responsible for autoregulatory programmed frameshifting. Identi®cation of an antizyme gene in Schizosaccharomyces pombe A search for DNA sequences encoding protein sequences homologous to Drosophila melanogaster antizyme (Ivanov et al., 1998b) and Homo sapiens antizyme 1 identi®ed the same S.pombe anonymous cDNA clone (DDBJ/EMBL/GenBank accession No. D89228). The similarity is limited (~10% identity, 24% similarity to both human antizyme 1 and D.melanogaster antizyme); however, it is highest in regions that are most highly conserved among the previously identi®ed antizymes ( Figure 1A ). Closer examination of the cDNA nucleotide sequence provided further evidence that it encodes an S.pombe homolog of antizyme. The initiating AUG codon for the ORF that is similar to higher eukaryotic antizymes (ORF2 of those genes) is not the 5¢-most AUG in this cDNA. In fact, there are eight AUGs closer to the 5¢ end. The ®rst or the second AUGs would initiate translation of an ORF (ORF1) that overlaps the longer downstream ORF (ORF2) such that a +1 translational frameshifting event in the overlap would generate a protein product analogous to the products of antizyme genes from higher eukaryotes. Furthermore, the last 12 nucleotides of ORF1 (UGG-UGC-UCC-UGA) are identical to the last 12 nucleotides of mammalian antizyme 1 ORF1s, including the frameshift site. Eleven of these 12 nucleotides are identical to the corresponding regions of all previously identi®ed antizyme genes ( Figure 1B ). Previous experiments with the mammalian frameshift sequence tested in S.pombe have shown that this short 12 nucleotide sequence, by itself, is suf®cient to stimulate measurable levels (up to 0.5%) of +1 frameshifting (Ivanov et al., 1998c) . To con®rm the ORF con®guration of the putative S.pombe antizyme gene, a region corresponding to the two overlapping ORFs plus~80 nucleotides of the 5¢ UTR and 370 nucleotides of the 3¢ UTR, was ampli®ed from both S.pombe genomic DNA and a cDNA library. The sequence of the ampli®ed DNA con®rmed that there are indeed two overlapping ORFs with the deduced con®guration. This sequence (DDBJ/EMBL/GenBank accession No. AF217277) differs from the previously sequenced cDNA clone by three nucleotides (two in the coding region and one in the 3¢ UTR); one changes an alanine codon to proline, another is a silent mutation within a proline codon. Since the sequences from the cDNA library and genomic DNA are identical, we conclude that the differences with clone No. D89228 are most likely due to strain variation. This gene contains no introns within the ampli®ed region. The S.pombe protein was tested for antizyme activity using a gene fusion with glutathione S-transferase (GST). In this construct, ORF1 and ORF2 of antizyme are fused in-frame by deleting the T nucleotide that encodes U of the stop codon of ORF1. This GST±antizyme fusion gene was expressed in Escherichia coli and the protein was puri®ed by af®nity chromatography. ODC inhibitory activity was tested by incubating the recombinant antizyme protein with an S.pombe crude extract and then assaying the mixture for ODC activity. The results ( Figure 2) show that the recombinant protein can inhibit S.pombe ODC. GST alone (1 mg) does not inhibit S.pombe ODC (data not shown). In light of these results, the S.pombe gene will be called S.pombe ODC antizyme (SPA). Interestingly, the S.pombe ODC was also inhibited by mouse antizyme 1 and antizyme 2 (both expressed as GST fusions); however, the yeast fusion protein did not inhibit mouse ODC (data not shown). Deletion and overexpression of SPA Although the effects of overexpression of antizyme on cellular physiology have been tested previously in mammalian cells, the physiological changes associated with complete absence of antizyme activity have not yet been investigated because of the complication of multiple antizymes. The single S.pombe antizyme provides the chance to explore a knockout. SPA deletion strains were I.P. Ivanov et al. generated by replacing the two ORFs of the gene with the ORFs of either URA4 or LEU2 (see Materials and methods). Complete deletion of SPA (both ORFs) did not affect the viability of S.pombe cells in rich (YE) or minimal (MM) media. Temperature had no differential effect on mutant and wild-type cell growth. Similarly, the growth rates, mating ef®ciencies and overall morphology of the knockout strains are apparently indistinguishable from those of wild-type cells (results not shown). In wild-type S.pombe cells the most abundant polyamine is spermidine followed by putrescine ( Figure 3 ). Spermine and cadaverine are found in much smaller amounts. This distribution of polyamine content is very similar to that in other fungi for which polyamine concentrations have been measured (for references, see review by Tabor and Tabor, 1985) . The effect of SPA deletion on cellular polyamine contents was examined in both exponentially growing and stationary phase cells ( Figure 3 ). The cellular concentrations of putrescine, spermidine and cadaverine (but not spermine) were higher in the knockout strains than in wild-type cells. The greatest effect was seen on putrescine and cadaverine content, with smaller effects on spermidine, presumably because eukaryotic ODC activity directly catalyzes decarboxylation of both ornithine and lysine to produce putrescine and cadaverine, respectively (Pegg and McGill, 1979) , but subsequent regulatory events affect homeostasis of spermidine and spermine. The effect of inactivating antizyme on the polyamine contents in exponentially growing cells is modest (<2-fold in all cases). The effect becomes very pronounced in cells in stationary phase with up to 40-and 10-fold increases of putrescine and cadaverine contents, respectively, in the knockout strains. To test overexpression of SPA, two versions of the gene were cloned into pREP3 expression vector behind a strong, thiamine-repressible promoter (nmt1). One had the wild- type SPA sequence while in the second, ORF1 and ORF2 are fused in-frame. SPA wild type and an SPA deletion strain were transformed with each of the overexpression constructs. Derepression of the nmt1 promoter is a gradual process since it requires dilution of the intracellular pool of thiamine (the repressor) through cell division. After 2.5 days of exponential growth under derepressed conditions, yeast strains transformed with either SPA overexpression construct show signi®cant increases in doubling time ( Figure 4A ). The growth inhibition is greater with the construct expressing the in-frame version of SPA and after prolonged incubation (5±7 days); these cells cease growth and accumulate in G 1 as determined bȳ ow cytometry (data not shown). The fact that the inframe overexpression construct, which differs by a single nucleotide from the wild-type construct, confers a more severe phenotype is consistent with the hypothesis that translational frameshifting is required for expression of SPA. The growth phenotype associated with SPA overexpression is only partially relieved by adding 100 mM putrescine to the media (1 mM had no further effect) (data not shown). To see whether the slower growth is correlated with aberrant polyamine levels the polyamine contents of the deletion strain carrying in-frame SPA overexpression vector were measured under derepressed and repressed conditions, in both cases after 2 days of exponential growth ( Figure 4B ). As expected, overexpression of SPA results in signi®cant reduction in the intracellular levels of all four polyamines. After longer (4±5 days) incubation under derepressed conditions, no putrescine and cadaverine can be detected (data not shown). Translational frameshifting during expression of SPA Previously, we developed an assay for measuring antizyme translational frameshifting in both S.cerevisiae (Matsufuji et al., 1996) and S.pombe (Ivanov et al., 1998c) . Brie¯y, the nucleotide sequence to be assayed is inserted between GST and lacZ, such that ORF1 of the assayed sequence is fused in-frame to GST, while ORF2 is fused in-frame to lacZ. b-galactosidase activity provides a measure of frameshifting ef®ciency. To determine whether translational frameshifting occurs in the overlap of ORF1 and ORF2 of SPA, a region of SPA including all but the ®rst codon of ORF1 plus 180 nucleotides downstream of the ORF1 stop codon was tested. +1 frameshifting occurred at 2.2% compared with a construct in which ORF1 and ORF2 are fused in-frame. This result is consistent with +1 frameshifting being crucial for expression of SPA. Previous experiments have shown that the frameshift cassette of mammalian antizyme 1 can direct ef®cient +1 frameshifting when tested in S.pombe. The reverse experiment was conducted here. The SPA gene was translated in vitro in rabbit reticulocyte lysate and its resulting frameshift ef®ciency measured. With no addition of polyamines, frameshifting ef®ciency is~1.5%. Addition of spermidine to the translation mixture to a ®nal concentration of 1 mM results in a 3.7-fold increase in frameshifting to~5.5%, a level even higher than that observed in the endogenous system in vivo (autoradiogram not shown). The observed ef®ciency of frameshifting with the SPA frameshifting cassette in vivo in S.pombe is signi®cantly more than that expected from its limited nucleotide similarity to the antizyme frameshift sites of higher eukaryotes. This prompted a search for additional stimulatory elements within the SPA frameshift cassette. The following experiments were done in a strain carrying deletion of SPA (high polyamines) because it gives higher frameshifting and higher b-galactosidase activity in general; however, we obtained similar ratios for mutant to wild-type frameshifting ef®ciency in a strain with the intact SPA gene. Deleting 5¢ sequences up to the third to last sense codon of ORF1 has little or no effect on frameshifting ef®ciency. Deleting all but the last sense codon (UCC) of ORF1 leads to a 4-to 5-fold reduction in frameshifting ef®ciency ( Figure 5A ). This implies that the conservation of the six nucleotides 5¢ of the UCC-UGA frameshift site is due to their importance for stimulating +1 frameshifting. It also suggests that no additional ORF1 sequences of SPA stimulate the +1 recoding event. The 180 nucleotide 3¢ region was searched for possible structure by computer RNA folding algorithms plus visual inspection. The algorithms predicted several minimal structures in that region. 3¢ deletion constructs (constructs del.3,3¢±81,3¢) tested the importance of any putative structure on the frameshifting ef®ciency. The results ( Figure 5B and C) show that all of these deletions lead to a signi®cant (~10-fold) reduction in +1 frameshifting, indicating the presence of a major 3¢ stimulatory element in the 180 nucleotide region immediately following the frameshift site of SPA. However, the results indicate that none of the putative RNA structures in this region are suf®cient for the activity of this element. Several additional 3¢ deletions delineated the boundaries of this stimulatory element from the frameshift site to 150 and 180 nucleotides downstream (since construct del.150,3¢ stimulates 5.5-fold more +1 frameshifting than del.129,3¢, 150 nucleotides downstream probably contain most of the 3¢ stimulator). In the experiments described above, two of the characteristics of the autoregulatory circuit of mammalian antizyme 1 were con®rmed: SPA inhibition of ODC and the +1 translational frameshifting. The key question left is whether the recoding event is responsive to polyamine levels in cells. As shown above, overexpression of SPA leads to signi®cant reduction of polyamine levels in S.pombe. An SPA + strain was co-transformed with an SPA wild-type overexpressing plasmid (cells overexpressing wild-type SPA grow slowly but continuously) and a construct that monitors the +1 frameshifting from an SPA frameshift sequence. The +1 frameshifting was compared with that in SPA non-overexpressing cells (in both cases frameshifting was measured relative to in-frame control). The results ( Figure 6 ) show a signi®cant reduction (6.5-fold) in frameshifting ef®ciency in SPA-overproducing cells that correlates with a decrease of polyamine content (4.5-fold for putrescine and 3.9-fold for spermidine). This indicates that polyamines modulate the frameshifting ef®ciency of SPA. An alternative but less likely possibility is that SPA overexpression reduces frameshifting because high levels of SPA transcript titrate some factor limiting for frameshifting. The SPA frameshift signals direct 2-fold more frameshifting in Dspa::LEU2 cells (4.4%) than in SPA + cells (in both cases the measurement is done during stationary phase); however, the relatively high standard deviations for both measurements make it dif®cult to draw ®rm conclusions from this particular result. A search of Caenorhabditis elegans expressed sequence tag (EST) sequences with mammalian antizyme 1 sequence identi®ed 20 clones. These sequences could be deconvoluted into a contiguous cDNA sequence. Primers designed on the basis of this sequence were used to PCR amplify and subclone this cDNA from a C.elegans cDNA library. The sequence of the subcloned cDNA was con®rmed (DDBJ/EMBL/GenBank accession No. AF217278); the subsequently released genomic sequence of this C.elegans gene (DDBJ/EMBL/GenBank accession No. AF040659) con®rms our cDNA data. The amino acid sequence deduced from the cDNA sequence revealed that the longer ORF has similarity to previously reported antizyme sequences (overall 27% identity, 39% similarity to human antizyme 1; 19% identity, 34% similarity to Drosophila antizyme). These similarities are higher than that of SPA to these two antizyme genes and again are concentrated in the regions most highly conserved among previously identi®ed antizymes ( Figure 1A ). Just like mammalian antizymes, the longer ORF (ORF2) lacks an appropriate in-frame initiation codon, and expression could be provided by initiation in a short upstream overlapping ORF (ORF1) leading to +1 ribosomal frameshifting in the overlap. The putative C.elegans antizyme frameshift site (the nucleotides proximal to the end of ORF1) has 18 of 26 nucleotides identical to the consensus sequence for antizyme frameshift sites ( Figure 1B) . Frameshifting for expression of C.elegans antizyme was investigated in heterologous systems. Two constructs containing the entire antizyme cDNA, one with the wildtype sequence and one with a single nucleotide deletion that fuses ORF1 to ORF2 in-frame (in-frame control), were transcribed in vitro and the RNA was translated in rabbit reticulocyte lysate. The products were examined by SDS±PAGE (Figure 7) . The main product from both constructs has an apparent M r of 21 kDa, slightly greater than the predicted M r of 17.7 kDa [aberrant, slower than expected, mobility is observed with antizyme proteins from other species (Ivanov et al., 1998a) ]. From the ratio of wild-type to in-frame product, we estimate that the ef®ciency of frameshifting of C.elegans antizyme in reticulocyte lysate is~0.8%, which is somewhat lower than SPA frameshifting in the same system. Addition of spermidine to the translation reactions almost doubles the ef®ciency of frameshifting to~1.5% (the exact numbers are not easy to determine because of dif®culty in de®ning background values). The frameshifting properties of C.elegans antizyme mRNA were also tested in vivo in S.pombe cells. A sequence including all but the ®rst codon of ORF1 plus 180 nucleotides downstream was inserted between GST and lacZ of the PIU-LAC plasmid. Comparison of the b-galactosidase activity of cells (Dspa::LEU2 strain) transformed with the wild-type construct and the in-frame control constructs indicated 3.5% +1 frameshifting. From the frameshifting observed in the heterologous systems, as well as the sequence considerations discussed above, we conclude that expression of this C.elegans gene requires ribosomal frameshifting. Searching the EST database with the newly discovered C.elegans antizyme identi®ed antizyme orthologs in four other nematode species. In two cases (Necator americanus and Haemonchus contortus), the cDNA sequences in the database were suf®cient to make contigs of the complete coding regions. In the other two cases [Onchocerca volvulus (DDBJ/EMBL/GenBank accession No. AF217279) and Pristioncus paci®cus (DDBJ/EMBL/ GenBank accession No. AF217280)] the complete cDNA sequences were obtained by PCR amplifying and sequencing the full genes from cDNA libraries. As with the previously identi®ed eukaryotic antizyme genes, the ORF con®guration of the newly found nematode orthologs implies the necessity for +1 frameshifting for synthesis of full-length protein. The C.elegans antizyme mRNA frameshift site UUU-UGA is unique, differing from the UCC-UGA of previously known antizyme mRNAs. The C.elegans antizyme gene shares this feature with N.americanus and H.contortus but not with P.paci®cus and O.volvulus antizymes. The phylogenetic tree of nematode antizyme protein sequences matches exactly the phylogenetic relationship (Blaxter, 1998) of the nematodes expressing them, indicating that these gene sequences are the result of divergent evolution within the nematode lineage (data not shown). These results also show that the UUU-UGA frameshift site evolved after the last common ancestor of P.paci®cus and C.elegans but before the divergence of C.elegans, N.americanus and H.contortus (probably 450± 500 million years ago). The ability of UUU-UGA sequence to direct +1 frameshifting was further tested in a mammalian system in the context of the mammalian antizyme mRNA (i.e. in the presence of the 3¢ RNA pseudoknot and 5¢ stimulator). A BMV-coat-protein±antizyme 1 gene fusion construct, which has a TCC-TGA to TTT-TGA substitution, was transcribed and then translated in a rabbit reticulocyte lysate. Eleven percent frameshift ef®ciency was seen in the absence of exogenously added polyamines, 2.2 times the ef®ciency seen with the UCC-UGA transcript. The frameshift ef®ciency becomes 18% when 0.6 mM spermidine is added, which is 1.3 times that with the wild type (Matsufuji et al., 1995) . Similar results were obtained in cultured mammalian (Cos7) cells transfected with TTT-TGA mutant construct, the frameshift being higher than that of wild-type construct in both high-and lowpolyamine conditions (our unpublished results). These results demonstrate that the putative C.elegans frameshift site (UUU-UGA) is, if anything, shiftier than UCC-UGA in the antizyme 1 context and is subject to polyamine stimulation. The results presented show that the yeast S.pombe has a homolog of mammalian antizyme. This is the ®rst documented example of antizyme-type regulation of ODC in a lower eukaryote. Deleting SPA from the yeast genome has no detectable effect on viability or any other overt phenotypic effect but, as expected, it results in altered accumulation of polyamines in the cell. Interestingly, the effect is most pronounced in cells in stationary phase, where the knockout cells accumulate up to 40 times more putrescine than wild-type counterparts. This compares with a <2-fold increase of putrescine in exponentially growing cells. A likely explanation for this observation is that the usual rate of ornithine decarboxylation in exponentially growing cells is close to capacity given`normal' concentrations of substrate, enzyme and product. At the same time, all newly synthesized polyamines are continuously diluted through Fig. 6 . Effect of polyamine depletion on SPA +1 frameshifting. Polyamine depletion is achieved by overexpression of the wild-type version of SPA. The same cultures were assayed both for frameshifting and polyamine content. Numbers above columns indicate fold reduction of frameshifting and polyamine content compared with cells that do not overexpress SPA. Antizyme genes in S.pombe and C.elegans cell growth and division at a rate that is almost identical to the rate of maximum capacity synthesis. Cells in stationary phase can no longer dilute newly synthesized polyamines, and more importantly lack an effective antizymeindependent mechanism of shutting off ODC. This suggests that SPA is the primary regulator of ODC activity in S.pombe, not only during cell growth (short term regulation) but also in non-dividing cells (longer term regulation). Overexpression of SPA (5±7 days derepression) leads to complete depletion of intracellular putrescine. This result implies that in S.pombe ornithine decarboxylation is the only source of putrescine synthesis (the pathway from arginine via agmatine is not utilized). The complete depletion of cadaverine in SPA overexpressing cells suggests that ODC is the only enzyme in S.pombe that can decarboxylate lysine, which is also the case in rat tissues (Pegg and McGill, 1979) . It is somewhat perplexing that addition of putrescine to the media leads to only partial relief of the growth phenotype associated with SPA overexpression. There are two likely explanations. (i) Perhaps S.pombe imports putrescine poorly. (ii) Alternatively, like the mammalian system, maybe SPA inhibits not only ODC but also the polyamine transporter. Further experiments will help to distinguish between these two models. It is unclear how widespread the antizyme gene is within the fungal kingdom. We have identi®ed and cloned antizyme homologs from two other ®ssion yeasts (Schizosaccharomyces octosporus and Schizosaccharomyces japonicus) and from two distantly related fungi (Botryotinia fuckeliana and Emericella nidulans) (our unpublished results). The antizyme frameshift site of the latter two fungi has evolved in a unique way different from all other known antizymes, but nevertheless even these two distantly related fungi have conserved the autoregulatory +1 frameshifting. The fact that the yeast S.pombe has an antizyme gene suggests the possibility that the higher eukaryotic metazoans may all have an antizyme gene. The only previously reported antizyme activity in unicellular organisms is from E.coli, but recent analyses suggest that E.coli does not have a true antizyme (Ivanov et al., 1998d) . This makes SPA the ®rst bona ®de antizyme in a unicellular organism. The remarkable similarity of the core sequence important for antizyme frameshifting from S.pombe to humans could be due to convergent or divergent evolution. The near identity of this sequence in worms, Drosophila, Xenopus, zebra®sh and humans argues against convergent evolution, as if antizyme frameshifting arose in a common ancestor perhaps more than one billion years ago. Three cis-acting RNA elements are known to stimulate mammalian antizyme 1 frameshifting. One is a 50 nucleotide sequence immediately 5¢ of the shift site (Matsufuji et al., 1995; our unpublished results) . A second stimulator is the UGA stop codon of ORF1 and the third is an RNA pseudoknot starting 3 nucleotides 3¢ of the UGA stop codon. Among frameshift sites of the previously identi®ed antizymes from mammals all the way to Drosophila, there is substantial similarity in the sequences immediately 5¢ of the shift site. Sixteen of the last 18 nucleotides of ORF1 are completely conserved in these genes. Schizosaccharomyces pombe and C.elegans antizymes have 9 of 9 and 6 of 9 (14 out of 19 in O.volvulus) nucleotides identical to the consensus, respectively. For the 5¢ sequences, generally, the more distantly related two antizymes are, the more the similarity is con®ned to the 3¢ end of that region. Our SPA ORF1 deletion data show that mutation of nucleotides that are part of the 5¢ consensus sequence leads to reduced frameshifting ef®ciency. This is another indication that conservation of nucleotide sequence in this region is because of its importance for stimulating ef®cient +1 frameshifting. It is quite striking that in all antizyme gene sequences identi®ed so far, including a number of unpublished ones, ORF1 ends with a UGA stop codon. This is particularly surprising since any of the other two stop codons can substitute for UGA to stimulate antizyme 1 frameshifting, although slightly less ef®ciently, in vitro (Matsufuji et al., 1995) and in vivo (our unpublished results). The 3¢ pseudoknot that stimulates frameshifting in antizyme 1 is highly conserved in all known vertebrate antizymes, including mammalian antizyme 2 ( Figure 1B) . None of the invertebrate antizyme mRNAs identi®ed so far, including those presented here, has a sequence in the equivalent region that can be simply folded to a comparable RNA structure. However, sequences immediately 3¢ of the frameshift site are conserved between invertebrates and vertebrates. The conservation of this region between Drosophila and the vertebrate counterparts has already been noted (Ivanov et al., 1998b) . The C.elegans antizyme gene contains the sequence YGYCCCYCA (Y = pyrimidine) in this region, which is identical to the consensus. The antizyme genes from the other four nematodes also have a similar sequence ( Figure 1B) . The signi®cance of this similarity is not clear [in fact, sequences in this region appear to play no role in antizyme 1 in vitro frameshifting outside of the RNA pseudoknot context (Matsufuji et al., 1995) ]. Only two examples are known where RNA elements 3¢ of the frameshift site stimulate +1 frameshifting. One is the RNA pseudoknot of mammalian antizyme 1 and the second is a short RNA sequence immediately following the frameshift site of Ty3 (Farabaugh et al., 1993) . Additional examples would be very helpful in deciphering the role such elements play in the mechanism of +1 frameshifting. It is currently not known how many and which of the invertebrate antizyme genes contain 3¢ frameshift stimulators. The results presented here show that an S.pombe 3¢ stimulator enhances frameshifting 10-fold. This stimulator appears completely different from the 3¢ RNA pseudoknot in vertebrates. Our deletion experiments indicate that none of the predicted RNA structures contained within the minimally required 3¢ region [up to 150±180 nucleotides downstream of the frameshift site ( Figure 5C )] are suf®cient to confer the stimulatory effect. The SPA 3¢ stimulator may act directly through sequence or may have an unusual RNA structure involving non-Watson±Crick base pairing. More detailed mutagenesis combined with phylogenetic analysis would be required to discern the nature of the 3¢ stimulator of SPA. The nematode antizymes were analyzed for the presence of possible 5¢ or 3¢ stimulators¯anking the core frameshift site. Computer RNA folding programs did not identify any potentially interesting structure. More importantly, phylogenetic analysis with the ®ve identi®ed nematode antizymes failed to identify any conservation of primary RNA sequence (or for that matter potential secondary structure) outside of the core region that is shared between two or more members. This could indicate that no such extra cis-acting stimulators exist in nematode antizymes or that they are located in a very different place within the mRNA, for example the 3¢ untranslated region (the latter suggestion is not supported by our sequence analysis). A common mechanism for frameshifting is re-pairing of the peptidyl tRNA in the new reading frame. However, an alternative mechanism whereby the peptidyl tRNA merely occludes the ®rst base of the next codon, has been documented for yeast Ty3 frameshifting (Farabaugh et al., 1993) . Results of experiments with some mutants of the mammalian antizyme 1 shift site pointed to an occlusion mechanism (Matsufuji et al., 1995) . However, the mechanism with the wild-type, UCC-UGA, shift site is not clear. For C.elegans antizyme the UUU-UGA sequence would be an obvious candidate for a re-pairing since Phe-tRNA could pair perfectly with UUU in both frames. But with UCC-UGA the Ser-tRNA ®rst reading UCC could at best pair two out of three with CCU. This important problem warrants further investigation. The frameshift ef®ciency of SPA frameshift site is lower than that observed with mammalian antizyme 1 even when both are tested in the same organism (S.pombe) [for the frameshift ef®ciency of antizyme 1 cassette in S.pombe, see Ivanov et al. (1998c) ]. It is possible that the observed ef®ciencies for S.pombe antizyme are arti®cially low because the constructs do not include all the cis-acting stimulatory elements. On the other hand there is no reason why a lower level of frameshifting does not correctly re¯ect the evolved balance with the other characteristics of the complex system such as relative protein stabilities. Like other core cellular processes, the antizyme polyamine regulatory scheme is conserved from yeast S.pombe to human. It is not obvious why this very special mechanism is so exquisitely preserved over vast evolutionary time. Perhaps there is another whole aspect to the system that our experiments do not yet detect. From this viewpoint it would seem very important to exploit the genetics systems of S.pombe and C.elegans to understand more thoroughly the physiological effects of perturbing the antizyme system. The SPA gene was ampli®ed using the following primers: 5¢-CAAAACAAGTTTTCATTATTGGTTTTTTTTAAATCAATCCCC (sense) and 5¢-CGTAAATCCAATCTAAATTTAATCTTCAACTAA-ATCATGAAAAGCCTC (antisense). The S.pombe cDNA library used as a template in the ampli®cation was kindly provided by R.Rowley (University of Utah). The C.elegans antizyme gene was ampli®ed using the following primers: 5¢-CCCAGGAATTCCTCGAGTATTTTGA-GTATAATTTTAC (sense) and 5¢-CGGCCGCTCGAGTTAGACCTT-GTAGCTCATGATG (antisense). This same ampli®ed DNA was used to make the constructs for in vitro transcription and translation of C.elegans antizyme by cloning it into pTZ18U plasmid using the SacI and HindIII sites incorporated in the two primers. The in-frame construct was made using a two-step PCR. The cDNA sequences of O.volvulus and P.paci®cus antizyme genes were obtained by performing 5¢ and 3¢ RACE PCR with cDNA libraries, which were kindly provided by Ralf Sommer (P.paci®cus) and Susan Haynes (O.volvulus). The SPA overexpression constructs were made by amplifying the gene with the primers 5¢-GCATCCGAATTCCCAAATCCAAGCATCATACGCC (sense) and 5¢-GCATCCGGATCCGCCAGTGTTCTTACTTTGAGA-TGC (antisense), and then inserting BamHI-digested product between the MscI and BamHI sites of pREP3 plasmid. The in-frame construct was made by two-step PCR and subsequently all in-frame SPA constructs described below were made by one-step PCR using this plasmid's DNA as a template. To make the constructs for frameshift assays in S.pombe, DNA fragments with a given nucleotide length (as described in the main text), were ampli®ed from both the SPA and C.elegans antizyme constructs described above. These fragments were then cloned between the KpnI and BstEII sites of PIU-LAC plasmid (Ivanov et al., 1998c) . The PCR primers included an`AC' spacer between the 5¢ cloning site (BstEII) and the antizyme sequences in order to correct the reading frame. The in vivo frameshifting assays in S.pombe (strains ura4-D18 leu1-32 ade6-M216 h ± and Dspa::LEU2 ura4-D18 leu1-32 ade6-M216 h ± ) were done as described (Ivanov et al., 1998c) . The plasmid for GST±SPA expression was made by PCR amplifying SPA (all but the ®rst codon of ORF1 through the downstream ORF2) from an in-frame template and cloning the product into the EcoRI and XhoI restriction sites of pGEX-5X-3 plasmid. The antizyme frameshift site in the BMV-coatantizyme fusion construct (C3NE) (Matsufuji et al., 1995) was mutated with a two-step PCR. To generate the two knockout strains, Dspa::URA4 and Dspa::LEU2, both ORFs of SPA were replaced exactly with the ORF of either URA4 or LEU2. To accomplish this, two pairs of primers ampli®ed URA4 and LEU2 such that 50±60 nucleotides, which normallȳ ank the two ORFs of SPA,¯ank the ORFs of the two genes. The ampli®ed DNA products were gel puri®ed and 2 mg of each were used to electroporate into ura4-D18 leu1-32 ade6-M216 h ± cells. URA + and LEU + transformants were selected by growth on URA ± and LEU ± media, respectively. PCR screen and partial sequencing, with primers¯anking the regions used for the homologous recombination, con®rmed the SPA disruptions. All DNA clones were sequenced with automated sequencing machines (ABI 100). Schizosaccharomyces pombe ODC active crude extracts were prepared as follows: S.pombe (strain 1519, leu1-32, h ± ) provided by R.Rowley was grown to OD 600 0.7 in 50 ml of minimal media + LEU. Ten milligrams of lysing enzymes (Sigma) were added, followed by continued incubation for 30 min at 30°C. Cells were harvested and washed once with cold homogenization buffer [25 mM Tris±HCl pH 7, 0.25 M sucrose, 1 mM dithiothreitol (DTT), 20 mM pyridoxal-5-phosphate, 2 mM EDTA] then resuspended in 0.75 ml of homogenization buffer. Cells were broken open and the lysate was clari®ed by centrifugation at 10 000 r.p.m. for 15 min at 4°C. Extracts were dialyzed overnight in dialysis buffer (25 mM Tris± HCl pH 7.4, 1 mM DTT, 20 mM pyridoxal-5-phosphate, 0.1 mM EDTA). A volume of 25 ml of extract was used for each ODC assay. ODC activity was assayed by measuring the release of 14 CO 2 from L-[1-14 C]ornithine (Amersham) as described (Nishiyama et al., 1988) . Each reaction took 1 h. Pre-incubation of S.pombe extract with 0.1 mM di¯uoromethyl ornithine (DFMO) for 15 min led to >99% inhibition of 14 CO 2 release. The cells were collected by centrifugation, washed twice with 1 ml of phosphate-buffered saline (PBS) and then the pellet was frozen at ±80°C until use. The pellet was resuspended in 0.1 ml of PBS. An aliquot of the suspension was mixed with an equal volume of 8% perchloric acid, vortexed for 1 min, kept on ice for 5 min and centrifuged at 15 000 r.p.m., 4°C for 5 min. Ten microliters of the supernatant were subjected to polyamine analysis using¯uorometry on high-performance liquid chromatography as described previously (Murakami et al., 1989) . Protein concentrations were determined with the BCA protein assay kit (Pierce). The experiments with the BMV-coat-antizyme fusion constructs were performed as described previously (Matsufuji et al., 1995) . All other plasmid DNA templates were prepared using QIAGEN Miniprep Kit and then digested with HindIII. Transcripts for SPA in vitro translation were made from PCR templates that had a T7 promoter incorporated into the PCR primers. Linearized DNA (1 mg) was used as a template for in vitro transcription with Ambion MEGAshortscript TM T7 Kit. The DNasetreated RNAs were recovered and resuspended in 40 ml of RNase-free water. One microliter of each speci®ed transcript suspension was used in each in vitro translation reaction [0.5 ml of 1 mM amino acid mix ±Met, 7 ml of reticulocyte lysate (Promega), 0.5 ml of [ 35 S]Met (Amersham)] to a total volume of 10 ml. The reactions were stopped by adding 1 ml of RNase (10 mg/ml). The frameshift ef®ciencies were quanti®ed as described (Ivanov et al., 1998a) ."
11
"Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus"
"Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant-negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering RNAs. Similar to the wild-type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild-type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA-dependent RNA synthesis."
"Introduction hnRNP A1 is an RNA-binding protein that contains two RNA-binding domains (RBDs) and a glycine-rich domain responsible for protein±protein interaction. It is involved in pre-mRNA splicing and transport of cellular RNAs (reviewed by Dreyfuss et al., 1993) . It is predominantly located in the nucleus, but also shuttles between the nucleus and the cytoplasm (Pin Äol-Roma and Dreyfuss, 1992) . The signal that mediates shuttling has been identi®ed as a 38 amino acid sequence, termed M9, located near the C-terminus of hnRNP A1 between amino acids 268 and 305 (Michael et al., 1995; Siomi and Dreyfuss, 1995; Weighardt et al., 1995) . Yeast two-hybrid screening with M9 as bait resulted in the discovery of a novel transportin-mediated pathway for nuclear import of hnRNP A1 (Pollard et al., 1996; Fridell et al., 1997; Siomi et al., 1997) . The function of the cytoplasmic hnRNP A1 has not been well de®ned. Studies have shown that cytoplasmic and nuclear hnRNP A1 exhibit different RNA-binding pro®les. Cytoplasmic hnRNP A1 is capable of high-af®nity binding to AU-rich elements that modulate mRNA turnover and translation (Hamilton et al., 1993 (Hamilton et al., , 1997 Henics et al., 1994) . It has also been shown to promote ribosome binding to mRNAs by a cap-mediated mechanism, and prevent spurious initiation at aberrant translation start sites (Svitkin et al., 1996) . MHV belongs to the Coronaviridae family of positivesense, single-stranded RNA viruses. MHV replication and transcription occur exclusively in the cytoplasm of infected cells via the viral RNA-dependent RNA polymerase (RdRp) (reviewed by Lai and Cavanagh, 1997) . Initially, the 5¢-most gene 1 of the viral genome is translated into the viral RdRp, which then replicates the viral genomic RNAs into negative-strand RNAs. Subsequently, the negative-strand RNAs are used as templates to transcribe mRNAs, which include a genomic-sized RNA and a nested set of subgenomic mRNA transcripts, all with an identical 5¢ non-translated leader sequence of 72±77 nucleotides and 3¢ co-terminal polyadenylated ends. The subgenomic mRNA transcription of MHV utilizes a unique discontinuous mechanism in which the leader sequence, often derived from a different molecule, is fused to RNAs at the intergenic (IG) sites (i.e. transcription initiation site) to generate subgenomic mRNAs (Jeong and Makino, 1994; Liao and Lai, 1994; Zhang et al., 1994) . The exact mechanism of how these mRNAs are made is still controversial. However, it has been shown that the process of discontinuous RNA transcription is regulated by several viral RNA elements, including the cis-and trans-acting leader RNA Zhang et al., 1994) , IG sequence (Makino et al., 1991) and 3¢-end untranslated sequence (Lin et al., 1996) . There is considerable biochemical evidence suggesting possible direct or indirect interactions between the various RNA regulatory elements. hnRNP A1 binds MHV negative (±)-strand leader and IG sequences (Furuya and Lai, 1993; Li et al., 1997) . Site-directed mutagenesis of the IG sequences demonstrated that the extent of binding of hnRNP A1 to the IG sequences correlated with the ef®ciency of transcription from the IG site (Zhang and Lai, 1995; Li et al., 1997) . Immunostaining of hnRNP A1 showed that hnRNP A1 relocated to the cytoplasm of MHV-infected cells, where viral RNA synthesis occurs (Li et al., 1997) . hnRNP A1 also mediates the formation of a ribonucleoprotein complex containing the MHV (±)-strand leader and IG sequences . These results suggest that hnRNP A1 may serve as a protein mediator for distant RNA regions to interact with each other. Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus The EMBO Journal Vol. 19 No. 17 pp. 4701±4711, 2000 ã European Molecular Biology Organization Many cellular proteins, including calreticulin (Singh et al., 1994) , polypyrimidine tract-binding protein (PTB) (Hellen et al., 1994; Wu-Baer et al., 1996) , La protein (Pardigon and Strauss, 1996), Sam68 (McBride et al., 1996) , poly(rC)-binding protein (Parsley et al., 1997) and nucleolin (Waggoner and Sarnow, 1998) , have been implicated to be involved in viral RNA transcription or replication. In addition to MHV, hnRNP A1 has also been reported to interact with human cytomegalovirus immediate-early gene 2 protein, which plays an important role in the regulation of virus replication (Wang et al., 1997) . Furthermore, a yeast protein related to human core RNA splicing factors, Lsm1p, has been shown to be required for the ef®cient replication of brome mosaic virus RNA (Diez et al., 2000) . Recently, Reddy and colleagues demonstrated an inhibition of HIV replication by dominant-negative mutants of Sam68 (Reddy et al., 1999) . However, none of these cellular proteins has been shown experimentally to participate directly in RNA-dependent RNA synthesis. In order to demonstrate the involvement of hnRNP A1 in MHV RNA replication and transcription, we established several DBT cell lines stably expressing either the wildtype (wt) hnRNP A1 or a C-terminus-truncated mutant lacking the M9 sequence and part of the glycine-rich domain. We showed that the mutant hnRNP A1, which was localized predominantly in the cytoplasm, exhibited dominant-negative effects on viral genomic RNA replication and subgenomic mRNA transcription. In contrast, overexpression of the wt hnRNP A1 accelerated the synthesis of all viral RNAs. Our results provide strong evidence that hnRNP A1 is directly or indirectly involved in MHV RNA synthesis in the cytoplasm and that the C-terminal part of the protein is important for its function. This ®nding thus reveals a novel function for hnRNP A1 in the cytoplasm. Characterization of stable cell lines expressing the wt and a C-terminus-truncated hnRNP A1 To explore a potential role for hnRNP A1 in MHV RNA synthesis, we established murine DBT cell lines stably expressing the Flag-tagged wt hnRNP A1 (DBT-A1) or a mutant hnRNP A1, which has a 75 amino acid deletion from the C-terminus (DBT-A1DC) ( Figure 1A ). This mutant lacks part of the glycine-rich domain and the M9 sequence responsible for shuttling hnRNP A1 between the nucleus and the cytoplasm. Immunoblot of the whole-cell lysates with an anti-Flag antibody detected a 34 kDa protein in DBT-A1 cells and a 27 kDa protein in three independent clones of DBT-A1DC cells ( Figure 1B ), whereas no protein was cross-reactive to the anti-Flag antibody in the control cell line stably transfected with the pcDNA3.1 vector (DBT-VEC). The amounts of the Flagtagged wt and truncated hnRNP A1 were comparable in these cell lines. A chicken polyclonal antibody against hnRNP A1 detected two endogenous hnRNP A1 isoforms or hnRNP A1-related proteins in the whole-cell lysates of all of the cell lines. The bottom band (34 kDa) overlaps the Flag-tagged wt hnRNP A1 in DBT-A1 cells. There was only a slight increase in the overall amount of hnRNP A1 in DBT-A1 cells as compared with DBT-VEC cells, indicating that the exogenous hnRNP A1 constituted a small fraction of the total hnRNP A1 in the cells. In DBT-A1DC cells, an additional band of smaller size (27 kDa) corresponding to the mutant hnRNP A1 was detected. The overall expression levels of the exogenous hnRNP A1 and hnRNP A1DC were~3-fold lower than that of the endogenous hnRNP A1 in whole-cell lysates ( Figure 1B ). Similar to the endogenous hnRNP A1 protein (Pin Äol-Roma and Dreyfuss, 1992) , the Flag-tagged wt hnRNP A1 was localized almost exclusively in the nucleus ( Figure 1C ). The mutant hnRNP A1, however, was localized predominantly in the cytoplasm ( Figure 1C) , consistent with the previous ®nding that the M9 nuclear localization signal is necessary to localize hnRNP A1 to the nucleus Weighardt et al., 1995) . Thus, hnRNP A1DC was much more abundant than the endogenous hnRNP A1 in the cytoplasm. The expression levels of the wt or mutant hnRNP A1 varied among individual cells based on immuno¯uorescent staining ( Figure 1C ). The growth rate ( Figure 1D ) and cell morphology (data not shown) were similar among the different cell lines. The effects of overexpression of the wt and mutant hnRNP A1 on syncytium formation and virus production We ®rst assessed the effects of hnRNP A1 overexpression on the morphological changes induced by MHV-A59 infection using several different clones of DBT cell lines. Virus infection was performed at a multiplicity of infection (m.o.i.) of 0.5 to detect the subtle morphological differences among the different cell lines. Syncytia appeared at~7 h post-infection (p.i.) in DBT-VEC cells and~1 h earlier in DBT-A1 cells. At both 8 and 14 h p.i., syncytia were signi®cantly larger and more spread out in DBT-A1 cells than those in DBT-VEC cells ( Figure 2A ). Similar differences were observed with two additional clones of DBT-A1 cells (data not shown). In contrast, no syncytium was observed in three different clones of DBT-A1DC cells, even at 14 h p.i. At 24 h p.i., almost all DBT-A1 cells detached from the plate, but~10±20% of DBT-VEC cells still remained on the plate (data not shown). Remarkably, there was no sign of syncytium formation in DBT-A1DC cells until 24 h after virus infection, when the overall morphology of the cells was similar to that of DBT-VEC cells at 7 h p.i. (data not shown). All of the DBT-A1DC cells were eventually killed at~48 h p.i., suggesting that the inhibition of viral replication was not a result of the disruption of the MHV receptor. Correspondingly, virus production from these cell lines was signi®cantly different. Between 6 and 14 h p.i., virus production from DBT-A1DC cells was 100-to 1000-fold less than that from DBT-VEC and DBT-A1 cells ( Figure 2B ). DBT-A1 cells produced twice as many viruses as those from DBT-VEC cells during that time period. Relocalization of hnRNP A1 during MHV infection MHV RNA synthesis occurs exclusively in the cytoplasm of infected cells. In order for hnRNP A1 to participate directly in viral transcription, it has to be recruited to the site of RNA synthesis. Although hnRNP A1 shuttles between the nucleus and the cytoplasm in normal cells (Pin Äol-Roma and Dreyfuss, 1992) , the level of cytoplasmic hnRNP A1 is very low. We have demonstrated previously that hnRNP A1 relocates from the nucleus to the cytoplasm of MHV-infected cells (Li et al., 1997) . To determine whether the overexpressed hnRNP A1 may participate in MHV RNA synthesis, we performed immunostaining experiments using an anti-Flag antibody to localize Flag-tagged hnRNP A1. In DBT-A1 cells, a signi®cant increase in the cytoplasmic level of hnRNP A1 and a corresponding decrease of nuclear hnRNP A1 were observed in virus-infected cell syncytia at 7 h p.i. ( Figure 3B ); these cells express the MHV nucleocapsid (N) protein in the cytoplasm ( Figure 3A ). By comparison, in the uninfected cells, which did not have N protein staining, hnRNP A1 was predominantly localized to the nucleus (arrow in Figure 3B ). In DBT-A1DC cells, very few cells were stained positive for the MHV N protein at 7 h p.i. ( Figure 3C ). Signi®cantly, the viral N protein was detected only in the cells that were stained weakly or not at all for Flag-hnRNP A1 ( Figure 3D ), suggesting that the expression of a high level of hnRNP A1DC interfered with viral replication. The effects of wt and mutant hnRNP A1 on MHV protein production We further investigated the effects of the wt and mutant hnRNP A1 on the production of MHV structural and nonstructural proteins. Cytoplasmic protein was extracted from infected cell lines at different time points after infection for immunoblot analysis to detect an open reading frame (ORF) 1a product, p22 (Lu et al., 1998) and the N protein. p22 expression in DBT-VEC cells was clearly detected at 6 h p.i. and peaked at~16 h p.i. ( Figure 4A ). In DBT-A1 cells, p22 appeared at 5 h p.i. and peaked at~8 h p.i. In DBT-A1DC cells, no p22 protein was detected until 16 h p.i. Similar patterns of differences were observed for the N protein in these three cell lines. Actin levels in different cell lines remained relatively constant throughout the infection, except that, in DBT-A1 cells, actin was not detected at 16 and 24 h p.i. due to the loss of the dead cells ( Figure 4A ). These results clearly demonstrated that overexpression of the wt hnRNP A1 accelerated viral protein production, whereas expression of the mutant hnRNP A1 delayed it. We also performed immuno¯uorescent staining of the N protein at 7 h p.i. to further con®rm the western blot results. As represented by images shown in Figure 4B , there were more DBT-A1 cells stained positive for the N protein than DBT-VEC cells. Very few cells were found to express the N protein in DBT-A1DC cells. The p22 and N proteins appeared as doublets in some of the lanes of Figure 4A , but the results varied from experiment to experiment. The N protein is known to be phosphorylated (Stohlman and Lai, 1979) . Whether p22 is post-translationally modi®ed is not known. Figure 5A ). DBT-A1 cells showed a signi®cantly higher level of [ 3 H]uridine incorporation, which peaked at~8 h p.i. DBT-A1DC cells did not show any detectable level of incorporation of the radioactivity. These results suggest that hnRNP A1 regulates MHV RNA synthesis. We further assessed the production of genomic and subgenomic MHV RNAs in these cell lines by northern blot analysis. The genomic and the six subgenomic RNA species were detected at 8 h p.i. in both DBT-VEC and DBT-A1 cells; there were signi®cantly higher steady-state levels of all of the RNA species in DBT-A1 cells ( Figure 5B ). In contrast, no viral RNA was detected in DBT-A1DC cells at that time point. At 16 h p.i., MHV RNA levels in DBT-VEC and DBT-A1 cells decreased generally because of the loss of the dead cells, while the smaller subgenomic RNAs became detectable in DBT-A1DC cells. By 24 h p.i., most viral RNA species became detectable in DBT-A1DC cells ( Figure 5B , lane 10), while most of the DBT-A1 cells were dead (lane 9). These results con®rmed that the synthesis of all of the viral RNA species is accelerated by overexpression of the wt hnRNP A1 and delayed by a dominant-negative mutant of hnRNP A1. In this analysis, we also detected an additional RNA species (arrow in Figure 5B ), which was determined to be a defective-interfering (DI) RNA by northern blot analysis using a probe representing the 5¢-untranslated region (without the leader), which is present only in genomic and DI RNAs (data not shown). Interestingly, this DI RNA was inhibited to a greater extent than other RNA species in DBT-A1DC cells. This result suggests that the replication of DI RNAs is more sensitive to the dominant-negative inhibition by cytoplasmic hnRNP A1. To demonstrate further that MHV RNA transcription machinery is defective in cells expressing the mutant hnRNP A1, we studied transcription of an MHV DI RNA, 25CAT, which contains a transcription promoter (derived from the IG sequence for mRNA 7, IG7) and a chloramphenicol acetyltransferase (CAT) reporter gene . CAT activity can be expressed from At 1 h p.i., serum-free medium was replaced by virus growth medium containing 1% NCS and 5 mg/ml actinomycin D. [ 3 H]uridine (100 mCi/ml) was added to the infected cells at 2, 3, 4, 5, 6, 7, 8, 9, 16 and 24 h p.i. After 1 h labeling, cytoplasmic extracts were prepared and precipitated with 5% TCA. The TCA-precipitable counts were measured in a scintillation counter. (B) Northern blot analysis of MHV genomic and subgenomic RNA synthesis in DBT cells. Cytoplasmic RNA was extracted from MHV-A59-infected cells at 8, 16 and 24 h p.i. for northern blot analysis. The naturally occurring DI RNA of MHV-A59 is indicated by an arrow. this DI RNA only if a subgenomic mRNA containing CAT sequences is produced . The 25CAT RNA was transfected into MHV-A59-infected cells 1 h after infection. At 8 h p.i., CAT activity in DBT-A1 cells was signi®cantly higher than that in DBT-VEC cells ( Figure 6A ). On the other hand, CAT activity was very low in DBT-A1DC cells. At 24 h p.i., CAT activity in DBT-A1 cells became slightly lower than that in DBT-VEC cells because of the loss of the dead DBT-A1 cells. The CAT activity in DBT-A1DC was still signi®cantly lower than that in DBT-VEC or DBT-A1 cells. These results established that mRNA transcription from the DI RNA was also inhibited by hnRNP A1DC. The results shown above ( Figure 5B ) also suggest that DI RNA replication is more sensitive to the inhibitory effects of the hnRNP A1 mutant. To con®rm this result, we further studied replication of another DI RNA during serial virus passages. DBT cells were infected with MHV-A59 and transfected with DIssE RNA derived from JHM virus (Makino and Lai, 1989) ; the virus released (P0) was passaged twice in DBT cells to generate P1 and P2 viruses. DBT cells were infected with these viruses, and cytoplasmic RNA was extracted for northern blot analysis using glyoxalated RNA for a better resolution of smaller RNAs. For DBT-A1DC cells, RNA was extracted at 36 h p.i. since viral RNA synthesis was delayed in this cell line. Cells infected with P0 viruses did not yield detectable amounts of DIssE, but contained the naturally occurring A59 DI RNA, whose replication was inhibited more strongly than the synthesis of MHV genomic and subgenomic RNAs in DBT-A1DC cells ( Figures 5B, lanes 8±10 and 6B, lanes 1± 3). However, this A59 DI RNA was not detectable in cells infected with P1 and P2 viruses ( Figure 6B , lanes 4±9). In contrast, DIssE appeared in cells infected with P1 viruses and further increased in cells infected with P2 viruses, indicating that the replication of the smaller DIssE may have an inhibitory effect on the replication of the larger A59 DI RNA (Jeong and Makino, 1992) . Similar to the A59 DI RNA, the replication of DIssE RNA was much more strongly inhibited than that of MHV genomic and subgenomic RNAs in DBT-A1DC cells ( Figure 6B , lanes 6 and 9). Our results thus suggest that MHV DI RNA replication is more dependent on the function of cytoplasmic hnRNP A1. The mechanism of dominant-negative inhibition by the C-terminal deletion mutant of hnRNP A1 To understand the underlying mechanism of the inhibition of MHV RNA transcription by the C-terminal-deletion mutant of hnRNP A1, we ®rst examined the RNA-and protein-binding properties of this mutant protein. Electrophoretic mobility shift assay demonstrated that hnRNP A1DC retained the ability to bind the MHV (±)strand leader RNA and to form multimers with itself, similar to the wt hnRNP A1 (data not shown); this is consistent with the fact that both of its RBDs are intact ( Figure 1A) . Furthermore, UV-crosslinking experiments showed that increasing amounts of puri®ed glutathione S-transferase (GST)±hnRNP A1DC ef®ciently competed with the endogenous hnRNP A1 for the binding of the MHV (±)-strand leader RNA ( Figure 7A ), indicating that the binding of hnRNP A1DC to RNA was not affected. These results suggest that the RNA-binding properties of hnRNP A1DC were intact. We next examined the protein-binding properties of hnRNP A1DC. Since hnRNP A1 has been shown to interact with the N protein, which also participates in MHV RNA synthesis (Compton et al., 1987; Wang and Zhang, 1999) , we ®rst determined whether the dominantnegative mutant of hnRNP A1 retained the ability to interact with the N protein in vitro. GST pull-down assay using various truncation mutants of hnRNP A1 showed that the N protein bound the N-terminal domain (aa 1±163) of hnRNP A1 ( Figure 7B) ; thus, the binding of hnRNP A1DC [equivalent to hnRNP A1(1±245)] to the N protein was not affected. We next examined the in vivo interaction of the wt and mutant hnRNP A1 with an MHV ORF 1a product, p22, which has been shown to co-localize with the de novo synthesized viral RNA (S.T.Shi and The viruses were passaged twice in wt DBT cells to obtain P1 and P2 viruses. Cytoplasmic RNA was extracted from the DBT cells infected with P0, P1 and P2 viruses and treated with glyoxal before electrophoresis and northern blot analysis using a 32 P-labeled (±)-strand mRNA 7 as a probe. The A59 DI RNA and DIssE RNA are indicated by arrows. M.M.C.Lai, unpublished results) and associate with the viral replicase complex (Gibson Bost et al., 2000) . Cytoplasmic extracts from MHV-A59-infected cells were immunoprecipitated with anti-Flag antibody-conjugated beads, followed by western blotting with a rabbit polyclonal antibody against p22. At 8 h p.i., p22 was co-precipitated with the Flag-tagged hnRNP A1 from DBT-A1 cells, whereas no precipitation of p22 was observed in DBT-VEC cells ( Figure 7C ). For DBT-A1DC cells, co-immunoprecipitation was performed at 24 h p.i., when abundant MHV proteins were synthesized. p22 was shown to co-precipitate with hnRNP A1DC, indicating that hnRNP A1DC still formed a complex with the viral polymerase gene product. These results suggest that the ability of hnRNP A1DC to interact with the N and polymerase proteins was not altered. We next investigated whether the mutant hnRNP A1 is de®cient in the interaction with any other cellular proteins in this RNA±protein complex. We labeled proteins in MHV-infected cells or mock-infected cells at different time points after infection and immunoprecipitated with the anti-Flag antibody. Signi®cantly, a cellular protein of 250 kDa was shown to be associated only with the wt hnRNP A1, but not the mutant hnRNP A1 ( Figure 7D ), suggesting that hnRNP A1 binds to this protein through its C-terminal domain. We propose that this cellular protein is another important component of the MHV RNA transcription/replication complex. There is an accumulating body of evidence signifying the importance of cellular factors in RNA synthesis of RNA viruses (reviewed by Lai, 1998) . Previous studies have shown that hnRNP A1 binds to the cis-acting sequences of MHV template RNA and that this interaction correlates with the transcription ef®ciency of viral RNA in vivo (Zhang and Lai, 1995; Li et al., 1997) . In addition, hnRNP A1 is also implicated in viral RNA replication by the recent ®nding that hnRNP A1 interacts with the 3¢-ends of both positive-and negative-strand MHV RNA (P.Huang and M.M.C.Lai, unpublished results). However, hnRNP A1 modulates cytoplasmic viral RNA synthesis the functional importance of hnRNP A1 in viral RNA synthesis has so far not been directly demonstrated. In the present study, we established that MHV RNA transcription and replication were enhanced by overexpression of the wt hnRNP A1 protein, but inhibited by expression of a dominant-negative hnRNP A1 mutant in DBT cell lines. Our results suggest that hnRNP A1 is a host protein involved in the formation of a cytoplasmic transcription/ replication complex for viral RNA synthesis. This represents a novel function for hnRNP A1 in the cytoplasm. Our results indicate that the inhibitory effects on MHV replication exhibited by the dominant-negative mutant of hnRNP A1 were relatively more prominent than the enhancement effects by overexpression of the wt hnRNP A1. This is consistent with the subcellular localization patterns of the wt and mutant hnRNP A1 proteins. The overexpressed exogenous wt hnRNP A1 in DBT-A1 cells was predominantly localized in the nucleus, similar to the endogenous hnRNP A1 ( Figure 1C ). The C-terminal-deletion mutant, however, was localized mainly in the cytoplasm. Thus, the level of hnRNP A1DC was much higher than the endogenous wt hnRNP A1 in the cytoplasm of DBT-A1DC cells, where MHV replication occurs. This result explains why hnRNP A1DC could have a strong dominant-negative inhibitory effect, despite the fact that it was expressed at a lower level than the endogenous hnRNP A1 ( Figure 1B) . The effects of the expression of the wt and mutant hnRNP A1 on virus production ( Figure 2B ), viral protein synthesis ( Figure 4A ) and viral RNA synthesis ( Figure 5A ) correlated with each other. Furthermore, hnRNP A1DC caused not only a global inhibition of genomic RNA replication and subgenomic mRNA transcription, but also a preferential inhibition of at least two DI RNA species. These results suggest that the inhibition of MHV replication by the hnRNP A1 mutant was most likely a direct effect on viral RNA synthesis rather than an indirect effect on other aspects of cellular or viral functions. Since hnRNP A1 binds directly to the cis-acting MHV RNA sequences critical for MHV RNA transcription (Li et al., 1997) and replication (P. Huang and M.M.C.Lai, unpublished results) , it is most likely that hnRNP A1 may participate in the formation of the transcription/replication complex. Indeed, our data show that hnRNP A1 interacts directly or indirectly with the N protein and a gene 1 product, p22, both of which are probably associated with the viral transcription/replication complex (Compton et al., 1987; Wang and Zhang, 1999; Gibson Bost et al., 2000) . hnRNP A1 may participate directly in viral RNA synthesis in a similar role to that of transcription factors in DNAdependent RNA synthesis, e.g. by maintaining favorable RNA conformation for RNA synthesis. Alternatively, hnRNP A1 may modulate MHV RNA transcription or replication by participating in the processing, transport and controlling the stability of viral RNAs. It has been reported that RNA processing of retroviruses, human T-cell leukemia virus type 2 (Black et al., 1995) and HIV-1 (Black et al., 1996) , is altered by the binding of hnRNP A1 to the viral RNA regulatory elements. It is also possible that hnRNP A1 may participate in MHV RNA synthesis indirectly by affecting the production of other host cell proteins, which may, in turn, regulate MHV RNA synthesis. Since hnRNP A1 is a dose-dependent altern-ative splicing factor (Caceres et al., 1994) , even small changes in the intracellular level of hnRNP A1 can alter the splicing of other cellular proteins. Regardless of the mechanism, our study established the importance of cellular factors in viral RNA-dependent RNA synthesis. The transcription from 25CAT RNA was strongly inhibited by the dominant-negative mutant of hnRNP A1, as shown by CAT assays ( Figure 6A ). In addition, the replication of the naturally occurring A59 DI RNA and the arti®cial DIssE RNA was completely abolished ( Figure 6B) . Surprisingly, the replication of MHV DI RNAs suffered a stronger inhibition by the dominantnegative mutant of hnRNP A1 than the synthesis of MHV genomic and subgenomic RNAs, suggesting that DI RNA replication may be more dependent on hnRNP A1. Although DI RNAs contain all of the cis-acting replication signals that are essential for their replication in normal cells (Kim and Makino, 1995) , the small size of DI RNA may cause it to require more hnRNP A1 to maintain a critical RNA structure. It has been shown that different DI RNAs require different cis-acting signals for RNA replication (Kim and Makino, 1995) . Our results demonstrate that the C-terminal domain of hnRNP A1, including the M9 sequence and the glycinerich region, is important for MHV RNA transcription and replication, but the mechanism of the dominant-negative effects of hnRNP A1DC is still not clear. hnRNP A1DC retains the RNA-binding and self-association ability and is capable of binding the viral proteins N and p22, which are associated with the transcription/replication complex. It is possible that hnRNP A1DC is not productive due to its inability to interact with other viral or cellular proteins that are involved in MHV RNA synthesis. We have found a protein of~250 kDa that binds only the wt, but not the mutant hnRNP A1 ( Figure 7D ). It remains to be shown whether this cellular protein is involved in MHV RNA synthesis. In our preliminary study, we found that MHV could replicate in an erythroleukemia cell line, CB3, which was reported to lack detectable hnRNP A1 expression as a result of a retrovirus integration in one allele and loss of the other allele (Ben-David et al., 1992) . Since hnRNP A1 protein is involved in a variety of important cellular functions, including RNA splicing, transport, turnover and translation, it is conceivable that other redundant gene products may substitute for the function of hnRNP A1 in CB3 cells. Indeed, UV-crosslinking assays using CB3 cell extracts detected two proteins comparable to hnRNP A1 in size that could interact with the MHV negative-strand leader RNA (data not shown). These proteins may represent hnRNP A1-related proteins, since many of such hnRNPs exist in the cells (Buvoli et al., 1988; Burd et al., 1989) . Therefore, multiple cellular proteins may have the capacity to be involved in MHV RNA synthesis. Based on previous ®ndings (Kim and Makino, 1995; Zhang and Lai, 1995; Li et al., 1997) and the results from this study, we propose a model for the regulation of transcription/replication of MHV RNA by hnRNP A1. We hypothesize that hnRNP A1 is one of the components of the MHV RNA transcription or replication complex, and the crosstalk between hnRNP A1 and another viral or cellular RNA-binding protein (designated X in Figure 8 ) is essential for MHV replication and transcription. The X protein binds to the C-terminus of hnRNP A1 and cooperates with hnRNP A1 to recruit more proteins to form the transcription or replication complex. The C-terminaldeletion mutant of hnRNP A1 loses the ability to interact with the X protein and to bring it into the initiation complex, resulting in an inhibition of MHV RNA transcription and replication. The residual replication and transcription activities of MHV RNA in the absence of functional hnRNP A1 may be due to a limited af®nity of the X protein to a cis-acting signal that is only present in MHV genomic RNA (site B). On the other hand, DI RNAs may lack this cis-acting signal. When the crosstalk between the X protein and hnRNP A1 is abolished by the dominant-negative mutant of hnRNP A1, the X protein can no longer participate in the formation of the initiation complex, resulting in a complete loss of DI RNA replication. In summary, our data provide direct experimental evidence that hnRNP A1 is involved directly or indirectly in MHV RNA synthesis, probably by participating in the formation of an RNA transcription/replication complex. This ®nding reveals a novel cytoplasmic function for hnRNP A1. Cells and viruses DBT cells, a mouse astrocytoma cell line (Hirano et al., 1974) , were cultured in Eagle's minimal essential medium (MEM) supplemented with 7% newborn calf serum (NCS) and 10% tryptone phosphate broth. MHV strain A59 (Robb and Bond, 1979) was propagated in DBT cells and maintained in virus growth medium containing 1% NCS. Plasmid construction and establishment of DBT stable cell lines The cDNA of the murine hnRNP A1 gene was ampli®ed by RT±PCR using RNA extracted from DBT cells and a set of primers representing the 5¢-and 3¢-ends of hnRNP A1-coding region, and cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). The 8 amino acid Flag tag was attached to the N-terminus of hnRNP A1 by including the Flag tag in the forward PCR primer. The truncated hnRNP A1DC was similarly constructed using a PCR-ampli®ed fragment that represents hnRNP A1 (aa 1±245). For the establishment of permanent DBT cell lines, pcDNA3.1 alone or the plasmid containing the Flag-tagged hnRNP A1 or hnRNP A1DC was transfected into 60% con¯uent DBT cells using DOTAP according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN) . After 4 h, the transfected cells were selected in DBT cell medium containing 0.5 mg/ml Geneticin (G418) (Omega Scienti®c, Tarzana, CA) for 10 days. Single colonies were then collected and cultured individually for 10 additional days before screening for the expression of Flag-tagged proteins. The polyclonal rabbit antibody against p22 was a gift from Dr Susan C.Baker at Loyola University, IL. The chicken polyclonal antibody against hnRNP A1 was produced by Aves Labs, Inc. (Tigard, OR) by immunizing chickens with the puri®ed mouse hnRNP A1 protein expressed in bacteria. The polyclonal anti-Flag antibody was purchased from Af®nity Bioreagents (Golden, CO). The goat polyclonal antibody against actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse monoclonal antibody against the N protein has been described previously (Fleming et al., 1983) . Examination of growth rate of permanent DBT cells Equal numbers (1 3 10 5 ) of DBT-VEC, DBT-A1 and DBT-A1DC cells were plated in 10-cm culture plates and maintained in culture medium for 4 days. Cells were trypsinized, stained with Trypan Blue (Gibco-BRL, Grand Island, NY) and counted at 24-h intervals with a hemacytometer (Hausser Scienti®c, Horsham, PA). Plaque assay DBT cells in 10-cm plates were infected with MHV-A59 at an m.o.i. of 2. After 1 h for virus adsorption, the cells were washed three times with serum-free MEM, which was then replaced with virus growth medium containing 1% serum. At 1, 6, 8, 10, 14 and 24 h p.i., 1 ml of medium was taken from each plate for plaque assay. [ 3 H]uridine labeling of MHV RNA Cells plated in 6-well plates were infected with MHV-A59 at an m.o.i. of 2. At 1 h p.i., 5 mg/ml actinomycin D was added to the virus growth medium to inhibit cellular RNA synthesis. To label newly synthesized MHV RNA, 100 mCi/ml of [ 3 H]uridine (NEN, Boston, MA) were added to the medium at hourly intervals. After 1 h of labeling, the cells were washed twice in ice-cold PBS and scraped off the plates in 1 ml of PBS. The cells were then collected by centrifugation and incubated in 200 ml of NTE buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA) containing 0.5% NP-40, 0.5 mM dithiothreitol (DTT) and 400 U/ml of RNasin on ice for 15 min. After centrifugation, 5 ml of the cytoplasmic extract were spotted on a piece of 3 mm paper and incubated with 5% trichloroacetic acid (TCA). The radioactivity remaining on the 3 mm paper was measured in a scintillation counter. Northern blot analysis DBT cells were infected with MHV-A59 at an m.o.i. of 2. At 8, 16 and 24 h p.i., cytoplasmic extract was prepared as described above and subjected to phenol/chloroform extraction and ethanol precipitation to purify cytoplasmic RNA. Approximately 10 mg of RNA were separated by electrophoresis on a 1.2% formaldehyde-containing agarose gel and transferred to a nitrocellulose membrane. For a better resolution of the DIssE RNA ( Figure 6B ), RNA was glyoxalated before being electrophoresed on a 1% agarose gel. An in vitro transcribed, 32 P-labeled negative-strand mRNA 7 of MHV-JHM was used as a probe to detect MHV genomic and subgenomic RNAs. For detecting DI RNA species, RNA blots were probed with an RNA representing a sequence complementary to the sequence of the 5¢-untranslated region of MHV-JHM RNA, but excluding the leader sequence. Western blot analysis DBT cells in 6-well plates were infected with MHV-A59 and cytoplasmic extracts were prepared as described previously (Li et al., 1997) at various hnRNP A1 modulates cytoplasmic viral RNA synthesis time points p.i. The extracts were electrophoresed on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane for western blotting. Immuno¯uorescence staining Cells were washed in phosphate-buffered saline (PBS) and ®xed in 4% formaldehyde for 20 min at room temperature, followed by 5 min in ±20°C acetone. Primary antibodies were diluted in 5% bovine serum albumin and incubated with cells for 1 h at room temperature. After three washes in PBS,¯uorescein-conjugated secondary antibodies were added to cells at 1:200 dilution for 1 h at room temperature. FITC-or TRITCconjugated secondary antibodies were used to generate green or red uorescence. Cells were then washed in PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA). UV-crosslinking assay UV-crosslinking assay was performed as described previously (Huang and Lai, 1999) . In brief, DBT cell extracts (30 mg protein), 200 mg/ml tRNA and 10 4 c.p.m. of an in vitro transcribed, 32 P-labeled negativestrand MHV 5¢-end RNA (182 bp) were incubated for 10 min at 30°C. Increasing amounts of puri®ed GST (0, 0.5, 1.5 and 5 ng) or recombinant GST±hnRNP A1 fusion protein (0, 1, 3 and 10 ng) were included in the reaction to compete with the endogenous hnRNP A1 for binding. The reaction mixture was placed on ice and UV-irradiated in a UV Stratalinker 2400 (Stratagene) for 10 min, followed by digestion with 400 mg/ml RNase A for 15 min at 37°C. The protein±RNA complexes were then separated on a 10% SDS±polyacrylamide gel and visualized by autoradiography. GST pull-down assay GST pull-down was performed as described previously (Tu et al., 1999) . In brief, GST±hnRNP A1 fusion proteins on glutathione beads (Pierce, Rockford, IL) were incubated with the in vitro translated, 35 S-labeled N protein in 0.3 ml of GST-binding buffer containing 0.1% NP-40 for 2 h at 4°C. The beads were washed ®ve times with the GST-binding buffer containing 0.3% NP-40. Proteins bound to beads were eluted by boiling in Laemmli buffer for 5 min and separated on a 10% polyacrylamide gel. [ 35 S]methionine labeling and immunoprecipitation DBT cells were infected with MHV-A59 at an m.o.i. of 2. The cells were incubated with methionine-free medium for 30 min before labeling and were labeled in 100 mCi/ml [ 35 S]methionine starting at 1.5, 7 or 24 h p.i. After labeling for 2 h at each time point, the cells were harvested for protein extraction as described previously (Li et al., 1997) . The protein extracts were immunoprecipitated with anti-Flag antibody-conjugated beads (Sigma, St Louis, MO) in Tm 10 buffer (50 mM Tris±HCl pH 7.9, 0.1 M KCl, 12.5 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.1% NP-40, 1 mM phenylmethylsulfonyl¯uoride) at 4°C for 2 h. The immunoprecipitates were washed and separated on a 4±15% gradient SDS±polyacrylamide gel and visualized by autoradiography. Plasmid 25CAT was linearized by XbaI and in vitro transcribed with T7 RNA polymerase to produce the DI RNA . The DI RNA was transfected into MHV-A59-infected DBT cells using DOTAP as described previously (Huang and Lai, 1999) . In brief,~80% con¯uent DBT cells were infected by MHV-A59 at an m.o.i. of 10. At 1 h p.i., the cells were transfected with 5 mg of in vitro transcribed DI RNA and incubated at 37°C for the desired lengths of time. To amplify the DI RNA, viruses (P0) were passaged twice in wt DBT cells to generate P1 and P2 viruses. Cells were harvested at 8 or 24 h p.i. and lysed by freezing and thawing for three times. After centrifugation at 12 000 r.p.m. for 10 min, the supernatant was used in a CAT assay as described previously (Lin et al., 1996) ."
12
"A Method to Identify p62's UBA Domain Interacting Proteins"
"The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays."
"p62 is a novel cellular protein which was initially identified in humans as a phosphotyrosine independent ligand of the src homology 2 (SH2) domain of p56 lck (1, 2) . p56 lck is a member of the c-src family of cytoplasmic tyrosine kinases that is found predominantly in cells of lymphoid origin (3, 4) . In addition to the interaction with p56 lck , p62 also associates with the Ser/Thr kinase (1, 2) , atypical protein kinase C (5, 6) , and ubiquitin (7) . In addition to the SH2 domain, p62 possesses several structural motifs, including a ubiquitin associated (UBA) domain that is capable of binding ubiquitin nonconvalently (8, 9) . Ubiquitin (Ub) is a small polypeptide of 76 amino acids that can be convalently attached to other proteins at specific lysine residues through chains composed of one (mono) or several ubiquitin moieties (poly). In addition to its classical role in protein degradation, ubiquitin is emerging as a signal for protein transport and processing (10) (11) (12) . Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin activating enzyme E1, a ubiquitin-conjugating enzyme E2, and a ubiquitin ligase E3. Initially, E1 activates ubiquitin by forming a high energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2, then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and ε-amino group of a lysine residue of the substrate. Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin are attached to lysine side chains of the previously conjugated moiety to form branched polyubiquitin chains. The fate of ubiquitinated substrates depends on the number of ubiquitin moieties conjugated, as well as, the lysine linkage of Ub-Ub conjugation. The conjugation of ubiquitin to eukaryotic intracellular proteins is one way in which those proteins are targeted to the proteasome for subsequent rapid degradation. This mechanism is particularly important for short-lived regulatory proteins such as cyclins, cyclin-dependent protein kinase-inhibitors, p53, the nuclear factor kappa B precursor, and IκB (13) . The ubiquitinproteasome system consists of two steps: 1) the target protein is conjugated with polyubiquitin molecules, which mark the substrate for degradation; 2) the target protein is transferred to the 26S proteasome, unfolded and degraded. The UBA domain is a conserved sequence motif among proteins that can bind polyubiquitin. It is comprised of ~45 amino acids (13) . The amino acids 386-434 of p62, which bind polyubiquitin, has been shown to possess homology to other recently described UBA domains (9) . Interestingly, proteins with UBA domains are more likely to bind polyubiquitin chains over monoubiquitin, such as the yeast UBA protein Rad23, a highly conserved protein involved in nucleotide excision repair (13) . Recently, it has been shown that yeast cells lacking two UBA proteins (Dsk2 and Rad23) are deficient in protein degradation and that the UBA motif is essential for their function in proteolysis (14) . In addition to the important role in recycling of amino acids from damaged or misfolded proteins, ubiquitin-protein conjugation also has functions unrelated to proteasomal targeting. For example, polyubiquitination is required for the internalization of several yeast and mammalian cell surface proteins into the endocytic pathway (15, 16) . Interestingly, p62 appears to sequester ubiquitinated substrates into a cytoplasmic structure referred to as a sequestosome, into which excess ubiquitinated proteins are segregated (17) . In addition, p62 is an immediate early response gene product for a variety of signals (18) . Thus, p62 appears to play a novel regulatory role for polyubiquitinated proteins and may have an essential function in cell proliferation and differentiation. We have developed a method that will enable identification of protein(s) that interact with p62's UBA domain. Human adult brain library 10×96 well plates with 100 cDNAs per well and Gold TNT SP6 To search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system. The human adult brain library 96 well plates with 100 cDNAs per well was transcribed and translated employing the Gold TNT SP6 Express 96 plate and [ 35 S] methionine. The TNT Quick-coupled transcription-translation system contained a rabbit reticulocyte lysate pre-mixed with most of the reaction components necessary to carry out transcription/translation in the lysate, including all of the amino acids except methionine. [ 35 S] Methionine was used to label newly synthesized proteins. The reactions were set up according to the manufacturer's instructions. Rabbit reticulocyte lysate has been shown to be capable of carrying out ubiquitination of proteins that were translated in such an in vitro translation system (19, 20) . The reactions mixtures also contained ubiquitin so that the newly synthesized proteins could be ubiquitinated. The reactions were incubated at 30°C for 2 hours. The resulting proteins were assayed to determine their binding ability with p62's UBA domain. Potential positive "hits" were further subdivided and reassayed to link individual clones to the protein of interest (Fig. 1 ). Each translated pool was resuspended in binding buffer (25 mM Tris pH 7.5, 125 mM NaCl, 0.1% NP-40) and used as a source of protein in p62 UBA pull down assays. Proteins that specifically interact with the UBA domain of p62 were isolated by interaction with agarose-immobilised p62-UBA peptide (amino acid 387-436 of p62) (5 µg) for 2 hours at 4ºC, then washed three times in washing buffer (25 mM Tris pH 7.6, 100 mM NaCl, 1% NP-40). Bound proteins were released by addition of SDS-sample buffer and separated by SDS-PAGE. The SDS-PAGE gels were fixed in 50% methanol, 10% acetic acid for 30 min, stained in 0.2% Commassie Brilliant Blue R-250, 45% methanol, 10% acetic acid for 15 min, destained in 10% acetic acid, 50% methanol overnight, and enhanced in autoradiography enhancer En 3 HANCE for 1 hr and exposed to X-Ray film. By combining 4 pools as one mixed pool, 96 protein pools were divided into 24 mixed protein pools for use in p62 UBA pull down assays. Positive mixed protein pools were selected and individual pools were retested for its ability to bind p62's UBA domain. The individual cDNA pool from which the positive protein pool was generated was transformed into JM109 competent cells and plated on LB ampicillin plate. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNA was purified from the cell culture and used for TNT Quick coupled in vitro transcription/ translation. The individual protein synthesized from each plasmid DNA chosen was screened for its ability to bind p62's UBA domain. To confirm the interaction with p62's UBA domain, the final resulting individual proteins were used in the coupled TNT/p62 UBA pull down assays. The cDNA inserts were sequenced in the Genomics Core Facility at Auburn University and the sequences were compared with known sequences in NCBI database by BLAST analysis. Human embryonic kidney 293 (HEK 293) cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum and transfected with myc-tagged HSP70 plasmid using the Mammalian Cell Transfection Kit. Cells were harvested and lysed in 1 ml of SDS lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM NaF, 0.5% TX-100, 1 mM Na 3 VO 4 , 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM PMSF, 1% SDS) for 30 min on ice, followed by centrifugation at 14000 rpm for 15 min at 4°C to remove the insoluble fraction. The protein concentration of the supernatant was determined using the Bio-Rad DC protein assay reagent with bovine serum albumin (BSA) as standard. Equal amount of protein (750 µg) was immunoprecipitated with anti-myc and collected with agarose-coupled secondary antibody. To the agarose beads containing the immunoprecipitated HSP70, 50 µl of reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP) was added containing 100 ng E1, 200 ng E2 (UbcH7), and 100 µg of E3 (Flag-tagged TRAF6) along with 5 µg GST-WT-Ub, GST-K29R Ub, GST-K48R Ub, GST-K63R Ub, or K63 Ub. Control samples without HSP70, E1, E2, E3, or GST-WT-Ub were also included. Reactions were carried out by continuous shaking at 37°C for 2 hours and then washed three times with reaction buffer. The proteins were released by boiling for 2 min in SDS-PAGE sample buffer, separated on 7.5% SDS-PAGE and Western blotted for anti-ubiquitin. To search for novel proteins that bind to the UBA domain of p62, we performed in vitro expression cloning (IVEC) using the ProteoLink IVEC system from Promega (Cat. No. L6500). The human adult brain library 96 well plates with 100 cDNAs per well were transcribed and translated employing the Gold TNT SP6 Express 96 plate in the presence of [ 35 S] methionine and ubiquitin (25 µg/µl, Sigma). By combining 4 protein pools as one mixed pool, 96 protein pools were divided into 24 mixed pools ( Fig. 2A, 2B ). Each lane contained more than 100 proteins (theoretically 400) with different molecular weight. Therefore, each lane appeared as a smear, indicating that the in vitro transcription/translation system from Promega worked successfully. In order to examine whether proteins synthesized in the IVEC system are also ubiquitinated, Western blot analysis was performed by blotting the newly synthesized proteins (in the presence of cold methionine instead of 35 S methionine) with ubiquitin monoclonal antibody. In the mixed protein pools, each of the 24 lanes appeared as a smear, indicating that proteins synthesized by the IVEC system are also ubiquitinated (Fig. 3A) . Furthermore, the rabbit reticulocyte lysate in the IVEC system can utilize different lysine linkages of ubiquitin (i.e., Ub K29, Ub K48, and Ub K63) for ubiquitination (Fig. 3B) . In order to investigate whether the agarose-immobilised p62 UBA peptide has binding specificity, a mixed protein pool synthesized by IVEC system was tested in a pull down assay in the presence of agarose beads alone or in the presence of p62 UBA agarose beads (Fig. 3C ). Our results revealed that proteins that bound to p62's UBA domain could not be pulled down by agarose beads alone, indicating that the agarose-immobilised p62 UBA peptide had binding specificity. In order to identify proteins that bind to p62's UBA domain, p62 UBA pull down assays were performed. Out of the 24 mixed protein pools, several pools contained [ 35 S] methionine-labeled bands in the primary p62 UBA pull down assays (Fig. 4) . We chose 6 pools (pool # 2, 4, 8, 14, 20, 21) because of their stronger signal to specifically identify which individual protein pool in the mixed pools has the ability to bind to p62's UBA domain. Therefore, a secondary screen was conducted on the 6 positive individual mixed pools (representing 24 individual protein pools) which bound with p62's UBA domain (Fig. 5) . Mixed protein pool # 2 generated a positive protein with molecular weight of 51 KDa (Fig. 4) , and only individual protein pool "c" out of the four protein pools (a, b, c, d) that comprised protein pool #2 had a protein with the same molecular weight (Fig. 5) . Depending on the size of the protein pulled down in the secondary screen compared to the primary screen (Fig. 4) , individual protein pools "c", "h", "i", "o", "t", and "v" were identified (Fig. 5) . To specifically identify which protein in the individual protein pool has the ability to bind p62's UBA domain, the cDNAs from the positive individual protein pools were then transformed into JM109 competent cells and plated out on LB ampicillin plates. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNAs were purified from the cell culture and used for TNT Quick coupled in vitro transcription/translation. The individual protein synthesized from each plasmid DNA was retested for its ability to bind p62's UBA domain. By synthesizing individual protein from individual plasmid using the Gold TNT Quick coupled in vitro transcription/translation system and subjecting them to p62 UBA pull-down assays, 11 positive clones were isolated from the 6 positive individual pools. It is not surprising that 5 more clones showed binding ability with p62's UBA domain since there are 100 cDNAs in each positive individual pool and some of them could have lower binding ability and therefore showed weak signal in the mixed protein pool. It is also possible that they are not as efficiently synthesized in the mixed TNT reaction as in the individual TNT reaction in which only one cDNA was used as template. The 11 positive plasmids were sequenced and compared with known cDNA sequences in NCBI database using BLAST analysis with results shown in Table 1 . Interestingly, the proteins identified in the screen fall into three distinct categories. One set are proteins that are associated with Alzheimer's disease, including myelin basic protein, 14-3-3 protein, syntaxin binding protein munc18, transketolase, heat shock protein HSP70, reelin, and calcium/calmodulin kinase II (Table 1 ). Significant decrease in the amount of myelin basic protein has been reported in the white matter of Alzheimer's disease patients, accompanied by increased quantities of βamyloid peptides (21) . The presence of β-amyloid peptides containing senile plaques and neurofibrillary tangles are the two major pathological features in the brain of patients with Alzheimer's disease (22) . Interestingly, 14-3-3 proteins have also been demonstrated to be components of neurofibrillary tangles of Alzheimer's disease brains (23) . Syntaxin binding protein munc18 can powerfully regulate amyloid precursor protein metabolism and β-amyloid secretion through direct and indirect interactions with X11 proteins (24) . The activity of transketolase has been reported to be reduced in dementia of Alzheimer's type brain (25) . Heat shock protein HSP70 expression is significantly increased in the temporal cortex of patients with Alzheimer's disease (26) . Besides HSP70, other heat shock proteins are also linked with Alzheimer's disease. For example, increased synthesis of HSP27 has been suggested to play a role in preventing neuronal injury in AD (27) , and alpha-crystallin heat shock protein has a close relationship with neurofibrillary tangles of AD brains (28) . Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation (29) . Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimer's disease (30) . Enhanced activity of calcium/calmodulin kinase II has been suggested to contribute to phosphorylation of tau protein and lead to neurofibrillary tangle deposition and neuronal death in Alzheimer's disease (31) . Although the relationship between p62 and neurofibrillary tangles or neuritic plaques is unclear, both neurofibrillary tangles and dystrophic neuritis of neuritic plaques are associated with ubiquitin (32) , suggesting that dysfunction in ubiquitin-mediated proteolysis and the resulting accumulation of ubiquitinconjugated proteins may contribute to the origination of dystrophic neuritis and neurofibrillary tangles. Furthermore, p62 has been recently reported to accumulate early in neurofibrillary tangles in Alzheimer's disease (33) , suggesting that p62 may play an important role in Alzheimer's disease by interacting with those proteins through its UBA domain. A second set of proteins identified in the screen that bind to p62's UBA domain are associated with brain development, including homeobox protein Meis2 and unc51 like kinase II (Table 1) . Although Meis proteins are not extensively studied in humans, these proteins have been shown to be required for hindbrain development in the zebrafish (34) . Unc51 like kinase II has been demonstrated to play a role in axonal elongation (35, 36) , which is needed for the formation of complicated neuronal networks. The third set of proteins that exhibit ability to bind p62's UBA domain are proteins that are linked with other neurodegenerative diseases, including FK506 binding proteins and nuclear receptor corepressor I (Table 1) . FK506 (tacrolimus) is a potent immunosuppressive drug used in the treatment of patients after organ transplantation and in selected autoimmune disorders (37) . FK506 is activated upon binding to members of the immunophilin family of proteins, which were designated as FK506 binding proteins (38) . Immunophilins are chaperone proteins and FK506 binding proteins have been suggested as therapeutics for neurological disorders (39, 40) . Nuclear receptor corepressor I has been suggested to play a role in Huntington's disease because it is able to interact with huntingtin (41) . The proteins identified here suggest that p62's UBA domain has the ability to interact with multiple proteins that play important roles in neurodegenerative diseases. Further screening from the whole genome-wide perspective will be necessary to define the important role that p62's UBA domain plays. It has been reported that polyubiquitin chains assembled through lysine 48 of ubiquitin act as a signal for substrate proteolysis by the 26S proteasome (42) (43) (44) . In order to understand whether the proteins identified in our screen bind to the p62's UBA domain through lysine 48 (K48), polyubiquitin K48 chains were added to the p62 UBA pull down assay (Fig. 6) . Inclusion of polyubiquitin K48 chains in the assay should compete for the binding of substrate to the p62's UBA domain and reduce the interaction of those proteins with the p62's UBA domain if those proteins are assembled through K48 chains. An alternative interpretation for polyubiquitin K48 chain competition is that the ubiquitin chains are competing for the same binding site as the binding partners which are either ubiquitinated or non-ubiquitinated. We randomly chose five proteins out of the 11 binding partners for the competition pull down (Fig. 6) . Out of the five proteins, four proteins (# 2, 3, 4, and 5) showed reduced binding ability with p62's UBA domain when polyubiquitin K48 chains were included (Fig. 6A, 6B ). However, K48 chains failed to compete with HSP70, suggesting that p62's UBA domain binds to HSP70 through a ubiquitin lysine linkage other than K48. Interestingly, it has been reported that heat shock protein 70 cognate (HSP70) is ubiquitinated by CHIP (carboxyl terminus of Hsc70-interacting protein) via ubiquitin chain synthesis that uses either K29 or K63 (45) . In order to examine which lysine linkage utilized by HSP70 binds to p62's UBA domain, in vitro ubiquitination assay was performed by incubating lysates from HEK cells expressing HSP70 with E1, E2, and E3 in reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP). As control, the ubiquitination of HSP70 utilizing the rabbit reticulocyte lysate was also investigated by Western blot analysis. Our results revealed that HSP70 was ubiquitinated in the IVEC system (Fig. 7A, 7B) , and the rabbit reticulocyte lysate contained enzymes such as TRAF6 (E3) and UbcH7 (E2) for in vitro ubiquitination (Fig. 7C ). TRAF6 was chosen as an E3 in this in vitro ubiquitination assay due to its RING domain, a common feature of E3 ligases, and the observation that p62 is a scaffold for TRAF6 interaction (46) . Therefore, in vitro ubiquitination assays using the E1-E2-E3 system were performed in the presence of either ubiquitin wild type or ubiquitin mutants (K29R, K48R, and K63R). If one lysine mutant blocks the ubiquitination of HSP70, it would suggest that the ubiquitination of HSP70 utilizes that specific lysine linkage. Our results revealed that HSP70 utilizes K63 linkage to assemble polyubiquitin chains to bind to p62's UBA domain since only the K63R ubiquitin mutant blocked the ubiquitination of HSP70 (Fig. 8A) . A similar result was also observed when reactions were conducted with wild type ubiquitin or mutant ubiquitin with all lysines mutated to arginines except K63 and the ubiquitination of HSP70 occurred only in the reaction that has either intact K63 ubiquitin or wild type ubiquitin (Fig. 8B ). This finding is consistent with previous reports (45) , demonstrating that HSP70 is K63-polyubiquitinated. Furthermore, the in vivo interaction of HSP70 and p62 was confirmed by transfecting myc-tagged HSP70 into HEK 293 cells in the presence of the proteasome inhibitor MG132 and subjecting cell lysates to p62 immunoprecipitation and Western blot with anti-myc antibody (Fig. 8C) . The interaction between HSP70 and p62 in vivo took place only when MG132 was included, suggesting that the interaction in vivo is dependent upon the ubiquitination of HSP70. The specific type of polyubiquitin chain recognized by p62's UBA domain is not yet known and studies are underway lab to determine p62's interaction with specific polyubiquitin chains, however, our preliminary studies suggest that p62's UBA domain may recognize K63 linked polyubiquitin chains. Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin, resolved on 10% SDS-PAGE gels, transferred to nitrocellulose membrane and western blotted with ubiquitin monoclonal antibody. B: HSP70 Protein was synthesized employing TNT Quick Coupled in vitro transcription/translation system in the presence of ubiquitin and 35 S-methionine, resolved on 10% SDS-PAGE and exposed to X-ray film. C: Western blot of rabbit reticulocyte lysate with TRAF6 (E3) and UbcH7 (E2). In summary, for the first time, we demonstrate a systematic approach to identify UBA domain binding proteins from a proteome wide perspective. This approach could be readily adapted to high throughput screening. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins in the human adult brain that interact with the UBA domain of p62, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer's disease. This is a very interesting finding since 9600 cDNAs have been screened and only 11 of them showed binding specificity with p62's UBA domain. Studies are underway to unfold the functional roles of p62 in the ubiquitin system. Our approach provides an easy route to the characterization of UBA domain binding proteins at the level of the whole proteome, its application will unfold the important roles that p62's UBA domain plays. This method could be easily adapted to identify proteins that interact with other UBA domains as well."
13
"Vaccinia virus infection disrupts microtubule organization and centrosome function"
"We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein–dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function."
"Intracellular bacterial and viral pathogens have evolved numerous mechanisms to appropriate and exploit different systems of the host during their life cycles in order to facilitate their spread during entry and exit from the host (Cudmore et al., 1997; Finlay and Cossart, 1997; Dramsi and Cossart, 1998) . In the case of viruses, perhaps the best studied example is the exploitation of the actin cytoskeleton by vaccinia virus during its exit from infected cells (Cudmore et al., 1997) . Vaccinia virus is a large DNA virus with a genome of~191 kb encoding 260 open reading frames (ORFs) that is a close relative of variola virus, the causative agent of smallpox (Johnson et al., 1993; Massung et al., 1993) . Vaccinia virus morphogenesis is a complex process which occurs in the cytoplasm of infected cells and results in the formation of the intracellular mature virus (IMV) and the intracellular enveloped virus (IEV). IMV consist of a viral core of DNA and protein enveloped in a membrane cisterna derived from the intermediate compartment (Sodeik et al., 1993) . The IMV core contains ®ve major proteins, A3L, A4L, A10L, F17R and L4R (Vanslyke and Hruby, 1994; Jensen et al., 1996a) , while 12 proteins, A12L, A13L, A14L, A14.5L, A17L, A27L, D8L, G4L, G7L, H3L, I5L and L1R, are associated with the membranes around the virus particle (Jensen et al., 1996a; Betakova et al., 2000) . Depending on the virus strain and cell type, a proportion of IMV can become enwrapped by a membrane cisterna derived from the trans-Golgi apparatus to give rise to IEV particles (Schmelz et al., 1994) . To date, six IEV-speci®c proteins, A33R (Roper et al., 1996) , A34R (Duncan and Smith, 1992) , A36R (Parkinson and Smith, 1994) , A56R (Payne and Norrby, 1976; Shida, 1986) , B5R (Engelstad et al., 1992; Isaacs et al., 1992) and F13L (Hirt et al., 1986) , have been identi®ed. Studies using recombinant viruses have shown that A33R, A34R, B5R and F13L play an important role in IEV assembly (Blasco and Moss, 1991; Engelstad and Smith, 1993; Wolffe et al., 1993 Wolffe et al., , 1997 Roper et al., 1998; Sanderson et al., 1998a; Ro Èttger et al., 1999) . Vaccinia virus is thought to leave the cell by fusion of the outer IEV membrane with the plasma membrane, to give rise to the extracellular enveloped virus (EEV) (Morgan, 1976; Payne, 1980; Blasco and Moss, 1991) or the cell-associated enveloped viruses (CEV) which remain associated with the outer surface of the plasma membrane (Blasco and Moss, 1992) . During the complex vaccinia infection process, the actin cytoskeleton is dramatically reorganized and numerous actin comet-like tails are induced by IEV particles (Cudmore et al., 1995; Ro Èttger et al., 1999) . Using actin polymerization as the driving force, IEV particles are propelled on actin tails until they contact the plasma membrane and extend outwards, thereby facilitating infection of neighbouring cells (Cudmore et al., 1995) . In addition, vaccinia infection results in stimulation of cell motility, loss of contact inhibition and changes in cell adhesion (Sanderson and Smith, 1998; Sanderson et al., 1998b) . Vaccinia virus-induced cell motility can be subdivided further into cell migration and extension of neurite-like projections, the latter of which is dependent on microtubules (Sanderson et al., 1998b) . The dependence of neurite-like projection formation on microtubules suggests that the microtubule cytoskeleton may also play a role during the life cycle of vaccinia virus. Indeed, recently, the vaccinia A27L protein and microtubules have been shown to be required for ef®cient IMV dispersion (Sanderson et al., 2000) . Furthermore, in the absence of vaccinia actin-based motility, cell to cell spread still occurs although it is less ef®cient (Wolffe et al., 1997 Sanderson et al., 1998a) , suggesting that additional transport mechanisms must exist. Given these observations, we wondered whether the microtubule cytoskeleton has a function during the life Vaccinia virus infection disrupts microtubule organization and centrosome function The EMBO Journal Vol. 19 No. 15 pp. 3932±3944, 2000 cycle of vaccinia virus. We now report that the microtubule cytoskeleton and the dynein±dynactin complex play an important role during the early stages of vaccinia infection. However, later during the infection cycle, loss of centrosome function and accumulation of viral-encoded microtubule-associated proteins (MAPs) result in a dramatic rearrangement of the microtubule cytoskeleton. Vaccinia localization in the vicinity of the MTOC depends on microtubules and the dynein±dynactin complex Indirect immuno¯uorescence labelling shows that by 6 h post-infection the majority of vaccinia virus particles are concentrated in the area coinciding with the centre of the microtubule aster ( Figure 1A and C). To examine whether this localization is indeed microtubule dependent, we infected cells pre-treated with nocodazole to depolymerize microtubules. In the absence of microtubules, virus particles were distributed throughout the cytoplasm ( Figure 1B and D) . The accumulation of virus particles in the area around the centre of the microtubule aster suggested that a microtubule minus end-directed motor may be involved in establishing the position of the virus in this location. To examine this possibility, we infected cells overexpressing p50/dynamitin which acts as a dominantnegative for dynein±dynactin function (Echeverri et al., 1996) . We found in cells overexpressing p50/dynamitin that virus particles did not accumulate at the centre of the microtubule aster but rather throughout the cytoplasm, as occurs in the absence of microtubules (compare Figure 2B with Figure 1D ). As vaccinia morphogenesis involves wrapping by host membranes, it was possible that the effects of nocodazole and p50/dynamitin on virus localization were in fact due to disruption of the intermediate compartment and Golgi apparatus by these reagents (Burkhardt et al., 1997) . However, two independent experiments showed that this is not the case. First, in cells infected in the absence of microtubules, the Golgi apparatus as well as vaccinia virus particles are dispersed throughout the cytoplasm but do not co-localize ( Figure 3F and O). Secondly, vaccinia particles remain in the vicinity of the microtubule-organizing centre (MTOC) when the Golgi but not the microtubules was disrupted by treatment with brefeldin A ( Figure 3G and P). Similar results were obtained using other markers: A17L for vaccinia, galactosyltransferase for the Golgi or ERGIC53 for the intermediate compartment (data not shown). Taken together, our data indicate that the microtubule cytoskeleton is required for the localization of newly assembled virus particles in the vicinity of the MTOC during vaccinia infection. Formation of functional IEV, but not IMV, is microtubule dependent Given the requirement for microtubules in vaccinia localization, we subsequently examined whether this localization has a role in morphogenesis of the two different intracellular forms of vaccinia virus, IMV and IEV. From electron microscopic examination of cells infected in the presence of nocodazole, it became clear that IMV particles which are morphologically indistinguishable from controls are formed ( Figure 4 ). Although IMV particles are assembled in the absence of microtubules, we wondered whether their number is reduced and whether those that are formed are infectious, since the integrity of the intermediate compartment depends on microtubules (Burkhardt et al., 1997) . To address this question, three independent virus stocks were prepared in the presence or absence of nocodazole. To simplify the interpretation of the data, we used the recombinant vaccinia virus mutant DF13L, which is unable to form IEV (Blasco and Moss, 1991) . The ®nal concentration of virus particles produced, Vaccinia uses and abuses the microtubule cytoskeleton as determined by the method of Joklik (1962) , was 30.2 6 5.2 3 10 10 particles/ml in the presence of microtubules and 9.0 6 6.7 3 10 10 particles/ml in the absence of microtubules. Although there is a 3-fold decrease in the number of virus particles formed in the absence of microtubules, the particles that are formed are infectious (data not shown). While infectious IMV are formed in the absence of microtubules, we found no evidence for IEV formation, based on electron microscope examination of cells infected in the presence of nocodazole ( Figure 4 ). We did, however, observe IMV particles partially wrapped in trans-Golgi membranes most probably in the process of abortive IEV formation ( Figure 4D ). Given these data, we examined by indirect immuno¯uorescence whether low amounts of IEV particles are formed in the absence of microtubules. However, we could ®nd no evidence for colocalization of the IEV protein markers A36R, A34R or A33R with vaccinia particles formed in the presence of nocodazole ( Figure 5F ). We also found no evidence for IEV formation, based on their ability to nucleate actin tails ( Figure 5O ). As IEV particle assembly involves wrapping by the Golgi apparatus (Schmelz et al., 1994) , we examined the effects of only disrupting this membrane compartment using brefeldin A. We could ®nd no evidence for IEV formation, based on co-localization of IEV protein markers with virus particles and actin tails in cells infected in the presence of brefeldin A ( Figure 5G±I and P±R). Indeed, in brefeldin A-treated cells, the IEV membrane proteins required for assembly were observed in the endoplasmic reticulum and not the trans-Golgi ( Figure 5H ). In summary, our data indicate that the microtubule cytoskeleton is required for ef®cient IMV assembly and is essential for IEV formation. In the course of our experiments, it became obvious that the Golgi apparatus becomes progressively dispersed during infection co-concominantly with disruption of the microtubule network ( Figure 6 ). Further analysis showed that during infection the normal morphology of the microtubule cytoskeleton is replaced by morphologically aberrant microtubule forms, which vary among each other but have in common the absence of a discrete MTOC ( Figure 7 ). These aberrant forms can be broadly classi®ed into three types: (i) cells with a disorganized microtubule network where microtubules seem randomly oriented ( Figure 7E ); (ii) cells in which microtubules form rings around the nucleus and throughout the cytoplasm ( Figure 7H ); or (iii) cells with long projections consisting of microtubule bundles ( Figure 7K ). We quanti®ed the appearance of the different morphological forms in ®ve independent infection experiments, in which 200 cells were counted for each time point for each experiment ( Figure 7C , F, I and L). Small compact cells, representing 20.7 6 2.6, 21.8 6 12.4 and 29.7 6 15.2% for 5, 8 and 24 h post-infection, respectively, in which the microtubule cytoskeleton morphology was not evident were not included in the analysis. Already by 5 h post-infection, when virus particle assembly has occurred, the normal aster microtubule con®guration has been disrupted and replaced in the majority of cells by microtubules without obvious organization from the MTOC ( Figure 7F ). Furthermore,~10% of cells have microtubule rings and 5% of cells have long projections by this time point ( Figure 7I and L). As the infection proceeds, microtubules become progressively more disrupted and bundled ( Figure 7I and L). From our observations, there seems to be no obvious connection between the disruption and changes in the actin and the microtubule cytoskeletons ( Figure 7) . Moreover, the same reorganization of the microtubule network occurs in cells infected with the vaccinia deletion mutants DF13L and DA36R which do not make actin tails (data not shown). The effects of vaccinia virus infection on the reorganization of the microtubule cytoskeleton were also observed in all cell lines we examined (BHK-21, C 2 C 12 , PtK2, RK 13 and Swiss 3T3) to varying degrees (data not shown). Our data show that vaccinia infection results in severe disruption of the normal morphology of the microtubule cytoskeleton. The formation of microtubule bundles and the loss of organization from the MTOC in vaccinia-infected cells is strongly reminiscent of the phenotype observed in cells overexpressing a MAP (Weisshaar et al., 1992; Togel et al., 1998) . As overexpression of MAPs stabilizes microtubules, we examined whether the microtubule cytoskeleton in infected cells was more resistant to depolymerization by nocodazole or cold treatment ( Figure 8 ). This was indeed the case, suggesting that the virus genome may encode viral proteins with MAP-like properties. To identify viral proteins which exhibit microtubule-binding properties, we performed microtubule co-sedimentation assays using extracts prepared from uninfected and vaccinia-infected cells (Figure 9 ). Initial experiments, however, revealed that intact virus particles in the extracts were prone to pellet even in the absence of microtubules, making identi®cation of viral MAPs impossible. To avoid this problem, we prepared extracts from cells infected in the presence of rifampicin, a drug that inhibits vaccinia virus particle assembly but does not affect viral protein expression (Moss et al., 1969; Tan and McAuslan, 1970) . The morphological effects of vaccinia infection on the microtubule cytoskeleton were the same in the presence or absence of rifampicin (data not shown). Comparison of the proteins present in pellets from microtubule co-sedimentation assays reveals that a number of additional prominent and minor bands are present in extracts prepared from vaccinia-infected but not from uninfected cells (Figure 9 ). Co-sedimentation assays Vaccinia uses and abuses the microtubule cytoskeleton performed in the presence of nocodazole or with coldtreated extracts reveal that the majority of these additional bands disappear in the absence of microtubules. To identify the viral proteins co-sedimenting with microtubules, we performed in-gel protease digestion followed by analysis of the resulting peptides by MALDI mass spectrometry. Using this approach, we identi®ed a number of potential vaccinia-encoded MAPs: A10L (a structural protein), I1L and L4R (which are DNA-binding proteins), all of which are associated with viral cores (Vanslyke and Hruby, 1994; Jensen et al., 1996a; Klemperer et al., 1997) , and A6L which is conserved in all poxvirus genomes but is of unknown function (Figure 9 ). A10L and L4R associate with microtubules in vivo and mediate binding of viral cores to microtubules in vitro Using available antibodies, we examined the localization of A10L, L4R and I1L in infected cells to see whether they associate with microtubules in vivo, in addition to their essential role in the virus core (Vanslyke and Hruby, 1994; Jensen et al., 1996a) . As a negative control, we also examined the localization of the A3L core protein which was identi®ed as the prominent 70 kDa protein pelleting in the absence of microtubules (Figure 9 ). Indirect immunouorescence analysis showed that A10L and L4R are associated with microtubules, in both the presence and absence of rifampicin ( Figure 10 ). As expected, A10L and L4R were also associated with viral particles (data not shown). In contrast, I1L and A3L were never observed in association with microtubules, regardless of the ®xation conditions, but were localized to viral factories and viral particles, respectively (data not shown). Interestingly, A10L and L4R were not associated with all microtubules but were co-localized with a subset of acetylated microtubules ( Figure 10) . The association of A10L and L4R with virus particles and microtubules raises the question of whether there is a role for this microtubule-binding activity during infection. We wondered whether these two proteins mediate the interaction of incoming viral cores with microtubules at the beginning of infection, as cores and not virus particles are released in the cytoplasm at the start of the infection cycle (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) . To examine this possibility, we investigated whether puri®ed viral cores would bind microtubules in vitro. We found that viral cores were able to bind microtubules, while protease-treated cores showed no association ( Figure 11A and B) . Pre-incubation of puri®ed viral cores with antibodies against A10L and L4R speci®cally inhibited the interaction of viral cores with microtubules ( Figure 11C and D); in contrast, IgG or antibodies against A3L had no inhibitory effect ( Figure 11E and F). Taken together, our data suggest that A10L and L4R have MAP-like properties and may play a role in mediating interactions of incoming viral cores with microtubules. The dramatic rearrangement of the microtubule cytoskeleton which occurs during vaccinia infection is unlikely to be attributed exclusively to the action of A10L and L4R since they only associate with a subset of microtubules ( Figure 10) . Furthermore, the loss of microtubule organization precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. We therefore wondered whether vaccinia infection disrupts centrosome function, given the loss of microtubule aster con®guration during infection (Figure 7 ). Since microtubules are nucleated by the centrosome in animal cells, we examined whether vaccinia infection affects g-tubulin, which is critically required for this process (Stearns and Kirschner, 1994) . We observed that g-tubulin labelling of the centrosome is greatly reduced from as early as 2 h post-infection ( Figure 12 ). The same result was obtained when we infected PtK1 cells stably expressing green¯uorescent protein (GFP)-labelled g-tubulin (Khodjakov and Rieder, 1999) . In addition, the centrosomal and centriolar components pericentrin, C-Nap 1, Nek 2 and centrin are reduced by immuno¯uorescence in the centrosomes/centrioles of vaccinia-infected cells ( Figure 12) . Furthermore, the reduction of centrosomal markers requires viral protein synthesis as their levels are not affected when cells are infected in the presence of cycloheximide (data not shown). The dramatic reduction of g-tubulin from the centrosome implies that vaccinia infection perturbs centrosome Vaccinia uses and abuses the microtubule cytoskeleton function. To test this hypothesis, we examined whether the centrosome in vaccinia-infected cells could re-nucleate microtubules, following their depolymerization by nocodazole. We found that by 2 h post-infection, when we already see a reduction in g-tubulin, microtubule nucleation from the centrosome was very inef®cient, as compared with uninfected controls, indicating that vaccinia has disrupted`normal' centrosome function (Figure 13 ). At later times post-infection, microtubule re-nucleation ef®ciency from the centrosome was even lower (data not shown). However, following nocodazole washout, microtubules eventually are repolymerized throughout the cytoplasm of infected cells but do not display any organization from the MTOC, as do controls (compare Figure 13I and K). The size of virus particles is such that they are unlikely to move within and between cells by diffusion alone, suggesting that their movements will require interactions with the host cytoskeleton. Previous data have shown that vaccinia virus both disrupts and hijacks the actin cytoskeleton to facilitate movement of the intracellular enveloped form of vaccinia virus (Cudmore et al., 1995; Ro Èttger et al., 1999) and of the infected cell itself (Sanderson and Smith, 1998; Sanderson et al., 1998b) . The data described here now show that vaccinia also uses and subsequently disrupts the microtubule cytoskeleton during its infection cycle. It is clear from our experiments and the previous observations of Ulaeto et al. (1995) that microtubules are required to maintain the integrity of the Golgi apparatus which is in turn required for IMV wrapping to form IEV (Schmelz et al., 1994) . In contrast, IMV are assembled in the absence of microtubules, albeit at reduced levels. While microtubules are not required for IMV assembly, they are required together with the dynein±dynactin complex for virion accumulation in the vicinity of the microtubule aster. One can envisage that minus enddirected microtubule-dependent movements of IMV particles from their site of assembly in the viral factory towards the MTOC, by the dynein±dynactin complex, would enhance the possibility of wrapping with the Golgi apparatus and subsequent IEV formation. Recently it has been shown that the IMV protein A27L and microtubules are required for ef®cient IMV dispersion from the viral factories (Sanderson et al., 2000) . In the absence of A27L, mature IMV particles accumulate at the periphery of the virus factory but do not subsequently wrap to form IEV, presumably because they are unable to move on microtubules (Sanderson et al., 2000) . The microtubule-and dynein±dynactin-dependent accumulation of vaccinia in the vicinity of the MTOC is analogous to the microtubule-dependent movements required for herpes simplex virus 1 (HSV-1) and adenovirus to reach their site of replication in the nucleus (Sodeik et al., 1997; Suomalainen et al., 1999; Leopold et al., 2000) . In the case of HSV-1, the UL34 protein, 9 . Vaccinia encodes proteins that co-sediment with microtubules. Analysis of pellets from in vitro microtubule co-sedimentation assays performed with protein extracts from vaccinia-infected (inf.) and uninfected (uninf.) cells. Twice the amount of pellet has been loaded in control assays performed in the absence of microtubules (nocodazole or 4°C). Proteins co-sedimenting with microtubules that were only present in extracts from infected cells are indicated by an asterisk. The identity of proteins determined by in-gel proteolysis MALDI mass spectrometry is indicated (arrowheads). which is associated with the incoming nucleocapsids, interacts with the intermediate chain of cytoplasmic dynein (IC-1a) (Ye et al., 2000) . It has also been reported that incoming nucleocapsids of pseudorabies virus, an alphaherpes virus closely related to HSV-1, are associated with and dependent on microtubules for their movement to the nucleus (Kaelin et al., 2000) . This interaction may be mediated by the UL25 protein, a minor but essential component of the capsid, which co-localizes with microtubules and accumulates at the MTOC (Kaelin et al., 2000) . The accumulation of UL25 at the MTOC is consistent with a possible interaction with the dynein± dynactin motor complex which is known to be localized at the MTOC (Echeverri et al., 1996) . It would not be surprising, based on observations with HSV-1 and pseudorabies, if microtubules and dynein±dynactin were also involved in establishing the infection cycle of cytomegalovirus (CMV), Epstein±Barr virus and varicella-zoster virus, all of which are herpes viruses. The other clear example of microtubule-dependent virus movements during the establishment of infection is that of incoming human foamy virus (HFV) which is dependent on microtubules and presumably a minus end-directed microtubule motor to get to its nuclear replication site (Saib et al., 1997) . In the absence of protein expression, HFV Gag proteins, which are associated with the viral genome, accumulate at the centrosome in a microtubuledependent fashion prior to nuclear import (Saib et al., 1997) . The centrosomal accumulation of Gag proteins of HFV, however, appears to be unique for this class of retroviruses as no similar localization has been reported for human immunode®ciency virus (HIV) or other retroviruses. On the other hand, the Gag protein of murine leukaemia virus and HIV has been shown to interact with KIF4, a microtubule plus end-directed kinesin motor, both in vitro and in vivo (Kim et al., 1998; Tang et al., 1999) , suggesting that additional roles may exist for microtubules and motors during the outward movement of virus particles. Indeed, vaccinia virus particles are able to reach the cell periphery in the absence of actin-based motility (see images in Wolffe et al., 1997; Sanderson et al., 1998a Sanderson et al., , 2000 Ro Èttger et al., 1999) , suggesting that viral particles can also move out on microtubules (Sanderson et al., 2000) . Microtubule-dependent motordriven movements of virus particles represent an ef®cient mechanism to achieve a peri-nuclear localization, required to facilitate entry into the nucleus during establishment of infection. They also provide an excellent way for newly assembled virus particles to reach the cell periphery, facilitating the continued spread of infection. Our data show that although vaccinia virus uses the microtubule cytoskeleton to achieve a peri-nuclear localization, microtubule and Golgi organization becomes disrupted later during the infection process. Interestingly, HSV-1 and CMV have also been reported to disrupt the microtubule cytoskeleton and Golgi organization in their infection cycles (Avitabile et al., 1995; Fish et al., 1996) . While disruption of the microtubule network might at ®rst sight not appear to be bene®cial to the virus, it may not actually hinder viral spread but could enhance it. First, extensive virus assembly and spread to the cell periphery have already occurred by the time the microtubule cytoskeleton and Golgi organization are disrupted. Secondly, disruption of microtubule organization may overcome potential microtubule motor anchoring effects at the MTOC, thus allowing viral spread to the periphery to occur more easily. Lastly, the formation of long projections of up to 200 mm supported by extensive microtubule bundles provides a means to achieve long range spread of virus particles (Sanderson et al., 1998b) . It is clear that disruption and reorganization of the microtubule cytoskeleton by vaccinia virus is mediated by the combined effects of viral proteins with MAP-like properties and loss of microtubule-organizing function from the MTOC. The same may also be true for HSV-1, although disruption of centrosome function remains to be established, as late in infection microtubules are organized in bundles around the nucleus and do not show MTOCorchestrated organization (Avitabile et al., 1995) . The identi®cation of viral proteins with MAP-like properties is not unique to vaccinia virus. The VP22 tegument protein from HSV-1 co-localizes with microtubules in infected cells and induces microtubule bundles when expressed in uninfected cells (Elliott and O'Hare, 1998) . Other examples of viral MAPs based on their in vivo localization Fig. 11 . Vaccinia cores bind directly to microtubules in vitro. Puri®ed viral cores labelled by DAPI (green) bind to rhodamine-labelled microtubules (red) in the absence of ®xation (A). Binding to microtubules is not observed if cores are pre-treated with protease (B) or pre-incubated with antibodies against the A10L (C) or L4R (D) proteins. In contrast, pre-incubation of puri®ed viral cores with control IgG (E) or antibody against the A3L protein (F) does not inhibit their interaction with microtubules. Scale bar = 5 mm. Vaccinia uses and abuses the microtubule cytoskeleton or in vitro association with microtubules are the N protein from murine coronavirus (Kalicharran and Dales, 1996) , the movement protein from tobamovirus (Heinlein et al., 1995) , the aphid transmission factor from cauli¯ower mosaic virus (Blanc et al., 1996) , the UL25 protein from pseudorabies virus (Kaelin et al., 2000) , the VP4 spike protein from rotavirus (Nejmeddine et al., 2000) and the M protein of vesicular stomatitis virus (VSV) (Melki et al., 1994) . The identi®cation of A10L and L4R, two viral core proteins, as MAP-like proteins was, however, unexpected given their previously characterized role in viral morphogenesis (Vanslyke and Hruby, 1994) . The interaction of A10L and L4R with microtubules in vivo, together with the in vitro microtubule-binding data, suggest a potential mechanism for the association of viral cores with microtubules. One could envisage that viral cores which are released into the cytoplasm at the beginning of infection (Ichihashi, 1996; Vanderpasschen et al., 1998; Pedersen et al., 2000) bind directly to microtubules in a manner analogous to adenovirus or HSV-1 nucleocapsids. Further work is required to determine whether incoming cores do in fact move towards the MTOC by the dynein± dynactin complex and/or use the complex for anchoring on microtubules. The loss of centrosome function must enhance disruption of the microtubule cytoskeleton during infection. Indeed, the loss of microtubule organization from the MTOC precedes detectable association of A10L and L4R with microtubules, which occurs from~8 h post-infection. Vaccinia-induced loss of centrosomal proteins is inhibited by cycloheximide, indicating that viral protein expression is required for disruption of the centrosome microtubule nucleation activity. To our knowledge, vaccinia virus infection represents the ®rst example of virus-induced disruption of centrosome function, although we would predict that HSV-1 may have a similar effect. The mechanism by which vaccinia virus disrupts the centrosome requires further study; nevertheless, it is clear that understanding the molecular basis of this disruption will provide important insights into the regulation and stability of centrosome function which currently is the subject of intense research (Ohta et al., 1993; Lane and Nigg, 1997; Karsenti, 1999) . HeLa cells (ATCC CCL2) were infected with the wild-type vaccinia virus strain Western Reserve (WR) or with the vaccinia deletion mutants DF13L (vRB12) (Blasco and Moss, 1991) or DA36R (Parkinson and Smith, 1994) at a multiplicity of infection of 1 p.f.u. (plaque-forming unit) per cell, as described previously (Ro Èttger et al., 1999) . Nocodazole dissolved in dimethyl sulfoxide (DMSO) and brefeldin A dissolved in ethanol were added to the culture medium to ®nal concentration of 10 mM and 5 mg/ml, respectively unless otherwise stated. In non-treated controls, an equal volume of DMSO or ethanol was added. Cells transfected with a myc-tagged p50/dynamitin expression construct (Echeverri et al., 1996) were infected 24 h later with WR and subsequently ®xed 6 h postinfection. All experiments described have been repeated 3±10 times. The following antibodies were kindly provided: anti-a-tubulin by Dr E.Karsenti, anti-centrin (20H5) by Professor J.L.Salisbury (Sanders and Salisbury, 1994; Paoletti et al., 1996) , anti-Nek2 and anti-C-Nap1 by Professor E.Nigg (Fry et al., 1998a,b) , anti-myc and anti-gp27 by Dr T.Nilsson (Fu Èllekrug et al., 1999) and antibodies against the corresponding vaccinia proteins: A3L, A10L and L4R by Professor D.Hruby (Vanslyke and Hruby, 1994) , I1L by Professor P.Traktman (Klemperer et al., 1997) , A27L (C3) by Dr M.Esteban (Rodriguez et al., 1985) , A33R, A34R and A36R (Ro Èttger et al., 1999) . In addition, the following antibodies were obtained from commercial sources: anti-a-tubulin (N356) (Amersham International, UK), anti-acetylated a-tubulin (6-11B-1) (Sigma, USA), anti-g-tubulin (GTU-88; Sigma), anti-pericentrin and anti-TGN46 (BAbCO, USA), and rabbit IgG (Sigma). Actin was visualized with¯uorescently labelled phalloidin derivatives (Molecular Probes, USA). Cells were ®xed in ±20°C methanol or in 5% paraformaldehyde in BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl 2 , 1 mM EGTA) followed by 0.1% Triton X-100 permeabilization. Fixed cells were processed for immuno¯uorescence, viewed and images recorded as described previously (Ro Èttger et al., 1999) . HeLa cells were pre-incubated with 25 mM nocodazole in the medium for 1 h to depolymerize microtubules, prior to infection with vaccinia DF13L at 1 p.f.u./cell. Nocodazole was kept in the medium throughout the infection, while an equal volume of DMSO was added to the controls. At 24 h post-infection, the cells were scraped from the¯asks into the medium and sedimented by centrifugation (300 g, 7 min, 4°C). The cell membranes were disrupted and the nuclei were removed by centrifugation. The resulting post-nuclear supernatant was centrifuged through a 36% sucrose cushion (76 000 g, 30 min, 4°C). The virus pellet was resuspended in 10 mM Tris pH 9; the virus was collected by centrifugation (76 000 g, 30 min, 4°C), resuspended in 10 mM Tris pH 9 and stored at ±80°C. The concentration of the virus (elemental bodies) was determined by OD 260 measurement (Joklik, 1962) . Fig. 13 . Vaccinia infection reduces centrosome microtubule nucleation ef®ciency. In uninfected cells, microtubules (A, E and I) nucleate from centrosomes (B, F and J) after nocodazole washout for the times indicated. In contrast, 2 h after infection with vaccinia, microtubules (C, G and K) are nucleated inef®ciently from centrosomes (D, H and L). All images were collected with identical camera settings, to allow comparison of uorescence intensity between centrosomes. Inserts (B, D, F, H, J and L) are adjusted as in Figure 12 to facilitate visualization of the weak g-tubulin centrosomal labelling. Arrowheads indicate the position of the centrosome. Scale bar = 10 mm. In vitro microtubule binding assays Puri®ed EEV particles were prepared as described previously (Ro Èttger et al., 1999) and subsequently were used to prepare virus cores following the method of Cudmore et al. (1996) . Rhodamine-labelled microtubules were prepared according to Hyman et al. (1991) . Vaccinia virus cores were incubated with rhodamine-labelled microtubules in BRB80 buffer containing 10 mM taxol for 5 min at room temperature. 4¢,6-diamidino-2phenylindole (DAPI) was added subsequently to a ®nal concentration of 0.1 mg/ml to label the virus cores. Finally, the mixture was diluted 1:1± 1:10 with antifade solution (0.1 mg/ml catalase, 0.1 mg/ml glucose oxidase, 10 mM glucose) and viewed without ®xation. Proteinase K or trypsin treatment of core particles prior to incubation with microtubules was performed as described previously (Roos et al., 1996) . Anti-A3L, A10L, L4R or control IgG antibodies were incubated with puri®ed cores for 1 h at room temperature prior to incubation with microtubules. Cell extracts and microtubule co-sedimentation assay Extracts from HeLa cells infected for 24 h or uninfected controls maintained in the presence of 0.1 mg/ml rifampicin were prepared as described previously (Ro Èttger et al., 1999) . The extract was clari®ed by centrifugation at 150 000 g for 20 min at 4°C and cytochalasin D added to a ®nal concentration of 1 mg/ml to depolymerize actin ®laments. Endogenous tubulin in the extract was polymerized in a two-step procedure. First, the extract supernatant was supplemented with protease inhibitors, 2 mM MgGTP and 5 mM taxol and incubated for 5 min at room temperature; subsequently, an additional 15 mM taxol was added to the mix and the reaction incubated at 33°C for 30 min. For controls, no taxol was added at any stage and microtubule polymerization was inhibited either by the addition of nocodazole to a ®nal concentration of 40 mM or by maintaining the extract at 4°C throughout the experiment. Following microtubule assembly, each 400 mg extract reaction was diluted 5-fold in BRB80 buffer (containing protease inhibitors and 20 mM taxol) and centrifuged through a 10% sucrose cushion containing protease inhibitors and 20 mM taxol at 165 000 g for 20 min at 25°C. The microtubule pellet was solubilized in SDS±PAGE sample buffer and analysed by SDS± PAGE. In-gel proteolytic cleavage was performed automatically in the`Progest' as described (Houthaeve et al., 1997) [Genomic Solutions Cambridge (http://www.genomicsolutions.com)] and the peptides obtained were analysed on a Bruker REFLEX MALDI mass spectrometer (Bruker Analytik, Germany) (Jensen et al., 1996b) . Proteins were identi®ed by peptide mass ®ngerprinting (Jensen et al., 1997) using the program PeptideSearch (http://www.narrador.embl-heidelberg.de/Services/ PeptideSearch/PeptideSearchIntro.html. At 1 h post-infection, nocodazole was added to the culture medium to a ®nal concentration of 25 mM to depolymerize microtubules. At 2 h postinfection, the cells were washed 3±4 times in warm medium to remove nocodazole. Washed cells were incubated in medium without nocodazole for the indicated time at 37°C to re-initiate microtubule polymerization; they were then washed brie¯y in warm phosphate-buffered saline (PBS) and immediately ®xed. In parallel, samples were also removed at the same time point, brie¯y rinsed in ice-cold PBS, ®xed and processed for immuno¯uorescence to con®rm complete microtubule depolymerization before initiation of microtubule assembly. Uninfected control HeLa cells were treated and processed in an identical fashion. The same numbers of images were integrated using identical camera settings to allow direct comparison between infected and uninfected samples from the same experiment."
14
"Multi-faceted, multi-versatile microarray: simultaneous detection of many viruses and their expression profiles"
"There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients."
"There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients. Microarray technology has been proven to be a powerful tool with great potential for biological and medical uses. In this technique, recombinant DNA fragments or synthesized oligonucleotides affixed on the surface of glass slides or nylon membranes are used for detecting complementary nucleic acid sequences (frequently representing a few hundred to >10,000 genes/expressed sequence tags) as well as for genotyping microorganisms and for profiling the gene-expression patterns in cells from higher organisms [1]. A new report by Ghedin, et al. [2] in Retrovirology describes the successful use of a multi-virus array (termed multivi-rus-chip) to detect multiple viral co-infections in cultured cells as well as to study viral gene expression and promoter activities (Figure 1 ). Ghedin's multivirus-chip contains genes from eight human viruses including human immunodeficiency virus type 1 (HIV-1). Conceptually, this chip can be used to detect viral co-infections in AIDS patients who are frequently rendered susceptible to additional opportunistic infections. In developing their multivirus-chip, Ghedin, et al. tested more than 250 ORFs from HIV-1, human T cell leukemia virus types 1 (HTLV-1) and 2 (HTLV-2), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human herpesvirus 6A (HHV6A) and 6B (HHV6B), and Kaposi's sarcoma-associated herpesvirus (KSHV) which were PCR-amplified and spotted on glass slides. They then hybridized their slides with Cy3-or Cy5labeled genomic DNA or cDNAs derived from various virus-infected cells. Their multivirus-chip was found to be highly specific and sensitive for detecting different viral genomic sequences in cell lines. Moreover, the chip could also detect the effect of various drugs on viral gene expression. In such instance, cell lines latently infected with HIV-1 and KSHV were used to generate profiles of viral gene expression in the presence of cyclin-dependent Schematic drawing of the multivirus-chip that possesses multiple functions kinase inhibitor (CKI), Roscovitine, which was applied to cells to suppress the reactivation of latently infected viruses. Ghedin, et al. [2] also studied the role of cellular chromatin structure on viral gene expression using their multivirus-chip. They employed the chromatin immunoprecipitation technique (ChIP) [3] to isolate cellular DNA fragments that were bound to phosphorylated histone H3 (P-H3). These DNA fragments were hybridized to the viral ORFs contained on the multivirus-chip to investigate the role of phospho-H3 on viral gene expression. They showed that whether transcriptionally active or silent the chromatin state played a role in regulating the expression of KSHV genes under the different cellular context. Current routine clinical diagnostics employ PCR, Southern blotting, Northern blotting, DNA sequencing and microarray hybridization to detect and characterize genes of interest in biomedicine. PCR is generally regarded as the most sensitive diagnostic method. However, Iyer, et al. [4] have shown that the sensitivity of cDNA-chip hybridization is comparable to that of TaqMan-driven quantitative PCR assay, and that the microarray hybridization technique is less likely to be complicated by high false positive rates due to carry-over contaminations. Furthermore, using microarrays, the viral gene transcripts in infected cells can be easily detected by hybridization without any prior amplification steps, and the microarray technique requires much less experimental material when compared to Southern or Northern blotting and can provide high sensitivity in the setting of large throughput. In view of the above, the multivirus-chip described in Retrovirology [2] holds several advantages over other more commonly used techniques (e.g. PCR, DNA sequencing) for the diagnosis of viral infections. First, this chip provides a more accurate diagnosis of viral infection by simultaneously evaluating the transcription of all viral genes, and can use such cumulative data to correlate infection with clinical disease manifestations. Second, the high throughput and flexible synthesis nature of DNA microarray construction can allow scientists to tailor-make and rapidly alter arrays to match evolving emergence of new pathogens. The SARS genome chip made by the US NIAID, NIH is a good example [5] of how diagnostic arrays can be developed quickly and be used in a timely manner. Finally, the most novel application described by Ghedin, et al. is their use of microarrays to correlate the cellular "histone code" [6] with the promoter activity of KSHV. Usually the transcription of a gene located on chromosomal DNA is influenced not only by the cis-acting ele-ments (or DNA-binding motifs), but also by the structure of chromatin. The latter can be vary depending on the post-translational modifications of histone proteins. Methylation, acetylation, and/or phosphorylation of certain amino acid residues at the amino terminal "tails" of histone H3 and/or H4 can indeed influence chromatin structure. Thus accumulating evidence has shown that chromatin-associated proteins and their modifications play vital roles in many physiological processes such as growth, differentiation, and development in mammals, plants and fungi [6, 7] . Many studies have used DNA array technology to investigate viral gene expression or to genotype viral isolates; however, none has used this technique to study the influence of cellular chromatin structure on viral gene expression [1]. Ghedin, et al. [2] demonstrated that only DNA fragments derived from ChIP of latent BCBL-1 cell genomic DNA captured using phospho-H3 antibody bound specifically to the KSHV ORF on the multivirus-chip. This result suggests that latent KSHV genome in BCBL-1 cells is packed into a nucleosomal structure and that histone H3 proteins near the viral promoter can be phosphorylated at serines to make the DNA at the promoter region less tightly packed with histones and more easily accessible to transcription factors. In conclusion, the multivirus-chip improvements developed by Ghedin, et al. [2] provide versatile clinical and basic uses. In the near future, such chips are likely to be used to detect viral co-infections in many different clinical settings. "
15
"Herpes simplex virus type 1 and normal protein permeability in the lungs of critically ill patients: a case for low pathogenicity?"
"INTRODUCTION: The pathogenicity of late respiratory infections with herpes simplex virus type 1 (HSV-1) in the critically ill is unclear. METHODS: In four critically ill patients with persistent pulmonary infiltrates of unknown origin and isolation of HSV-1 from tracheal aspirate or bronchoalveolar lavage fluid, at 7 (1–11) days after start of mechanical ventilatory support, a pulmonary leak index (PLI) for (67)Gallium ((67)Ga)-transferrin (upper limit of normal 14.1 × 10(-3)/min) was measured. RESULTS: The PLI ranged between 7.5 and 14.0 × 10(-3)/min in the study patients. Two patients received a course of acyclovir and all survived. CONCLUSIONS: The normal capillary permeability observed in the lungs argues against pathogenicity of HSV-1 in the critically ill, and favors that isolation of the virus reflects reactivation in the course of serious illness and immunodepresssion, rather than primary or superimposed infection in the lungs."
"In some critically ill patients herpes simplex virus (HSV)-1 is isolated from the upper or lower respiratory tract [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . Immunodepressed patients may be susceptible to transmission and acquisition of viral diseases; alternatively, viral reactivation may occur and may contribute relatively little to morbidity and mortality. Indeed, reactivation of human herpesvirus-6 is common in critically ill patients and does not worsen outcome [16, 17] . In immunocompetent patients, however, isolation of HSV-1 may be associated with viral pneumonia, even if reactivation rather than primary infection is responsible [6, 8, 18] . HSV-1 has been associated with acute respiratory distress syndrome (ARDS) and ventilator-associated pneumonia in the critically ill [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] , as either a primary or a superimposed infection. However, there are few reports of the virus eliciting an infectious host response, as demonstrated by a rise in serum antibodies, by bronchoscopic airway disease, by 'typical' findings on computed tomography of the lungs, or by the presence of giant cells or nuclear inclusion bodies on cytology or biopsy of the lower respiratory tract [3, 5, 9, 10, 18] . Indeed, Tuxen and coworkers [4] observed that prophylactic antiviral therapy in ARDS prevented respiratory HSV-1 emergence but it had no impact on duration of mechanical ventilation or on patient outcome. The pathogenicity of the virus therefore remains unknown, and the rare association in the critically ill of HSV-1 isolation with mortality may represent reactivation of the virus in immunodepressed patients with multiple organ failure and poor outcome [1, 2, 11, 14, 15] , rather than a symptomatic primary infection or superinfection contributing to death. Assessing pulmonary capillary protein permeability noninvasively at the bedside to yield the pulmonary leak index (PLI) could help in determining the extent of tissue injury, as was previously described [18] [19] [20] . This radionuclide technique involves gallium-67-labelled transferrin ( 67 Gatransferrin) and technetium-99m-labelled red blood cells ( 99m Tc-RBCs). In bacterial pneumonia, for instance, the PLI is elevated and the increase above normal directly relates to the severity of pneumonia, expressed as the lung injury score (LIS) [19] . In patients with acute lung injury (ALI) or ARDS during the course of bacterial pneumonia, the PLI is uniformly and greatly elevated above normal (up to 14.1 × 10 -3 /min) when LIS is greater than 2.5; the PLI is also elevated in 80% of patients with mild injury and a LIS between 1.5 and 2.5 [19] . Hence, the technique is a direct measure of permeability and an indirect measure of capillary injury in the lungs. The PLI is also elevated in interstitial lung disease [21] . In order to help differentiate between symptomatic and asymptomatic viral shedding and spread, which could inform the decision regarding whether to institute antiviral therapy and help in determining the pathogenicity of the virus, we measured the PLI in four consecutive critically ill patients with persistent pulmonary infiltrates of unknown origin on ventilatory support, in whom a HSV-1 had been isolated. We studied a small series of consecutive patients in whom respiratory secretions, sent for viral culture because of persistent pulmonary infiltrates of unknown origin, were found to be positive for HSV-1 (Table 1) . Tracheal aspirates or bronchoalveolar lavage fluid were transported directly to the microbiology laboratory or placed in viral transport medium (Copan Diagnostics Inc., Corona, CA, USA). For isolation of HSV-1, specimens were inoculated using standard procedures in triplicate flat bottom tubes on human embryonal lung fibroblasts and incubated at 37°C. Cultures were studied three times weekly for 10 days to identify the presence of a cytopathic effect. If a cytopathic effect, indicating the presence of HSV-1, was apparent or otherwise at days 2 and 7, the cells were fixed in methanol:acetone (1:1) and typed by immunofluorescence with labelled specific HSV-1 and HSV-2 antibodies (Syva Mikrotac HSV-1/HSV-2 typing kit, Palo Alto, CA, USA). In the four patients studied, the results were available within 3 days after samples had been inoculated in culture medium. On the day of specimen collection for viral culture, demographic, chest radiographic and respiratory data were recorded, as were clinical features. In three out of four patients on mechanical ventilation after intubation, the total respiratory compliance was calculated from ventilator settings as follows (ml/cmH 2 O): tidal volume/(plateau -end-expiratory pressure). From the radiographic score (ranging from 0 to 4 depending on the number of quadrants with radiographic opacities), the ratio of arterial oxygen tension to fractional inspired oxygen, the level of positive end-expiratory pressure and the compliance, the LIS was calculated [22] . (LIS ranges between 0 and 4, with values up to 2.5 denoting ALI and those above 2.5 ARDS.) None of the patients had visible oropharyngeal vesicles. To characterize further the persistent pulmonary infiltrates, the PLI was measured using a modification to a method described previously [19, 20] . Because this is a routine procedure, informed consent was waived. Autologous RBCs were labeled with 99m Tc (11 MBq, physical half-life 6 hours; Mallincrodt Diagnostica, Petten, The Netherlands), using a modified in vitro method. Ten minutes after injection of the labelled RBCs, transferrin was labelled in vivo, following intravenous injection of 67 Ga-citrate (6 MBq, physical half-life 78 hours; Mallincrodt Diagnostica). Patients were in the supine position, and two scintillation detection probes were positioned over the right and left lung apices. The probe system (manufactured by Eurorad C.T.T., Strasbourg, France) consists of two small cesium iodide scintillators (15 × 15 × 15 mm 3 ), each in a 2-mm tungsten and 1-mm aluminium housing cover (35 mm in diameter and 40 mm in height). The front end of each probe has an aluminium flange attached (3 mm in thickness and 70 mm in diameter) to facilitate easy fixation to the patient's chest with tape. Each probe weighs approximately 255 g. The probe signals are led into a dual amplifier, from which the output is fed into a multichannel analyzer system connected to a personal computer. Because the probes have separate channels, there is no electronic crossover. Starting at the time of the intravenous injection of 67 Ga, radioactivity was measured each minute for 1 hour. For each measurement interval, the entire spectrum of photon energies was stored on disk. During processing, the 99m Tc and 67 Ga -and plotted against time. The PLI was calculated, using linear regression analysis, from the slope of increase of the radioactivity ratio divided by the intercept, in order to correct for physical factors in radioactivity detection. By taking pulmonary blood volume and thus presumably surface area into account, the radioactivity ratio represents the ratio of extravascular to intravascular 67 Ga radioactivity. The PLI represents the transport rate of 67 Ga-transferrin from the intravascular to the extravascular spaces in the lungs, and it is therefore a measure of pulmonary capillary permeability to transferrin [19, 20] . The mean PLI from the two lungs was taken. The upper limit of normal PLI is 14.1 × 10 -3 /min. Where appropriate, numbers are summarized as median (range). Patient data are presented in Table 1 . The patients had stayed for some time in the hospital or intensive care unit before HSV-1 was isolated, and they had been admitted primarily because of respiratory insufficiency during the course of pneumonia. Patient 4 was admitted into the coronary care unit a few days before intensive care unit admission for cardiogenic pulmonary oedema. All patients had been dependent on mechanical ventilatory support for some time before sampling. They had received adequate antibiotic therapy for pneumonia and had ALI at the time of sampling, which was of otherwise unknown origin. Table 1 shows that patients had radiographic abnormalities but without an increased PLI. Central venous pressure was not elevated, which suggests that the persistent pulmonary infiltrates were not caused by overhydration. In patients 1 and 3 a high-resolution computed tomography scan of the lungs with contrast was obtained; the findings were nonspecific, however, with alveolar consolidations and pleural fluid, even in the presence of interstitial abnormalities with a ground glass appearance in patient 3. In patient 1 a bronchoscopy was performed and there were no mucosal lesions. There was a normal distribution of lymphocyte subtypes in the lavage fluid. A transbronchial biopsy revealed interstitial inflammation with many macrophage deposits, and immunohistochemical staining for HSV-1 was negative. No multinucleated cells or cell inclusions were observed, either in bronchoalveolar lavage fluid from patient 1 or in tracheal aspirates from the other patients. In patients 1-3 concomitant isolation of bacteria by culture was regarded as bacterial colonization. Antibody testing was not done in patients 2-4 but was found to be positive for anti-HSV-1 IgG in patient 1, which is indicative of prior HSV-1 infection. The antiviral agent aciclovir (10 mg/kg three times daily) was started when cultures became positive in two patients, at the discretion of the treating physician. Aciclovir was withheld in the other two patients because it was presumed that the pulmonary infiltrates were not caused by HSV-1, on the basis of a normal PLI among other findings. In patient 1, who had a normal PLI, a course of steroids was initiated on the day after the PLI was measured, and was continued despite positivity for HSV-1, reported 5 days later. All patients survived until discharge from the intensive care unit. The 67 Ga-transferrin PLI is a sensitive and specific measure of pulmonary capillary permeability, which is utilized for noninvasive assessment of severity of a broad range of pulmonary conditions [19] [20] [21] . The PLI roughly parallels clinical severity (i.e. the LIS) [19, 20] . Although it involves the use of relatively routine equipment, the diagnostic method has not gained broad application, partly because of its laborious nature [20] . It has the advantage that bedside measurements are possible in mechanically ventilated critically ill patients, who cannot easily be transported. Pulmonary inflammation, of whatever cause, increases the PLI up to four times normal values in the most severe forms of lung injury, including ARDS. In less severe injury, such as impending ARDS and interstitial lung disease, the PLI is also elevated, albeit to a lesser extent, as reported by us and other groups [20, 21] . The patients had in common a prior infectious episode, followed by a relatively prolonged period of respiratory insufficiency. They had persistent and nonspecific pulmonary infiltrates of unknown origin, after treatment of their primary disease, which prompted viral culture. The normal PLI observed suggests the involvement of a relatively harmless reactivation of HSV-1, rather than the presence of a primary and damaging infection. Indeed, critically ill patients with sepsis may have late immunodepression, with lymphocytic apoptosis, lymphocytopenia and T-cell anergy, promoting viral reactivation [23, 24] . Apparently, the virus must have been latent in the nerve endings of the mucous membranes of the upper respiratory tract in these patients [2, 15] . Herpesviruses (HSV-1) have frequently been isolated in vivo from respiratory secretions of patients with ARDS [3, 4] and detected in surveillance cultures from the respiratory tract of patients following burns, trauma, transplantation, major surgery and others. However, these viruses are detected in only 3% of lung biopsies from patients with prolonged and unresolving ARDS [3, 7, [9] [10] [11] [12] [13] 15] . The literature is thus widely divergent on the precise role of the virus in pulmonary disease in the critically ill and its contribution to patient morbidity and mortality [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . We believe that the tracheal aspirates were representative of lower respiratory tract secretions, in the absence of herpes orolabialis and oral epithelial cells in smears for Gram stain of the secretions. Concurrent colonization with other pathogens has previously been described [5, 13] . Because there was no overlap in the duration of stay of the patients, transmission of the virus from one patient to another can be excluded. This further suggests that respiratory HSV-1 infections in the critically ill may result from relatively harmless endogenous reactivation. Although the normal PLI argues against pulmonary parenchymal pathogenicity, tracheobronchitis caused by the virus [18, 25] cannot be ruled out, even in the absence of orolabial lesions, because bronchoscopy was not performed in three of the four patients, even though it was unremarkable in patient 1. The persistent pulmonary infiltrates in our patients may thus relate to slow radiographic resolution of prior bacterial or aspiration pneumonia, rather then superimposed infection. Moreover, computed tomographic images of the lung may be largely nonspecific [26] , and so the precise diagnostic criteria for HSV-1 pneumonia remain unclear. When properly standardized, for instance with respect to cell numbers in bronchoalveolar fluid or tracheal aspirates, quantitative cultures, viral RNA and DNA by polymerase chain reaction, could be helpful together with the PLI in further studies to quantitate viral load and the ratio of replication to shedding, and therefore the pathogenicity of the virus in the lower respiratory tract. In conclusion, the anecdotal data presented here suggest that isolation of HSV-1 from respiratory secretions in the critically ill patient with a persistent pulmonary infiltrate may warrant evaluation of tissue injury potentially caused by the virus to judge its pathogenicity. This could be done using a radionuclide PLI measurement, and would help to inform decisions regarding antiviral therapy, which may have adverse effects. In some patients a normal PLI may argue against viral pathogenicity, and withholding of aciclovir in such patients may be safe."
16
"Logistics of community smallpox control through contact tracing and ring vaccination: a stochastic network model"
"BACKGROUND: Previous smallpox ring vaccination models based on contact tracing over a network suggest that ring vaccination would be effective, but have not explicitly included response logistics and limited numbers of vaccinators. METHODS: We developed a continuous-time stochastic simulation of smallpox transmission, including network structure, post-exposure vaccination, vaccination of contacts of contacts, limited response capacity, heterogeneity in symptoms and infectiousness, vaccination prior to the discontinuation of routine vaccination, more rapid diagnosis due to public awareness, surveillance of asymptomatic contacts, and isolation of cases. RESULTS: We found that even in cases of very rapidly spreading smallpox, ring vaccination (when coupled with surveillance) is sufficient in most cases to eliminate smallpox quickly, assuming that 95% of household contacts are traced, 80% of workplace or social contacts are traced, and no casual contacts are traced, and that in most cases the ability to trace 1–5 individuals per day per index case is sufficient. If smallpox is assumed to be transmitted very quickly to contacts, it may at times escape containment by ring vaccination, but could be controlled in these circumstances by mass vaccination. CONCLUSIONS: Small introductions of smallpox are likely to be easily contained by ring vaccination, provided contact tracing is feasible. Uncertainties in the nature of bioterrorist smallpox (infectiousness, vaccine efficacy) support continued planning for ring vaccination as well as mass vaccination. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity) and should be accompanied by increased public awareness and surveillance."
"Concerns about intentional releases of smallpox have prompted extensive preparations to improve our ability to detect and respond to an outbreak of smallpox [1, 3, 4, 2] . Many factors contribute to the public health challenge of understanding and preparing for smallpox, including the age and quality of epidemiological data on native smallpox and the smallpox vaccine, the difficulty of extrapolating that data to our current populations, the possible terrorist use of altered smallpox, our ignorance of terrorist methods of release, and the relatively high risk of adverse events caused by the smallpox vaccine. The Centers for Disease Control and Prevention (CDC) established ring vaccination (selective epidemiological control [5] ), a strategy in which contacts of cases are identified and vaccinated, as the preferred control measure in the event of a smallpox outbreak (interim plan). The successful use of ring vaccination during the smallpox eradication campaign and its logical emphasis of case-contacts for immediate vaccination support its use (though the attribution of the success of the eradication program to ring vaccination has been challenged [6] ). Health Officers should initiate ring vaccination upon identification of the first cases of smallpox. However, there are legitimate concerns regarding the ability of public health practitioners to mount a quick, comprehensive and successful ring vaccination program, particularly in the face of a moderatesized or large smallpox outbreak. To guide preparation efforts and inform incident decision-making, we attempt to identify outbreak characteristics and response capacities that significantly impact the ability of ring vaccination to control a smallpox outbreak and to determine whether ring vaccination is useful in the presence of a mass vaccination campaign. Our analysis uses a newly developed mathematical model: a continuous-time, event-driven network simulation model of smallpox ring vaccination. Mathematical models can advance our understanding of how a smallpox outbreak might progress. Several mathematical and computer models address the question of smallpox transmission [7] [8] [9] [10] [11] [12] [13] . The first model to appear [8] concluded that ring vaccination would be effective, but did not treat response logistics in detail; the model was linear and did not treat the depletion of susceptibles as the epidemic progressed (appropriate, however, for assessing control early in an epidemic, when the number infected is small compared to the number of susceptibles, e.g. [14] ). The innovative model by Kaplan et al. [9] emphasized the importance of resource limitation and the logistics of smallpox response, but assumed that full infectiousness began before the onset of symptoms (and the subsequent identification and removal), and did not separately monitor close epidemiological contacts of patients (which are at greatest risk, but also easiest to find and vac-cinate); the conclusions were highly critical of ring vaccination. The model by Halloran et al. [11] , a stochastic, discrete-time network model omitted the explicit inclusion of response logistics while otherwise used parameter values similar to those in Kaplan et al. [9] ; the inclusion of residual immunity from individuals vaccinated prior to the discontinuation of routine vaccination, however, led to a more favorable view of ring vaccination. The model by Bozzette et al. [12] assumed that ring vaccination would reduce the number of transmissions and focused on health care workers (but did not explicitly include the network structure of the population nor the response logistics of ring vaccination). The model by Eichner [15] did not explicitly include the network structure of the population nor the logistics of ring vaccination, but did use parameters based on data from an outbreak in Nigeria, and did distinguish close and casual contacts, case isolation, and surveillance of contacts; it concluded that case isolation and contact tracing could prevent the spread of smallpox. Finally, the individual-based model by Epstein et al. [16] presented scenarios illustrating certain alternatives to pure mass vaccination and ring vaccination of contacts of cases in preventing smallpox transmission in small populations of 800 individuals; this model includes no homogeneity assumptions, but did not analyze tracing of contacts of contacts. Because none of the available models includes both network structure (with explicit contact tracing) and response logistics limited by the number of available disease control investigators [9] , we included these features in a continuous-time event-driven network simulation model of smallpox ring vaccination. Specifically, the model we developed includes the following features: exposed individuals and vaccinate them in time, resulting in a "race to trace" [9] . Mild, ambulatory cases of smallpox may spread disease because such cases may be harder to recognize. Vaccination of individuals prior to the discontinuation of routine vaccination may provide some, possibly considerable, protection against infection [11, 23, 24] , although it may also result in more mild cases which may be harder to detect. Public awareness may lead to more rapid detection of cases. We use this model to determine what factors promote or hinder the success of ring vaccination during a smallpox outbreak, and whether ring vaccination is useful in the presence of a mass vaccination campaign. In particular, the goal of this paper is to examine the control of smallpox by contact tracing and ring vaccination using a network model which includes response logistics [9] . Natural history of smallpox We briefly review relevant features of the natural history and epidemiology of smallpox [17, [25] [26] [27] 8, 28] . Following infection by the variola virus, individuals exhibited an incubation period of approximately 7-19 days with 10-14 being most typical. Sudden onset of fever and malaise, often with accompanying headache and backache, began the initial (or pre-eruptive) phase of smallpox. After 2-3 (or perhaps 4) days, individuals with the most common form, ordinary type smallpox, developed the characteristic focal rash, preceded in many cases by oropharyngeal lesions. In fatal cases of ordinary smallpox, death often occurred between the tenth and sixteenth day of symptoms; among survivors, most scabs had separated by day 22-27 of illness [26] . The course of smallpox varied widely between individuals, and several different clinical classifications were developed [29] [30] [31] 17, 26] . Consideration of the clinical features and severity of smallpox is important from the standpoint of mathematical transmission modeling because (1) the clinical features affect the ease of diagnosis (and thus of case identification), (2) more severe forms of smallpox may result in more transmission, (3) vaccinated individuals may develop less severe disease. We utilize a modified or simplified version of the classification system developed by Rao [32, 31, 26] ; for the mathematical model, we will classify smallpox into five categories: early hemorrhagic, flat and late hemorrhagic, ordinary, modified, and mild. However, the clinical features and severity of smallpox in different populations may have been affected by underlying host factors, differences in viral strains, or differences in the infectious dose owing to different prevailing modes of transmission, and thus robust and precise quantitative estimates of the effects of (pre-or post-exposure) vaccination on the resulting smallpox severity, or of the infectivity differences between individuals exhibiting different forms of smallpox, are not available. The significance of such differences will be revealed through sensitivity analysis. Further details are given in Appendix 1 [see Additional file: 1]. Vaccination with vaccinia virus provided substantial protection against infection. Dixon assessed the risk of infection for an individual successfully vaccinated 3 years prior to exposure to be 0.1% the infection risk of an unvaccinated individual [17] . However, smallpox vaccination did not always take when applied, and moreover, in many instances, individuals who experienced a repeated vaccination failure developed severe smallpox upon exposure. The probability of a successful take depended on the vaccination method used; we assume that the take rate is between 95% and 100% [22, 28] . In addition to protection against infection, vaccination could in many cases modify the course of infection and reduce the severity. Vaccine protection waned over time, but individuals vaccinated 20 years prior to exposure were believed to still have half the infection probability that an unvaccinated person had [17] , and to have some protection against the most severe manifestations of smallpox. Dixon [17] believed that vaccine protection had at least three components, which decayed at different rates; for the purpose of this paper, we will assume that the severity of smallpox in previously any (recently or otherwise) vaccinated individuals follows the same distribution as for the vaccinated subjects seen in the case series observed by Rao in Madras [26] , except that anywhere from 0 to 5% of vaccinated subjects develop smallpox too mild to diagnose without special surveillance or awareness. Observe that the vaccinated cases studied by Rao were vaccinated (at some point in their lives) before exposure, rather than after exposure to smallpox. Smallpox was largely a disease of close contacts [17, 26, 33] , spread primarily through face to face contact with an infected person (or occasionally through contaminated clothing). Individuals in the incubation period of smallpox were not infectious, and long term carriers did not exist. Patients were believed to be infectious following the development of oropharyngeal lesions, which could precede the rash by 24 hours [26] . However, patients were believed to be most infectious during the first week of the rash [26] ; Dixon (1962) believed that patients could be infectious from the onset of acute viremia, but most evidence suggested that little transmission occurred prior to the development of the rash [26, 33] . The more severe the case, the more infectious they appeared to be [34] ; mild cases were believed to have very little infectiousness. While scabs contained infectious material and patients were considered to be infectious until the last scab fell off, in practice patients were not highly infectious during the scabbing phase. Importantly, patients who had been vaccinated were found to cause fewer secondary cases [34] . Very severe cases, such as hemorrhagic or flat smallpox, occasionally resulted in considerable transmission, owing to diagnostic difficulties; mild cases, in which the patient remained ambulant during the course of the disease, could cause considerable spread as well [35, 36] . Within a household or family dwelling, the secondary attack rate of unvaccinated susceptibles depended on the time and place, occasionally below 50% [29] , but often approaching 100% [37] . Drier conditions were often believed to favor transmission [17, 27] , so that lower rates of transmission derived from tropical regions may not be applicable to the temperate zone [38] . The number of secondary cases resulting from a given importation into Europe varied widely [39] , with most importations yielding few cases, but with the occasional large outbreak being seen. Mathematically, we represent the course of smallpox according to Figure 1 . We distinguish eight epidemiologically relevant states: (1) just following exposure, during which time vaccination could afford complete protection against disease, (2) a period of several days during which vaccination will not prevent disease, but may still reduce the severity of disease, (3) still prior to the development of symptoms, but too late for vaccination, (4) the beginning of the pre-eruptive period, during which the patient exhibits fever, malaise, and possibly other symptoms, but is not yet infectious, (5) a short period prior to the appearance of the rash, during which the appearance of oropharyngeal lesions will permit variola transmission, (6) the first week of the rash, during which time the patient is most infectious, (7) and (8), succeeding stages of the rash, during which time the patient is less infectious. For each of these states, we assume that conditional on surviving, the waiting time until the next stage is chosen from a uniform distribution as indicated in Appendix 2 [see Additional file: 2], except that the incubation period (the time from infection until Stage 4) is derived from estimates of the incubation distribution of smallpox based on importation cases in Europe [26] (see Appendix 2 [see additional file 2] for details). We chose to sample from a uniform distribution as a simple way to ensure a minimum waiting time in each state; many alternatives to this choice are possible. We simulate the transmission of smallpox on a "smallworlds" network (highly clustered, but with short characteristic path lengths) [40] . Specifically, we assume that each person is located in a single household, and that the transmission rates were greatest in the household. We also assume that a fraction of the population are grouped into workplace or social groups, in which transmission may also occur, but with a lower rate per unit time than for household contacts. Finally, we assume that with a still smaller probability, any individual may transmit infection to any other individual in the population (casual contacts). In general, in a network-structured model, the number of secondary cases caused by an index case in a completely susceptible population is not a useful index of epidemic potential [41, 42] (for a simple example, see [43] ), since (for instance) an individual could infect everyone in his or her household, and not cause a widespread epidemic unless between-household transmission were sufficiently frequent. Rather than constructing the appropriate generalized basic reproduction number for our model (leading to highly cumbersome expressions), we chose an alternative (ad hoc) index of epidemic potential. For any given scenario of interest, we simulated the introduction of 10 index cases at random into a population of size 10000, and operationally defined "containment" to occur whenever the final size of the epidemic was less than 500 cases within 250 days (we showed, in the discussion of Figure Smallpox stages used in the simulation model Figure 1 Smallpox stages used in the simulation model. Flat and ordinary smallpox rashes are indicated with more dots than modified and "mild" smallpox, suggesting potentially greater infectiousness. Hemorrhagic smallpox is indicated by horizontal line shading. Further details are provided in Table 6 . 5A below, that in nearly all cases, the 250-day window differs very little from a 1000-day window). Because we simulate a disease with a finite duration on a finite and nonrenewing population, epidemic extinction always occurs in finite time. We assume that even in the absence of specific case investigations, the presence of smallpox symptoms will prompt patients to be diagnosed; we assume, however, a higher diagnosis rate for all forms of ordinary smallpox than for the severe flat and hemorrhagic forms, or for the mildest form. We assume that once an individual is diagnosed, their household and workplace contacts are investigated and detected with some probability; we assume that a high fraction (such as 95%) of household contacts are assumed to be traceable (see below). We assume that the fraction of workplace/social contacts that are traceable is less than the fraction of household contacts that are traceable; we assume that no casual contacts are traceable. High contact-finding rates may be plausible; we examined San Francisco Department of Public Health records of contact investigations for meningococcal disease (like smallpox, a potentially fatal disease for which rapid intervention may prevent mortality and morbidity). Records were available from December 2001 to April 2002; 13 such investigations during this period resulted in identification of 62 household contacts, all of which were contacted; out of 38 workplace/social contacts identified, 32 were contacted (84%). In our model, we assume that identified asymptomatic contacts are vaccinated, quarantined, and monitored for symptom development, while symptomatic patients are isolated and treated as necessary [9] ; thus, the modeled interventions include more than ring vaccination alone. Finally, we include the possibility that all contacts (of both symptomatic and asymptomatic) traced and the same procedure applied, so that all contacts of contacts would be investigated. We assume that uninfected or asymptomatic individuals who are visited or traced individuals will be diagnosed more rapidly than if they had not been traced; in fact, such individuals would be isolated and would not be able to continue a chain of transmission. We follow previous models [9] in assuming a limited vaccination capability of K r per day for ring vaccination. We assumed one of two strategies for contact tracing: (1) tracing only of direct contacts of diagnosed cases, and (2) tracing of contacts of contacts of diagnosed cases as well as direct contacts. The contact structure of the network is illustrated in Figure 2 . Observe that individuals b and c are household contacts of individual a, so that if individual a were a smallpox case, an attempt would be made to find and vaccinate individuals b and c as household contacts of a case. If individuals a and b were both cases, then two attempts could be made to find individual c. We have modeled the effect of multiple contact-finding attempts conservatively in the sense that if the first attempt to find an individual as a household contact (of a case or of a contact) is determined to fail, no further attempts will be made. This maintains the failure rate of contact tracing (looked at from the standpoint of finding individuals) even in large households. Similar considerations apply to workplace/social groups. Figure 2 Network structure shown for households (joined by thick lines) of size 3 and workplace/social groups of size 4 (joined by thin lines); a small portion of the network is shown. Individual a has two household contacts (b and c), and three workplace/social contacts (d, e, and f). If individual a were a smallpox case, the household contacts would be at highest risk for acquiring smallpox, followed by workplace/social contacts; all individuals in the population are at a low risk of casual transmission from individual a. Case investigation of individual a would identify the direct contacts b-f with probabilities that depend on whether the contact is household or workplace/social; if such individuals are identified, they will be vaccinated. If contacts of contacts are being traced, the investigation will subsequently identify individuals g-p. We analyzed the model in three ways. First, we selected a Latin Hypercube sample [44] [45] [46] of parameters chosen uniformly from the parameter ranges given in Appendix 2 [see additional file 2] , and simulated the transmission and control of smallpox to determine which parameters were most important for contact tracing and ring vaccination to be effective. Second, we used the same Latin Hypercube Sample of input parameters, but assumed that all disease control efforts were inactive. We used these parameters to simulate smallpox transmission, but then iteratively selected transmission parameters so that (1) between 1% and 10% of new infections resulted from casual (random) transmission, and (2) each index case resulted in between two and five secondary cases (thought to be plausible for historic smallpox; [8] suggest three secondary cases). For each of the resulting smallpox parameter sets using 100 stochastic simulations per set, we determined the daily ring vaccination/case tracking capacity needed to contain all simulated smallpox epidemics (i.e., keep the total number of cases below 500 within 250 days). Third and finally, we chose parameter values to yield an moderately large smallpox epidemic (with each index case causing approximately six secondary cases), and present illustrative scenarios for ring vaccination. These scenarios are intended to complement the simulations which were calibrated to historic smallpox, since the characteristics of smallpox that may be used in a deliberate release are not known. It is important to realize that in our model, the case finding time determines the fraction of contacts that will become infected, and that our model parameters have been chosen to yield quite rapid transmission to close contacts; in reality, much transmission of natural smallpox occurred through "sickbed" routes which would not occur in a modern setting [47] , so that in this sense our model errs considerably on the side of caution and pessimism. To determine which of the input parameters were most important in determining the total number of smallpox cases, we selected a Latin Hypercube sample of size 1000 from the input parameter ranges indicated in Appendix 2 [see additional file 2] and simulated the mean number of cases within 250 days in a population of 10000. We then computed the partial rank correlation coefficient [46] (PRCC; see Appendix 2 [see additional file 2]) between each input parameter and the number of smallpox cases; when the PRCC is close to zero, the value of the parameter has little relation to the simulation output; when the PRCC is close to +1.0 or -1.0, the value of the parameter is highly important in determining the simulation output. Neglecting the number of index cases (which is directly related to the number of new cases), those parameters whose PRCC exceeds 0.1 are shown in Table 2 . Most of these parameters identified as important are related to the density of available contacts (mean household size, prior vaccination fraction, and protection due to prior vaccination) or the transmission rate and infectivity (including the length of the pre-eruptive infectious period (stage 5 in Figure 2 )). Note, however, that the speed of ring vaccination (household tracing delay) and faster diagnosis due to awareness of the outbreak are important parameters. Additionally, the infectivity of mild cases appears as an important parameter as well. To explore factors which contribute to the success of ring vaccination, we chose smallpox scenarios which resulted in severe and fast-moving epidemics in the absence of disease control; these simulated epidemics are considerably more severe than is believed likely under present circumstances. We used these parameters to simulate smallpox epidemics beginning with 10 cases, for a variety of levels of ring vaccination capacity per day (contact tracing capacity per day), as shown in Figure 3A . In this Figure, we assume that the population size is 10000, and that the epidemic began with 10 infected individuals. The mean household size is assumed to be 4, the mean size of the workplace/social contact group is 8, and contacts of contacts are traced. We assume that each day, the number of contacts that can be traced and vaccinated as a result of case investigation is 0, 10, 20, 30 and 40 per day; the probability of finding a workplace/social contact is assumed to be 80%. The Table 1 . Because we assumed nonzero diagnosis probabilities during the prodromal period for all individuals in Figure 3A , we repeated the simulation assuming no diagnosis in the prodromal period unless individuals were under specific surveillance. The results were nearly identical: assuming 30 contact tracings (ring vaccinations) per day, we found 26% of the scenarios in Figure 3A exhibited decontainment, and 28% assuming no diagnosis during the prodromal period; assuming 40 contact tracings per day, we found 1 out of 100 scenarios showed loss of containment in Figure 3A and when we repeated the scenario of Figure 3A assuming no diagnosis during the prodromal period. In Figure 3B , we illustrate control of an epidemic for which all parameters are identical to Figure 3A , except that the workplace/social group size is 12 (instead of 8, as in Figure 3A ), and the probability of finding workplace/ social contacts is 60% (instead of 80%, as in Figure 3A ). In this case, the larger size of the workplace/social groups and the lower contact finding probability makes it necessary to have a higher ring vaccination capacity to attain a high probability of containing the epidemic, and on average it takes longer for eradication to finally occur. Finally, in Figure 3C , we show control of an epidemic in a population of 100,000, beginning with 1000 initial infectives, keeping all other parameters the same as in Figure 3A . Each curve corresponds to the indicated number of possible ring vaccinations per day. This figure shows that assuming sufficient capacity, ring vaccination is in principle capable of containing even epidemics beginning with very many infected individuals. However, mass vaccination in such cases is justified because of the far larger number of individuals at risk and the inability to perform such extensive contact tracing. In Figure 3D , we compare the effect of tracing contacts of contacts (as described in Appendix 2 [see additional file 2]) at different levels of ring vaccination capacity. Thin Figure 5A , 5B φ Prob. of finding household contact 0.95 Delay, tracing household contacts 1-5 days Expanding severe smallpox epidemic Figure 3 3A -Expanding severe smallpox epidemic beginning with 10 initial cases, assuming 0, 10, 20, 30, and 40 possible ring vaccinations per day. The household size is 4 and the workplace/social group size is 8; we assume 95% of household contacts are traceable (with a mean delay of 1 day) and 80% of workplace/social contacts are traceable (with a mean delay of 2 days). We also assume that 25% of the population have 50% protection from infection resulting from vaccination prior to the discontinuation of routine vaccination. We assume that infection will be transmitted to close contacts with a mean time of 0.2 days, and that each person while infective causes on average 0.15 casual (untraceable) infections per day. We assume that individuals are 20% as infectious in the day just before the appearance of the rash as they will be during the first week of the rash, and that individuals are 20% as infectious as this (4% as infectious as during the first week of the rash) during the prodromal period. We assume that diagnosis rates will increase by a factor of 50% after smallpox becomes known to the community; we assume that each individual contacted during an investigation has a additional diagnosis or removal rate of 0.75 per day following the onset of symptoms (reflecting enhanced surveillance or contact isolation). Important parameters are summarized in Table 1 ; the full set of parameter choices is outlined in Tables 8-11 in Appendix 2 [see additional file 2] . Diagnosis times are discussed in Appendix 2 [see additional file 2]. 3B -An expanding severe smallpox epidemic under inadequate ring vaccination is shown for parameters identical to Figure 3A , except that workplace/social group sizes are 12 (instead of 8), and the probability of tracing workplace/social contacts is 0.6 (instead of 0.8). 3C -A severe smallpox epidemic is controlled by ring vaccination despite the large number of initial cases. The parameters are identical to Figure 3A , except that 1000 index cases inaugurate the attack in these scenarios (and ring vaccination capacity is much greater, as indicated). While not recommended, ring vaccination may ultimately halt epidemics beginning with many index cases if sufficient vaccination capacity were available, contact finding feasible, and follow-up sufficient. 3D -Tracing contacts of contacts (red) is beneficial when sufficient contact tracing/ring vaccination capacity exists (dotted lines). In these scenarios, all parameters are the same as in Figure 3A ; the number of contact tracings possible per day is either 20 or 40 per day. Contacts of contacts are traced in two scenarios; in the other two, only direct contacts of cases are traced. For low levels of ring vaccination (20 per day), tracing contacts of contacts is harmful; for high levels (40 per day) of ring vaccination, it is beneficial to trace contacts of contacts. When the contact tracing/ring vaccination capacity is too small to adequately cover contacts of the cases themselves, diversion of resources to contacts of contacts is harmful; however, provided that sufficient capacity exists, tracing contacts of contacts helps outrun the chain of transmission. Each line corresponds to the average of 100 realizations. The average number infected on each day is plotted in the Figure. The figure illustrates that when ring vaccination capacity is low, tracing contacts of contacts (as modeled) yields a more severe average epidemic; when ring vaccination capacity is large, tracing contacts of contacts results in a less severe average epidemic; if the contact tracing/ring vaccination capacity is too low to cover adequately the contacts of contacts in addition to the contacts of cases, extension of tracing to the contacts of contacts (the second ring) is harmful; however, if there is sufficient capacity to cover the contacts of contacts, then the tracing of contacts of contacts is beneficial. Finally, in Figure 4 , we illustrate the considerable variability that may be seen from simulation to simulation. This figure shows twenty simulations when contacts of contacts are not traced. Stochastic variability between realizations is considerable, even when all parameters are held constant; this variability is expected to limit the ability to make inferences based on observation of a single realization of the process. Because our baseline hazard for infection of individuals may be larger than would be expected for naturally occurring smallpox, we examined the effect of more realistic values of this hazard. In particular, we chose different levels of ring vaccination capacity (10, and 20) , and of the relative hazard for workplace/social contacts, and then chose values of the baseline hazard for infection varying from 0.5 per day (for a mean time to infection of 2 days) to 2 per day (for a mean time to infection of one half day), Table 3 : Estimated decontainment probability for different levels of ring vaccination capacity (Kr) and relative hazard for infection due to workplace/social contacts (h2), for different levels of the baseline hazard for infection from household contacts λ (based on replications of 100 simulations for each level). For each scenario, 10 index cases were introduced into a population of size 10000. All other parameters were the same as for Figure 3A . As before, we define decontainment to mean that the total number of cases from 10 index cases eventually exceeded 500 by day 250. and introduced 10 index cases into a population of 10000. We then repeated this 100 times, and reported the fraction of scenarios in which the number of infections ultimately exceeded 500 (as before, chosen as a cutoff to indicate the ultimate "escape" of containment of the epidemic). These results, shown in Table 3 , support the idea that ring vaccination can easily control introduced smallpox provided there is sufficient capacity and efficacy of tracing. Because of considerable uncertainty in the model parameters, we chose a collection of parameter values, and for each, estimated the containment probability (operationally defined as fewer than 500 total cases as a result of 10 index cases, within 250 days). We estimated this containment probability by simulating the smallpox epidemic 100 times for the same parameter values, and computing the frequency out of these 100 realizations for which fewer than 500 index cases resulted within 250 days. (Using a 1000 day window produces slightly smaller containment estimates; for 3 out of 1000 parameter set choices, this difference was greater than 0.06; the maximum difference seen was 0.23; the mean absolute difference was 0.0029; in only one case out of 1000 did we see containment in all 100 cases for the 250-day window, but not in all 100 cases for the 1000-day window). One thousand scenarios chosen from a Latin Hypercube sample were analyzed, and as indicated before, we chose the hazard for close contact transmission and the hazard for random transmission to guarantee that between 2 and 5 secondary cases per case occur, and that no more than 5% of cases are attributable to random transmission (we refer to this set as the "calibrated" scenarios further in this text). Having chosen this collection of 1000 parameter sets, we considered two levels of two different control parameters which were applied to each (so that each of the 1000 parameter sets were simulated under four different control conditions). The first of the two control parameters was the probability of workplace/social group contact finding; we chose values of 0.8 and 0.9 for this parameter (the household contact finding probability was 0.95 in all cases). The second of the control parameters was the rate of diagnosis (and effective removal) from the community of cases developing among previously identified and traced contacts who were initially asymptomatic (we refer to this as the monitored diagnosis rate); we assumed first a low level corresponding to a mean diagnosis time of one day from the onset of symptoms, and a high level corresponding to a mean time of 3 hours from the onset of symptoms (high levels of the monitored diagnosis rate correspond effectively to isolation of contacts). Finally, we assumed a doubling of the diagnosis rate after the beginning of widespread community awareness of smallpox. We then computed the containment fraction at different levels of ring vaccination capacity (contact tracing capacity per day). Thus, for each of 1000 scenarios (parameter set choices), we assigned the workplace/social group contact tracing success probability (υ 2 ), the monitored diagnosis rate φ (Appendix 2 [see additional file 2]), and the contact tracing/ring vaccination capacity per day (K r ). We then performed 100 realizations beginning with 10 index cases, and computed the containment fraction (fraction showing fewer than 500 cases in 250 days, beginning with 10 index cases). Thus, for each of the two choices each of υ 2 and φ, and for each value of K r we examined, we obtained 1000 values of the containment fraction. We use the resulting distributions in Figure 5A (averaging over these 1000 containment fractions), and Figure 5B (displaying the minimum value of the 1000 containment fractions). In Figure 5A , we plot the mean containment fraction (averaging the containment fraction over all 1000 scenarios), as ring vaccination capacity varies, for the two levels of workplace/social group contact finding probabilities (0.8 and 0.9), and for the two levels of monitored diagnosis rate among initially asymptomatic contacts (1 day -1 and 8 day -1 ). For low levels of ring vaccination (traceable contacts per day), the epidemic is almost never contained, but for ring vaccination levels near 50-60 per day (5-6 per index case per day), the average containment fraction Figure 4 Stochastic variability is illustrated by plotting the number of infectives over time over multiple replications. In this example, most simulations exhibit rapid containment of smallpox. The mean number of cases (averaging over simulations) is influenced by a small number of simulations exhibiting an uncontained epidemic. The parameters are the same as in Figure 3A , except that contacts of contacts are not traced in these replications. The mean containment probability Figure 5 5A -The mean containment probability increases as the number of ring vaccinations per day is increased. For this figure, the 1000 "calibrated" parameter sets were chosen, and for each parameter set, 100 realizations were simulated and the fraction of these for which the epidemic was contained to fewer than 500 cases was determined. The average of these 1000 containment fractions is plotted on the vertical axis. We assumed a household contact finding probability of 95% and that the diagnosis rates double after community awareness of the epidemic. We considered high levels of workplace/social (w/s) contact finding (0.9), as well as moderate levels (0.8). We also considered two levels of diagnosis of smallpox among investigated (alerted) contacts: high levels (corresponding to a 3 hour mean delay, indicated by "high contact isolation"), and moderate levels (corresponding to a one day delay, and indicated by "less contact isolation"). The figure shows four such conditions, a. high workplace/social contact finding probability and high contact isolation, b. moderate workplace/social contact finding probability and high contact isolation, c. high workplace/social contact finding probability and less contact isolation, and d. moderate workplace/social contact finding probability and less contact isolation. All other parameter values were chosen from the uncertainty analysis (the 1000 "calibrated" parameter sets). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, Table 1 , Table 8 ). 5B -The minimum containment probability out of the same 1000 scenarios chosen in Figure 5A . Whereas in Figure 5A , we averaged the simulated containment frequency (out of 100 realizations for each scenario), in this figure we determined which of the 1000 scenarios led to the lowest containment frequency, and we plotted this single worst (out of 1000) containment frequency, at different levels of ring vaccination capacity, for the same four conditions as in Figure 5A : a. high workplace/ social contact finding probability (0.9) and high contact isolation (effective 3 hour delay following symptoms), b. moderate workplace/social contact finding probability (0.8) and high contact isolation, c. high workplace/social contact finding probability (0.9) and less contact isolation (effective one day delay), and d. moderate workplace/social contact finding probability (0.8) and less contact isolation. All parameters are the same as in Figure 5A (the household contact finding probability is 0.95 for all scenarios, and the diagnosis rates are doubled after the onset of community awareness). In this figure, "contact isolation" refers to the monitored diagnosis rate, i.e. the rate at which previously asymptomatic contacts who subsequently develop disease will be diagnosed (φ, c. d. became close to 1. However, this average conceals the fact that for some scenarios (parameter sets chosen from the calibrated uncertainty analysis), control remains difficult or impossible even at high levels of ring vaccination. Therefore, in Figure 5B , we plotted the single lowest containment fraction seen out of the 1000 computed; focusing on the single worst scenarios reveals a different picture, and shows that isolation of asymptomatic contacts and very high probabilities of finding workplace or social contacts would be needed to control smallpox under these most pessimistic parameter choices. Rapid contact tracing in ring vaccination may play an important role in suppressing the epidemic, since the longer it takes to trace a contact, the less likely the vaccine is to be efficacious, and the more opportunities the infected individual may have to transmit disease before they are finally located, isolated, and vaccinated if appropriate. We illustrate this possibility in Figure 6 by examining the same scenario we showed earlier in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social groups traceable, an average time to infection for a household contact of an infective given by 0.2 days). We assume in one case that contacts may be traced quickly (1 day for a household contact, 2 days for a workplace/ social contact), and in the other that the contacts are on average found slowly (5 days for a household contact, 10 days for workplace/social contacts); we assumed 30 ring vaccinations (traceable contacts) possible per day. In this scenario, the epidemic is more severe and containment (as we have been defining it) less likely when contact tracing is slow: in the fast scenario, 238 infections occurred on average and the (estimated) containment probability was 99%; for the slow scenario, on average 3587 infections occurred and the (estimated) containment probability was only 1%. While Figure 6 illustrates the possibility that rapid contact tracing may be of decisive importance in some scenarios (parameter set choices), this is not always the case. For some parameter sets, the probability of tracing contacts (household or workplace/social) may be too low, or the transmission rate too high, for more rapid contact tracing to make any difference. Conversely, for other parameter sets, the smallpox transmission rate may be so low that smallpox is easily contained even with slow contact tracing. While rapid contact tracing is never harmful, overall, how typical are the results of Figure 6 (in which rapid contact tracing was important in ensuring the efficacy of ring vaccination)? To address this question, we simulated the growth of smallpox for the 1000 "calibrated" scenarios we used in Figure 5A and 5B. As before, we assumed ten initial cases, and (as in Figure 6 ) that 30 ring vaccinations were possible per day; then we simulated 100 epidemics assuming one day to find a household contact (and 2 days to find a workplace/social contact). We then simulated 100 epidemics assuming that it takes five days to find a household contact and 10 days to find a workplace/social contact (as in Figure 6 ). For each of these 1000 scenarios, we calculated the fraction of simulations for which the total number of cases is 500 or less within 250 days, i.e. the containment fraction. For nearly all scenarios (parameter set choices), the containment fraction was smaller (sometimes much smaller) when the contact finding time is faster (since faster contact finding, all else being equal, improves smallpox control, as illustrated in Figure 6 ). However, for 64.5% of the scenarios (parameter set choices) examined, the difference was less than 2.5% in absolute terms (smallpox was either contained or not contained depending on other factors, and rapid contact tracing did not make the difference). On the other hand, for 18.7% of the scenarios examined, the absolute difference in the containment probability was 20% or more; thus, a substantial difference in containment probability is occa-Faster contact tracing Figure 6 Faster contact tracing may improve the efficacy of ring vaccination. We assume the same baseline parameters as in Figure 3A (e.g. households of size 4, workplace/social groups of size 8, 95% of household contacts traceable, 80% of workplace/social contacts traceable), and 30 ring vaccinations available per day (with contacts of contacts not traced). The fast scenario corresponds to an average one day delay for household and two days for workplace/social contacts (as in Figure 3A ); the slow scenario corresponds to an average five day delay for household and ten day delay for workplace/ social contacts. This figure shows the average of one hundred realizations starting with ten index cases. Effect of more rapid diagnosis Public awareness of smallpox, leading to more rapid isolation and identification, may play an important role in eliminating the epidemic, as illustrated by the scenarios in Figure 7 . We assumed 20 ring vaccinations possible per day, a capacity too small to contain the epidemic in the absence of increased surveillance or diagnosis; the black line in the figure shows the steeply rising average number of cases for the first 100 days. If, however, surveillance or public awareness of the symptoms of smallpox increases the diagnosis rate by 50% (multiplies the baseline diagnosis rates by 1.5), containment becomes possible (blue line); with a doubling of the diagnosis rate (red line) the peak number of cases is lower still. In these scenarios, increased diagnostic rates markedly improve the ability of ring vaccination to control the epidemic, this suggest that any ring vaccination effort be accompanied by increased public awareness and surveillance. In many cases, however, more rapid diagnosis was not required for ring vaccination to be effective. As before, we simulated smallpox epidemics for each of 1000 calibrated scenarios, performing 100 realizations each beginning with 10 index cases, and computed the fraction of scenarios for which the epidemic was always contained (as defined earlier), assuming no change in diagnosis rates. We assumed 80 ring vaccinators per day, contact finding probabilities of 0.95 for households and 0.8 for workplace/social contacts (as in Figure 3A ). Under these assumptions, for 83.4% of the scenarios, the epidemic was contained within 500 total cases in each of the 100 realizations, even with no change in diagnosis rates. Uncertainty analysis (using the 1000 calibrated scenarios, and based on the fraction of 100 replications showing decontainment) revealed the most important parameters which predict the failure of ring vaccination without more rapid diagnosis were the same as we found in the earlier uncertainty analysis; a higher fraction vaccinated before the epidemic, smaller households or workplace/social groups, less transmissibility, lowered infectivity prior to the rash, more rapid diagnosis, and a higher rate of diagnosis for alerted individuals all contribute to a greater containment probability even without an overall increase in the diagnosis rate. We have been assuming that whenever an individual is contacted during an investigation, the individual will be diagnosed more quickly should they subsequently develop symptoms. When transmission is assumed to be very rapid (smallpox is assumed to be highly contagious), most individuals may already be infected when identified through contact tracing from an infective. Using the scenario we examined in Figure 3A , we see that continued surveillance of contacts is an essential component of effective ring vaccination designed to control rapidly spreading smallpox: if smallpox in a contact is not diagnosed any more quickly than for a non-contact, containment by ring vaccination requires over 98% contact finding probabilities for both household and workplace/ social contacts -even if unlimited numbers of ring vaccinators are available; containment cannot be guaranteed by adding additional ring vaccination capacity if the contact finding rates are too low and/or the follow-up for contacts is insufficient. Smallpox which is transmitted less rapidly to contacts would, however, be containable with a lower contact finding probability (results not shown). Finally, we used the "calibrated" scenarios (parameter set choices) to explore the levels of contact finding probability needed to contain the epidemic (as before, defined to mean 500 or fewer cases ultimately resulting from ten initial cases) ( Table 4 ). In these scenarios, we assumed that all traceable contacts were followed up very More rapid diagnosis Figure 7 More rapid diagnosis due to public awareness or increased surveillance may lead to far more effective epidemic control. We assume the same baseline parameters as in Figure 3A , and averaged 100 realizations of the epidemic beginning with 10 index cases and assumed a ring vaccination capacity of 20 per day (and contacts of contacts not traced). For the black line, the diagnosis rate of cases does not change after the first case is identified (the multiplier is 1.0); for the blue line, the diagnosis rate increases by 50% (multiplier 1.5) after the first case is identified (as in Figure 3A ), resulting in substantially fewer cases; and for the red line, the diagnosis rate is doubled (multiplier 2.0) after the first case is identified, resulting in still fewer cases. quickly (1/a = 1 hour, so that cases arising in previously contacted persons almost never transmit the infection further). We chose different levels of household and workplace/social contact finding probabilities and different levels of ring vaccination capacity, and performed 100 replications of each of the 1000 different scenarios. In Table 4 we report the fraction of scenarios for which all 100 replications exhibited containment. Scenarios in which smallpox is highly contagious require high contact finding probability to ensure the containment of the epidemic. Transmission prior to the rash makes epidemic control more difficult. In Figure 8 , we show an expanding smallpox epidemic assuming differing levels of infectivity prior to the rash (adding increased infectivity prior to the rash, keeping constant the infectivity after the rash). We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). We consider three scenarios: a relative infectivity during entire period is one (i.e., infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash), b the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A ) of this value, and c the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value. The figure shows that increased infectivity just prior to the rash leads to a larger epidemic (comparing b and c); in case b (high infectivity just prior to onset of rash), loss of containment occurs 36% of the time (but in none of the 100 realizations shown in case c (low infectivity prior to rash)). Scenario a (full infectivity during entire the prodromal period) showed loss of control in every realization. Increasing the ring vaccination capacity from 40 per day to 80 per day (results not shown) led to containment in all of the realizations with high infectivity just prior to the rash and low infectivity during the prodromal period (case b), but made no difference if the infectivity was as high during the prodromal period as during the rash (case a). While intuitively adding additional infectiousness must increase the number of secondary cases and make control more difficult, these results do illustrate that even a small amount of increased infectiousness prior to the rash (when diagnosis is more difficult) may substantially increase the difficulty of smallpox control. Finally, in Figure 9 , we present scenarios in which each of four other parameters are modified from the baseline values of Figure 3A , assuming 40 contact tracings (ring vaccinations) are possible per day (line a in the figure) . Specifically, we assume that severe smallpox (hemorrhagic and flat) on average takes four times longer to diagnose and isolate than ordinary smallpox (case b), Table 4 : Containment of severe smallpox at different levels of contact finding. The first three columns are assumed levels for the probability of finding a household contact, the probability of finding a workplace/social (W/S) contact, and for the number of contact tracings/ring vaccinations possible per day; the last two columns express (as percentages) the resulting probability of containment given the assumed contact finding probabilities and contact tracing capacities; two containment probabilities are given: the containment probability when only contacts of cases are traced (first column, "Contacts"), and the containment probability when contacts of contacts of cases are traced in addition to the contacts of cases (second column, "Contacts of Contacts"). All other parameters are the same as in Figure 3A . that no one in the population has prior vaccination protection (from before the discontinuation of routine vaccination, case c), that 10% more smallpox is too mild to diagnose (but still contagious, case d) compared to baseline, and finally that the vaccine is completely ineffective (case e). Each of these scenarios will be discussed further below. Scenario b was motivated by the possibility that individuals with severe forms of smallpox may be more difficult to diagnose, and thus remain infectious in the community longer (despite the much greater degree of illness of such patients), or that such patients may be more infectious. In this particular case, quadrupling the mean diagnosis time led to one additional replication out of 100 in which containment was not achieved (2/100, compared to the baseline of 1/100). However, we assumed that community awareness of smallpox leads to the same relative rate of increased diagnosis among severe cases as for ordinary cases, and that the most severe forms are relatively rare. In addition to the scenario shown in the figure, we also replicated the same 1000 "calibrated" simulations, assuming that in each case 40 contact tracings per day are possible and that the diagnosis time for severe cases was four times that of ordinary cases. Finally, we repeated each "calibrated" scenario 100 times assuming long diagnosis times for severe cases, and not making this assumption, and found that the difference in the decontainment fraction was not large (results not shown). Scenario c illustrates that vaccination prior to the discontinuation of routine vaccination does play a role in smallpox control by ring vaccination; there were more decontainment scenarios (5/100) when no prior Transmission prior to the rash Figure 8 Transmission prior to the rash makes epidemic control more difficult. The figure shows a expanding smallpox epidemic assuming differing levels of infectivity prior to the rash. We assume all parameters are the same as in Figure 3A (and that the ring vaccination capacity is 40 per day). Infectivity prior to the rash is modeled as the relative infectivity during the short (1 day) period of oropharyngeal lesions just prior to the rash (compared to the infectivity during the first week of the rash), and as the relative infectivity during the prodromal period (relative to the period just prior to the rash). For scenario a, relative infectivity during the prodromal period and just prior to the rash is the same as during the first week of the rash, for scenario b, the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% (as in Figure 3A ) of this value, and for scenario c, the relative infectivity just prior to the rash is 20% of the infectivity during the first week of the rash, and during the prodromal period is 20% of this value (these two parameters are the same as in Figure 3A ). Additional scenarios, assuming 40 ring vaccinations or con-tact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated Figure 9 Additional scenarios, assuming 40 ring vaccinations or contact tracings possible per day, and that contacts of contacts are traced; all parameters are identical to those in Figure 3A unless otherwise indicated. The figure shows the average of 100 replications of five scenarios (Case a repeats the result from Figure 3A for reference); the numbers in parentheses in the legend are the corresponding fraction of the 100 scenarios for which decontainment occurred. For case b, we assumed that flat and hemorrhagic smallpox cases took four times as long on average to diagnose as ordinary cases; for case c., we assumed that no one in the population had prior protection (as opposed to 25% for Figure 3A) ; for case d, we assumed that an additional 10% of individuals (13% instead of 3%) would develop mild smallpox (with 75% developing ordinary smallpox instead of 85% as in Figure 3A ); and for case e, we assumed that the vaccine is completely ineffective and provides no protection against infection. protection exists in the population. The results suggest that prior vaccination aids in the control of smallpox, but that it is not strictly necessary for control (in this scenario, 95% of the replications exhibited containment). In Figure 3A , we assumed 25% of individuals had protection due to vaccination prior to the discontinuation of routine vaccination; in scenario c of Figure 9 , we assumed this fraction was zero. Scenario d demonstrates that if 10% more smallpox infections (in absolute terms, i.e. 13% compared to 3% in Figure 3A ) lead to mild cases among individuals with no prior protection, the epidemic is more difficult to contain (13/100 replications showed loss of containment). Finally, scenario e demonstrates that containment is still possible even when the vaccine is completely ineffective in everyone -because of case isolation and isolation of contacts (and of contacts of contacts). Here, with 40 contact tracings possible per day, 55% of the replications nevertheless exhibited containment even with a vaccine which offered no protection whatever. With 90 contact tracings possible per day, all replications exhibited containment even assuming no vaccine protection. Although less efficient than ring vaccination in the sense that more vaccinations must be delivered to eliminate infection, comprehensive mass vaccination following the introduction of smallpox is sufficient to eliminate the infection. In Figure 10 , we show the probability of achieving containment (defined to be fewer than 500 total cases resulting from 10 index cases) for different levels of ring vaccination (0, 5, 10, and 20 vaccinations per day) and mass vaccination (0, 0.5%, 1%, and 2%; compare with the 10%-20% per day many jurisdictions in the United States are planning to vaccinate). Specifically, for each level of ring vaccination and mass vaccination, we used the same 1000 parameter sets used in Figure 5 , and performed 100 simulated epidemics for each parameter set. On the vertical axis, we plot the fraction of the 1000 scenarios for which each of the 100 simulated epidemics was contained. We further computed the fraction of scenarios for which none of the 100 simulated epidemics was contained; this is indicated by the colored segment in the small pie chart at each symbol. When the mass vaccination rate was 2% per day, the mean number of deaths (averaging over all scenarios and all simulations within each scenario) was 47.7, 33.7, 26.4, and 20.1 for a ring vaccination level of 0, 5, 10, and 20 per day (respectively) out of a population of 10000. Moreover, when we increased the mass vaccination level to 3%, an average of 28.9 deaths occurred when no ring vaccination was used, but this fell to 22.3 deaths when only 5 ring vaccinations per day were available (again assuming a population of 10000, and 10 index cases). With a mass vaccination level of 5% per day, an average of 18.8 deaths occurred without ring vaccination, and 15.8 deaths occurred when only 5 ring vaccinations per day were possible. (At a mass vaccination rate of 3% per day, containment as defined above was achieved in all 100 replications for 95% of the scenarios even without ring vaccination; at a mass vaccination rate of 5% per day, containment was achieved in all replications for all scenarios.) These results show that over a wide range of simulated epidemics, even seemingly small levels of ring vaccination (coupled with follow-up) may have a substantial effect in preventing epidemic spread and reducing deaths from smallpox, even during a mass vaccination campaign. Note that many jurisdictions in the United States are planning mass vaccination campaigns which could reach 10%-20% of the population per day, far greater than the mass vaccination levels examined here; it is interesting to note that mass vaccination cam- Figure 10 Mass and ring vaccination together. Low-level mass vaccination programs are improved substantially by the addition of ring vaccination. The shaded pie segments represent the fraction of 1000 scenarios for which containment (as defined in the text) was never realized; the vertical position of the pie chart represents the fraction of the 1000 "calibrated" scenarios for which containment was always achieved. As the fraction of the population mass vaccinated increases or the ring vaccination capacity increases, the probability of containment increases. paigns may be effective in preventing a widespread epidemic even at much lower levels than are being planned for. Where feasible, such rapid mass vaccination rapidly eliminates smallpox transmission in our model; vaccination of contacts is still beneficial, since we are assuming that earlier vaccination yields a greater probability of preventing or ameliorating infection (results not shown). We constructed a simple network model of smallpox transmission, and addressed the question of what circumstances contribute to the success of a ring vaccination campaign designed to control smallpox. Our analysis focused on the use of contact tracing/ring vaccination to prevent a widespread epidemic following a deliberate release. We conducted a sensitivity analysis based on particular, but reasonable, ranges for the unknown parameters. Our results are consistent with prior vaccination models in identifying prior vaccination and ring vaccination capacity as significant factors in determining the spread of smallpox. Unsurprisingly, we also find that household size and ring vaccination speed are particularly important parameters; these results are intuitively plausible. The contact finding probability did not appear important in this analysis only because a narrow range of values was chosen. We illustrated smallpox control by presenting scenarios based on control of moderately severe smallpox epidemics. We find that swift, aggressive contact tracing and ring vaccination is is usually sufficient to bring the infection under control. Provided that there is sufficient capacity, vaccination of contacts of contacts is beneficial, and results in fewer infected individuals and more rapid elimination of infection; investigating contacts of contacts allows the chain of transmission to be outrun to some extent. When ring vaccination capacity is small, diversion of crucial resources away from contacts is harmful; contacts of contacts should only be traced and vaccinated provided that no resources are diverted away from contacts of cases. The increased surveillance (or isolation) of contacts, together with improved rates of diagnosis due to community awareness, play important roles in smallpox control; we note that in some cases, lowered diagnosis rates among severe cases contributed to a small extent to loss of epidemic control, and suggest that any public awareness campaign include information to help the public be more aware of the full spectrum of the clinical features of smallpox. One limitation of our analysis is that we chose not to explicitly incorporate the specific epidemiology of health care workers (or mortuary workers), who are likely to be exposed to infected individuals during any smallpox epidemic (e.g. [17, 22] ), and who may then infect further members of the community [22] (as was also seen in the recent outbreak of SARS, e.g. [48] ). Transmission to health care workers may be considered to amplify the initial attack or to be simply accounted among the exposures we considered (and thus be approximated by the behavior of our model), since health care workers and their household contacts are in all likelihood traceable contacts, and ring vaccination/contact tracing would identify and halt these chains of transmission as in our model. The disruption of smallpox control and patient care that may occur is not accounted for in our analysis, however, causing our model in this sense to err on the side of optimism. The appropriateness of pre-event vaccination of health care workers or other first responders has been addressed by other analyses [12, 49] , and is beyond the scope of our model. While we analyzed the effect of contact tracing, case and contact isolation, and ring vaccination (together with mass vaccination), in a real smallpox epidemic, in practice, control efforts are unlikely to be limited strictly to vaccinating contacts (and health care workers, as likely contacts) and isolating cases. Indeed, making vaccine available to individuals who believe they live near cases or to others on a voluntary basis occurred in smallpox control efforts in the past [22] . Vaccination of such individuals can only harm the disease control effort if it hinders or delays the diagnosis of cases or the investigation and vaccination of contacts; our results show that even relatively low levels of vaccination of the general population may have a beneficial effect in preventing the epidemic from escaping control. More serious is the possibility that individuals who should be vaccinated or isolated would be missed; this could occur either because individuals or institutions did not cooperate with the disease control effort, or because the individuals simply could not be found. Our analysis suggests that ring vaccination need not be perfect to successfully contain the epidemic, and yet, under conditions where there is a high rate of infection among contacts, or a relatively high rate of casual transmission, high rates of contact finding (in excess of 90%), together with increased surveillance and contact isolation, are needed to contain the epidemic. Finally, the vaccination of individuals at low risk of contracting smallpox will cause harm due to adverse events of the vaccine; in our model, the assumed death rate due to vaccination was small compared to the probability of death from smallpox, and played essentially no role in the analysis. In practice, individuals suspected to be at high-risk for vaccine complications, but at relatively low risk for contracting smallpox, might simply be isolated or closely monitored even during an outbreak; while the presence of individuals in the population at higher risk for vaccine complications would increase the death rate during an outbreak, such individuals are unlikely to impair the containment of the epidemic (the primary focus of this analysis). Our results support ring vaccination against epidemics of smallpox (even assuming high rates of transmission to close contacts), but do note that stochastically, for severe (rapidly transmissible) smallpox, scenarios of loss of control are seen, with resulting widespread epidemics. In scenarios in which the transmission potential of smallpox is smaller, such loss-of-control scenarios occur less frequently (results not shown). Mass vaccination campaigns, when conducted quickly and with very high coverage, do not result in loss of control in our model. Nevertheless, fewer deaths due to smallpox result when ring vaccination is conducted along with mass vaccination. Simulated smallpox epidemics with ring vaccination suggest that aggressive, fast ring vaccination can control epidemics of smallpox. To do so, however, smallpox must be identified quickly and contacts vaccinated promptly. We also identify public awareness of smallpox -leading to prompt identification of cases -as a major factor in smallpox control; in some simulations, it may play a role as significant as ring vaccination itself [15] . However, we also found that uncertainty in (1) transmission from mild cases, (2) the household size, and (3) casual transmission contributed to the overall uncertainty in the epidemic size. Other parameters to which the number of infections were highly sensitive were the prior vaccination fraction, parameters related to infectiousness, and parameters related to transmission prior to the rash. Because our model combines network structure with response logistics, our results support and complement the results of other investigators. Our results support the notion that prior vaccine protection may play an important role in slowing the epidemic [11] , despite the possibility that some vaccinated individuals may develop mild cases which are harder to identify, but which nevertheless transmit disease. Likewise, our results provide support for the view that ring vaccination should play a central part in smallpox control. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity to cover all contacts of cases), and should be accompanied by a vigorous campaign of public awareness which can facilitate more rapid identification and isolation of cases. We assumed that ring vaccination could be fast (little delay between identification of a case and vaccination of the contacts), effective (nearly all household contacts can be found, and most of workplace and social contacts), and available (there is sufficient capacity). To be effective, ring vaccination planning must yield a system capable of meeting these benchmarks; we should not only be able to assess the number of contact vaccinations that will be possible per day, but should have a plan in place to (1) identify contacts by working with individuals, employers, schools, community representatives, and authorities or businesses who may have access to information facilitating contact tracing, (2) rapidly investigate and vaccinate such individuals, perhaps using field teams managed by central dispatch. It is important to realize that for highrisk, transient, or unstably housed populations where reliable contact tracing is impossible, the conclusions of the model we present cannot be applied. It is important to note that while our model suggests that ring vaccination together with contact tracing and isolation is likely to be successful, we found that for some scenarios (where smallpox was more transmissible, or was relatively more transmissible before the rash), epidemic containment required not only ring vaccination, but increased public awareness, the isolation of contacts, and tracing of contacts of contacts. For scenarios in which the smallpox was less transmissible, epidemic containment was possible at lower contact finding probabilities. Thus, while our simulations suggest that contact tracing/ring vaccination need not be perfect to succeed, because of uncertainties in our knowledge of the behavior of bioterrorist smallpox, it is impossible to know in advance how good it will have to be. Thus, that high contact finding rates, mass public awareness leading to early identification of cases, isolation of contacts, and investigation of contacts of contacts should all be conducted with maximum effectiveness to reduce the probability of a widespread epidemic. While the possibility of smallpox uncontrollable by ring vaccination has made mass vaccination preparations wise, and while mass vaccination may be unavoidable in the event of a deliberate release of smallpox, we believe that ring vaccination is essential in any case. This is not only because individuals recently exposed to smallpox may be protected if they are vaccinated promptly, but because each contact identified potentially lies in the immediate future of the transmission chain. From the standpoint of epidemic control, it is far more valuable to vaccinate individuals next in the transmission chain than to vaccinate other persons. Our results support the idea that ring vaccination/case isolation may in many, if not most cases, eliminate smallpox even without mass vaccination, but also support planning for mass vaccination (so that the vastly more costly and difficult policy of mass vaccination will be available in the event of an explosive epidemic). When faced with the unknown, multiple redundant prep-arations are appropriate; case investigation/isolation may control smallpox even if the vaccine does not work at all, but mass vaccination is useful in the event of an explosive epidemic for which case tracking becomes impossible."
17
"Protection of pulmonary epithelial cells from oxidative stress by hMYH adenine glycosylase"
"BACKGROUND: Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1, hMYH, hNTH and hMTH. METHODS: The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H(2)O(2). Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05. RESULTS: Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O(2 )toxicity did lead to a reduced endogenous expression of hNTH in A549 cells. CONCLUSION: Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O(2 )and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O(2 )and its metabolites."
"Oxidative stress leading to the overproduction of free radicals in the lungs is present in many clinical situations. Such clinical settings include acute respiratory distress syndrome (ARDS), infants of prematurity going on to develop bronchopulmonary dysplasia (BPD), pathogenesis of chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, ischemia-reperfusion injury, druginduced lung toxicity, cancer and aging [1] [2] [3] [4] . Although the use of oxygen may be clinically indicated in hypoxemic situations, one must consider the potential longterm toxic side effects. For example, we know that oxygen creates cellular damage by a variety of mechanisms. Normal cellular metabolism of oxygen involves the transfer of electrons from NADH to O 2 molecules to form water (H 2 O). At normal partial pressure, 95% of oxygen molecules (O 2 ) are reduced to H 2 O and 5% are partially reduced to toxic byproducts by normal metabolism in the mitochondria [5] . These metabolites include the superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals ( • OH) all of which make up what are known as Reactive Oxygen Species (ROS) [6] . Exposure to conditions of hyperoxia as well as ionizing radiation (IR) leads to increased amounts of these ROS and their damaging effects. ROS are known to attack the lipids, proteins, and nucleic acids of cells and tissues [5, 7] . Lipids, including pulmonary surfactant, react with ROS to produce lipid peroxides, which cause increased membrane permeability, inactivation of surfactant, and inhibition of normal cellular enzyme processes. Proteins reacting with ROS result in decreased protein synthesis due to inhibition of ribosomal translation or destruction of formed proteins. This ultimately leads to inactivation of intracellular enzymes and transport proteins resulting in impaired cellular metabolism and accumulation of cellular waste products. Lastly, ROS cause damage to nucleic acids by leading to modified purine and pyrimidine bases, apurinic (AP) / apyrimidinic sites, and DNA protein cross-links which can lead to single strand breaks [8] . Several defense mechanisms exist to combat the damaging effects of ROS. Intracellular enzymatic systems include superoxide dismutase which eliminates the superoxide anion, catalase which catalyzes the reduction of H 2 O 2 directly to H 2 O without the production of the hydroxyl radical, and glutathione peroxidase which directly reduces H 2 O 2 and lipid peroxides. Free radical scavengers, which stop free radical chain reactions by accepting electrons, include α-tocopheral (vitamin E), ascorbic acid (vitamin C), niacin (vitamin B), riboflavin (vitamin B 2 ), vitamin A, and ceruloplasmin [1, 2, 9] . These systems usually provide enough protection against oxygen metabolism under normal conditions, but may become depleted under conditions of increased oxidative stress [7, 10] . The defense mechanism of interest in this paper involves the repair of oxidative damage through the human DNA base excision repair pathway (BER). BER is the most important cellular protection mechanism that removes Base excision repair pathways for Oxidative DNA damage oxidative DNA damage [11] . Damaged bases are excised and replaced in a multi-step process. Lesion-specific DNA glycosylase repair genes initiate this process. After removal of the damaged base, the resulting AP site is cleaved by APendonuclease generating a 3'OH and 5'deoxyribose phosphate (dRP). β-polymerase, which possesses dRPase activity, cleaves the dRP residue generating a nucleotide gap and then fills in this single nucleotide gap. The final nick is sealed by DNA ligase [12] [13] [14] ( Figure 1A ). The oxidative repair genes that we have analyzed in this study include 8-oxoguanine DNA glycosylase (hOgg1), human Mut Y homologue (hMYH), human Mut T homologue (hMTH), and endonuclease III (hNTH) all of which are present in human cells and involved in the protection of DNA from oxidative damage. The repair enzyme hOgg1 is a purine oxidation glycosylase that recognizes and excise 8-oxoguanine lesions (GO) paired with cytosine. GO can pair with both cytosine and adenine during DNA replication [15] . If repair of C/GO does not occur, then G:C to T:A transversions may result [5, [15] [16] [17] . The repair enzyme hMYH is an 8-oxoguanine mismatch glycosylase that removes adenines misincorporated opposite 8-oxoG lesions that arise through DNA replication errors [5, [18] [19] [20] . The repair enzyme hMTH hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-GTP, 8-oxo-dATP, and 2-hydroxy-dATP, effectively removing them from the nucleotide pool and preventing their incorporation into DNA ( Figure 1B ) [21] . Lastly, the repair gene endonuclease III (hNTH) is a pyrimidine oxidation and hydration glycosylase that recognizes a wide range of damaged pyrimidines [22] . hNTH has also been shown to have a similar DNA glycosylase/AP lyase activity that can remove 8-oxoG from 8-oxoG/G, 8-oxoG/A, and 8-oxoG/C mispairs [23, 24] . Subsequent steps following hNTH are identical to those following hOgg1 ( Figure 1A ). A previous study has shown that over-expression of the DNA repair gene hOgg1 leads to reduced hyperoxiainduced DNA damage in human alveolar epithelial cells [25] . The primary goal of our present study was to compare the protective effects of the four main lesion-specific DNA glycosylase repair genes by individually overexpressing each in lung cells and determining which of these provides the greatest degree of protection under conditions of increased oxidative stress. The human alveolar epithelial cell line A549 (58 year old Caucasian male), was purchased from ATCC Cat No CCL-185. The cells were grown in DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and penicillin (100 U/ml)/ streptomycin (100 µg/ml) (Gibco, Grand Island, NY). Passaging of cells was performed every 3-4 days with cells grown to 80% confluency in a 10 cm cell culture dish (Corning Incorporated, Corning, NY). Cells were kept at 37°C in a humidified, 5% CO2 incubator. The retroviral vector pSF91.1, a gift from Dr. C. Baum from the University of Hamburg in Germany, was constructed with an internal ribosome entry site (IRES) upstream to the gene expressing enhanced green fluorescent protein (EGFP) as previously described [26] . Four DNA repair genes were individually ligated into the retroviral vector pSF91.1. hOgg1-6pcDNA3.1 was initially amplified by PCR by primers to introduce a kozak sequence at the 5' end [27] . Digestion of this product with EcoRI and SalI was performed and then hOgg1 was subcloned into digested plasmid vector pSF91.1, with T4 DNA ligase. DNA sequencing was performed to confirm integrity of the hOgg1 gene. hMYH/PGEX4T-1 and hMTH/PGEX4T-1 hMYH was a gift from Dr. A. McCullough (University of Texas Medical School, Galveston, TX) and hMTH was cloned in Dr. Kelley's lab. Plasmid DNA was prepared as above by digestion with EcoRI and SalI and ligated into pSF91.1 as above and sequencing was performed to confirm integrity of the genes. PGEX-6PI-hNTH1-wild type this gene was a gift from Dr. S. Mitra (University of Texas Medical School, Galveston, TX). Digestion with BamHI and SalI was performed and the hNTH1-wt fragment was ligated into the empty plasmid vector PUC18. The hNTH1-wt fragment was then excised with both sides flanked by EcoRI restriction sites and ligated into pSF91.1. Proper orientation of the gene was confirmed and sequencing was performed to determine the integrity of the gene. 2.5 × 10 5 A549 cells were suspended with the viral supernatant and plated in 1 well of a 6-well plate along with polybrene (Sigma, St. Louis, MO). This exposure was performed 6 hours per day for three days. At approximately five days from the beginning of the infection, the infected cells were analyzed using flow cytometry and sorted for EGFP expression. Cell pellets of sorted cells were resuspended in NuPage buffer (Invitrogen, Carlsbad, CA) and protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). 20 ug of protein were loaded into individual lanes of a NuPage Bis-Tris Gel (Invitrogen, Carlsbad, CA). The gel was then transferred to nitrocellulose paper (Osmonics Inc, Gloucester, MA). The membranes were then blocked with 1% blocking solution (Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibodies to hOgg1 (Novus Biologicals, Littleton, CO), hMTH (Novus Biologicals, Littleton, CO), hMYH (Oncogene Research Products, Darmstadt, Germany) and hNTH (Proteintech Group Inc, Chicago, IL) all at a dilution of 1:1000 except hNTH which was diluted 1:2500. They were then washed 2 times with TBST and 2 times with 0.5% blocking solution, 10 minutes per wash. The membranes were incubated with anti-rabbit secondary antibodies at 1:1000 for 1 hour at room temperature. Lastly, the membranes were washed 4 times with TBST, 15 minutes per wash. The membranes were briefly soaked in BM chemiluminescence blotting substrate (Roche Diagnostics, Indianapolis, IN) and then exposed to high performance autoradiography film (Amersham Biosciences, Buckinghamshire, England). Kodak Digital Science 1D Image Analysis software was utilized to quantify the region of interest (ROI) band mass of individual bands on films where visualized differences were detected. Sorted EGFP positive A549 cells infected with the above DNA repair genes were counted and seeded into 96-well plates at a density of 1000 cells/well, 6 wells per gene. Six hours after seeding, individual plates were placed into an oxygen chamber supplied by Dr. L. Haneline (Wells Center for Research, Indianapolis, IN) located in a 37°C incubator. The oxygen chamber was then infused with 95% O 2 and 5% CO 2 . Individual plates were removed after 12, 24, 48, and 72 hours of exposure. Control A549 cells were incubated in a normal 37°C humidified-5% CO 2 incubator. O 2 concentrations were monitored with a MAXO 2 analyzer (Maxtec, Salt Lake City, UT). Four days from the beginning of the exposure, cells were assessed for cell growth/survival using the sulforhodamine B assay (SRB assay). The SRB assay (Sigma, St. Louis, MO), developed by the National Cancer Institute, provides a sensitive measure of drug-induced cytotoxicity through a colorimetric endpoint that is non-destructive, indefinitely stable, and visible to the naked eye. This assay was used to assess the cell growth/survival of over-expressed cells [28] . Cold 10% TCA was used to fix the cells to the plate. After incubation for 1 hour at 4°C, the individual wells were rinsed with water. After air-drying, SRB solution was added to each well and cells were allowed to stain for 20-30 minutes. 1% acetic acid wash was used to rinse off unincorporated dye. Incorporated dye was then solubilized in 100 µl per well of 10 mM Tris. Absorbance was measured by a tunable microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 565 nm. Background absorbance measured at 690 nm was subtracted from the measurements at 565 nm. Sorted EGFP positive A549 cells were seeded into 96-well plates at a density of 1000 cells/well. Six hours after seeding, individual plates were then exposed to radiation at doses of 250, 500, 1000, and 1500 Rads or 0. well. All the plates were placed into a 37°C humidified-5% CO 2 incubator. Every 24 hours for 4 days, 1 plate was removed and the cells were fixed and analyzed by the SRB assay looking at cell growth under non-toxic conditions. Growth curves and exponential growth equations were determined to look at the doubling time (DT) of cells infected with each repair gene of interest compared to vector infected and uninfected wild type cells. All drug exposure experiments were performed at least three times and individual drug doses included 6-8 wells for each group of infected cells. Analysis of cell growth and exponential growth equations were determined using Microsoft Excel. All experiments involving drug exposures were normalized to the zero dose. Data are expressed as means ± SE. The significance of differences were calculated using the paired Student's t test with significance being accepted for p < 0.05. The DNA repair genes hOgg1, hMYH, hMTH, and hNTH were ligated into the retroviral vector pSF91.1 ( figure 2 ). This vector, derived from a murine stem cell virus backbone, along with each individual repair gene, was used for transfection of phoenix amphotropic cells. Viral supernatant was then collected and used to stably infect A549 Western analysis of A549 cells over-expressing individual repair genes and effect on endogenous glycosylase level contained the genes of interest integrated into their DNA (data not shown). Western blot analysis was performed on sorted cells in order to verify over-expression of the four genes of interest. hOgg1, hMYH, hMTH, and hNTH were all detected at their correct position on western blots (data not shown). Western analysis was also utilized to assess whether overexpression of each individual repair gene resulted in altered endogenous expression of the other repair genes under both non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad). Cells over-expressing the repair genes hOgg1, hMYH, hMTH, and hNTH did not lead to altered expression of the other endogenous repair genes under the above conditions when compared to each other or pSF91.1 vector control cells ( Figure 3A ,3B,3C and 3D). hOgg1's endogenous expression was below the level of detection. The pattern of endogenous expression of hNTH was consistent for each condition when comparing cells over-expressing hOgg1, hMYH, hMTH, and pSF91.1. Reduced expression of hNTH after exposure to 95% O 2 was noted. Lastly, we assessed endogenous expression of each individual repair gene in cells infected with pSF91.1 following non-toxic and toxic conditions (24 hrs of 95% O 2 and 1000 Rad) at 24 and 48 hrs after the onset of exposure. Endogenous hMYH and hMTH were expressed to the same degree. hOgg1's endogenous expression was below the level of detection using western analysis (results not shown). When analyzing endogenous hNTH expression, it was noted that hyperoxia at 24 hrs and 48 hrs resulted in reduced protein expression by 93% and 64% respectively. There also was a small increase in expression of hNTH noted after 1000 Rad one day post exposure that was back to baseline by two days post exposure. ROI band mass quantification demonstrated this finding ( Figure 4A and 4B). Two or more replicates were performed for each western analysis to determine consistency of the results. A549 cells expressing hMYH demonstrated increased survival after exposure to conditions with elevated levels of oxygen compared to cells expressing only the pSF91.1 vector ( Figure 5A ). Results were highly significant at all time points except after 12 hours O 2 where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12% after 12 hours O 2 exposure to 7% after 72 hours O 2 exposure. A549 cells expressing hMYH also demonstrated increased survival after exposure to all doses of radiation in comparison to pSF91.1 ( Figure 5B ). These results were also highly significant at all doses of radiation except at 250 Rads where it almost reached a highly significant value. The differences between pSF91.1 and hMYH varied from 12%-14% for all doses of radiation. Also noted in these experiments was that vector control cells demonstrated no Experiments looking at the effects of H 2 O 2 on cells expressing the repair genes did not demonstrate increased survival for any of these repair genes when compared to vector control cells ( Figure 5C ). This data demonstrates that over-expression of hMYH has the ability to improve cellular survival under conditions of hyperoxia and radiation but may not be able to overcome the toxicity of H 2 O 2 . Cell growth under normal conditions was ascertained to determine if over-expression of any of the repair genes caused an alteration in the growth of cells in the absence of oxidative stress. Wild type A549 cells and cells expressing pSF91.1, hNTH, hOgg1, and hMTH appeared to grow at similar rates with doubling times within the same range. A549 cells expressing hMYH did show a slower growth rate that resulted in significant differences in cell number by day 3. The calculated doubling time for the cells over expressing hMYH is > 3 hrs longer than the cells with the other repair genes and vector alone ( Figure 6 ). This slowing of growth may allow for more time to repair Cell survival analysis following O 2 , IR, and H 2 O 2 treatments Oxidative stress to the lung leads to cellular DNA damage as evidenced by the release of specific gene products known to regulate DNA base excision repair pathways such as p53 and p21 [29] [30] [31] . Alterations in pro-inflammatory mediators, transcription factors, and other related gene products are also observed [32] . This injury has been shown to be associated with features of both cellular necrosis and apoptosis [33] [34] [35] . The resultant cellular inflammation and death from oxidative stress has a dramatic impact on the outcome of patients in the clinical setting [7, 36] . Most of our current clinical therapy towards oxidative stress in the lung involves both supportive measures and prevention. Research dealing with oxidative lung injury has focused mainly on enhancing antioxidant enzymatic processes and free radical scavengers [37] [38] [39] [40] . The ability to alter cellular survival by increasing specific DNA repair mechanisms may add another approach to the treatment of oxidant-mediated lung injury. Many investigators have used hydrogen peroxide as a substitute for hyperoxia since it is known to be one of the metabolites produced by the metabolism of oxygen. ROS such as H 2 O 2 and those produced by hyperoxia clearly lead to DNA damage but questions exist as to whether H 2 O 2 leads to the same deleterious effects upon DNA as hyperoxia. Analysis of our growth curves after exposure to H 2 O 2 in comparison to hyperoxia and IR clearly indicates that cellular protection by oxidative DNA repair genes is specific to the agent used. Because no protection was observed with over-expression of any of the repair genes following exposure to H 2 O 2 , we speculate that the damage it causes is dissimilar. It may be that its damage not only involves oxidized bases, but may also include other forms of DNA, lipid, and protein damage that are not corrected by oxidative DNA repair genes. Alternatively, the amount and type of damage evoked by H 2 O 2 could be beyond that which can be corrected by over-expressing these repair genes. Another form of stress known to induce damage through the formation of ROS is IR. Radiation induced free radical damage to DNA has substantial overlap with that of oxidative damage [41] [42] [43] . The protection provided by specific oxidative DNA repair genes under conditions of IR, was notable throughout our experiments only with the repair enzyme hMYH. The primary agent utilized to induce the formation of ROS was an oxygen rich environment. The use of oxygen as a stressor leading to the formation of ROS, offers a distinct advantage over IR and H 2 O 2 by mimicking the clinical situation where constant exposure to hyperoxia leads to cumulative cellular damage which further compromises repair. We determined that survival of A549 cells was also enhanced to a small degree with increased expression of the repair enzyme hMYH. This was an unexpected finding as we anticipated the repair gene hOgg1 would demonstrate the greatest protection in response to oxidative stress based on previous studies, however these experiments utilized the colony forming assay (CFA) to detect improvements in survival [25] . Additionally, the CFA may provide different results compared to the SRB assay, which allows for growth analysis over a shorter window of time. Furthermore, their study did not look at the repair enzyme hMYH and its impact on survival. Another study has investigated the repair function of hMYH in MYH-deficient murine cells. It was demonstrated that transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:GO repair [44] . An interesting observation noted while doing our experiments lead us to look at individual growth characteristics of cells over-expressing each of the oxidative repair enzymes. Cells over-expressing the repair enzyme hMYH clearly grow at a slower rate when compared with the other enzymes. The mechanism behind this is not understood at this point in time. The repair action of Cell growth curve and associated doubling times (DT) Figure 6 Cell growth curve and associated doubling times (DT hMYH is known to remove adenines misincorporated opposite 8-oxoG lesions. This lesion occurs when a C/GO lesion is allowed to replicate before being corrected by hOgg1. Repair by hMYH is not a final corrective measure. The product of hMYH activity is the lesion C/GO, which allows hOgg1 to have another opportunity to remove 8-oxoG opposite cytosine. We know that A549 cells possess the hOgg1 gene based on a previous study demonstrating the presence of this gene after amplification by genomic PCR [45] . We also have demonstrated endogenous activity of hOgg1 in A549 cells by using an 8-oxoguanine bioactivity assay. Therefore, our explanation of these results is that the slowed growth created by hMYH may provide a wider window of opportunity for the repair process to take place, which ultimately grants endogenous hOgg1 another opportunity to remove the 8-oxoG lesion created by oxidative stress. As noted in the methods section, the SRB assay provides a sensitive measure of drug-induced cytotoxicity that is used to assess cell proliferation/survival. The reduced cell proliferation of A549 cells over-expressing hMYH under nontoxic conditions may likely underestimate the magnitude of the protective effect of this particular repair enzyme. This may in fact make the results even more significant. Recent studies have discovered hereditary variations of the glycosylase hMYH that may predispose to familial colorectal cancer [46, 47] . Others have looked for hMYH variants in lung cancer patients and have not identified any clear pathogenic biallelic hMYH mutations or an overrepresentation of hMYH polymorphisms [47] . The A549 cell line has not demonstrated somatic mutations in hMYH, but a single nucleotide polymorphism (SNPs) has been noted [45] . The impact on function by this SNP is unknown. It would appear that the function of hMYH is very important in preventing somatic mutations leading to cancer in the gastrointestinal tract. Although studies to date have not demonstrated this same relationship with lung cancer, we do know that the lungs are subjected to large quantities of ROS under certain conditions as discussed earlier. The formation of mutations from oxidative stress does have other deleterious effects on cells including cellular death by necrosis and apoptosis. Tissue viability is dependent upon mutation correction and replication of the surviving cells to replace those that have died. The ability to enhance cellular survival, after specific oxidative exposures, is evident after increased production of the hMYH repair gene in these experiments. We additionally wanted to determine the level of endogenous expression of the glycosylase repair genes in the pulmonary epithelial A549 cell line. Others have demonstrated how different stressors lead to alterations in the endogenous production of specific repair genes. For example, it has been shown that endogenous gene expression of hOgg1 was elevated following exposure to crocidolite asbestos which is known to cause an increase in 8-oxoG levels [48] . It has also previously been reported that treatment of A549 cells with sodium dichromate, a prooxidant, leads to a reduction of hOgg1 protein expression that was not observed with H 2 O 2 [49] . One additional study demonstrated a dose dependent down regulation of hOgg1 protein expression in rat lung after exposure to cadmium, a known carcinogen associated with the formation of intracellular ROS [50] . In our experiments we were able to demonstrate that both hyperoxia and IR do not appear to impact the endogenous expression of hOgg1, hMYH, and hMTH at 24 and 48 hours following exposure. It was noted that endogenous hNTH was reduced after hyperoxia at 24 and 48 hours after the onset of exposure. One would speculate that this reduction in endogenous hNTH secondary to hyperoxia is related to either decreased production or increased destruction in response to O 2 exposure. Over-expression of this repair enzyme did not result in improvements in survival after O 2 exposure based on our experiments. It may be that endogenous levels are adequate to correct this specific mutational burden for these experiments. Furthermore, no previous studies have determined how cells over-expressing specific repair genes may impact endogenous expression of the other oxidative BER genes under both normal and oxidative stress conditions. We were also able to demonstrate that endogenous expression of glycosylase repair genes were not altered under these conditions secondary to the over-expression of any of these genes. This is an important finding for interpretation of survival data; protection of cells is due to the overexpression of the specific gene and not due to enhancement of other endogenous repair enzyme levels, at least for the genes studied under these conditions. Some limitations may exist in using a lung carcinoma cellline, which likely differs both in proliferative properties as well as in response to oxidative stress in comparison to primary epithelial cells. The enhanced cell growth observed with cell lines may be more reflective of undifferentiated alveolar type II cells which are likely to replace terminally differentiated alveolar type I cells after injury/ death due to oxidative stress. This may not be a true reflection of growth under non-toxic conditions when very little cell division is occurring. This is an inherent problem observed when comparing cell lines with primary cells and results need to be interpreted in a way that considers this. It is difficult to know how this will translate to pulmonary epithelial cells in vivo at this stage. It certainly would appear that the protection observed is modest in degree in this pulmonary epithelial cell line. Further experiments assessing the function of the repair enzyme hMYH in this model will be important to perform in order to delineate the findings of slowed growth under normal conditions and improved survivability under conditions of O 2 and IR. More research looking at the potential for combination therapy, including DNA repair mechanisms in conjunction with other antioxidant defense mechanisms may be another approach to enhancing cell survival, which may lead to better clinical outcomes. Alternatively, cell survival may not be the most important end point for hyperoxia studies. Given that 8-oxoG, if left unrepaired, leads to G:C to T:A transversions, there may be an increase in mutational burden by these cells that isn't reflected in cell survival. Further experiments studying the impact on mutation production is underway. Ultimately, experiments need to be done in animal models to determine the translation to in vivo pulmonary cells. In summary, we have demonstrated that over-expression of the DNA glycosylase repair enzyme hMYH may enhance survival of a pulmonary epithelial cell line after exposure to conditions of IR and hyperoxia. We have also demonstrated that over-expression of hMYH leads to a slowing of growth of A549 cells under non-toxic conditions, which may in part play a role in this enhancement of survival by providing a wider window of opportunity for repair of oxidized lesions to occur. Lastly, we demonstrated that over-expression does not lead to altered endogenous expression of these repair genes. As the understanding of DNA repair mechanisms continues to grow and the evolution of gene therapy takes place, more treatment options may be available in the clinical setting to help with many disease processes including the damaging effects of oxygen and its metabolites."
18
"Bioinformatic mapping of AlkB homology domains in viruses"
"BACKGROUND: AlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily. In Escherichia coli the protein protects RNA and DNA against damage from methylating agents. 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins. Similar domains have also been observed in certain plant viruses. The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants. We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g. in Eukaryotes. RESULTS: Profile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the Flexiviridae family. Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations. This pattern seems to be more consistent with increased environmental pressure, e.g. from methylating pesticides, than with interaction with the PTGS system. CONCLUSIONS: The AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment."
"The purpose of this study has been to identify domains with homology to AlkB in viral genomes, in order to get a better understanding of distribution and possible function of such domains. The AlkB protein of E. coli, and probably most of its homologues, is involved in repair of alkylation damage in DNA and RNA. It repairs 1-methyl-adenine and 3-methylcytosine by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Recently the protein was identified as a member of the 2-oxoglutarate (2OG)-and Fe(II)dependent oxygenase superfamily [1] [2] [3] . The catalytic reaction requires molecular oxygen, Fe 2+ and 2-oxoglutar-ate, which is subsequently converted into succinate, CO 2 and formaldehyde [4] . The 2OG-FeII oxygenase superfamily is widespread in Eukaryotes and bacteria [1] , and is currently the largest known family of oxidising enzymes without a heme group [5] . The 3D structure of several of these oxygenases is known, and they share a common fold with a structurally conserved jelly roll β-sheet core with flanking α-helices. Very few residues are totally conserved across these structures, basically just the residues involved in coordination of the Fe(II) ion and the 2-oxoglutarate. AlkB-like genes are widespread in most types of organisms except Archaea. However, whereas bacteria normally have just one or at most two AlkB homologues [6] , multicellular Eukaryotes tend to have several homologues. In the human genome at least 8 different AlkB homologues (ABHs) have been identified [7] . These homologues seem to have slightly different properties with respect to substrate preference and subcellular localisation, and this may be a reason for the proliferation of ABHs e.g. in humans. However, a detailed functional mapping of all ABHs has not yet been carried out. A sequence alignment of known ABHs identifies very few residues as totally conserved, basically just a HxD motif, a H and a RxxxxxR motif. These residues are also conserved in the more general 2OG-FeII oxygenase superfamily as described above, except for the final R. The first three residues (HxD and H) are involved in Fe(II)-coordination, whereas the first R is involved in 2OG-coordination. The final R is most likely involved in AlkB-specific substrate binding. In addition to DNA repair, it has been shown that E. coli AlkB and the human AlkB homologue hABH3 may be involved in RNA repair. When expressed in E. coli both AlkB and hABH3 reactivate methylated RNA bacteriophage MS2 in vivo. This illustrates that direct repair may be an important mechanism for maintenance of RNA in living cells [4] . RNA repair proceeds by the same mechanism as DNA repair. Repair of damaged RNA was previously considered very unlikely, due to the natural redundancy of RNAs in a cell [8] . However, RNA is essential for cell function: unrepaired RNA can lead to miscoded or truncated proteins, and alkylated RNA could signal cell cycle checkpointing or apoptosis [9] . Consequently the occurrence of RNA repair does not come as a great surprise. The mechanism of direct reversal of methylation as used by AlkB homologues is particularly important for RNA repair, as it means that single-stranded regions may be repaired without introducing strand breaks. Repair of alkylation damage in DNA and RNA has recently been reviewed [10] . AlkB homologues have also been found in plant viruses. It has been suggested that methylation may be used in host-mediated inactivation of viral RNAs, and that AlkB homologues in some plant viruses may be used to counteract such defence mechanisms [1] . However, no detailed study of this has been published. The research project reported here has focused on a better understanding of the distribution and potential function of putative AlkB homology domains by using in silico mapping of viruses in which such domains have been found, as well as related viruses. The general mapping strategy of the project was to identify viral genomes with AlkB homology domains, identify common features of these genomes, and subsequently find additional genomes with similar features, but without AlkB homology domains. This data set could then be used to analyse the properties and distribution of AlkBlike domains in viruses, as a basis for generating hypotheses about the evolution and function of these domains. The PSI-Blast search for viruses in the NCBI nr protein sequence database was initiated with ALKB_ECOLI (NCBI gi113638), restricted to residues 110 to 210 and using the default inclusion threshold of 0.005 on E-values. The [11] . In all of these viruses the AlkB domain is a part of the replicase polyprotein, which normally consists of a viral Other Pfam domains -Peptidase_C21, C23, C33, C34, C35 and C41, A1pp and OTU -were also identified in subsets of sequences. A1pp is a member of the Appr-1-p processing enzyme family, and the domain is found in a number of otherwise unrelated proteins, including non-structural proteins of several types of ssRNA viruses. OTU is a mem-ber of a family of cysteine proteases that are homologous to the ovarian tumour (otu) gene in Drosophila. Members of this family are found in Eukaryotes, viruses and pathogenic bacteria. The MT, HEL and RdRp domains identified by Pfam as described above were extracted from the library sequences, aligned by ClustalX, and combined into a new alignment consisting of only these domain regions. This turned out to be necessary in order to get robust alignments. The intervening regions between the conserved domains are extremely variable in these sequences, and this tended to confuse alignment programs in the sense that conserved regions were not correctly aligned. The combined sequence alignment of domains from Closteroviridae, Flexiviridae and Tymoviridae was then used as input for building a phylogenetic tree with MEGA2. The final tree is shown in Figure 2 , with polyproteins containing AlkB-like domains indicated. A second alignment was generated from all sequences with AlkB-like domains, using only the regions corresponding to MT, AlkB, HEL and RdRp Pfam domains. The domains were aligned individually, and the combined alignment was used as input for MEGA2. However, this data set did not give a reliable phylogeny (data not shown), and the separate domains of this alignment were therefore analysed individually and compared. This analysis is summarised in Tymoviridae measures (including SJA) for comparison of random trees [12] . The SJA values shown in Table 2 for comparisons between MT, HEL and RdRp NJ trees were 14.2 -17.1 standard deviations from the expectation value of 0.665 for a tree with 22 nodes, whereas the corresponding values for the AlkB NJ tree were 4.4 -5.4 standard deviations from the expectation value. Similar ranges were observed for the ML trees as well as for alternative distance measures, e.g. the Symmetric Difference (SD) measure (data not shown). Although this means that the SJA value for comparing AlkB trees to MT, HEL and RdRp trees were significantly better than for random trees, it also shows that the MT, HEL and RdRp trees were clearly more similar to each other than to the AlkB tree. The alignment of the AlkB domain seemed to be of comparable quality to the other alignments. In fact the AlkB domain had the highest average pairwise sequence identity, as seen in Table 2 (see Figure 3 for the actual alignment). In other words, these AlkB domains were as similar to each other as the other three domains with respect to sequence identity, but they did not represent a consistent evolutionary history when compared to the other domains of this polyprotein. This may indicate that the AlkB domains have evolved separately from the other domains, and possibly as several independent instances. The degree of co-evolution was analysed by computing pairwise distances between sequence regions in the alignment of MT, AlkB, HEL and RdRp domains described above. In Figure 4 selected results are shown as scatter plots, where the Blosum 50 score value between e.g. the MT domains in a pair of sequences is plotted against the score value for AlkB domains in the same pair of sequences. Plots for the MT, HEL and RdRp domains show that they are strongly correlated for MT vs. RdRp (r 2 = 0.95), MT vs. HEL (r 2 = 0.87) and HEL vs. RdRp (r 2 = 0.81). The plot of the AlkB domain vs. these three domains for the same set of sequences shows a very low degree of correlation for AlkB vs. RdRp (r 2 = 0.10), AlkB vs. MT (r 2 = 0.12) and AlkB vs. HEL (r 2 = 0.16). As mentioned above the genome organisation of these replicase polyprotein sequences seems to be very flexible. In order to analyse domain organisation the location of identified Pfam domains were plotted for a number of sequences, as shown in Figure 5 . The results described above may indicate that the AlkB domains have been integrated into the replicase polyprotein relatively recently (see Discussion). In order to test for potential sources selected AlkB domains were compared to non-viral sequences. PSI-Blast was used to search the NCBI nr database, removing all viral hits in the final search report. Most of the remaining top-scoring hits were from bacteria. This included two different strains of Xanthomonas, X. axonopodis pv citri and X. campestris pv campestris. Xanthomonas attacks plants such as citrus, beans, grapevine, rice and cotton [13] . The search also returned high-scoring hits from another plant pathogen, Xylella fastidiosa. This bacterium infects a great variety of plants, including grapevine, citrus, periwinkle, almond, oleander and coffee [14] . Pfam searches obviously will only identify known domain types in protein sequences. In order to identify potential similarities in regions that were not recognised by Pfam, systematic PSI-Blast searches were performed, using the polyprotein regions between the MT and HEL domains and searching against the NCBI database of reference sequences [15] , excluding all viral entries. A maximum of 5 PSI-Blast iterations were allowed, with an inclusion threshold of 0.005. The expected homologues of the AlkBdomain were found with high confidence, as most of the E-values were < 1 × 10 -50 . Homologues of typical viral domains like the viral peptidases were obviously not found, as all viral database entries were excluded. Very few Multiple alignment of sequence regions corresponding to the AlkB domains Figure 3 Multiple alignment of sequence regions corresponding to the AlkB domains. The alignment was generated with ClustalX. The residues involved in coordination of the essential Fe 2+ ion are completely conserved, except in one of the Vitivirus sequences. These residues are the HxD motif, a single H, and the first R in the RxxxxxR motif. The function of the remaining conserved residues is unclear, but at least some of them may be involved in coordination of the substrate [10] . Pairwise distances between sequence regions corresponding to methyltransferase (MT), RdRp and AlkB domains. Each data point corresponds to e.g. RP-RP and MT-MT distances for the same pair of sequences, and sequences showing similar evolutionary distance in these two regions will fall on the diagonal. The pairwise distances were estimated from multiple alignments using the Blosum50 score matrix [47] . Trend lines were estimated with Excel. The trend line for AlkB vs. RdRp is heavily influenced by the point at (675, 670). It represents two Foveavirus sequences (NCBI gi3702789 and gi9630738), they are 98% identical over the full polyprotein sequence. Alignment score (AlkB) Alignment score (RdRp) r 2 = 0.10 new similarities were found by these searches. Pepper ringspot virus (Tobravirus, NCBI gi20178599) showed significant similarity to site-specific DNA-methyltransferase from Nostoc sp (E = 1 × 10 -74 ), as well as other cytosine 5Cspecific DNA methylases. Bamboo mosaic virus (Potexvirus, NCBI gi9627984) showed similarity to aggregation substance Asa1 from Enterococcus faecalis (E = 6 × 10 -34 ). A small number of additional similarities seemed to be caused by biased sequence properties (e.g. proline-rich regions), and were probably not significant. This included matches against mucin and cadherin-like proteins from Homo sapiens and multidomain presynaptic cytomatrix protein (piccolo) from Rattus norvegicus. In general the variable regions seemed to be truly variable, with very little similarity to other proteins, except for the Pfam domains already identified. As seen in Figures 2 and 5 , some closely related sequences are lacking specific domains in the sense that HMMER does not find a significant similarity to the Pfam entries for these domains. In order to understand the degree of sequence variation associated with this domain loss, as well as the general sequence variation in conserved vs. non-conserved regions of typical polyproteins, several dot plots were generated. The dot plot for two Carlavirus sequences, Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191), is shown in Figure 6 . The dot plot confirms that these two sequences are closely related in the MT, HEL and RdRp domains. However, there are significant differences in the region between MT and HEL. Potato virus M is lacking the AlkB domain whereas Aconitum latent virus is lacking the OTU domain. As seen from the dot plot, short regions of similarity close to the diagonal shows that both domains may have been present in an ancestral sequence. However, this region shows a high degree of sequence variation, and as indicated by the dot plot they are almost exclusively mutations. Non-essential or non-functional domains are probably rapidly lost. In this particular case, none of the typical AlkB motifs seem to be conserved in Potato virus M, indicating that this indeed is a non-functional AlkB domain. The N-terminal domains of Flexiviridae and Tymoviridae are methyltransferases As described above the Pfam methyltransferase motif (Vmethyltransf) did not match any of the putative methyltransferase domains of Flexiviridae and Tymoviridae, despite the fact that they had been identified via PSI-Blast searches starting with known methyltransferases. Therefore an additional Pfam-type profile was generated. It is obviously a possibility that these domains in Flexiviridae and Tymoviridae are not methyltransferases, and that they are false positives from PSI-Blast. However, the essential residues of a typical viral methyltransferase motif are conserved in the alignment of these domains (data not shown) [16] . In Bamboo mosaic virus, which belongs to Flexiviridae, the residues H68, D122, R125 and Y213 have been identified as putative active site residues with similarity to the Sindbis virus-like methyltransferase [17] , and it has been demonstrated that this region of the Bamboo mosaic virus has methyltransferase activity, as it catalyses the transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP or guanylylimidodiphosphate (GIDP). The corresponding sequence positions are almost completely conserved in the alignment of Flexiviridae and Tymoviridae N-terminal domains. This is most likely significant, as only 7 positions in total are completely conserved in this alignment, which means that the majority of the conserved positions are known to be essential for methyltransferase activity. Work e.g. by Hataya et al. seems to support the assumption that this sequence region is a methyltransferase domain [18] . It therefore seems likely that all the sequences with AlkB domains also contain functional MT, HEL and RdRp domains. The MT Location of Pfam domains in the variable region of Flexiviridae 2 sequences Figure 5 Location of Pfam domains in the variable region of Flexiviridae 2 sequences. The regions have been extracted directly from Pfam output, and sequences and regions are drawn to scale. The black bar at each end of a motif indicates that a fulllength motif has been found, for partial motifs the bar at the truncated end would be missing. domains are probably involved in capping of genomic and subgenomic RNA [19] . Based on the bioinformatic evidence generated here, it seems reasonable to assume that the viral AlkB domains identified by Pfam are functional. All the essential residues found in 2-oxoglutarate-and Fe(II)-dependent oxygenases are conserved, in particular the putative Fe 2+ coordinating H, D and H residues at alignment positions 19, 21 and 91 of Figure 3 , and the 2-oxoglutarate coordinating R at position 100. The conserved R at position 106 is also very characteristic of AlkB homologues [10] . The fact that all AlkB-like domains identified in these viral genomes are full-length, compared to the Pfam profile, also seems to support the hypothesis that these domains are functional. The Pfam searches show that AlkB domains are found only in a subset of the viral genomes. This subset is phylogenetically consistent (see Figure 2 ), as it is mainly restricted to the Flexiviridae, and in particular to a subset of the Flexiviridae consisting of Viti, Capillo, Tricho, Fovea and Carlavirus. This subset is well separated from the remaining Flexiviridae in the phylogenetic analysis. The split seems to be robust from bootstrap analysis, therefore this family will be discussed here as two subfamilies, Flexiviridae 1 and 2. The same split was observed by Adams et al. in their recent analysis of the Flexiviridae family [20] . Most of the AlkB domains (15) are found in Flexiviridae 2. The remaining AlkB domains are found in Flexiviridae 1 (5) and Closteroviridae (2) . In general, all the Flexiviridae 2 sequences have at least one extra domain in addition to MT, HEL and RdRp: either AlkB, OTU-like cysteine protease or a peptidase. Most other plant viruses that are included in this survey do not have additional domains, except for Tymoviridae where a peptidase domain seems to be common. For the remaining plant virus families included here (excluding Tymoviridae and Flexiviridae 2), only 14% seem to have additional domains. The observed distribution of AlkB domains could most easily be explained by assuming that an ancestral AlkB domain was integrated into the genome of the last common ancestor of the Flexiviridae 2 subfamily. Subsequent Figure 6 Dot plots for Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191). To the left the full sequences are shown, using the program default for similarity threshold, and to the right the region with AlkB, OTU and peptidase integration, using a slightly lower (more sensitive) threshold for sequence similarity. The Pfam regions corresponding to MT (magenta), AlkB (red), OTU (green), peptidase (blue), HEL (yellow) and RdRp (cyan) domains are indicated. virus generations derived from this common ancestor would then also contain an AlkB domain, except in those cases where the domain was lost again. This scenario could also include subsequent transfer to a small number of other virus families e.g. by recombination. If this scenario was correct, then one would expect the different domains of the polyprotein to have a similar evolutionary history. From the phylogenetic analysis (Table 2) this seems to be confirmed for the MT, HEL and RdRp domains, but not for the AlkB domain. This indicates that the AlkB domain may not have co-evolved with the other domains, at least until relatively recently. This seems to be confirmed by looking at the degree of co-evolution, which was analysed by computing pairwise distances between alignment regions representing the relevant domains ( Figure 4 ). In the case of perfect co-evolution all points should fall on a diagonal. This seems to be the case for the MT, HEL and RdRp domains. However, the plot of the AlkB domain vs. these three domains for the same set of sequences does not show a similar correlation. Only some of the closely related sequence pairs in the upper right quadrant of the plot in Figure 4 show some degree of correlation for AlkB vs. RdRp. The most likely explanation seems to be that most of the AlkB domains have not coevolved with the other domains for any significant period of time. This seems to rule out the possibility of ancient integration of the AlkB domain, except if we assume that an ancient viral AlkB domain has frequently recombined with other AlkB domains. However, it is difficult to distinguish a scenario with frequent recombination of AlkB domains from de novo integration, and the net effect on the properties observed here would be the same. As seen in Figure 4 , the range of score values is generally smaller for the AlkB domains than e.g. the RdRp domains, particularly if we exclude a couple of very high-scoring cases (see figure caption) . On the other hand, the degree of sequence variation within the collection of AlkB domains is significant, average sequence identity for pairwise alignments is 38%, and only 10% of the positions are totally conserved. This can be consistent with a recent integration if we assume that several different AlkB-type vectors have been used for integration (see below for details). An increased mutation rate after integration could also have contributed to sequence diversity in this region. Moving the AlkB domain into a novel structural and functional context would have removed many of the original evolutionarily constraints, as well as introduced some new ones. This could have created a "punctuated equilibrium" type of situation, potentially leading to a very rapid evolution that could have introduced significant differences between the AlkB domains, independent of the evolution in the other domains. A high mutation rate seems to be the case for this region in general, as indi-cated in Figure 6 . Although the MT, HEL and RdRp domains seem to be well conserved from the dot plot, there are very large sequence variations in the intervening region. One sequence in Figure 6 has a well conserved AlkB domain, the other an OTU domain. The fact that there are very weak sequence similarities in these two domains in the dot plot indicates that both sequences originally had both domains. However, the fact that this similarity now is very weak and without any of the typical AlkB active site motifs also indicates a high mutation rate where non-essential domains are rapidly lost. Therefore the conservation of AlkB domains is a strong indication that they are functional, as already mentioned. If we assume that AlkB domains have been integrated relatively recently, then either de novo integration or recombination (horizontal gene transfer) may have been the main driving force for spreading the AlkB domain to new genomes. In the first case a large number of individual integrations could have lead to the present situation. If horizontal gene transfer was the main driving force, the initial number of integrations might have been quite small. It is not easy to differentiate between these two situations. The map of Pfam motifs in the variable region between the MT and HEL domains in Flexiviridae 2 polyproteins ( Figure 5) shows that they have a very similar domain organisation, basically an AlkB domain followed by an OTU domain and a peptidase domain, located towards the C-terminal part of the sub-sequence. The relatively constant domain organisation seems to be consistent with a small number of initial integrations that were subsequently diffused to related genomes e.g. by homologous recombination. However, this is not fully consistent with the fact that the viruses with AlkB domains have been collected from hosts at very different locations, e.g. Canada, USA, Russia, Italy, Germany, France, India, Taiwan, China and Japan. Although import of virus-infected species or transmission by insects may transport viruses over significant distances, it is not obvious that this is enough to explain the observed distribution of AlkB-like domains. Therefore several independent integrations, mainly from closely related hosts, have to be considered as an alternative explanation. This explanation seems to be supported by the apparent lack of any consistent evolutionary relationships between the various AlkB domains, as seen in Table 2 . It is not easy to see how this model can be consistent with the observed similarities in domain organisation in Flexiviridae. Assuming that this region has a high degree of variability, one would expect the variability to affect localisation of integrated domains as well. However, it is possible that conserved regions e.g. in the polyprotein play a significant role in integration of novel domains. It may be relevant in this context that preliminary simulations indicate that e.g. the AlkB domains tend to form independent folding domains in the folded RNA structure of the polyprotein RNA (F. Drabløs, unpublished data). This property may possibly facilitate the insertion of such domains into the viral genome. There are many groups of organisms that can act as vectors and spread viruses, including bacteria, fungi, nematodes, arthropods and arachnids. The plant viruses may have acquired the AlkB domain either from the vector or from the host itself. As already mentioned, searching with viral AlkB domains in protein sequence databases resulted mainly in bacterial sequences, including the plant pathogens X. fastidiosa and campestris. It is therefore a reasonable possibility that AlkB domains in plant viruses have originated from bacterial mRNA. It is also possible that the mRNA originated from other vectors or from the host itself, but at the present time this is not easily verified or disproved because of the limited number of insect and plant genomes that have been sequenced. It has previously been suggested that the viral AlkB domain may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system of the host [1] . PTGS is known as one of a plant's intrinsic defence mechanisms against viruses [21] . Gene silencing can occur either through repression of transcription (transcriptional gene silencing -TGS) or through mRNA degradation, PTGS. The PTGS-mechanism in plants shows similarities to RNA interference (RNAi) in animals [22] . This mechanism results in the specific degradation of RNA. Degradation can be activated by introduction of transgenes, RNA viruses or DNA sequences homologous to expressed genes [23] . Many viruses have developed mechanisms to counteract PTGS in order to successfully infect plants [24] . Two of these suppressors of PTGS have been identified as Hc-Protease and the 2b protein of Cucumber mosaic virus [25] . Although both proteins suppress PTGS, it is likely that they do so via different mechanisms. Could the AlkB-like domain found in some of the plant viruses also be a suppressor of PTGS? Previously reported research indicates that methylation of transcribed sequences is somehow connected with PTGS, and the methylation can be mediated by a direct RNA-DNA interaction [26] . This RNA-directed DNA methylation has been described in plants, and leads to de novo methylation of nearly all cytosine residues within the region of sequence identity between RNA and DNA [27] . Both RNA methylation and methylation of host proteins that are essential for viral replication would be detrimental to the virus. It has already been mentioned that AlkB repairs 1methyladenine and 3-methylcytosine by oxidative demethylation. It is therefore possible that AlkB demethylates the nucleotides methylated by the PTGS mechanism, helping the virus to overcome one of the major defence mechanisms of the plant. As shown here, only a subset of plant viruses have the AlkB domain. However, other viruses may be utilising naturally occurring AlkB proteins in the host. Viruses have to rely on a number of host proteins in order to replicate [28] . In some cases it is probably beneficial for the virus to integrate such genes into their own genome in order to ensure that they are accessible, although there will be a trade off between this advantage and the increased cost of maintaining a larger genome [29] . However, there is an alternative hypothesis with respect to the AlkB integration that also has to be considered. As discussed above, the AlkB domain seems to have been integrated relatively recently in viruses found at very different geographical locations, and the only obvious connection seems to be that most viruses belong to a subset of the Flexiviridae. However, the source of these viruses points at another common feature. As seen from the table given in Additional file 1, AlkB domains are often found in viruses associated with grapevine, apple, cherry, citrus and blueberry -crops where the usage of pesticides is common. It is known that several common pesticides (e.g. methyl bromide and some organophosphorus compounds) may cause methylation of DNA and RNA [30] [31] [32] [33] . An integrated repair domain for methylation damage as part of the viral replication complex would therefore give the virus a competitive advantage in a highly methylating environment. The application of such pesticides would probably also stimulate AlkB production e.g. in co-infecting bacteria, giving these viruses easy access to AlkB mRNA for integration into their RNA genome. It could be argued that a more active PTGS system in these plants would give a similar effect. However, in that case we would expect to see more ancient integrations of AlkB domains. It could also be argued that the presence of AlkB domains may be an artefact caused by promiscuous viral domains picking up available mRNA sequences during cultivation of viruses in the laboratory. However, given the large number of different laboratories involved, and the number of different hosts used (data not shown), this seems to be a very unlikely explanation. The hypothesis that environmental compounds, in particular pesticides, may have provoked the integration of AlkB domains into the viral genomes depends upon a high mutation rate and frequent integrations of non-viral domains. The integrations have to be recent, not only in relative terms, compared to other domains in the same genome, but also in absolute terms, compared to the progress of modern agriculture. The integrations also have to be frequent, in the sense that it is likely that integration could have happened several times, in different biotopes. It is difficult to estimate mutation rates in RNA viruses. They evolve very rapidly, and it is often difficult to assign reliable phylogenies. However, recent studies indicate that most ssRNA viruses have a mutation rate close to 10 -3 substitutions per site per year [34] , e.g. the SARS virus has 1.16-3.30 × 10 -3 non-synonymous substitutions per site per year, which is considered to be a "moderate" ssRNA mutation rate [34] . If we assume that most ssRNA viruses have effective mutation rates within the same order of magnitude, a realistic mutation rate for the viruses included here might be something like 2.0 × 10 -3 . In that case, the MT, HEL and RdRp trees shown in Additional file 2 represent approximately between 325 and 750 years of evolution. In general the NJ trees estimate a slightly shorter evolutionary history (between 325 and 450 years) compared to the ML trees (between 550 and 750 years). In this estimate the Ampelovirus sequences have not been included, as they seem to have diverged from the remaining AlkB-containing viruses at a much earlier stage. If we believe that the AlkB integrations happened after the divergence of most sequence included here, as indicated by the lack of co-evolution in Figure 4 , it does not seem unrealistic to assume that most of these integrations happened within the last 50 -100 years or so. This estimate is of course very approximate, in particular since we do not know the true mutation rate of these viruses. However, it shows that a likely time span for AlkB integration is compatible with the evolution of modern agriculture. Unfortunately, because of the lack of any robust phylogeny for the viral AlkB sequences it does not make sense to do a similar estimate for that domain. Although it is generally accepted that viruses frequently use recombination to acquire functionality [35] , it is less well known how often this includes nonviral sequences. However, there are some well-documented examples, and in particular the properties of the ssRNA positive-strand Pestivirus may be relevant in this context. There are two biotopes of the pestiviruses, cytopathogenic (cp) and noncytopatogenic (noncp). The host is infected by the noncp form which is converted into the cp form by integration of a fragment of a cellular gene into the viral genome [36] . This introduces a protease cleavage site in the polyprotein. However, the important point here is that this happens as part of the normal infection process. It has been suggested that the integration is facilitated by the viral polymerase undergoing two subsequent template switches during minus-strand synthesis [37] , although nonreplicative RNA recombination also may be a possibility [38] . Inte-gration of cellular sequences have also been observed in other viruses, e.g. in influenza virus [39] . This shows that at least some viruses do have efficient mechanisms for recruitment of host genes into the viral genome. Therefore a recent and rapid integration of AlkB domains into selected plant virus genomes does not seem to be an unlikely scenario. This study has focused on the AlkB domain, mainly as an attempt to get a better understanding of potential functions associated with this domain. However, it is likely that additional information about integration patterns and the relative importance of de novo integration vs. recombination can be achieved by a closer investigation of the other variable domains, e.g. by looking at how they correlate with the evolution of the AlkB domains. We believe that the viral AlkB-like domains are conventional repair domains targeted towards the viral RNA. The integration of AlkB domains into viral genomes may have been provoked by environmental methylating agents, e.g. the introduction of DNA/RNA-methylating pesticides in farming. The hypothesis [1] that the domain interferes with the PTGS system of plants can not be excluded, but seems to be less consistent with observed features of the AlkB integration. and Tymoviridae was generated from a ClustalX alignment, using hmmbuild and hmmcalibrate from the HMMER package. Visualisation of motif positions in viral sequences was generated directly from the HMMER output files using a local tool as an interface to the GNU [50] groff software. Systematic large scale searches with polyprotein subsequences were done locally with PSI-Blast and the NCBI reference sequence database [15] . Dot plots for comparison of viral protein sequences were generated with Dotter version 3.0 [51] . "
19
"Managing emerging infectious diseases: Is a federal system an impediment to effective laws?"
"In the 1980's and 1990's HIV/AIDS was the emerging infectious disease. In 2003–2004 we saw the emergence of SARS, Avian influenza and Anthrax in a man made form used for bioterrorism. Emergency powers legislation in Australia is a patchwork of Commonwealth quarantine laws and State and Territory based emergency powers in public health legislation. It is time for a review of such legislation and time for consideration of the efficacy of such legislation from a country wide perspective in an age when we have to consider the possibility of mass outbreaks of communicable diseases which ignore jurisdictional boundaries."
"The management of infectious diseases in an increasingly complex world of mass international travel, globalization and terrorism heightens challenges for Federal, State and Territory Governments in ensuring that Australia's laws are sufficiently flexible to address the types of problems that may emerge. In the 1980's and 1990's HIV/AIDS was the latest "emerging infectious disease". Considerable thought was put into the legislative response by a number of Australian jurisdictions. Particular attention had to be given to the unique features of the disease such as the method of transmission, the kinds of people who were at risk, and the protections needed by the community and the infected population to best manage the care of those infected and to minimize new infections. Health workers and researchers began to find that "the most effective strategies that we have so far found to help promote reduction of the spread of HIV involve the adoption of laws and policies which protect the rights of people most at risk of infection" [1] . A good example of a legislative response which adopts this approach is found in section 119 and 120 of the Victorian Health Act 1958. These sections emphasize the need to protect the privacy of the infected individual and to undertake a staged response which is proportional to the risk presented by the infected individual. The legislation has been very effective with HIV and has been praised for its progressive approach [2] . In 2003 the community has been faced with the emergence of two new infectious diseases, SARS and Anthrax. Whilst there were no cases of either disease in Australia, the threat of a possible outbreak had to be acknowledged and a response planned. Anthrax is not a new infectious disease. Humans can become infected with anthrax by handling products from infected animals or by breathing in anthrax spores from infected animal products (like wool, for example). People also can become infected with gastrointestinal anthrax by eating undercooked meat from infected animals. However, its manufacture and use as a weapon for bioterrorism forces us to rethink its management in a new context. These two infectious diseases have very different features from HIV which spreads only via transmission of infected bodily fluids such as blood or semen. SARS, by contrast is transmitted via droplets from infected cases which, as a result of coughing, carry the virus to close contacts [3] Thus, the infection profile of SARS requires planning for the possible overrun of Intensive Care Units and the likely infection of a number of ICU staff affecting both morale and capacity to cope. Anthrax raised different problems. These include the possible investigation of terrorist suspects alongside investigation of the outbreak of the infectious disease. Difficulties are also raised by likelihood of public panic, and the flooding of public health officials with reports of suspicious white powder. In early 2004 the media reported the spread of avian influenza across South East Asia. This disease has different features from HIV/AIDS and SARS and an approach to an Australian outbreak would also be different. The main difference is in the source of transmission of the virus, that is, from infected birds to humans. There is very little difference [from ordinary influenza] in the symptoms (though these may vary in severity) or treatment of the virus [4] It is too early to predict whether this may be the next "emerging infectious disease", but its current spread has given rise to concern about such a possibility [5] Australia is a federal system. There are two parallel sets of laws in operation. The Commonwealth Constitution sets out the legislative powers of the Commonwealth. Specific powers are listed in the Commonwealth constitution but State constitutions have broad powers covering matters such as peace, order and good governance. As the Commonwealth has no specific power to legislate with respect to health, other than the quarantine power, national legislative schemes in public health which rely upon a cooperative approach from all States and Territories are cumbersome and difficult. Without a specific head of power, the Commonwealth has limited ability to legislate with respect to health. "That is, the legislative powers of the Commonwealth are specified in the Constitution and do not include expressly most of the activities that together comprise the field of public health" [6] For this reason, there are no Commonwealth emergency health powers except quarantine powers. Quarantine powers are currently restricted to isolation at the border of the country of people, plants, and animals to prevent the spread of disease. There is a real possibility that quarantine laws could have a broader scope. It depends on how widely the High Court would interpret section 51(x) of the Commonwealth Constitution. A quarantine law could override state laws as long as it remained a law "with respect to quarantine". However, "the power is potentially a colossus so far as the expansion of legislative authority in the fields of public health is concerned". [6] The quarantine power would be the most likely candidate for a head of power on which to base development of commonwealth laws for the management of public health emergencies. Another possibility may be the external affairs power, if there was a relevant treaty or international agreement which could be given effect to in domestic law. However the legislation would have to be limited to laws giving effect to the treaty. States and territories have a range of emergency powers available to them in their existing public health legislation. Some are relatively old. For example, the Health Act 1911 (WA), Public Health Act 1952 (NT) based on an 1898 Ordinance (Both these Acts are currently under review). Health emergency powers vary from one jurisdiction to another, but include powers to support disease surveillance, contact tracing and orders to restrict behavior or movement of individuals with an infectious disease in certain circumstances. There are also powers to recall food, search premises and seize property, close buildings and a range of other substantial and intrusive powers. It is suggested that it is time to consider whether state and territory public health legislation contains sufficient measures to manage the outbreak of an infectious disease in a modern environment which includes mass travel, swift spread of infection and additional complexity raised by fears of bioterrorism. Currently, in a public health emergency caused by the spread of an emerging infectious disease, Australia could need to rely on a patchwork of legislative measures to assist it to cope. Commonwealth quarantine laws and State and Territory powers in public health legislation may all be needed to address the problem. If an outbreak occurred on a border, or in some area where jurisdiction may be in doubt such as airspace or offshore and a state or territory response was required in addition to any quarantine measures, there could be confusion over jurisdiction for the application of State and Territory powers. State and Territory public health acts do not adequately provide for interjurisdictional communication and cooperation. There could also be difficulties if an infectious disease caused overseas deaths of people from more than one State or Territory in circumstances where an Australian coronial investigation was considered desirable. In such a situation, the jurisdiction of more than one Australian coroner would be triggered. Several State and Territory coronial laws could apply and there could be different inquests under different laws undertaken by different coroners into the same incident. It is suggested that it is time to look at the efficiency of the emergency powers laws of Australia as a whole: to map the laws in each jurisdiction and the Commonwealth quarantine laws and to consider their effectiveness in the face of the outbreak of a fast moving, easily spread infectious disease. The efficacy of Australia's laws should also be considered in relation to bioterrorism. While there were no infections from anthrax in 2003 despite a great deal of media coverage and infections and deaths in the US, a responsible legislature ought to acknowledge the possibility and ensure that the law is ready to support a swift and effective response. It is not enough to consider whether the individual pieces of legislation are up to the task of managing outbreaks of newly emerging infectious diseases. Indeed many of the jurisdictions are currently reviewing their public health legislation and will no doubt give proper consideration to this issue as part of the review. But who is thinking about how the legislation of all jurisdictions and the Commonwealth quarantine fits together? What powers enable communication and cooperation between jurisdictions about the outbreak of infectious disease? What kind of opportunity is there for a coordinated response? Can public health orders made in one jurisdiction travel to another jurisdiction when the infected individual travels? What arrangements can be made if an outbreak occurs on or close to a interstate border? What if there is an outbreak on a bus carrying passengers from Victoria, through South Australia to the Northern Territory? It is encouraging to note that, even without specific legislation, there has been a mechanism to achieve communication and cooperation between jurisdictions through the Communicable Disease Network of Australia (CDNA). This Network has in fact been quite successful in fostering regular communication between the Communicable Disease Units across the country and has been involved in coordinated actions during a number of multistate outbreaks. Despite the existence of this network and other good working relationships between government officials and various agencies in different jurisdictions, a serious outbreak of communicable disease would require the existence of legislative powers. Public health emergencies generate confusion, even panic. Clarity of powers and the way those powers interact with each other would be crucial in an emergency. It became apparent after the Bali tragedy in 2002 that coroner's jurisdiction was triggered differently in different jurisdictions and some acts did not support communication and cooperation when inquests might be needed for deaths of people ordinarily resident in several jurisdictions. The time to find the shortcomings in the legislation is well before the crisis. A review of the efficacy of how these laws work together to protect the public health of all Australians should be undertaken. It has been possible to overcome the hangovers of federation for the betterment of all Australians in relation to corporations law. When doubts were recently raised about the constitutional basis of the corporations law scheme, the States and Territories were able to cooperate and refer the necessary powers to the Commonwealth to provide certainty about the laws which govern our corporations. Is our public health any less important than governance of our corporations? Could we cooperate to give ourselves certainty, flexibility and a consistent approach which protects the rights of those subject to some very broad powers? The States and Territories are generally reluctant to refer powers to the Commonwealth. It may be time to seriously discuss referral of powers in the context of health emergency powers. At the very least, it is time that the Commonwealth, States and Territories recognised the need for the laws to work as a set of laws to protect the whole country, not simply individual laws to protect individual jurisdictions. There has been work done internationally in this area. A model State Emergency Health Powers Act has been developed in the US in 2001 [7] In the preamble to this Act a rationale for its development is set out: "In the wake of the tragic events of September 11, 2001, our nation realizes that the Government's foremost responsibility is to protect the health, safety and wellbeing of its citizens. New and emerging dangers including emergent and resurgent infectious diseases and incidents of civilian mass casualties -pose serious and immediate threats to the population. A renewed focus on the prevention, detection, management and containment of public health emergencies is thus called for." The US, like Australia, is a Federal system. The model was intended to be taken up by those US states which wished to do so. To date, it has been passed in over half the US states. This bill would be an excellent starting point for development of an Australian model. There are a number of legislative mechanisms which could be used to support a nationally uniform approach to health emergency powers legislation in Australia. The development and adoption of the model food legislation provides a useful model of a cooperative uniform approach. A model act was developed in consultation with all jurisdictions. It covered areas agreed to be core areas of the Act which ought to be the subject of a national approach and other provisions which were considered to be administrative and were to be adopted at the discretion of each jurisdiction. An intergovernmental agreement was signed as a mechanism to protect the uniformity of the legislation. The agreement sets up a Ministerial Council, supported by a Food Regulation Standing Committee. The Council has responsibility for deciding on proposals to amend the model [8] If a decision is made in favor of amendment, States and Territories will use their best endeavors to submit to their respective Parliaments, legislation which gives effect to the amendment. The law is an important tool in supporting the management of the outbreak of infectious diseases. The existence of our Federal system has meant that we have a different approach in each State and Territory together with Commonwealth control of quarantine. Newly emerging infectious diseases creating real threats to public health in an era of easy mass travel, and the present threat of bioterrorism mean that it is time Australia examined all laws to contain and manage infectious disease outbreak. The laws should be examined both for their effectiveness in the areas they cover, and as part of a whole which ought enable a response which protects the health of all Australians, and crosses borders as easily as SARS or avian influenza."
20
"Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production"
"Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species."
"Lactic Acid Bacteria (LAB) are anaerobic Gram positive bacteria with a GRAS (Generally Regarded As Safe) status. They are also food grade bacteria, and therefore, they can be used for the delivery of proteins of interest in foodstuff or in the digestive tract. A last advantage compared to other well-known protein producers is that L. lactis does not produce LPS or any proteases as Escherichia coli or Bacillus subtilis do, respectively. In the last two decades, genetic tools for the model LAB, Lactococcus lactis, were developed: transformation protocols, cloning-or screening-vectors [1, 2] , and mutagenesis systems [3] are now available. Moreover L. lactis genome is entirely sequenced [4] . Many protein expression-and targeting-systems have also been designed for L. lactis [5] [6] [7] . These systems have been used to engineer L. lactis for the intra-or extra-cellular production of numerous proteins of viral, bacterial or eukaryotic origins (Table 1) . To produce a protein of interest in fermentors, secretion is generally preferred to cytoplasmic production because it allows continuous culture and simplifies purification. To use L. lactis as a protein delivery vehicle in the digestive tract of humans or animals, secretion is also preferable because it facilitates interaction between the protein (e.g. enzyme or antigen) and its target (substrate or immune system). In LAB, like in other Gram positive bacteria, secreted proteins are synthesized as a precursor containing an N-terminal extension called the signal peptide (SP) and the mature moiety of the protein. Precursors are recognized by the host secretion machinery and translocated across the cytoplasmic membrane (early steps). The SP is then cleaved and degraded, and the mature protein is released in the culture supernatant (late steps). Sometimes, secreted proteins require subsequent folding and maturation steps to acquire their active conformation [8] . In most of the works describing heterologous protein production by recombinant lactococci, only one cellularlocation (i.e. cytoplasm, external media or surface anchored) is described. Only a few works report the production of a given protein in different locations using the same backbone vector, the same induction level and or promoter strength, allowing thus a rigorous comparison of the production yields of cytoplasmic and secreted forms. Here, six examples of different heterologous proteins produced in L. lactis in both secreted and cytoplasmic forms are reviewed and discussed. Our major conclusion is that the best production yields are observed in most of these cases with secretion (up to five-fold higher than with cytoplasmic production). Moreover, engineering the expres-sion cassette to enhance the secretion efficiency (SE, proportion of the total protein detected as mature form in the supernatant) resulted in increased overall amounts of the protein. L. lactis is able to secrete proteins ranging from low-(< 10 kDa) to high-(> 160 kDa) molecular mass through a Sec-dependant pathway. Altogether, these observations suggest that i) heterologous proteins produced in L. lactis are prone to intracellular degradation whereas secretion allows the precursor to escape proteolysis, and ii) conformation rather than protein size is the predominant feature that can impair SE. New perspectives are now opened in the studies of heterologous protein production in L. lactis. Indeed, there is a need for food grade systems and for a better understanding of the host factors influencing heterologous protein secretion in L. lactis . For example, HtrA-mediated proteolysis (HtrA is the unique housekeeping protease at the cell surface) is now well-characterized in L. lactis [9] and can be overcome by use of a htrA L. lactis strain designed for stable heterologous protein secretion [10] . However, intracellular proteolysis (involving Clp complex -the major cytoplasmic housekeeping protease-, and probably other cellular components) remains poorly understood and is also discussed here. Genetic tools to target a given protein in different cellular compartments were developed using several reporter proteins [6, [11] [12] [13] (Table 1 ). The staphylococcal nuclease (Nuc) is a well-characterized secreted protein whose activity is readily detectable by petri plate assay and it has been used as a reporter protein for secretion studies in several Gram positive hosts [14] [15] [16] . In L. lactis, Nuc was used to develop protein targeting- [6] and SP screening-systems [1, 2] . Nuc was chosen to develop the pCYT and pSEC vectors for controlled production in L. lactis of cytoplasmic or secreted forms of a protein of interest, respectively ( Fig. 1 ) [5] . The pCYT and pSEC plasmids, where expression is controlled by a nisin inducible promoter, should be used in L. lactis NZ9000 (hereafter referred to as NZ) strain bearing a nisR,K chromosomal cassette, required for the nisin signal transduction [17] . In each case described below, protein sample concentration was adjusted to the cell density of the producing culture (for details see [18] ). At similar induction levels in lactococcal strains containing pCYT:Nuc and pSEC:Nuc vectors, the highest production yields were observed with the secreted Nuc form ( Table 2) . Similar results were obtained with constitutive nuc expression cassettes for cytoplasmic and secreted forms. Nuc was the first heterologous protein where highest protein yields were obtained with the secreted form. Similar results were obtained for the production of a Brucella abortus ribosomal protein. B. abortus is a facultative intracellular Gram negative bacterial pathogen that infects Unpublished results Bacteriocins human and animals by entry through the digestive tract. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate for the development of oral live vaccines against brucellosis using L. lactis as a delivery vector. L7/L12 was produced in L. lactis using pCYT and pSEC vectors [19] . Similarly to Nuc production, the production yield of secreted L7/L12 was reproducibly and significantly higher than that of the cytoplasmic form (Table 2) . Another example of higher protein yields in secreted vs cytoplasmic form is the production the human papillomavirus type 16 (HPV-16) E7 antigen, a good candidate for the development of therapeutic vaccines against HPV-16 induced cervical cancer. The E7 protein is constitutively produced in cervical carcinomas and interacts with several cell compounds. E7 was produced in a cytoplasmic and a secreted form in L. lactis [20] . Using similar induction level in exponential phase cultures, E7 production 1: protein samples were adjusted to the cell density and protein quantification was performed as described in the references either by western blot or by ELISA. *: E7 was not quantified but ratio was calculated by scanning the western blot signals and comparing their intensity as described in the corresponding reference. nd: not determined was higher for the secreted form than for the cytoplasmic form (Table 2) . This difference was even higher when induction occurred in late-exponential phase, where intracellular E7 was detected at only trace amount whereas secreted E7 was accumulated in NZ(pSEC:E7) culture supernatant (see below). Thus, production of E7 clearly illustrates the fact that secretion results in higher yields in L. lactis. Production of ovine interferon omega (IFN-ω) further illustrates this observation. In the case of poorly immunogenic antigens, co-delivery of an immuno-stimulator protein can enhance the immune response of the host. In order to optimize the use of lactococci as live vaccines, the production of cytokines was investigated in L. lactis [5, 21, 22] . IFN-ω is a cytokine able to confer resistance to enteric viruses in the digestive tract by reduction of viral penetration and by inhibition of intracellular multiplication of the viruses. Delivery of ovine IFN-ω in the digestive tract by recombinant L. lactis strains could therefore induce anti-viral resistance and could protect the enterocytes. Ovine IFN-ω cDNA was cloned into pCYT and pSEC plasmids for intracellular (pCYT:IFN) and secreted (pSEC:IFN) production respectively [5] . Induction of recombinant NZ(pCYT:IFN) and NZ(pSEC:IFN) strains were performed at equal level and IFN-ω production was measured. The levels of IFN-ω activity showed that i) an active form of IFN-ω was produced in both strains, and ii) the activity of IFN-ω found in the supernatant and cell fractions of NZ(pSEC:IFN) strain was about two-fold higher than that observed for the cytoplasmic form (Table 2) . Similarly to what was observed for Nuc and E7, secretion leads to higher heterologous protein yields. L. lactis has been engineered to secrete of a wide variety of heterologous proteins from bacterial, viral or eukaryotic origins (Table 1) . There are reports about secretion bottlenecks and biotechnological tools for heterologous secretion in model bacteria such as Escherichia coli and Bacillus subtilis [23, 24] , but only few data are available concerning this aspect in L. lactis. Protein size, nature of the SP and presence of a propeptide are parameters that may interfere with protein secretion. Some data available about these features are compiled here. To optimize secretion and thus production yields, the nature of the SP was the first parameter to modify on heterologous precursor as previously shown using Nuc as a reporter protein. The replacement of the native staphylococcal SP Nuc by the homologous lactococcal SP Usp45 to direct the secretion of Nuc in L. lactis led to an increased SE [25] (Table 3) . On the other hand, the replacement of SP Nuc by SP Usp45 did not enhance the SE of NucT (a truncated mature moiety of Nuc devoid of N-terminal propeptide) suggesting the importance of the propeptide in the SE for Nuc [25] (Table 3) . However, in several cases, the use of a homologous SP (and especially SP Usp45 ) allows a better SE compared to a heterologous one. Screening vectors were thus developed to search for new homologous secretion signals in L. lactis [1, 2] . These screening works offer now a panel of SPs that are suitable for heterologous secretion. However, when compared to SP Usp45, the newly described SPs were less efficient to direct secretion of Nuc [1] . Even after a direct mutagenesis on SP310, one of these new SPs identified using a screening strategy [1] , the enhanced SE was still lower than the one measured with SP Usp45 [26] . However, a recent study by Lindholm et al. showed that a Lactobacillus brevis SP (originated from a Slayer protein) drove the secretion of the E. coli FedF Schematic representation of Nuc cassettes for controlled and targeted production in L. lactis adhesin more efficiently than SP Usp45 [27] . High SE might thus result, at least in part, from good adequacy between the mature protein and the SP used to direct secretion. The fusion of a short synthetic propeptide between the SP and the mature moiety is another innovative biotechnological tool to enhance protein secretion. One such propeptide (composed of nine amino acid residues, LEISSTCDA) was developed and was shown to enhance the SE of several heterologous proteins in L. lactis: NucB, NucT, (Table 3 ) [18] , the B. abortus L7/L12 antigen (Table 3 ) [19] , and the α-amylase of Geobacillus stearothermophilus (Table 3 ) [18] . Directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety [25] . Furthermore, the enhancement effect does not depend on the nature of the SP, since the secretion of NucB fused to either SP Nuc or SP Usp45 was enhanced by LEISSTCDA insertion [25] . Strikingly, the enhancement of SE was reproducibly accompanied by an overall increase of protein yields as determined in Western blot experiments. This observation suggests that heterologous precursors are degraded by intracellular proteases when they are not efficiently secreted and that a higher secretion could be a way to escape proteolysis. Proteins with molecular mass ranging from 165 kDa (size of DsrD, the Leuconostoc mesenteroides dextransucrase, [28] ) to 9.8 kDa (size of Afp1, a Streptomyces tendae antifungal protein; Freitas et al., submitted) have been successfully secreted in L. lactis. This suggests that protein size is not a serious bottleneck for heterologous protein secretion in L. lactis. In contrast to protein size, conformation may be a major problem for heterologous secretion in L. lactis as illustrated by some recent examples. The first example is the production of the non-structural protein 4 (NSP4) of the bovine rotavirus, the major etiologic agent of severe diarrhea in young cattle. In order to develop live vaccines against this virus, the NSP4 antigen was successfully produced in L. lactis [29] . Derivatives of pCYT and pSEC plasmids were constructed to target NSP4 into cytoplasmic or extracellular location. The highest level of production was obtained with the secreted form. However, no secreted NSP4 was detected in the supernatant and both SP Usp45 -NSP4 precursor and NSP4 mature protein were detected in the cell fraction. Two degradation products were detected in addition to the NSP4 precursor and mature protein. These results suggest that the cytoplasmic form of NSP4 was probably totally degraded inside the cell whereas fusion to the SP Usp45 protected NSP4 protein against intracellular proteolysis. Similar results were obtained when pCYT and pSEC vectors were used to produce the B. abortus GroEL chaperone protein: only pSEC:GroEL plasmid was obtained and subsequently the fusion SP Usp45 :GroEL was detected in Western blot experiments (V. Azevedo, unpublished data). In this case, B. abortus GroEL is likely to interact with lactococcal cytoplasmic proteins leading to severe cellular defects and thus to a lethal phenotype. On the other hand, fusion of SP Usp to GroEL might keep the chimeric protein in an unfolded and/or inactive state allowing thus its heterologous production. Another example is the production of the bovine β-lactoglobulin (BLG) in L. lactis [30, 31] . BLG, a 162 amino acid residues globular protein, is the dominant allergen in cow's milk and was produced in L. lactis to test the immunomodulation of the allergenic response in mice when BLG is delivered by a bacterial vector [30] . Western blot and ELISA showed that BLG production was significantly higher when BLG was fused to SP Usp45 although the SE was very low, with no detectable BLG in the supernatant of pSEC:BLG strains [30] . Further studies revealed that a fusion between the LEISS propeptide and BLG could not enhance the SE of BLG above ~5%, as determined by ELISA [31] . For rotavirus NSP4, B. abortus GroEL, and BLG (which are medium-sized compared to DsrD or Afp1), either very low secretion yields or absence of secretion was observed in L. lactis. In all cases, fusion to a SP stabilizes heterologous protein production even though they are not efficiently secreted. These results could be due either to the SP itself that reportedly acts as an intramolecular chaperone or to the protection of the chimeric precursor from intracellular proteolysis by the cytoplasmic chaperones of the Sec-machinery. GroEL (a cytoplasmic chaperone), NSP4 (a structural protein), and BLG (a globular protein) have dramatically different primary sequences. A higher affinity of intracellular housekeeping proteases for these particular sequences cannot be hypothesized since the fusion of a SP leads to the stabilization of the protein. Change of conformation is therefore the predominant criterion involved in the stabilization of the precursors and the higher yields observed. On the other hand, these proteins might undergo rapid folding right after their synthesis, which interferes with (or hampers) the secretion process. Such interferences between protein conformation and SE were previously shown in E. coli and B. subtilis [32, 33] . Altogether, these results suggest that protein conformation rather than protein size is a major problem for heterologous protein secretion in L. lactis as well. It was clearly demonstrated that the secreted form of E7, a reportedly labile protein, can be stabilized by fusion to Nuc [20, 34] . Nuc is reportedly a stable protein and its use, as a fusion partner, does not affect its enzymatic activity. The production of the resulting chimerical protein is thus easy to follow. The cytoplasmic form of E7 was stabilized by the fusion to Nuc even when the production was induced in stationary phase ( Fig. 2A) , whereas cytoplasmic E7 alone was degraded (see below; Fig. 3 ). Thus, fusion to the stable Nuc could rescue E7 production in L. lactis and allowed higher protein yields compared to E7 alone [20] . Stabilization by fusion to Nuc was observed for several secreted proteins as well. First, a Nuc-E7 fusion on a pSEC backbone resulted in higher production yield although the SE was altered (Fig. 2B) . Fusion to the synthetic propeptide LEISSTCDA in a pSEC:LEISS:Nuc:E7 construction restored an efficient secretion yield [34] . Second, in an attempt to increase the protein yield of the secreted L7/L12, a fusion to Nuc (pSEC:Nuc:L7/L12) resulted in a 2.5-fold increase in production yield (Fig. 2B ) [19] . Recent results concerning the production of BLG provide a third example of yield enhancement by fusion to Nuc. A pSEC:Nuc:BLG construction allowed a 2-fold increase in BLG yields compared to pSEC:BLG [31] . These results show that Nuc is a stable carrier protein and has a protective effect on labile heterologous chimerical proteins by reducing its sensitivity to intracellular proteolysis. To our knowledge, Nuc is the fusion partner most commonly tested so far for stabilization in L. lactis. Bernasconi et al (2002) fused the Lactobacillus bulgaricus proteinase PrtB to BLG, which was subsequently stabilized by the PrtB carrier [13] . It is thus difficult to postulate any rule concerning the stabilization effect. Different results (i.e. no stabilization) could perhaps be observed with a different partner and thus could help to determine the mechanism of the stabilization effect. In biotechnological use of recombinant L. lactis strains for protein production, fusions can also facilitate purification (e.g. His-tag strategy). Protein fusion has also been successfully used to optimize the production of the two subunits of heterodimeric complexes as demonstrated with murine interleukin-12 in L. lactis [22] or with heterodimeric enzymes in E. coli [35] . In both cases, the resulting fusion had the expected properties. In other cases however, such fusions might dramatically interfere with the conformation of one or both of the proteins, which might be deleterious for the expected activity. Nevertheless, when L. lactis is used as an antigen delivery vector, fusions can be envisioned since it was demonstrated that both moieties of the chimerical protein are still recognized by the corresponding antiserum [10, 20, 34] and are immunogenic [36] . Several of the results mentioned above suggest that secretion could be an efficient way to escape intracellular proteolysis. This hypothesis was particularly tested in E7 production [20] . E7 was indeed degraded when intracellular production was induced in late exponential or early stationary growth phase (Fig. 3) . E7 production was then tested in a clpP deficient strain (ClpP is reportedly the major house keeping protease in L. lactis; [37] ) and in a dnaK deficient strain (DnaK is an intracellular chaperone that may promote proteolysis by maintaining the protein in an unfolded state; [38] ). In exponential or stationary phase cultures, no significant difference in E7 patterns was observed between wild type and clpP - (Fig. 3 ) or dnaK -(not shown) strains: E7 was equally degraded in the cytoplasm and remained unchanged in supernatants samples. Altogether, these results indicate that E7 intracellular proteolysis is ClpP-and DnaK-independent. Until recently, only two cytoplasmic proteases, ClpP and FtsH [39] , have been identified in L. lactis. The existence of a third, as yet unidentified protease was postulated by studies of a clpP mutant suppressor [40] . E7 may thus be a useful screening target to identify a putative L. lactis protease that, as suggested by our data, is activated in stationary phase. Besides the features of the precursor itself, these results also rise that host factors are involved in protein stability and SE (Fig. 4) . Research efforts are now focusing on the analysis of host factors involved in protein production and secretion by either directed or random mutagenesis in L. lactis [41] . Although L. lactis possesses a wide range of enzymes (peptidases, housekeeping proteases) dedicated to intracellular proteolysis, it possesses only one extracellular housekeeping protease (HtrA) [9] and its major extracel-lular scavenger protease, PrtP, is plasmid encoded [42] . Thus, a plasmidless strain does not present any protease activity in the medium. Better production yields could then be expected when secretion is used versus cytoplasmic production. These results give clues and provide the research workers with target proteins to study intracellular proteolysis and protein stability inside and outside the host strain. Such studies already led to the development of htrA deficient L. lactis strains. Heterologous protein secretion and anchoring in a htrA deficient strain allowed Fusion to Nuc rescue E7 in intracellular production and increase protein yields for the secreted forms of E7 and L7/L12 Native E7 production in wt L. lactis depends on growth phase Figure 3 Native E7 production in wt L. lactis depends on growth phase. E7 production and secretion were analyzed by Western blot from cultures induced at different times so that, 1 hour after nisin induction, the samples are harvested at exponential (OD 600 = 0.5-0.6, upper panels) or stationary phase (OD 600 = 1.5, lower panels). wt/pCYT-E7, NZ(pCYT-E7) strain (encoding native E7, cytoplasmic form). wt/pSEC-E7 NZ(pSEC-E7) strain (encoding the precursor preE7). Positions of E7 mature and precursor forms are given by arrows. C, cell lysates; S, supernatant fraction. ClpP is not involved in the intracellular degradation of E7 in L. lactis. Analysis by western blot shows that a strain of L. lactis deficient in the intracellular protease ClpP cannot rescue cytoplasmic E7 production. Induced cultures samples of wt L. lactis or L. lactis clpP mutant strain containing pCYT-E7 (clpP/pCYT-E7) or pSEC-E7 (clpP/pSEC-E7) taken at exponential-(upper panel) or stationary-(lower panel) phase. Stationary-phase higher protein stability at the cell surface for several heterologous proteins [10] . Current research works are now focusing on other host factors that affect protein production and secretion in L. lactis. L. lactis complete genome sequence analysis revealed indeed that the Sec machinery comprises fewer components than the well-characterized B. subtilis Sec machinery. Notably, L. lactis does not possess any SecDF equivalent and complementation of the lactococcal Sec machinery with B. subtilis SecDF results in better secretion yields as determined for Nuc reporter protein (Nouaille et al., submitted) . Random mutagenesis approaches also revealed that features of some cell compartment, such as the cell wall, play an important role in the secretion process [41] . Similar approaches allowed the identification and characterization of genes of unknown functions specifically involved in production yields of the secreted proteins in L. lactis (Nouaille et al., in preparation) . Many molecular tools are now available to direct heterologous protein secretion in L. lactis and the list of heterologous proteins produced in this bacterium is regularly increased. The reports where cytoplasmic and secretion production can be compared mostly show that secretion allows better protein yields compared to intracellular Schematic presentation of the molecular tools and the cellular events that can affect the production yields of heterologous pro-tein in L. lactis Figure 4 Schematic presentation of the molecular tools and the cellular events that can affect the production yields of heterologous protein in L. lactis. Thicknesses of the arrows are proportional to the final production yields. All the host factors involved in the cellular events are not identified and or characterized yet. SP, signal peptide (encoded in pSEC constructions), +Nuc, fusion between the protein of interest and the stable Nuc protein. production; and allow a better understanding of the protein production and secretion process in L. lactis. Future works should investigate the L. lactis capacities for protein modifications. For example, we showed that proteins that require a disulfide bond (DSB) to acquire their native conformation can be efficiently produced and secreted in L. lactis [5, 22, 27] . However, no equivalent of E. coli dsb or B. subtilis bdb, the genes involved in DSB formation, was found by sequence comparison in L. lactis. Similarly, other folding elements (i.e. PPIases, so-called maturases...) are still to be identified and the L. lactis capacities for post-translational modifications are still to be investigated. Altogether, these works will contribute to the development and the improvement of new food-grade systems for L. lactis [43] and should lead, in a near future, to the construction of lactococcal strains dedicated to high-level production of proteins of interest. The GRAS status of L. lactis and LAB in general, is a clear advantage for their use in production and secretion of therapeutic or vaccinal proteins."
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"Detection and characterization of horizontal transfers in prokaryotes using genomic signature"
"Horizontal DNA transfer is an important factor of evolution and participates in biological diversity. Unfortunately, the location and length of horizontal transfers (HTs) are known for very few species. The usage of short oligonucleotides in a sequence (the so-called genomic signature) has been shown to be species-specific even in DNA fragments as short as 1 kb. The genomic signature is therefore proposed as a tool to detect HTs. Since DNA transfers originate from species with a signature different from those of the recipient species, the analysis of local variations of signature along recipient genome may allow for detecting exogenous DNA. The strategy consists in (i) scanning the genome with a sliding window, and calculating the corresponding local signature (ii) evaluating its deviation from the signature of the whole genome and (iii) looking for similar signatures in a database of genomic signatures. A total of 22 prokaryote genomes are analyzed in this way. It has been observed that atypical regions make up ∼6% of each genome on the average. Most of the claimed HTs as well as new ones are detected. The origin of putative DNA transfers is looked for among ∼12 000 species. Donor species are proposed and sometimes strongly suggested, considering similarity of signatures. Among the species studied, Bacillus subtilis, Haemophilus Influenzae and Escherichia coli are investigated by many authors and give the opportunity to perform a thorough comparison of most of the bioinformatics methods used to detect HTs."
"It is now widely admitted that actual genomes have a common ancestor (LUCA, Last Universal Common Ancestor). Their current diversity results from events that have modified genomes during evolution. While some of these events happen at the nucleotide level (point mutation, indel of few nucleotides), others [strand inversion, duplications, repetitions, transpositions and horizontal transfers (HTs)] may concern significant parts of the genome. It has been postulated that HTs (exchange of genetic material between two different species) were very frequent during the first stages of evolution and are essentially subsisting nowadays in prokaryotes (1) (2) (3) (4) . As a consequence, the detection of HTs appears crucial to the understanding of the evolutionary processes and to the qualitative and quantitative evaluation of exchange rate between species (5) (6) (7) (8) (9) . The recent complete sequencing of several genomes allows to systematically search for the presence of DNA transfers in species, especially in prokaryotes where the probability of occurrence is higher (10) (11) (12) (13) (14) . It has been reported in particular that (i) HTs in bacteria account for up to 25% of the genome (8, (14) (15) (16) ; (ii) archaebacteria and non-pathogenic bacteria are more prone to transfers than pathogenic bacteria (15, 16) ; and (iii) operational genes are more likely transferred than genes dealing with information management (15) (16) (17) . The HT concept has been originally coined to explain the dramatic homologies between genes of unrelated species (18, 19) . An 'unusual' match is subsequently the criteria for the detection of HTs (20, 21) . While this approach allows detection of gene transfers with only a partial knowledge of genomes, it requires the sequencing of homologous genes in a number of species and consequently cannot be used for HT screening. Genes from a given species are very similar to one another with respect to base composition, codon biases and short oligonucleotide composition (15, 16, (22) (23) (24) . As a general rule, usage of oligonucleotides varies less along genomes than among genomes (24) (25) (26) (27) . In addition, it has been observed that transferred DNA retains (at least for some time) characteristics from its species of origin (8, 14) . These particularities are used alone or in conjunction to detect DNA transfers between species (8, 12, 13) . Transferred DNA is consequently detected on the basis of some of its singularities with respect to the sequence characteristics of the recipient species. However, these techniques suffer several drawbacks and weaknesses (28) (29) (30) that led us to consider generalizing the above approach for the screening of atypical regions in sequences. In fact, the genomic signature that accounts for all possible biases in DNA sequences has been shown to be speciesspecific (26, 27, 31, 32) . The signature is approximately invariant along the genome in such a way that the species of origin of DNA segments as small as 1 kb could be identified with a surprisingly high efficiency by means of their signatures (25, 27) . As a consequence, the sequence signature may be most often (at least in bacteria) considered a valuable estimation of the genomic signature. Assuming that (i) transferred DNA fragments exhibit signature of the species they come from and (ii) recipient and donor signatures are different, the screening of local variations of signature along genomes is expected to reveal regions of interest where HTs might be located. In addition, the status of HT is strongly suggested if the signatures of these regions of interest are found close to the signature of other species. The sequence signature is defined as the frequencies of the whole set of short oligonucleotides observed in a sequence (26, 31) . It can be easily obtained thanks to a very fast algorithm derived from the Chaos Game Representation (CGR) (33) , which allows coping with a 1 Mb sequence in a few seconds on a laptop computer. Signatures may be visualized as square images where the color (or gray level) of each pixel represents the frequency of a given oligonucleotide (called word thereafter) (31) (for examples of signatures, see Supplementary Materials 2, 4 and 6). DNA sequences are gathered from GenBank. The genomes of 22 prokaryotes are scanned for HTs, B.subtilis, E.coli and H.influenzae genomes being given a special attention to illustrate our approach. In particular, B.subtilis and E.coli provide valuable benchmark thanks to the set of previous works addressing that very issue (12, 14, 16, (34) (35) (36) (37) . Signatures of about 12 000 species are obtained from genomic sequences longer than 1.5 kb. Sequences derived from the same species are concatenated for accuracy purposes. Species from the three domains of life, archaea ($260 species), bacteria ($3950 species) and eukarya ($6750 species) as well as viruses ($1300 species), are represented for a total amount of 1.0 Gb. The detection of atypical regions is based on the observation of deviation of local signatures (i.e. signature of small fragments of DNA) from the genomic signature of the recipient species. Genomes are consequently sampled by means of a sliding window with an appropriate size. In fact, it would be interesting to have windows the smallest as possible for highest sampling accuracy. However, intra-genomic variability of signature increases for small windows. In addition, variability depends on species and word length. Base composition (1-letter word), 2-and 3-letter words are poorly speciesspecific: they do not allow a good discrimination between species (25, 27) . As a general rule, the longer the words (up to 9-letter long), the higher the specificity of the signature (25, 27, 31) . However, counts of long words in small windows are too low to allow a reliable estimation of the parameters. In our hands, the analysis of 4-letter words in a sliding window of 5 kb (with a 0.5 kb step) offers a good trade-off between reliability of count, file size and computational charge, whatever the species. In addition, a double-strand signature (called local signature thereafter) is computed for each window to get rid of variations induced by strand asymmetry (38) (39) (40) (41) (42) . For illustration purposes, local signatures are developed as vertical vectors and stacked together in genome order to give an overall picture of word usage variations along each genome. In such plots, horizontal lines show the variation in frequency of words along the genome, whereas local changes in word usage appear as vertical breaks ( Figure 1 ). Figure 1 . Signatures (4-letter words and 5 kb windows) along genome for Clostridium acetobutylicum, Deinococcus radiodurans and Mycobacterium tuberculosis. In this kind of displays, lines represent the frequency of words along genome, columns represent signature of windows. Considering that the greatest part of the genome is speciestypical, the signature of the recipient species might have been estimated from the analysis of the whole sequence. Although the vast majority of local signatures look mostly the same (believed to be instances of the recipient species signature), some of them may greatly differ. In order to avoid potential biases linked to these outliers, it has been subsequently decided to select typical local signatures on the basis of their similarities, observed after clustering. The underlying idea is that typical local signatures aggregate in few large groups, whereas outliers are found in small complementary groups at a great distance from the recipient genome signature. Groups were consequently determined with the K-means clustering tool, using every scheme of clusters between 3 and 8 for each species. Finally, the best scheme of clusters was obtained by a decision tree-based partition [CART algorithm (43) ]. The purpose of the CART algorithm is to predict values of a categorical dependent variable (clusters of local signatures in this work, each signature being characterized by its distance to the estimated genomic signature) from one or more continuous and/or categorical predictor variables [the different clustering schemes (3-8 clusters) in this work]. The CART algorithm thus provides an optimal split between groups collecting signatures close to the estimated recipient genome signature and the others groups. For each species, a clustering scheme is selected (e.g. the 5-group clustering) and a partition offered (continued example: group 2 and 3 on one side; 1, 4 and 5 on the other). The recipient species signature is subsequently calculated as the mean of the signatures of the groups belonging to the partition with the smallest distance to the estimated genomic signature. Comparison of signatures is made possible, thanks to an Euclidian metric, accounting for differences in word usage. It must be pointed out that distances between signatures are calculated for high dimensional data (256 dimensions corresponding to the 256 different 4-letter words) and are consequently subjected to the so-called 'concentration of measure phenomenon' (44) . All distances in a high dimension space seem to be comparable since they increase with the square root of the dimension of the space, whereas the variance of their distribution remains unchanged. In fact, the radius of the hyper sphere holding 99% of the signatures of our database is only seven times the nearest neighbor distance (smallest distance between two species). Small differences in distance may consequently be considered highly significant. For each species, a set of recipient-specific distances is obtained, every local signature belonging to the large clusters being given a distance to the host signature. In order to select outlying signatures, a cut-off distance is chosen on the basis of the distribution of distances observed for each species. It appears that the 99% percentile offered a good trade-off between sensibility and specificity for outlier detection (for impact of the threshold on detection of atypical regions, see Results). Most signatures from minority clusters are detected in this way. Isolated signatures are detected as well, while very few signatures from the recipient species clusters are selected (1%). Outliers together with the flanking regions on the genome are later on reanalyzed with smaller window and step (1/10 th of the original size typically) in order to more accurately determine their limits, when signal-to-noise ratio allows it. Finally, the gene content of all detected regions is analyzed with the help of species dedicated databases [Genome Information Broker, http://gib.genes.nig.ac.jp/]. A BlastN search (GenBank, default settings) is carried out for each atypical region in order to identify the origin of potential HTs if homology is high enough. Search for the origin of atypical regions About 12 000 species (including chromosomal, plasmidic, mitochondrial and chloroplastic DNA) from GenBank are found eligible for a genomic signature. Given the signature of an atypical DNA fragment, species with a close signature might be considered as potential donors. Such a screening is performed for every atypical region of the 22 species under consideration. The first five nearby species are retained when their distance to the outlier was donor-compatible. A total of 22 genomes are screened for atypical regions (Table 1 and Supplementary Material 1). On the average, the 6-cluster scheme offers the best partition. However, in a single case (Aeropyrum pernix), nine clusters are required. In general, a single cluster is devoted to rRNA. The mean distance of windows to host varies over species from 121 to 145 (mean = 132, coefficient of variation = 3%). It is tightly correlated (P-value for the Pearson correlation coefficient <10 À4 ) with the cut-off distance that varies from 178 to 289 (mean = 234, coefficient of variation = 14%). Such large variations can hardly be explained on the mere basis of statistical fluctuations. As already observed (31, 45, 46) , variation of oligonucleotides usage along genome depends on species and can consequently be considered as a species property. Segmentation quality of atypical regions can be tested using rRNA genes. About 94% of rRNA is detected as atypical ( Table 1) . Borders of rRNA genes are accurate to within 130 nt (0.5 kb window and 50 bp step, threshold 99%). Meanwhile, adjacent tRNAs are identified as well. As a general rule, it can be concluded that rRNA has a specific signature that is consistently at variance with the host signature. In this context, it is worth noticing that rRNA and the remaining outliers lie at comparable distances from the species they belong to, but they are clearly different from one another, rRNAs being consistently found in their own cluster. The percentage of RNA-free outliers (at the nucleotide level) varies from 1.3 to 13% as a function of species (threshold 99%, Table 1 ). B.subtilis shows the highest percentage of atypical regions, whereas Pyrococcus abyssi has the lowest. Percentages among species are found correlated with the cut-off distance: the higher the cut-off distance, the lower the percentage of outliers (P = 0.007). In fact, a high cutoff distance takes place in species that display a high intragenomic variability, also expressed by a high mean distance to the host (Table 1) . Whether the actual percentage of atypical DNA is an intrinsic property of the species or a mere consequence of the resolution power of nucleotide biases-based methods remains consequently an open question. In addition, as already observed (13, 14) , the percentage of outliers is significantly higher for longer genomes (P = 0.004), whereas the cut-off distance is not related to the length of the genome (P = 0.69). The mean cut-off distance for the 22 species is 234 (Table 1) . This value is chosen to select credible donors. About 50% of atypical regions are subsequently given credible donors (Supplementary Material 1). Each species has it own set of (Table 1) . Many plasmids and viruses are also found in agreement with the known molecular mechanisms of horizontal transfer (Table 1 and Supplementary Material 1). A clustering with three classes allows assessing the signature of B.subtilis. The most populated class (collecting 84% of the segments) is chosen to represent B.subtilis. For this subpopulation, the mean distance (arbitrary unit) to the recipient (centroid of the class) and the cut-off distance are 126 and 204, respectively ( Table 1 ). Runs of contiguous outlying windows sharing the same cluster are considered as single transfer events. As a consequence, 58 regions (Figure 2a and Supplementary Material 2) fall beyond the cut-off distance and are thus potential candidates for hosting foreign DNA (for a segmentation of the B.Subtilis genome in terms of genes, see Supplementary Material 3). Figure 2b illustrates the accuracy of segmentation of an atypical region obtained by using a sliding window of 0.5 kb with a 50 bp step. rRNA genes make up $1.1% of B.subtilis genome ( Table 1) . All rRNA genes are found in the outlier population. In addition, all windows containing rRNA are assigned to a specific cluster. In fact, it is known that rRNA has its own signature, which is at variance from the host signature (12) . rRNA genes account for 7% of the outliers (tRNAs are not considered in this study, because their size is too small to generate a significant deviation from the host signature if they are isolated). A total of 86% of the B.subtilis genome should be considered as B.subtilis typical (Table 1) . When looking for the origin of B.subtilis segments in the 12 000 signature database, B.subtilis appears in the 10 first potential donors for 84% of the whole set of 5 kb sequences that can be derived from its genome. This result confirms that segments having signatures belonging to the predominant clusters are good representatives of the recipient species signature. The 49 rRNA-free atypical regions vary in size from 1.5 to 135 kb and make up 13% of the total genome (Table 1) . About 50% of atypical regions are less than (or around) 6 kb long. Distances of outlier from first potential donor often fall within the intra-genomic range ( However, in some instances, the outlier-to-donor distance is too great to consider the 'closest' species as potential donor. In contrast, unusual small values deserve a specific attention. In particular, the very small distance between bacteriophage SPBc2 and '2150751-2285750' atypical region (d = 2) allows to spot the part of B.subtilis genome where bacteriophage SPBc2 is incorporated (12, 47) . Other regions in the genome are also found similar (in terms of signature) to bacteriophage SPBc2. Most of them correspond to bacteriophages, imbedded in B.subtilis genome, whose free forms are not sequenced (12, 47) . Observed similarities with SPBC2 are, however, expected since signatures of phages usually share some characteristics with the species they infect (48) . The SPBc2 sequence is the only foreign sequence identified in B.subtilis, using homology as criterion (BlastN, with parameters set to default). In fact, Blast analysis of B.subtilis outliers leads to contrasted results. Besides SPBc2 and 7 out of 9 prophages imbedded in the genome, the only atypical regions identified are those containing the 30 rRNA genes coded in B.subtilis genome. The only few genes that are homologous to parts of atypical regions are found in species belonging to the Bacillus genus. It is interesting to note that no house-keeping genes (except rRNA) are detected in atypical regions. In fact, a great number of genes in atypical regions (except bacteriophage genes and rRNA) have no known function. A clustering with five classes is required to determine the recipient species signature of H.influenzae. The three most populated classes (collecting 94% of the segments) are chosen to calculate the H.influenzae signature. Mean distance to host and cut-off distance is subsequently found equal to 130 and 239, respectively (Table 1) . Similarly to B.subtilis, one cluster (1.5% of H.influenzae genome) is devoted to the 18 rRNA gene copies (Table 1) . A total of 91% of rRNA is labeled atypical and account for 29% of the outliers. Analysis of Table 1 shows that 95% of the H.influenzae genome should be considered as H.influenzae typical. In fact, H.influenzae is one of the 10 first potential donors for 92% of all 5 kb sequences that can be derived from its genome. As already observed for B.subtilis, the concordance of these two percentages corroborates the partition procedure used for the selection of typical/atypical fragments. The 13 rRNA-free atypical regions vary in size from 1.5 to 19.5 kb and make up 3.3% of the genome (Table 1 , Annex 4 and Figure 3 , see Annex 5 for a segmentation of the H.influenzae genome in terms of genes). About 50% of atypical regions are less than (or around) 2.5 kb long. Numbers for H.influenzae are clearly at variance with those for B.subtilis: a smaller percentage of the genome qualifies as atypical and the average size of atypical regions is also smaller. This result is examined below in the context of intra-species signature variability (see Discussion). A clustering with six classes is required to determine the recipient species signature of E.coli. The main features are summarized in Table 1 . The potential donors of the 84 RNAfree atypical regions are given in Annex 6 (for a segmentation of the E.coli genome in terms of genes, see Annex 7). It is worth noticing that 56% of E.coli potential donors belong to the Enterobacteriales family. Segmentation in terms of genes is displayed in Annex 7. The analysis of this genome is particularly useful for the comparison with literature (see below). Numerous approaches for detecting horizontal gene transfers have been proposed in the last 2 decades. Phylogenetic trees of protein or DNA sequences, unusual distribution of genes, nucleotide composition (including codon biases) are some of the HT features that are considered within the framework of these models (16, 34) , Hidden Markov Models (HMMs) (12, 14, 35) and Factorial Correspondence Analysis (FCA) (37) are some criteria that are currently employed. Each of the resulting models has its own advantages and caveats (28) (29) (30) . As it has been recently pointed out by Ragan (49) and Lawrence and Ochman (50) , each approach deals with a particular subset of HTs, being for example more efficient for detecting recent transfers, or more effective for the detection of ancient HTs. Our approach, which is clearly based on oligonucleotide composition, assumes that different species have different signatures but does not rely on any other assumption. It is not surprising, therefore, that the genomic signature approach provides results (in terms of % of DNA transferred) in reasonable agreement with those proposed by Garcia-Vallve (16) and Nakamura et al. (14) for the 22 species that were analyzed in common. Correlations between percentages of HTs found by these three methods are highly significant Two species are extensively studied for HT content: B.subtilis (five methods including ours) and E.coli (six methods including ours). H.influenzae is also analyzed by Garcia-Vallve (16) and Nakamura (14) . Comparisons of methods are presented in Tables 2-4 and detailed in Supplementary Materials 3, 5 and 7. A voting procedure (majority rule) has been implemented to determine the status of genes with respect to atypicality. For that task, our initial analysis is converted in terms of genes (Supplementary Materials 3, 5 and 7). Degree of agreement between methods is subsequently observed using the statistical Kappa coefficient (51) . Kappa measures the degree of agreement on a scale from minus infinity to 1. A Kappa of one indicates full agreement, a Kappa of zero indicates that there is no more agreement than expected by chance and negative values are observed if agreement is weaker than expected by chance (a very rare situation). (14, 13, 11, 13 and 15%, respectively). The number of detected genes per method is close, ranging from 457 for Nakamura (14) to 599 for this work (median 537). Detailed votes are given in Table 2 . Among the 4100 genes of B.subtilis genome, 1011 genes are detected by at least one method (about 25% of B.subtilis genes). The number of 'single vote' genes ranges from 116 for Garcia-Vallve (16) to 47 for Nicolas (12) . A total of 470 genes make up the majority consensus set and we detected 453 of them, which is the best score of the five methods. The best agreement with the majority consensus (in terms of Kappas) is reached by Nicolas (12), followed by our method and Moszer (36) ( Table 2 ). Our method gets the best agreement with Nicolas (12) and the worst with the other HMM method used by Nakamura (14) (pairwise Kappa comparison, Table 2 and Supplementary Material 3). In fact, Nakamura approach is at variance with every other approach (14) . It gets the lowest Kappa with the Garcia-Vallve (16) Hayes (35) Lawrence (34) Nakamura (14) Medigue (49) This work majority consensus or with whatever other methods. From Table 2 , the probable number of HT genes in B.subtilis would range from 230 to 1011 with a 'reasonable' estimation around 470 corresponding to the majority consensus. It is to be noted that our method is unable to find two genes that are detected by every other methods (Supplementary Material 3) . These genes are 338 and 236 nt long, respectively, as compared with 2500 nt, the median size of atypical regions detected by our method (Table 1) . Clearly, our method is not appropriate for detecting short isolated atypical genes. H.influenzae. Garcia-Vallve (16), Nakamura et al. (14) and we are the voters concerned with the analysis of the H.influenzae genome (Supplementary Material 5 and Table 3 , H.influenzae). The originality of results obtained by Nakamura (14) is the salient feature of this comparison. The number of detected HT genes is more than twice higher for Nakamura et al., whereas the part belonging to the majority consensus is the smallest ( Table 3) . Eleven genes are detected both by Garcia-Vallve and Nakamura (14, 16) but not by our method; however, the small number of voters precludes any specific comment in this respect. The probable number of HT genes in H.influenzae would range between 11 and 273, with a 'reasonable' estimation around 60 (majority consensus of 57) ( Table 4 ). The results obtained by Hayes and Borodovsky (35) are clearly at variance with the others (Table 4 ). Although the proportion of claimed outliers is within the range of published numbers for E.coli (14, 16, 24, 34, 35, 37) , 37% of them are method-specific, and the agreement with other methods is weak (Table 4 ). Hayes and Borodovsky have obviously developed an approach based on HMM dealing with specific outliers. Lawrence and Ochman (34) also get a poor rating especially because they detect about twice as many genes as the other authors do (Table 4) . It is worth noting that if the cut-off distance for our method is lowered, i.e. 95% instead of 99% for instance, some of the 'single vote' genes are dug out (for details about the impact of the cut-off distance, see Supplementary Material 7). Meanwhile, the percentage of outliers as reported by our approach rises to 20% and the percentage of 'single vote' genes reaches 24%. As expected, a high cut-off distance provides few single vote genes at the risk of missing some potentially transferred genes. Lowering the cut-off increases the proportion of single vote genes with the advantage of detecting most of the potential transfers (Supplementary Material 7) . There is obviously a continuous grading in gene 'atypicality'. It is suggested to first consider most 'consensual' genes as potential HTs and then apply amelioration models to explain the grading. It is difficult to assess the relevancy of proposed donors, because genes detected as potential HT have generally undergone amelioration (8) . The comparison of recently diverged genomes (species or strains) provides the opportunity to find recent HTs, for which corresponding homologous genes in the donor species may be detected (52) . Such a study is performed for five E.coli strains (two K12 strains: E.coli MG1655, E.coli W3110, one uropathogenic strain: E.coli CFT073, two enterohaemorrhagic strains: E.coli O157-H7 RIMD 0509952, E.coli O157-H7_EDL933) and two Shigella flexneri strains (S.flexneri 2a 2457T, S.flexneri 2a 301). These seven strains/ species have recently diverged, genome sizes are different and the proportion of horizontally transferred genes varies from one strain/species to another (14, 52) . For instance, only $40% of the non-redundant set of proteins is common to E.coli strains CFT073, 0157-H7 EDL 9333 and MG1655 (53) . These strains/species can be clustered in four groups with respect to phylogeny (Table 5) . Two criteria are used to searching for 'recent horizontally transferred genes': atypical regions (window size 1 kb, step 0.5 kb) (i) must have a signature that differs greatly from that of the host [distance to host must be at least >325, 2.5 times the E.coli intrinsic mean distance (Table 1) ] and (ii) must be present in a limited number of strains/species to ascertain their recentness. In fact, outliers meeting the first criterion generally aggregate into several heterogeneous clusters (K-means clustering) that usually include samples from each strain/species. In some instances, however, some strains/species were absent from the cluster. It was subsequently considered that the corresponding regions might have been recently acquired by the relevant strains/ species. Table 5 shows a selection of potential recently transferred genes. Each cluster of atypical regions contains genes present in a specific set of strains. Some atypical genes are strainspecific, some are only absent in the non-pathogenic K12 strains and intermediate situations are also encountered. FASTA and Blast searches confirm that these genes are absent from some of the tested strains as already observed in the analysis complete genomes (53) (54) (55) . In a large number of cases, we are able to find a well-conserved homologous gene in another species (Table 5) . It is interesting to note that some of the suggested donors using our 12 000 signature database are in agreement with the species found by alignment methods. When no homologous gene is found, the proposed donors give credit to the known mechanisms of gene transfer (bacteriophages or plasmids) ( Table 5) . It is worth noticing that most of the selected genes that are absent in K12 strains are involved in the pathogenicity of the other strains (52) . E.coli 0157-H7 is the strain exhibiting the greatest number of genes absent in K12 strains [about 1400 (54) ]. It has the greatest number of genes for which no homolog can be found (Table 5) . Moreover, we are unable to propose a donor for a great part of these genes (Table 5) . Many selected genes for E.coli 0157-H7 lie in the Ter region of the genome (between positions 2 000 000 and 2 500 000) in agreement with the published results (56). We have observed that most genomic regions are typical of the genome they belong to, using the signature as endpoint. Considering that the genomic signature is species-specific, atypicality of a region in terms of oligonucleotide usage has been promoted as a criterion for the detection of HTs. However, atypicality-based methods suffer several caveats that reduce their effectiveness in such a way that only a part of HTs can be detected. In fact, transfers between species with close signatures cannot be detected: significant differences between characteristics of transferred DNA and recipient species DNA are required. For similar reasons, HTs that were drastically ameliorated following their introduction cannot be detected either (8, 14) . The most stringent constraint, however, results from the size of the screening window. On the one hand, ideally, the best signal-to-noise ratio would be obtained when windows and HTs have a comparable size. On the other hand, the window size must be large enough to provide significant word counts, a requirement that strengthens with the size of the words under consideration and the intrinsic variability of the genomic signature along the genome. All together, the trade-off that has been implemented in this paper allows detecting atypical regions as small as 1 kb. In fact, rRNA regions sharing this characteristic were consistently detected. It must be pointed out that smaller fragments can be eventually detected if their signatures are radically atypical. G+C% atypicality has often been considered as criterion for detecting HTs (8, 24) , but this approach suffered several drawbacks (28) (29) (30) . It is to be noted that our signature-based method detects regions for which the G+C% lies within one standard deviation from the mean G+C% of the species (for instance, regions 2675251-2676250 in B.subtilis or 534751-535250 in H.Influenzae, see also Supplementary Materials 2 and 4). As already observed by Nicolas et al. (12) for B.subtilis, rRNA has definitely an atypical signature. It is systematically classified as outlier, whatever the species (Table 1) . Although transfer of rRNA from one species to another is unlikely (11, 57) , it cannot be firmly ruled out. However, it is clear that the atypical signature of rRNA does not imply that they are horizontally transferred. The signature approach has an interesting property (that it shares with HMM) (7, 12, 28) : detection is not bound to any specific function in the genome. In contrast with most other methods, the signature approach not only detects genes, but whole transferred regions as well, in agreement with the described mechanisms of DNA exchange between species. It is to be noticed that the method allows detecting several atypical non-coding regions (Supplementary Materials 3, 5 and 7). One major difference between HMM and signature method lies beyond the time required for the learning process, in the few resources that HMM can mobilize to deal with a short 'one of its kind' HT. On the other hand, HTs shorter than 1 kb can hardly be detected by a signature-based approach. An innovative HT detector is likely to result from an adequate fusion of both methods. Several factors contribute to the efficiency of the search for donors. Of course, distance between putative HT and donor signatures is essential. Accuracy of signatures, linked to the length of available sequences, density of signatures in the 'vicinity' of HT, amount of amelioration sustained by HT during its presence in the host are also of importance [P. Deschavanne, S. Lespinats and B. Fertil, unpublished results; (25, 27, 31) ]. Distance between the signature of a putative HT and the closest species varies to a large extent, but usually the shortest ones fall within the intra-genomic range ( Table 1 , Supplementary Materials 1, 2, 4 and 6) . In some cases, the distance between the closest donor signature and the atypical segment signature is so great that no potential donor can be proposed (Supplementary Materials 1, 2, 4 and 6) . When strong similarities between a given DNA sequence and a foreign species are observed, the hypothesis for an underlying transfer is highly strengthen. However, the 'true' donor has to be previously sequenced and included in our bank of signatures to allow such a situation to occur. Moreover, we must take into account the intrinsic variability of short DNA segment signature (which is a function of their size, but also species-specific) when compared with the signature of a complete genome or any other large species sample (25, 27, 31) . In the present state, our signature database is in no way representative of the diversity and richness of life. However, it must be noticed that there is already an obvious structure (in terms of distances between signatures) expressing taxonomy relationships between species in our signature database (31, (58) (59) (60) (61) . Related species are often found close to one another. Clusters of potential donors may consequently provide pertinent information about the origin of HTs. The diversity of signatures of putative HTs that can be observed for most of the species analyzed in this paper reveals the multiplicity of transfer events and donors (Supplementary Materials 2, 4 and 6). However, several outliers, not necessarily neighbors in the genome, are given the same set of potential donors (Table 1 , Supplementary Materials 1, 2, 4 and 6). In general, the potential donors belong to few sets of taxonomically close species (Table 1 ) and share the biotope of the host (Supplementary Materials 1, 2, 4 and 6). For instance, B.subtilis, H.Influenzae and E.coli live in distinct biotopes; their potential donors do so as well. It is particularly encouraging to find that most of the potential donors that our approach has pointed out have had the opportunity to exchange DNA material with the recipient species. Numerous viruses and plasmids qualify as potential donors (Tables 1 and 5 , Supplementary Materials 1, 2, 4 and 6 ). It is not really surprising since they are known as HT vectors. They are often totally or partially inserted together with transferred genes in the host genome (14) . Some atypical DNA segments are particularly peculiar. They are isolated, have a specific signature (distances from neighbors are great), so that they cannot be given a credible set of donors (Supplementary Materials 1, 2, 4 and 6) . Lack of data in the search domain, shift of signature features after a substantial amelioration process, structural constraints serving special functions or roles (14,62) (as it is for rRNA coding regions) are some of the tracks that remain to explore in these circumstances. It would be interesting to localize the region the transfer may come from when the complete genome of the donor is available. However, homology (at the DNA level) is not a pertinent criterion for the comparison of sequences as soon as amelioration has taken place (8, 14) . In fact, homology is sometimes weak, e.g. between genes of Escherichia and Salmonella although these species have 'recently' diverged (34) . It is clear that a more powerful search for the origin of putative HTs would have to embody models of amelioration [such as the one designed by Lawrence and Ochman (8) ]. When searching for very recent horizontally transferred genes, in different strains of a species for instance, it was possible to find a great homology between detected genes and some genes from other species (Table 5 ). In numerous cases, the selection of donors is consistent with FASTA results ( Table 5 ). This confirms the pertinence, beyond the similarity of signature between putative HTs and donors, of the proposed method to retrieve the species of origin of a transferred region. It seems that the search for origin of HTs on the basis of genomic signature is a powerful approach to understand some of the mechanisms of evolution (13, 63) . Oligonucleotide usage is known to be species-specific and to suffer only minor variations along the genome (25, 27) . Considered together, these properties allow searching for atypical local signatures that may point out DNA transfers. Results obtained with the 22 genomes analyzed in this paper are found in good agreement with literature (Tables 2-4 , Supplementary Materials 3, 5 and 7) (12, (14) (15) (16) 24, 34, 35) . The species specificity of signature allows searching for donor species. Quite often, sets of donor species with common taxonomic features are obtained. With the help of environmental considerations, it is subsequently possible to identify (or collect clues about) potential donors. The search for donor makes use of non-homologous sequences. Partially sequenced species become consequently eligible, inasmuch 1.5 kb of the genome is available (25, 27) . Thanks to the exponentially growing rate of nucleotide databanks, the search for donor species by means of the sequence signature will turn more and more pertinent and fruitful in the future. In this context, it is worth noticing that computational power is clearly not an issue since the CGR algorithm described in this paper is fast and of 0 order (calculation time is proportional to the number of nucleotides). Several methods are proposed to look for HTs. The signature method, based on different hypotheses, is complementary to those already described. It seems that each method detects preferentially certain types of HTs (49, 50) . In agreement with many authors (1, 16, 49, 50, 64) , it appears that the conjunction of several methods is required to obtain an overview of HT extent in a genome. The signature method described in this paper generalized many approaches that ground the detection of outliers on the basis of the bias in oligonucleodides. The strong species specificity of the signature not only allows detecting various kinds of outliers but also provides clues about their possible origin. Obviously, the detection of HTs remains an open question; a consensus has still to emerge. Additional materials and experimentation with the genomic signature are available from the GENSTYLE site (http:// genstyle.imed.jussieu.fr)."
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"Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays"
"Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis."
"Oligonucleotide microarrays are a powerful technological platform for large-scale screens of common genetic variation and disease-causing mutations (1) (2) (3) (4) (5) . In most published studies (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) , oligonucleotide microarrays are designed to screen specific sequence tracts, up to megabases in length (11, 15, 22, 23) , for all possible single nucleotide substitutions. With some exceptions (24) (25) (26) (27) (28) (29) (30) (31) , the same emphasis was not placed on identifying all possible small insertions and deletions in the heterozygous state. Nevertheless, it is crucial to detect such small insertions and deletions since they can play a major role in inactivating or altering gene function by disrupting functional elements (e.g. splice junctions, cis-acting elements and open reading frames) and also represent another class of common genetic variation. Two fundamental approaches are commonly used to analyze data sets from oligonucleotide microarrays tailored to identify genetic variation in specific DNA segments purely by hybridization (1, (3) (4) (5) 9) . One approach involves identifying statistically significant gains of target hybridization signal to oligonucleotide probes complementary to specific sequence variants (9) . In theory, the gain of signal approach has the advantage of both detecting the presence of genetic variation and identifying the nature of the sequence change in the target. However, it is not feasible to screen for virtually all possible insertions and deletions due to the overwhelming The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org number of mutation-specific probes needed for this analysis. Furthermore, little effort has been made to systematically access the hybridization properties of probes complementary to these small insertions and deletions. The second approach involves identifying losses of hybridization signal to perfect match (PM) probes that are fully complementary to the DNA segment of interest (8, 25, 27, 30, 31) . In theory, the loss of signal approach allows one to screen for all possible sequence changes, including insertions and deletions, that cause a given target nucleic acid sequence to contain mismatches with specific PM probes. However, this necessitates the sequencing of specific DNA regions to identify the nature of the sequence changes (8, 25, 27, 30, 31) . Thus, a combination of the gain and loss of hybridization signal analysis could provide the most robust means of identifying and characterizing mutations using non-enzymatic oligonucleotide microarray assays. Here, we analyze the specificity and reproducibility of nucleic acid hybridization to oligonucleotide microarrays used in the large-scale mutational analysis of the ATM (ataxia telangiectasia mutated) gene that is responsible for autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition and is also commonly mutated in certain lymphoid malignancies (32, 33) . These microarrays include 25mer oligonucleotide probes complementary to all possible single base substitutions and insertions as well as one and two base deletions on both strands of the ATM coding region. This provides the first comparative analysis of the hybridization properties of substitution, insertion and deletion probes in an oligonucleotide microarray-based mutational analysis of a large gene. A series of 120 DNA samples derived from biopsies of lymphoma patients were previously screened for all possible ATM mutations using oligonucleotide microarrays (30) . Here, we have selected a total of 68 samples that showed robust amplification signals in all 62 coding exons for further analysis (30) . A total of 17 unique mutations, each in a one-to-one mixture with wild-type sequence, occurred once in these samples. The impact of any given mutation in a single sample is minimal given that 67 other samples with wild-type sequences in the region encompassing a given mutation are included in this analysis. Several single nucleotide polymorphisms (SNPs) were present multiple times: 735 C/T, 2572 T/C and 4258 C/T in two samples; 3161 C/G in four samples; and 5557 G/A in five samples. Likewise, these SNPs have a minimal effect on our global analyses given the large number of samples and bases interrogated in this study. As previously described (30) , individual ATM coding exons were amplified from genomic DNA using primers containing T3 and T7 RNA polymerase tails, pooled, and then in vitro transcribed using T3 or T7 RNA polymerase to create biotinlabeled sense and antisense strand targets, respectively. Fluorescein-labeled reference target was made using genomic DNA from an unaffected individual. Reference and test sample targets were fragmented, diluted in hybridization buffer [3 M TMA-Cl (tetramethylammonium chloride), 1· TE, pH 7.4, 0.001% Triton X-100] and hybridized to the ATM microarrays as described previously (30) . Afterwards, the microarray was stained with a phycoerythrinstreptavidin conjugate and digitized hybridization images from both reference and test targets were acquired using the Gene Array Scanner (Hewlett Packard, Palo Alto, CA) equipped with the appropriate emission filters. Custom software was used to quantify hybridization signals for each probe and subtract background hybridization signals. We exclusively focused on raw data from the biotin-labeled test targets since they provide approximately seven times the hybridization signal of the fluorescein-labeled wild-type reference target in this system (28) . This enhanced signal provides greater sensitivity toward detecting weak hybridization. For each sample, for each base and for each potential type of mutation (i.e. substitution, one or two base deletion or one base insertion), the specificity was calculated as the ratio of the PM probe hybridization signal of the wild-type target to their cognate insertion, deletion or single base substitution probes on each strand. The logarithm of these ratios was plotted as a function of the position within the gene. To illustrate the special patterns and to smooth out random variation, running averages of data from 10 bases were used. To capture the variability, at each base, the sample-to-sample standard deviation was again calculated using data derived from a running average of 10 bases for each sample. To estimate the mean hybridization specificity for each type of mutation, the geometric mean (i.e. the antilog of the average of the logged ratios) over all bases and over all specimens was calculated (Table 1) . To further examine the variability of the specificity ratios, the coefficient of variation (cv) was calculated in two ways. The cv is the ratio of the standard deviation divided by the mean; it is useful for understanding the amount of variability relative to the magnitude of the mean or typical value. For the intra-sample cv, the cv was calculated for each of the 68 samples (using the running average of 10 at each ATM base) and the average of the 68 coefficient of variations was taken. For the inter-sample cv, at each of the bases, the cv a Hybridization specificity ratio is defined as the ratio of PM probe hybridization signal to that of the brightest mismatch probe within a given category. The global average of all hybridization specificity ratios for each base in all samples for a given probe type is provided. b Determined for hybridization specificity ratios averaged across windows of 10 bases either within (intra) or across (inter) samples. was calculated using the 68 samples, and the average of the coefficient of variations was taken. For both calculations, the moving average of 10 was used, instead of the original value, since the goal was to understand how the specificity varied over bases and across samples, rather than to estimate the experimental (or measurement) error. In order to determine the relative specificity of the hybridization of complex nucleic acid targets to oligonucleotide probes complementary to single base substitutions, insertions and deletions, we analyzed data generated from oligonucleotide microarray-based mutational analysis of the 9168 bp ATM coding region (30) . These studies used a pair of oligonucleotide microarrays (Affymetrix, Santa Clara, CA) containing over 250 000 probes (25 nt in length) specifically designed to screen the sense and antisense strands of the ATM coding region for genetic variation (27, 30) . Collectively, the ATM sense and antisense microarrays contain 55 008 probes complementary to all possible single base substitutions, 73 344 probes complementary to all possible one base insertions, and 18 336 probes complementary to all possible one base deletions and 18 336 probes complementary to all possible two base deletions in the ATM coding sequence (Figures 1 and 2 ). These microarrays have been used to screen for sequence variation in the ATM gene in over 100 DNA samples (30) . SNPs and gene inactivating mutations were uncovered by screening for localized losses of hybridization signal to PM probes complementary to every 25 nt segment of the ATM coding region (8, 25, 27, 30) . However, hybridization data from deletion and insertion probes were not relied upon in this analysis. Therefore, this data set provides a unique opportunity to examine the relative hybridization specificity of nucleic acid targets to each of these classes of mismatch probes. In order to gain a global overview of hybridization specificity, we determined the average ratio of PM probe hybridization signal of wild-type target (see Materials and Methods) to their cognate insertion, deletion and single base substitution probes on each strand (Table 1 ). In these calculations, we considered data for all 9168 interrogated bases in all 68 DNA samples (see Materials and Methods). For example, we report the ratio of the PM probe signal to the signal from its cognate 1 or 2 bp deletion probe. However, for single base substitutions, we report the ratio of the PM probe signal to that of the cognate substitution probe with the highest hybridization signal. This provides the most rigorous assessment of cross-hybridization to single base substitution probes. Likewise, for single base insertion probes, we report the ratio of the PM probe signal to that of the cognate insertion probe with the highest hybridization signal. For both sense and antisense strands, we found that the two base deletion probes had the highest average PM to cognate MM hybridization specificity ratio (3.26-fold sense and (Table 1) . To provide a finer-scale analysis of hybridization specificity, we determined the relative frequencies of hybridization specificity ratios in defined bins. There was a similar distribution of specificity ratios for single base substitution and two base deletion probes on both strands ( Figure 3 ). The overall lower hybridization specificities of single base deletion and insertion probes are reflected by the increased frequencies of probes within the lower specificity bins (i.e. <2-fold ratio) and decreased frequencies of probes within higher specificity bins (i.e. >3-fold ratio) on both strands. Next, we sought to uncover underlying trends in the hybridization specificity of different classes of mismatch probes across the entire ATM coding region within a given sample (intra-sample variation). This provides insights into sequence context effects that may influence the hybridization specificity of each class of mismatch probe. To approach this problem, we plotted the average hybridization specificity ratios of substitution, deletion and insertion probes for all 1168 bases across the 68 samples (Figure 4 and Supplementary Figure 1) . We analyzed data determined over running averages of 10 bases in order to maximize our ability to detect trends and minimize the effect of randomly dispersed confounding factors (e.g. intra-or intermolecular secondary structure) that may skew data for any given base. As expected from Table 1 and Figure 3 , the two base deletion probes consistently showed a higher average hybridization specificity ratio followed by single base substitution, single base deletion and single base insertion probes on both strands of exon 50 ( Figure 4) . Nevertheless, the hybridization specificity ratios for all classes of mismatch probes fluctuate across the exon 50 sequence (Figure 4 ). For example, two base deletion probes showed a peak value of 6.76 (unlogged) centered at base 7071 and a trough value of 1.90 (unlogged) centered at base 7002 on the sense strand. We also found similar fluctuations in specificity ratios for all mismatch probe types in the remaining 61 ATM coding exons (Supplementary Figure 1 ). To assess intra-sample variability in hybridization specificity by a different means, we determined the average cv for substitution, deletion and insertion probes within a given experiment (Table 1) . Again, we analyzed data from running average of 10 bases in order to maximize our ability to detect trends and maintain consistency in our data analysis. Substitution probes had the lowest average intra-sample cv, 0.31 and 0.23 for sense and antisense strands, respectively. One base deletion, two base deletion and insertion probes showed comparable intra-sample coefficients of variation on the sense strand, 0.37, 0.39, and 0.38, respectively. However, insertion probes showed relatively higher variability than the deletion probes on the antisense strand. Coupled with plots shown in Supplementary Figure 1 , it is evident that of all the mismatch probe types, the hybridization specificities of base substitution probes were least affected by target sequence context. Intrigued by the above observations, we next searched for specific target sequence tracts that produced the lowest hybridization specificity among and between the different classes of mismatch probes. To approach this problem, we determined how many mismatch probes within running windows of 10 bases gave poor hybridization specificity, previously defined as a hybridization specificity ratio <1.2 (26). In Table 2 , we report nucleotide tracts where at least 8 probes within a given 10 base window showed poor hybridization specificity ratios. A comprehensive listing of probes with poor hybridization specificity is provided in Supplementary Table 1 . Repetitive sequence tracts, including homopolymer, homopurine and homopyrimidine, are highly represented in Table 2 . Upon closer inspection, it became apparent why the cross-hybridization is strong for probes in homopolymeric regions. In these sequence contexts, substitution and deletion probes can form duplexes with wild-type target that are longer than 12 bp in length. For example, the probe designed to detect a single base deletion at position 633 is designed to form one 12 bp and one 13 bp duplex with wild-type target. However, this probe can form duplexes that range from 12 to 18 bp in length with wild-type sense strand target due to slippage ( Figure 5 ). This type of ambiguity leads to increased stability of these DNA-RNA heteroduplexes (34) . In principle, the homopurine and homopyrimidine tracts uncovered have the capacity to form higher order structures, such as triple helices (35) . These tracts are known to alter the conformation and stabilities of RNA-DNA heteroduplexes (36, 37) , such as those formed between RNA targets and DNA probes in our system. Finally, we expect the ATM target to be especially rich in such sequence tracts given that both strands of the 3 0 -splice acceptor sequences, typically containing homopyrimidine tracts, for all 62 coding exons are included in the ATM target. This increases the likelihood that highly related sequence tracts in the ATM target can cross-hybridize to probes interrogating a particular homopurine or homopyrimidine sequence tract and reduce the overall hybridization specificity in this region. Next, we screened for potential structures that can form in the PM probes listed in Table 2 or their targets that could explain their poor hybridization specificity. To do this, we used Mfold (38) to calculate Gibbs free energies for intramolecular structures that can form in these PM probes and targets. Based on these Gibbs free energy values, we classified the probes and targets as having strong (S) [DG < (À3 kcal/mmol)], medium (M) [(À1 kcal/mmol) > DG > (À3 kcal/mmol)] and weak (W) [G > (À1 kcal/mmol)] potential for secondary structure. We found that several target and probe sequences could form substantial secondary structures, as displayed in Figure 6 . This could artificially lower the affinity of target to PM probes and thus lower the hybridization specificity. It is more difficult to model intermolecular structure in the solution-phase complex target and in the solidphase oligonucleotide probes. However, it appears likely that such structures could also have a similar negative impact on hybridization specificity. The relative variability in hybridization specificity ratios across samples (inter-sample variability) represents another important issue that should be considered in resequencing analysis (9) . To uncover general trends in inter-sample variability for each type of mismatch probe, we calculated an average cv for mismatch probe hybridization specificity ratios determined over running windows of 10 bases (Table 1) . Interestingly, on both strands, the single base substitution probes showed the lowest inter-sample cv. The one and two base deletion probes showed at least 2-fold higher coefficients of variation on both strands, relative to the substitution probes. Surprisingly, the one base insertion probes showed significantly higher coefficient of variations than any of the other classes of mismatch probes across samples. In fact, they are 3.5-fold higher than the corresponding substitution probes on each strand. The relative levels of inter-sample variation for all mismatch probes across exon 50 are displayed graphically in Figure 4 . The error bars represent one standard deviation from the mean of the hybridization specificity ratio determined over a running window of 10 bases in each of the 68 samples. Note that the substitution probes show lower inter-sample variability than one base deletion, two base deletion and . Hybridization specificities of mismatch probes. A 10-base running window of the log 10 hybridization specificity ratios of substitution (red), one base deletion (green), two base deletion (blue) and one base insertion (black) was plotted for the sense (A) and antisense (B) strands of ATM exon 50. The light red, light green, light blue and gray shaded areas represent -1 SD of the log 10 hybridization specificity ratios for the substitution, one base deletion, two base deletion and one base insertion probes, respectively. one base insertion probes, in agreement with Table 1 . The variability in hybridization specificity measurements is consistent across all 62 ATM coding exons (Supplementary Figure 1) . Overall, our analyses indicate that, on average, single base insertion probes show substantially lower reproducibility across experiments than base substitution, one base deletion and two base deletion probes. The increased inter-and intrasample variability in hybridization specificity of single base insertion and deletion probes relative to single base substitution and two base deletion probes should be considered when designing and interpreting microarray-based screens for genetic variation. For a given microarray design, substantially more control hybridization experiments may be needed to determine baseline fluctuations in the hybridization specificities of insertion and deletion probes relative to those of substitution probes. In contrast to single nucleotide mismatches, detailed thermodynamic analyses of double helical nucleic acids with bulged nucleotides have only recently been conducted (34, (39) (40) (41) . In such cases, the bulged nucleotide is unpaired on only one of the nucleic acid strands. These studies are relevant to understanding the properties of the deletion and insertion probes since they can form duplexes containing bulges with target nucleic acid. For deletion probes, the bulged nucleotide is located on the target strand ( Figure 7) . Conversely, the insertion probes contain the bulged nucleotide in duplexes with wild-type target (Figure 7) . Although subject to sequence context effects, duplexes containing a single base bulge are predicted to be more stable than those containing single nucleotide mismatches (34, (39) (40) (41) . This is reflected in the lower average hybridization specificity of single base deletion and insertion probes relative to that of substitution probes (Table 1 and Figure 4) . Conversely, duplexes containing two base bulges are predicted to be generally less stable than those containing a single base mismatch (40, 41) . In part, this is due to the assumption that helical stacking is interrupted by bulges of two or greater bases in length while it is preserved for one base bulges (40, 41) . The higher average hybridization specificity ratios of two base (38) was used to predict the intramolecular structures with the lowest Gibbs free energy (DG) for either the 25-30 base stretches that encompass each listed sequence tract in the target or for the PM probes complementary to each sequence tract. We use these DG values to predict the stability of these structures. DG > (À1 kcal/mmol) = weak (W); (À1 kcal/mmol) > DG > (À3 kcal/mmol) = medium (M); and DG < (À3 kcal/mmol) = strong (S). c Type of mismatch probe that provided poor hybridization specificity ratios. d Low hybridization specificity found on both sense and antisense strands. e Immediately following the 3 0 end of this segment is a (T) 5 sequence tract. deletion probes relative to substitution probes are in agreement with the predicted properties of these probes ( Table 1) . The considerably lower average inter-sample variability of substitution probes relative to deletion and insertion probes was unexpected given that the same target was hybridized to all mismatch probes simultaneously in the same experiment. The sources of inter-sample variation include sample preparation, hybridization conditions and the microarrays themselves. It is reasonable to assume that the microarrays themselves are not the major source of variability since the combinatorial manufacturing processes should lead to roughly equivalent synthesis quality for all the arrayed probes (42, 43) . It seems more likely that the insertion and deletion probes are more sensitive to subtle changes in target preparation (e.g. amount of fragmentation and dye incorporation) and hybridization conditions (e.g. target concentration, temperature and wash conditions) than the substitution probes. However, a definitive explanation for our observations will require further investigations (44) (45) (46) (47) (48) (49) (50) (51) (52) . In addition to their potential value, it is important to note some of the caveats when relying upon mismatch probes for mutation detection. For example, it is important to screen for all possible sequence changes, including multiple base insertions and deletions, in mutational analyses of disease-related loci, such as the ATM, BRCA1 and BRCA2 genes. Given that 4 N probes per base per strand are needed to screen for insertions of length N in a mixed sequence, it is unlikely that oligonucleotides complementary to insertions of two or more base pairs will be represented on microarrays screening large sequence tracts for mutations in the near future. Deletions represent a more tenable situation since only one probe per base per strand is needed to screen for a deletion of a given length in a mixed sequence. Nevertheless, there will still be limitations as to the number of deletion probes that can be realistically represented in a given microarray. Finally, it is often critical to precisely determine the nature of a sequence change within a given sample in order to properly assess its functional significance. Thus, it is important to consider error rates when assigning the identity of a mutation based on mismatch probe data. When dealing with clinical samples, it will be especially important to confirm the identity "
23
"A Gene Encoding Sialic-Acid-Specific 9-O-Acetylesterase Found in Human Adult Testis"
"Using differential display RT-PCR, we identified a gene of 2750 bp from human adult testis, named H-Lse, which encoded a putative protein of 523 amino acids and molecular weight of 58 kd with structural characteristics similar to that of mouse lysosome sialic-acid-specific 9-O-acetylesterase. Northern blot analysis showed a widespread distribution of H-Lse in various human tissues with high expression in the testis, prostate, and colon. In situ hybridization results showed that while H-Lse was not detected in embryonic testis, positive signals were found in spermatocytes but not spermatogonia in adult testis of human. The subcellular localization of H-Lse was visualized by green fluorescent protein (GFP) fused to the amino terminus of H-Lse, showing compartmentalization of H-Lse in large dense-core vesicles, presumably lysosomes, in the cytoplasm. The developmentally regulated and spermatogenic stage-specific expression of H-Lse suggests its possible involvement in the development of the testis and/or differentiation of germ cells."
"Sialic acids are a diverse family of acidic nine-carbon sugars that are frequently found as terminal units of oligosaccharide chains on different glycoconjugates in higher invertebrates and vertebrates [1, 2] . As a part of determinants in many glycoproteins [3, 4] , sialic acids play an important role in intercellular and/or intermolecular recognition [5] . The 9-O-acetylation and de-Oacetylation are the most common modifications of sialic acids found in mammalian cell surface sialoglycoconjugates, which can alter its size, hydrophobicity, net charge, and antigenicity [2, 6, 7] . These modifications can regulate a variety of biological phenomena, including endogenous lectin recognition, tumor antigenicity, virus binding, and complement activation [8, 9] . Enzymes specifically capable of removing O-acetyl esters from the 9-position of sialic acids are sialic-acidspecific 9-O-acetylesterase. The enzymes in mammals have two forms, one is cytosolic sialic-acid-specific 9-O-acetylesterase (Cse) in the cytosolic fraction and another is lysosome sialic-acid-specific 9-O-acetylesterase (Lse) in the lysosomal/endosomal compartment [10] . Lse is likely to participate in the terminal lysosomal degradation of 9-O-acetylated sialoglycoconjugates, while Cse is likely to salvage any 9-O-acetylated molecules that escape the initial action of the Lse enzyme. The process of de-O-acetylation of sialic acid, which is catalyzed by sialicacid-specific 9-O-acetylesterase, has been implicated in organogenesis and cellular differentiation [2, 5] . Spermatogenesis is a complicated process of germ cell differentiation in adult testis, which is established during testicular development. There are five types of germ cells, each at a specific developmental stage, found in the seminiferous tubules: spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and sperms. They can be divided into three groups according to their DNA content: 4N DNA content cells (4C cells), 2N DNA content cells (2C cells), and 1N DNA content cells (1C cells). The separation of these cells enables researchers to investigate the molecular mechanisms underlying testicular development and/or spermatogenesis. In the present study, we separated the 2C and 4C cells of seminiferous tubules in human adult testis by flow cytometry, and identified human H-Lse by differential display RT-PCR. The expression pattern of H-Lse was found to be developmentally regulated and stage-specific, indicating its possible role in testicular development and/or germ cell differentiation. Human testes were obtained from Donation Center of Nanjing Medical University with consent of relatives. The seminiferous tubules were collected in DMEM/F12, which contained collagenase, and washed to remove the Leydig cells as well as interstitial cells. Trypsin treatment and a brief treatment with DNase I were used to release the spermatogenic cells from seminiferous tubules. The suspension of cells was filtered with nylon mesh. Disaggregated spermatogenic cells were suspended at 1 × 10 6 cells/mL in 0.5 M sodium citrate solution (PH 2.35) with fresh 0.1% DEPC overnight at room temperature and at 4 • C for two days; they were centrifuged and resuspended in 0.5 M sodium citrate solution (PH 4.5) with fresh 0.1% DEPC for at least 1 day. The day before use, the cells were centrifuged and resuspended in PBS with 10 mM HEPES (PH 7.0), 0.1% BSA, and fresh 0.1% DEPC. Then the cells were spun down and resuspended in PBS with 100 µg/mL PI (propidium iodide) and fresh 0.1% DEPC. The cells were stained overnight at 4 • C [11] . The flow cytometry (FCM) used in this research was FACSVantage SE (Becton Dickinson, Calif) equipped with argon laser (power: 200 mW, wavelength: 488 nm); a 585 nm/42 nm filter set was used before the FL2 detector. Cellquest (Becton Dickinson) was used for sorting and the sorting mode was Normal-R. Drops per sort were 3 and drop delay was 13.6. The density of cells for sorting was about 1 × 10 6 cells/mL. Isolation of total RNA from 2C cells and 4C cells was performed with Trizol Reagent (Gibco BRL, Ontario). One hundred nanograms of total RNA was used for differential display RT-PCR [12] . The first chain cDNA was synthesized by using T12G, T12C, and T12A oligo (dT) primers, and then was used as template in PCR. PCR was performed as follows: 94 • C, 1 minute; 37 • C, 1 minute; 72 • C, 2 minutes for 40 cycles. Ten microlitres of the PCR products from the two cells were run on a 1.5% agarose gel. The fragments highly or specifically displayed in 4C cells were excised and purified. This DNA was reamplified with the same combination of primers and then subcloned into Pinpoint Xa1-T vector (Promega, USA). The colonies of full-length cDNA were screened by PCR. Human Testis Large-Insert cDNA Library (Clontech, Calif) was first converted into plasmid cDNA Library, and then an arrayed cDNA library in 96-well plates was made according to the method of Munroe [13, 14] . In this arrayed cDNA library 1.54 × 10 6 colonies were screened by PCR. Multiple tissue northern (MTN) blots (Clontech) were hybridized with the 32 P-labeled probes. The probe corresponding to 1378-1634 bp of H-Lse was used for hybridization. After stringent wash, the blot was placed on the storage phosphor screen (Packard, USA) and exposed for 3 hours in the dark. The signal was detected at the Cyclone storage phosphor system (Packard). The Stanford TNG Radiation Hybrid Panel (Research Genetics, Huntsville, Ala) was used to map the chromosomal localization of HSE with primers HSEmapF (5 -ATGAACACCGTCTCCACC-3 ) and HSEmapR (5 -AAATCTGAAGGACCCATC-3 ), according to the manufacturer's instructions. After 35 cycles of amplification, the reaction products were separated on a 1.5% agarose gel. The positive amplification was labeled as 1 and the negative one was labeled as 0. The results were analyzed through the Stanford genome center web server to determine the probable chromosomal location. RNA DIG-labeled probes were made by in vitro transcription. T7 and SP6 promoter sequences were incorporated into the two sides of the templates (195-553 bp of H-Lse) by PCR, sense and antisense probes were made using DIG-RNA labeling mix (Roche, USA) according to the manufacturer's instructions. After fixation, paraffin embedding, mounting, and sectioning, sections of human embryonic and adult testes were prehybridized in hybridization buffer (DIG Easy Hyb, Roche, Germany) at 42 • C for 2 hours. Hybridization was carried in hybridization buffer containing appropriate probes at 65 • C for 16 hours in humidity chamber. Subcellular localization of HSEI and HSEII was performed by the method of green fluorescent protein. pEGFP-C2-HSEI AND pEGFP-C2-HSEII were constructed using two sets of primers (HSEI: 5 -GGGGAATT CAATGATATGGTGCTGCAG-3 and 5 -GGGGTCGACAT TTAGCAACATTGCTCTG-3 ; HSEII: 5 -GGGGAATTCA TGGTCGCGCCGGGGCTTG-3 and 5 -GGGGTCGACA TTTAGCAACATTGCTCTG-3 ) and EcoRI/SalI restriction sites of pEGFP-C2. Recombinant vectors were transfected into BxPC-3 cells (BxPC-3 cell is a cell lineage of adenocarcinoma from pancreas) by Lipofectin reagent (Gibco BRL). Cells were imaged 40 hours after transfection on the fluorescence microscope. After being stained with PI and measured by the FCM, three groups of cells in seminiferous tubules of human adult testis were detected (Figure 1 ), 2C and 4C cells were subsequently sorted. A clone was identified by differential display RT-PCR, which was highly expressed in the 4C cells ( Figure 2 ) and with high homology (86%) to a mouse lysosome sialic acid 9-O-acetylesterase. The clone was named H-Lse. In the two rounds of screening in the arrayed cDNA library, the plasmid containing full-length H-Lse (GenBank accession number: AF303378) was found. H-Lse is 2750 bp in length, encoding a putative protein of 523 amino acids with a molecular weight of 58 kd. Its isoelectric point is 7.19. The N terminus (1-18 aa) of the protein is a region containing hydrophobic amino acid residues, which may be a signal peptide. By comparison of the protein sequences (Figure 3 ), we hypothesized that H-Lse is the human counterpart of mouse lysosome sialic acid 9-O-acetylesterase. After PCR amplification, the results can be shown as a pattern (00000000100010100000011000001000000011 000001000001000001001000000010000000000100100100 0001). Retrieving results from the Stanford genome center web server shows that HSE is localized in the human 11q24 ( Figure 4) . The distribution of H-Lse in various human tissues was analyzed by Northern blot ( Figure 5 ) and the results showed the presence of three distinct mRNA species at approximately 2.7 kb, 6.0 kb, and 7.5 kb. The expected transcript of H-Lse was approximately 2.7 kb and it was consistently expressed in all the tissues examined with high expression found in the testis, prostate, and colon. The transcript of approximately 7.5 kb was exclusively expressed in the colon. The transcript of approximately 6.0 kb was distributed in the testis, colon, small intestine, prostate, and thymus, with the highest level of expression found in the testis. To examine a possible role of H-Lse in testicular development and/or spermatogenesis, in situ hybridization experiments were conducted to compare H-Lse expression in human embryonic and adult testes since spermatogenesis is not initiated in the embryo and there is no meiosis in embryonic seminiferous tubules. The results showed that no signal was detected in the embryonic testis, while positive signals were detected in spermatocytes but not spermatogonia in the seminiferous tubules of adult testis. Signals were associated with germ cells but not other somatic cells in the testis, that is, Sertoli and Leydig cells. Negative control of sense probes confirmed the specificity of the results ( Figure 6 ). The subcellular localization of H-Lse fusion proteins was visualized by transiently transfecting H-Lse gene fused with GFP into BXPC-3 cells. As shown in Figure 7 , the control cells transfected with GFP protein alone exhibited fluorescence evenly distributed throughout the cytoplasm, while GFP-H-Lse fusion protein was compartmentalized in numerous large dense-core vesicles in the cytoplasm. Spermatogenesis is a developmental program that occurs in mitotic, meiotic, and postmeiotic phases. In the mitotic phase, spermatogonia proliferate to expand the quantity of germ cells; in the meiotic phase, spermatocytes accomplish chromosomal synapsis and genetic recombination before two meiotic divisions; and in the postmeiotic phase, haploid spermatids are remodeled into spermatozoa by the processes of acrosome formation, nuclear condensation, flagellar development, and loss of the majority of cytoplasm. Under the control of intrinsic and extrinsic factors, spermatogenesis is characterized by the expression of a spectrum of genes that are celltype-specific or stage-specific. They are thought to play an essential role in spermatogenesis at particular stages. For example, MutS homologue 5 is required for chromosome pairing, CPEB and SCP3 are required for synaptonemal complex assembly and chromosome synapsis in primary spermatocytes [15, 16, 17] . In the present study, we have identified a gene, H-Lse, from human adult testis with high homology to m-Cse 1 [19] . Similarly, it can inhibit binding of sialoadhesin, a macrophage-restricted and sialic-acid-dependent adhesion molecule [20] . On the other hand, 9-O-acetylation of sialic acids can form novel epitopes. Influenza virus C haemagglutinin specifically requires 9-O-acetylated sialic acids for binding to host cells [21] . Incubation of red blood cells with sialate 9-Oacetylesterase rendered the erythrocytes resistant against agglutination by influenza C virus [22] . O-acetylation of disialoganglioside GD3 by human melanoma cells has been reported to create a unique antigenic determinant [23] . Modifications of sialic acids may be an important mechanism underlying the interaction/cross-talk between different types of cells. The essential role of sialic acids modification in cellular communications may explain the presently observed wide distribution of H-Lse in all examined tissues. The present study suggests that the expression of H-Lse is developmentally regulated and spermatogenic stage-specific. The evidence for this includes: (1) lack of expression in embryonic testis; (2) association of high level of mRNA detected by DD-RT-PCR with the 4C but not 2C cells in adult testes; and (3) detection of in situ hybridization signal in spermatocytes but not spermatogonia or other somatic cells. In the absence of spermatogenesis, embryonic testis contains only two distinct cell types, spermatogonia and Sertoli cells, while the seminiferous epithelium of adult testis consists of germ cells at different stages of spermatogensis. The 4C cells found in adult testis include the primary spermatocytes and spermatogonia of G 2 /M stage, while 2C cells include spermatogonia of G0/G1 stage, secondary spermatocytes, and Sertoli cells. The absence of H-Lse mRNA in embryonic testis and the high level of its mRNA in the 4C cells of adult testis suggest that its expression is restricted to spermatocytes, particularly the primary spermatocytes. Together with the in situ hybridization results showing mRNA of H-Lse restricted to spermatocytes, but not spermatogonia, Sertoli cells or interstitial cells, these data suggest that H-Lse is likely to be involved in the process of spermatogenesis, although its role in testicular development cannot be entirely ruled out. Unfortunately, due to the deformation of the available human testes, we were not able to make further distinction between primary and secondary spermatocytes. What has been clearly shown by the present data is that H-Lse is only present at a stage beyond spermatogonia, suggesting its possible role in the differentiation of germ cells. G N F T Y M S A V C W L F G R Y L Y D T L Q Y P I G L V S S S W G G T Y I E V W S S R R T L K A C G V P N T 143 m-Lse 181 A G N L G H G N F T Y M S A V C W L F G R Y L Y D T L Q Y P I G L V S S S W G G T Y I E V W S S R R T L K A C G V P N T 240 h-Lse 181 S E N L G H G Y F K Y M S A V C W L F G R H L Y D T L Q Y P I G L I A S S W G G T P I E A W S S G R S L K A C G V P K Q 240 m-Cse 144 R D E R V G Q P E I K P M R N E C N S E E S S C P F R V V P S V R V T G P T R H S V L W N A M I H P L Q N M T L K G V V 203 m-Lse 241 R D E R V G Q P E I K P M R N E C N S E E S S C P F R V V P S V R V T G P T R H S V L W N A M I H P L Q N M T L K G V V 300 h-Lse 241 G S _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I P Y D S V T G P S K H S V L W N A M I H P L O N M T L K G V V 274 m-Cse 204 W Y Q G E S N A D Y N R D L Y T C M F P E L I E D W R Q T F H Y G S Q G Q T D R F F P F G F V Q L S S Y M L K N S S D Y 263 m-Lse 301 W Y Q G E S N A D Y N R D L Y T C M F P E L I E D W R Q T F H Y G S Q G Q T D R F F P F G F V Q L S S Y M L K N S S D Y 360 h-Lse 275 W Y Q G E S N I N Y N T D Interestingly, the processes of 9-O-acetylation and de-O-acetylation of sialic acid have been implicated in organogenesis and cellular differentiation, since alteration of these processes could lead to interruption of cellular development such as embryogenesis. Transgenic mice constitutively overexpressed the 9-O-acetyl-sialicacid-specific esterase of influenza C that has been found to arrest embryo development at the two-cells stage. It has also been reported that in vitro development of embryonic stem cells shows that the expression level of Lse is low at the initiation of the development, and followed by an increase at later stages [24] . In transgenic mice with selective expression of 9-O-acetyl-sialic-acid-specific esterase in retina and the adrenal gland, these organs showed various abnormalities in organization, while all other tissues appeared normal [25] . Lse has also been considered to play a key role in the differentiation of B lymphocyte [2] , since it is expressed in late but not early B lymphocyte. The presently observed developmentdependent pattern of H-Lse expression is consistent with that found in other cell types: absence or low expression at early stage of differentiation but high at later stages. Taken together, 9-O-acetyl esters in sialic acids appear to be important for development or cellular differentiation. Spermatogenesis is a multiple-staged continuous progress of cellular differentiation. It has been reported that some cell surface glycoconjugates are modified during the early steps of spermatogenesis, and influence the differentiation of spermatogenic cells [26] . As ninecarbon sugars commonly found in many glycoproteins of spermatogenic cells, sialic acids represent a target for cell surface modification, that is, removal of 9-O-acetyl esters by enzymes such as Lse. Modification of sialic acids may result in alteration in cell-cell communication, that is, Sertoli cells and germ cells interaction, thereby influencing the differentiation of spermatogenic cells. Thus, future studies on the presently identified H-Lse may provide insight into molecular mechanisms underlying testicular development and/or germ cell differentiation during spermatogenesis in humans."
24
"The role of mast cells in the pathogenesis of pain in chronic pancreatitis"
"BACKGROUND: The biological basis of pain in chronic pancreatitis is poorly understood. Mast cells have been implicated in the pathogenesis of pain in other conditions. We hypothesized that mast cells play a role in the pain of chronic pancreatitis. We examined the association of pain with mast cells in autopsy specimens of patients with painful chronic pancreatitis. We explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS) -induced chronic pancreatitis in both wild type (WT) and mast cell deficient mice (MCDM). METHODS: Archival tissues with histological diagnoses of chronic pancreatitis were identified and clinical records reviewed for presence or absence of reported pain in humans. Mast cells were counted. The presence of pain was assessed using von Frey Filaments (VFF) to measure abdominal withdrawal responses in both WT and MCDM mice with and without chronic pancreatitis. RESULTS: Humans with painful chronic pancreatitis demonstrated a 3.5-fold increase in pancreatic mast cells as compared with those with painless chronic pancreatitis. WT mice with chronic pancreatitis were significantly more sensitive as assessed by VFF pain testing of the abdomen when compared with MCDM. CONCLUSION: Humans with painful chronic pancreatitis have an increased number of pancreatic mast cells as compared with those with painless chronic pancreatitis. MCDM are less sensitive to mechanical stimulation of the abdomen after induction of chronic pancreatitis as compared with WT. Mast cells may play an important role in the pathogenesis of pain in chronic pancreatitis."
"Although pain is the presenting symptom of most patients with chronic pancreatitis, its neurobiological basis remains poorly understood [1] . In the past, investigators have focused on the role of anatomical abnormalities such as a strictured pancreatic duct or narrowed intraparenchymal ducts. However, mechanical decompression techniques such as endoscopic stent placement or even surgical pancreatojejunostomy frequently do not provide a permanent solution to the pain [1] . More recently, investigators have begun focusing on the role of neurotransmitters and neurotrophins such as substance P and nerve growth factor with known or suspected roles in nociceptive signaling and/or sensitization and have reported an increased expression of several of them in the pancreas of patients with painful chronic pancreatitis [2] . Mast cells are also increased in both acute and chronic pancreatitis [3, 4] but their role in the generation of pain in pancreatitis has not been investigated. We hypothesized that mast cells are involved in the pathogenesis of pain in chronic pancreatitis. This hypothesis is based on the following observations. First, mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological findings [5, 6] . In both conditions, an increase in the number of mast cells has been described in the bladder and the colon, respectively [5, 6] . Further, mast cells are frequently found in close proximity to nerves in the intestinal mucosa and the bladder [7] [8] [9] . This has also been observed in the pancreas -the total number of mast cells was significantly higher in pancreatic tissue from patients with chronic pancreatitis than in the normal pancreatic controls [3] . One of the preferential locations of mast cells was around and within the perineurium of nerve fibers in tissue samples of patients with chronic pancreatitis, suggesting the potential for interactions between mast cells and the nervous system. Lastly, there is evidence for bi-directional functional communication between mast cells and nerves [10] [11] [12] . Mast cells can not only release mediators that increase excitability of neurons but in turn, neurotransmitters such as substance P can trigger mast cell degranulation [10] . Mast cells may therefore contribute to the pathogenesis of pain in pancreatitis through degranulation products that can sensitize pancreatic afferent neurons in an ongoing vicious circle of neuronally mediated mast cell degranulation. Our first aim was to analyze the presence and distribution of mast cells in autopsy specimens of chronic pancreatitis and study the correlation, if any, with historical documentation of pain. We then explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis in both wild type and Kit W /Kit W-v mice, a strain deficient in mast cells (MCDM). Autopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term "pancreatitis." One-hundred sixtysix patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26) . Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/ 26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis (amylase, lipase, calcifications on abdominal plain film, CT-scan, ultrasound or ERCP). Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch. All mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-Kit W /Kit W-v (MCDM) and the respective littermate control mouse strain, Kit W-v -+/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25-30 gram. Experimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health. Mice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi [13] ). The canula was removed and the abdomen closed. Control mice were treated in the exact same fashion but were perfused with saline instead (Figure 1 ). Mice were sacrificed 8 weeks after surgery. VFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain. More recently, VFF testing has been used as a surrogate marker for visceral pain [14, 15] . Mice were placed in a cage with a mesh floor and habituated to the environment for 30-60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery ( Figure 1 ). VFF filaments of various caliber were applied to the mid-abdomen in ascending order 10 times, each for 1-2 seconds with a 10 second interval. A response was defined as: a) sharp retraction of the abdomen; b) immediate licking or scratching of site of application of hair; or c) jumping. The response frequency was defined as the total number of responses out of 10 applications (expressed as a percentage) to the skin per filament. An investigator blinded to the different treatment groups performed the behavioral testing. Fresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgCl 2 at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups [16] . Comparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test. For each behavioral experiment (see figure 1), the average response frequency was calculated as the mean of the mean response frequencies for each mouse (across four measures). The "post-pre response frequency" was calculated by subtracting the pre-surgical average response frequency from the post-surgical average response frequency. To assess the independent effect of TNBS on VFF response (ie. to control for the effect of the surgery itself), the postpre response frequency for TNBS infusion was compared with the post-pre response frequency for saline infusion. This comparison was performed using analysis of variance for a two-factor experiment with repeated measures on time at each level of force for each type of mice (WT and MCDM). The two factors were induction of pancreatitis or not (TNBS or saline, respectively) and time (pre-surgical or post-surgical). TNBS infusion was considered to have had an independent effect on the VFF response if the postpre response frequency was greater for TNBS than for saline infusion. Fisher's least significant difference procedure was used for multiple comparisons of least squares means with Experimental design Figure 1 Experimental design All mice underwent pre and post surgical VFF testing. For the VFF testing, 4 measures were taken for each mouse. WT and MCDM were randomized to either saline or TNBS perfusion into the pancreatic duct. Patient demographics are summarized in Table 1 . Alcohol abuse was the most common cause for pancreatitis in both groups. Analysis of our results, using the Mann-Whitney U test, revealed significantly more mast cells in patients with a history of painful chronic pancreatitis (n = 8) when compared to patients with either non-painful chronic pancreatitis (n = 6) (33.8 vs 9.4 average mast cell count/10 high power fields; p < 0.01) or controls (n = 8), (33.8 vs 6.1 average mast cell count/10 high power fields; p < 0.01) ( Figure 2 ). The increased number of mast cells in patients with painful pancreatitis was noted predominantly in interstitial areas and, to a lesser degree, in the periacinar space. Figure 3 shows the post-pre surgical response frequency for both WT and MCDM. TNBS had a significant independent effect on abdominal VFF response in WT mice at the force levels 4 and 8 mN (p = 0.007 and 0.037, respectively) ( Figure 3A ). There was a trend towards a significant effect at the force level of 16 mN (p = 0.066). In contrast, for MCDM, TNBS had no significant effect on abdominal VFF response at any force level ( Figure 3B ). There was no significant TNBS effect on VFF response in the left A g e Pancreatic histology confirmed the presence of chronic pancreatitis in both WT and MCDM with marked fibrosis, inflammatory infiltrates and ductular proliferation mimicking changes seen in human chronic pancreatitis ( Figure 5A ). The pancreas of saline treated controls was normal. There was no significant difference in the overall inflammatory scores between the WT and MCDM ( Figure 5B ). An increased number of mast cells were counted in WT mice with chronic pancreatitis compared to saline Histology (Giemsa) of mice with chronic pancreatitis (Figure 6 ). As to be expected, no mast cells were present in pancreas of MCDM. Chronic pancreatitis has been defined as a continuing inflammatory disease of the pancreas characterized by irreversible morphologic changes that typically cause pain and/or permanent loss of function [17] . The pathogenesis of pain in this condition remains to be satisfactorily established. We examined the association, if any, of pain with mast cells as quantified in autopsy specimens of patients with a history of painful and non-painful chronic pancreatitis and normal controls. Significantly more mast cells were present in pancreatic tissue from patients with a history of painful chronic pancreatitis, indicating an association with this condition and a potential role for these cells in the pathogenesis of pain in painful chronic pancreatitis. There are clearly limitations to a retrospective, autopsybased study such as the one we report here. For instance, we do not know whether pain was present at the time of death and there was incomplete information on the different patterns of pain. Also, our findings pertain mainly to patients with a history of alcoholic pancreatitis. Nevertheless, our findings do suggest an association of painful chronic pancreatitis with an increased number of mast cells. This observation provided the rationale for further experimental testing, which we performed in mice. We first developed a model of chronic pancreatitis in mice following a modified protocol first described by Puig-Divi et al. [13] . Histological changes consisted of periductal and lobular fibrosis, duct stenosis, chronic inflammatory cell infiltrates, and gland atrophy, mimicking features of chronic pancreatitis in humans. Significantly more mast cells were present in WT mice with chronic pancreatitis, adding to the validity of this model for use in studies on the role of mast cells in pancreatitis. Both WT and MCDM developed histological changes consistent with chronic pancreatitis, indicating that the elimination of mast cells did not modulate the animals' ability to mount an inflammatory response. Therefore, any changes observed in pain behavior are unlikely to stem from differences in underlying inflammation. Next we determined whether this mouse model could be used to study behavioral differences associated with chronic pancreatitis. The assessment of spontaneous pain in a visceral organ presents significant difficulties. We have used a behavioral method to assess this, which relies on the association of visceral pain with sensitization of somatic regions of the body that share segmental innervation at the level of the spinal cord (referred pain). This somatic sensitization can be quantified using VFF to stim-ulate the somatotopically appropriate abdominal region and measuring the abdominal withdrawal response. Thus, VFF testing of the anterior abdominal wall can be used as a surrogate marker for visceral pain. Although this is the first time that this technique has been used for the measurement of referred visceral hyperalgesia in a mouse model of chronic pancreatitis, this method has previously been described and validated to assess the severity of referred visceral pain for models of colonic hypersensitivity [14] as well as rat models of acute necrotizing pancreatitis [15] and chronic pancreatitis [18] . The abdominal VFF response was compared to the hind paw response to assess the specificity of the interventions to the pancreas. TNBS treated mice, but not the saline control, developed increased abdominal wall withdrawal responses to VFF testing when compared to baseline, suggesting the development of force-dependent referred hyperalgesia of the abdominal wall in WT mice. There was no evidence of referred hyperalgesia in the hindpaws, suggesting that the measured effect on abdominal withdrawal is specific for an intra-abdominal origin of the pain. Vera-Portocarrero et al. previously described similar findings, increased withdrawal frequency after VFF stimulation to the abdominal area, in a rat model of chronic pancreatitis [18] . These behavioral changes were abrogated by morphine. Rats that demonstrated behavioral changes also expressed increased substance P expression in the nociceptive layers of the spinal cord, suggestive of central nociceptive changes. Mast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. The neurotrophin growth factor (NGF) is one such product [19] [20] [21] [22] . NGF is released in the setting of inflammation and can not only function as a chemoattractant for other mast cells, but it can also trigger mast cell degranulation [23] . We are speculating that NGF production in the inflamed pancreas is responsible for plastic changes in the sensory neurons by activating proalgesic receptors and channels such as the NGF receptor tyrosine kinase A (TrkA) and Transient Receptor Potential Family V receptor 1 (TRPV1; previously known as VR1) thereby contributing to the generation of pain [24] [25] [26] . Similarly, other mast cell degranulation products such as tryptase and histamine are capable of modulating neuronal function [27] [28] [29] [30] [31] [32] . Tryptase may directly activate the proteinase-activated receptor-2 (PAR-2), a G-protein coupled receptor expressed by pancreatic nerves, important in the pathogenesis of pain in pancreatitis [33, 34] . Although the role for mast cells in the mediation of visceral nociceptive signaling needs to be explored further, we speculate that mast cell products released in pancreatitis, contribute to the development of pain by direct effects on nociceptors located on pancreatic afferent neurons (Figure 7 ). However, before concluding a definite role for mast cells from our experimental data, it should be noted that MCDM carry a spontaneous mutation for tyrosine kinase receptor c-kit which not only produces a deficiency of mast cells but may have an independent effect on the function of sensory neurons, which are known to express it [35] . Therefore, it remains to be determined whether the detected differences in nociceptive responses is due to the absence of mast cells per se or a yet unknown change in the responsiveness of sensory neurons due to a congenital lack of the c-kit receptor. Reconstitution of mast cells into the MCDM mice should restore their nociceptive responses close to the wild type phenotype. Our data should increase awareness of the importance of mast cells in the pathogenesis of painful inflammatory Proposed involvement of mast cells in nociceptive signaling in pancreatitis Figure 7 Proposed involvement of mast cells in nociceptive signaling in pancreatitis In pancreatitis, mast cells may migrate to sites of inflammation, in response to release of mast cell chemoattractants. Mast cell degranulation products may modulate neurotransmission directly by activating proalgesic receptors and channels such as trka (NGF), TRPV1 (NGF) and PAR-2 (tryptase and trypsin). The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-230X/5/8/prepub"
25
"Recombination Every Day: Abundant Recombination in a Virus during a Single Multi-Cellular Host Infection"
"Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment—based on data on the timing of coat protein detection—the per base and replication cycle recombination rate was on the order of 2 × 10(−5) to 4 × 10(−5). This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus."
"As increasing numbers of full-length viral sequences become available, recombinant or mosaic viruses are being recognized more frequently [1, 2, 3] . Recombination events have been demonstrated to be associated with viruses expanding their host range [4, 5, 6, 7] or increasing their virulence [8, 9] , thus accompanying, or perhaps even being at the origin of, major changes during virus adaptation. It remains unclear, however, whether recombination events represent a highly frequent and significant phenomenon in the everyday life of these viruses. Viruses can exchange genetic material when at least two different viral genomes co-infect the same host cell. Progeny can then become hybrid through different mechanisms, such as reassortment of segments when the parental genomes are fragmented [10] , intra-molecular recombination when polymerases switch templates (in RNA viruses) [11] , or homologous or non-homologous recombination (in both RNA and DNA viruses). Quantification of viral recombination in multicellular organisms has been attempted under two distinct experimental approaches: in vitro (in cell cultures) [12, 13, 14, 15] , and in vivo (in live hosts) [16, 17, 18] . The in vitro approach, which has so far been applied only to animal viruses, allows the establishment of the ''intrinsic'' recombination rate in experimentally co-infected cells in cell cultures [14, 15, 19] . However, it does not necessarily reflect the situation in entire, living hosts, where the frequency of coinfected cells is poorly known and depends on many factors such as the size of the pathogen population, the relative frequency and distribution of the different variants, and host defense mechanisms preventing secondary infection of cells. The in vivo experimental approach is closer to biological conditions and may thus be more informative of what actually happens in ''the real world.'' However, as discussed below, numerous experimental constraints have so far precluded an actual quantification of the baseline rate of recombination. First, many experimental designs have used extreme positive selection, where only recombinant genomes were viable (e.g., [13, 20, 21] ). Other studies did not use complementation techniques but detected recombinants by PCR within infected hosts or tissues [18, 22, 23, 24, 25] , which provides information on their presence but not on their frequency in the viral population. So far, no quantitative PCR or other quantitative method has been applied to evaluate the number of recombinants appearing in an experimentally infected live host. Finally, recent methods based on sequence analysis inferred the population recombination rate, rather than the individual recombination rate [1, 26, 27] . While results from these methods certainly take in vivo recombination into account, there are other caveats: isolates have often been collected in different hosts-sometimes in different geographical regions-and sometimes the selective neutrality of sequence variation on which these estimates are based is not clearly established. Estimates from such studies by essence address the estimation of the recombination rate at a different evolutionary scale. Taken together, the currently available information indicates that no viral recombination rate has ever been estimated directly at time and space scales corresponding to a single multi-cellular host infection, although this level is most significant for the biology and evolution of viruses. This study intends to fill this gap by evaluating the recombination frequency of the cauliflower mosaic virus (CaMV) during a single passage in one of its host plants (the turnip Brassica rapa). CaMV is a pararetrovirus, which is a major grouping containing hepadnaviruses (e.g., hepatitis B virus), badnaviruses (e.g., banana streak virus), and caulimoviruses (e.g., CaMV). Pararetroviruses are characterized by a non-segmented double-stranded DNA genome. After entering the host cell nucleus, the viral DNA accumulates as a minichromosome [28] whose transcription is ensured by the host RNA polymerase II [29] . The CaMV genome consists in approximately 8,000 bp and encodes six viral gene products that have been detected in planta ( Figure 1 ) [30] . Viral proteins P1 to P6 are expressed from two major transcripts, namely a 19S RNA, encoding P6, and a 35S RNA corresponding to the entire genome and serving as mRNA for proteins P1-P5 [31] . Using the pre-genomic 35S RNA as a matrix, the protein P5 (product of gene V) reverse-transcribes the genome into genomic DNA that is concomitantly encapsidated [30] . The detection of CaMV recombinants in turnip hosts has been reported numerous times. Some studies have demonstrated the appearance of infectious recombinant viral genomes after inoculation (i) of a host plant with two infectious or non-infectious parental clones [21, 32, 33, 34, 35] or (ii) of a transgenic plant containing one CaMV transgene with a CaMV genome missing the corresponding genomic region [36] . While the former revealed inter-genomic viral recombination, the latter demonstrated that CaMV can also recombine with transgenes within the host's genome. Another study based on phylogenetic analyses of various CaMV strains has clearly suggested different origins for different genomic regions and, hence, multiple recombination events during the evolution of this virus [37] . Indirect experimental evidence has indicated that, in some cases, CaMV recombination could occur within the host nucleus, between different viral minichromosomes, presumably through the action of the DNA repair cellular machinery [21, 35] . Nevertheless, the mechanism of ''template switching'' during reverse transcription, predominant in all retroviruses, most certainly also applies to pararetroviruses. For this reason, and on the basis of numerous experimental data, CaMV is generally believed to recombine mostly in the cytoplasm of the host cell, by ''legal'' template switching between two pre-genomic RNA molecules [21, 35, 36, 38, 39] , or ''illegal'' template switching between the 19S and the 35S RNA [36, 40] . Under this hypothesis, recombination in CaMV could therefore be considered as operating on a linear template during reverse transcription, with the 59 and 39 extremities later ligated to circularize the genomic DNA (position 0 in Figure 1 ). The above cited studies clearly demonstrate that CaMV is able to recombine. However, since these studies are based on complementation techniques, non-quantitative detection, or phylogenetically based inferences of recombination, they do not inform us on whether recombination is an exceptional event or an ''everyday'' process shaping the genetic composition of CaMV populations. In the present work, we aimed at answering this question. To this end, we have constructed a CaMV genome with four genetic markers, demonstrated to be neutral in competition experiments. By co-inoculating host plants with equal amounts of wild-type and marker-containing CaMV particles, we have generated mixed populations in which impressive proportions of recombinants-distributed in several different classes corresponding to exchange of different genomic regions-have been detected and quantified. Altogether, the recombinant genomes averaged over 50% of the population. Further analysis of these data, assuming a number of viral replications during the infection period ranging from five to 20, indicates that the per nucleotide per replication cycle [44] ) indicates the origin of replication via reverse transcription, which occurs in the direction indicated by the dotted outermost circle-like arrow. Reverse transcription is accomplished by the viral reverse transcriptase, using the 35S RNA as template [49] . DOI: 10.1371/journal.pbio.0030089.g001 recombination rate of CaMV is of the same order of magnitude, i.e., on the order of a few 10 À5 , across the entire genome. We thereby provide the first quantification, to our knowledge, of the recombination rate in a virus population during a single passage in a single host. From Figure 1 , and supposing that all marker-containing genomic regions can recombine, we could predict the detection and quantification of seven classes of recombinant genotypes: þbcd/aþþþ, aþcd/þbþþ, abþd/þþcþ, abcþ/þþþd, þþcd/abþþ, aþþd/þbcþ, and aþcþ/þbþd. Indeed, all classes were detected, and their frequencies in the ten CaMV populations analyzed are summarized in Table 1 . Altogether, the proportion of recombinant genomes found in the mixed viral populations was astonishingly high and very similar in the ten co-infected plants analyzed (Table 1 , last column), ranging between 44% (plant 5) to 60% (plants 7, 12, and 20), with a mean frequency (6 standard error) of 53.8% 6 2.0%. This result indicates that recombination events are very frequent during the invasion of the host plant by CaMV and represents, to our knowledge, the first direct quantification of viral recombination during the infection of a live multi-cellular host. The inferred per generation recombination and interference rates, assuming that CaMV undergoes ten replication cycles during the 21 d between infection and sampling, are given for each of the ten plants in Table 2 . Recombination rates between adjacent markers are large, on the order of 0.05 to 0.1. Taking the distance in nucleotides between markers into account yields an average recombination rate per nucleotide and generation on the order of 4 3 10 À5 . Interestingly, this recombination rate does not vary throughout the genome (Kruskal-Wallis test, p = 0.16). To relax the assumption of the number of replications during the 21 d, we calculated the recombination parameters assuming five or 20 generations. The effect of the number of generations on the estimates is linear: doubling the number of generations results in a halving of the recombination rate (detailed results not shown). For example, the average recombination rates r 1 , r 2 , and r 3 assuming 20 generations were equal to 0.05, 0.04, and 0.025, respectively (compare with values in Table 2 ), yielding per nucleotide per generation recombination rates of 1.9 3 10 À5 , 2.2 3 10 À5 and 1.6 3 10 À5 . Inspection of Table 2 also shows that first-order interference coefficients were in general negative, indicating that a crossing over in one genomic segment increases the probability that a crossing over will occur in another genomic segment, while the second-order coefficient parameter had an average value close to zero with a large variance. The mechanism leading to these results will be discussed in the following section. One major breakthrough in the work presented here lies in the space and time scales at which the experiments were performed. Indeed, the processes occurring within the course of a single infection of one multi-cellular host are of obvious biological relevance for any disease. Previous studies on viral recombination suffered from major drawbacks in this respect, basing their conclusions on experiments relying on complementation among non-infectious viruses or between viruses with undetermined relative fitness, on phylogenetically based analyses, or on experiments in cell cultures. For reasons detailed in the Introduction, the first two methods either do not provide information on the frequency of recombination, but only its occurrence, or address the question at a different temporal, and often spatial, scale. Results from cell cultures, on the other hand, impose cell coinfection by different viral variants, potentially overestimat- ing the frequency of recombination events. Our study circumvents these limitations by analyzing viral genotypes sampled from infected plants after the course of a single infection, and therefore the invasion and co-infection of cells in various organs and tissues is very close to natural. More than half of the genomes (53.8% 6 2.0%; see Table 1 ) present in a CaMV population after a single passage in its host plant were identified as recombinants, and these data allowed us to infer a per nucleotide per generation recombination rate on the order of 2 3 10 À5 to 4 3 10 À5 . The time length of one generation, i.e., the time required for a given genome to go from one replication to the next, is totally unknown in plant viruses. The only experimental data available on CaMV are based on the kinetics of gene expression in infected protoplasts, where the capsid protein is produced between 48 and 72 h [40] . The reverse transcription and the encapsidation of genomic DNA being two coupled phenomena [30] , we judged it reasonable to assume a generation time of 2 d and, thus, an average of ten generations during our experiments. In case this estimate is mistaken, we have verified a linear relationship between r and the number of generations, thereby allowing an immediate adjustment of r if the CaMV generation time is more precisely established. At this point, we must consider that all cloned genomes may not have been through all the successive replication events potentially allowed by the timing of our experiments. It was previously shown that about 95% of CaMV mature virus particles accumulate in compact inclusion bodies [41] , where they may be sequestered for a long time, as such inclusions are very frequent in all infected cells, including those in leaves that have been invaded by the virus population for several weeks. The viral population may thus present an age structure that could bias the estimation of the recombination rate. In order to minimize this bias, the clones we analyzed were collected in one young newly formed leaf, where the chances of finding genomes from ''unsequestered lines'' were assumed to be higher. In any case, our data analysis is conservative, since this age structure can only lead to an underestimation of the recombination rate. Our results show that interferences between pairs of loci are negative: a recombination event between two loci apparently increases the probability of recombination between another pair of loci. We believe that the most parsimonious explanation of these negative interferences is based on the way the infection builds up within plant hosts. Indeed, one can divide infected host cells into those infected by a single virus genotype and those infected by more than one viral genotype. In the former, analogous to clonal propagation, recombination is undetectable. In the latter, recombination is not only detectable but, as our results indicate, very frequent. Samples consisting of viruses resulting from a mixture of these two types of host cell infections will thus contain viruses with no recombination and viruses with several recombination events, thus yielding an impression of negative interference. These conceptual arguments are supported by mathematical models. It is indeed easy to show (detailed results not shown) that if a proportion F of the population reproduces clonally, analogous to single infections, while the remaining reproduces panmictically, negative interferences could be inferred even if they do not exist. For example, assuming a three-locus model with real recombination rates r 1 and r 2 and interference i 12 , the ''apparent'' recombination and interference parameters, would be r 1 = (1 À F)r 1 , r 2 = (1 À F)r 2 , and i 12 = À(F À i 12 )/(1 À F). Interestingly, this example also shows that our estimates of the recombination rate are conservative: that a fraction F of host cells are singly infected while others are multiply infected leads to an underestimation of the recombination rate. As judged by r 1 , r 2 , and r 3 , calculated between markers a-b, b-c, and c-d, respectively, we found evidence for recombination through the entire CaMV genome. The values for r 1, r 2 , and r 3 are remarkably similar, hence the recombination sites seem to be evenly distributed along the genome. We considered the template-switching model as the major way recombinants are created in CaMV. As already mentioned in the Introduction, hot spots of template switching have been predicted at the position of the 59 extremities of the 35S and 19S RNAs [21, 36, 42] . If other recombination mechanisms, such as that associated with second-strand DNA synthesis or with the host cell DNA repair machinery, act significantly, hot The various parameters are as follows: r1, recombination rate between markers a and b; r2, recombination rate between markers b and c; r3, recombination rate between markers c and d; i12, interference between crossovers in segments a-b and b-c; i23, interference between crossovers in segments b-c and c-d; i13, interference between crossovers in segments a-b and c-d; i123, second-order interference accounting for residual interference. The recombination rates are the maximum likelihood estimates (6 95% confidence intervals). The interference parameters were obtained numerically as explained in the Materials and Methods. DOI: 10.1371/journal.pbio.0030089.t002 spots would be expected at the positions of the sequence interruption D1, D2, and D3 [43] . Due to the design of our experiment and the position of the four markers, we have no information on putative hot spots at positions corresponding to the 59 end of the 35S RNA and to D1 (at nucleotide position 0). Nevertheless, the putative hot spots at the 59 end of the 19S RNA and at D2 and D3 (nucleotide positions 4,220 and 1,635, respectively) fall between marker pairs c-d, b-c, and a-b, respectively. Our results indicate that either these hot spots are quantitatively equivalent-though predicted by different recombination mechanisms-or, more likely, that they simply do not exist. Whatever the explanation, what we observe is that the CaMV can exchange any portion of its genome, and thus any gene thereof, with an astonishingly high frequency during the course of a single host infection. To our knowledge, the viral recombination rate has never previously been quantified experimentally for a plant virus [3] . In contrast, retroviruses and particularly HIV-1 have been extensively investigated in that sense. As we have already discussed for these latter cases, the quantification of the intrinsic recombination rate was carried out in artificially coinfected cell cultures. The estimated intrinsic per nucleotide per generation recombination rate in HIV-1 is on the order of 10 À4 [14, 15, 19] , less than one order of magnitude higher than our estimation for CaMV. Because for various reasons detailed above we probably underestimate the within-host CaMV recombination rate, we believe that the intrinsic recombination rate in CaMV is higher and perhaps on the order of that of HIV. Other pararetroviruses such as plant badnaviruses or vertebrate hepadnaviruses have a similar cycle within their host cells, including steps of nuclear minichromosome, genomic size RNA synthesis, and reverse transcription and encapsidation. Nevertheless, vertebrate hepadnaviruses (e.g., hepatitis B virus) infect hosts that are very different from plants in their biology and physiology, and this could lead to a totally different frequency of cell co-infection during the development of the virus populations. Thus, even though our results can be informative for other pararetroviruses because of the viruses' shared biological characteristics, they should not be extrapolated to vertebrate pararetroviruses without caution. Viral isolates. We used the plasmid pCa37, which is the complete genome of the CaMV isolate Cabb-S, cloned into the pBR322 plasmid at the unique SalI restriction site [44] . To analyze recombination in different regions of the genome, we introduced four genetic markers: a, b, c, and d, at the positions 881, 3,539, 5,365, and 6,943, respectively, thus approximately at four cardinal points of the CaMV circular double-stranded DNA of 8,024 bp ( Figure 1 ). All markers, each corresponding to a single nucleotide change, were introduced by PCR-directed mutagenesis in pCa37, and resulted in the duplication of previously unique restriction sites BsiWI, PstI, MluI, and SacI in a plasmid designated pMark-S. Because, in this study, we targeted the possible exchange of genes between viral genomes, all markers a, b, c, and d were introduced within coding regions corresponding to open reading frames I, IV, V, and VI, respectively. Another important concern was to quantify recombination in the absence of selection, i.e., to create neutral markers. Consequently all markers consist of synonymous mutations (see below). Production of viral particles and co-inoculation. To generate the parental virus particles, plasmids pCa37 and pMark-S were mechanically inoculated into individual plants as previously described [33] . All plants were turnips (B. rapa cv, ''Just Right'') grown under glasshouse conditions at 23 8C with a 16/8 (light/dark) photoperiod. Thirty days post-inoculation, all symptomatic leaves were harvested and viral particles were purified as described earlier [45] . The resulting preparations of parental viruses, designated Cabb-S and Mark-S, were quantified by spectrometry using the formula described by Hull et al. [46] . We fixed the initial frequency of markers to a value of 0.5, and a solution containing 0.1 mg/ml of virus particles of both Cabb-S and Mark-S at a 1:1 ratio was prepared. Plantlets were co-infected by mechanical inoculation of two to three leaves with 20 ll of this virus solution, using abrasive Celite AFA (Fluka, Ronkonkoma, New York, United States). The mixed CaMV population was allowed to grow during 21 d of systemic infection. Estimation of marker frequency within mixed virus populations. We designed an experimental protocol for quantifying marker frequency within a mixed Cabb-S/Mark-S virus population after a single passage in a host plant. Twenty-four individual plants, inoculated as above with equal amounts of Cabb-S and Mark-S, were harvested 21 d post-inoculation, when symptoms were fully developed. The viral DNA was purified from 200 mg of young newly formed infected leaves according to the protocol described previously [47] . After the precipitation step of this protocol, the viral DNA was resuspended and further purified with the Wizard DNA clean-up kit (Promega, Fitchburg, Wisconsin, United States) in TE 1X (100 mM Tris-HCl and 10 mM EDTA [pH 8]). Aliquots of viral DNA preparations were digested by restriction enzymes corresponding either to marker a, b, c, or d and submitted to a 1% agarose gel electrophoresis, colored by ethydium bromide and exposed to UV. Each individual restriction enzyme cut once in Cabb-S DNA and twice in Mark-S, thus generating DNA fragments of different sizes attributable to one or the other in the mixed population of CaMV genomes. After scanning the agarose gels, we estimated the relative frequency of the two genotypes in each viral DNA preparation and at each marker position, by densitometry using the NIH 1.62 Image program. The statistical analyses of the frequency of the four markers are described below. Isolation of individual CaMV genomes and identification of recombinants. To identify and quantify the recombinants within the CaMV mixed populations, aliquots from ten of the 24 viral DNA preparations described above were digested by the restriction enzyme SalI, and directly cloned into pUC19 at the corresponding site. In each of the ten viral populations analyzed, 50 full-genome-length clones were digested separately by BsiWI, PstI, MluI, and SacI, to test for the presence of marker a, b, c, and d, respectively. In this experiment, with the marker representing an additional restriction site, we could easily distinguish between the Cabb-S and the Mark-S genotype at all four marker positions, upon agarose gel (1%) electrophoresis of the digested clones. Clones with none or all four markers were parental genotypes, whereas clones harboring 1, 2, or 3 markers were clearly recombinants. Due to the very high number of recombinants detected, markers eventually appearing or disappearing due to spontaneous mutations were neglected. Statistical analysis. Here we present the different methods we used to quantify recombination in the CaMV genome. Because all these methods assume that the different markers are neutral, we first discuss assumption. We used two datasets to test the neutrality of markers, both resulting from plants co-infected with a 1:1 ratio of Mark-S and Cabb-S. The first consisted of viral DNA densitometry data derived from 24 plants (described above), where for each plant we have an estimate of the frequency of each marker in the genome population. The second consisted of the restriction of 50 individual full-genome-length viral clones obtained from one co-infected plant (described above), yielding an estimate of the frequency of each marker, and this was repeated on ten different plants. The frequencies of the different markers were 0.508, 0.501, 0.516, and 0.507 for markers a, b, c, and d in the first dataset and 0.521, 0.518, 0.514, and 0.524 in the second dataset. We tested whether these frequencies were significantly different from the expected value under neutrality, 0.5, using either t-tests, for datasets where normality could not be rejected (seven out of eight cases), or Wilcoxon signed-rank non-parametric tests otherwise (marker c in the first dataset). In all cases p-values were larger than 0.05. There are several cautionary remarks regarding these analyses. First, in all cases we found an excess of markers. Unfortunately, the two datasets cannot be regarded as independent because, even though the methods through which the frequency estimates were obtained were different, the plants used in the second dataset were a subset of the plants of the first. We thus have only four independent estimates in each case, and there is minimal power to detect significant deviations from neutrality with such a small sample size. It should be noted at this stage that deviations from the expected value could also be caused either by slight deviations from the 1:1 ratio in the infecting mixed solution, or by deviations from that ratio in the frequency of the viral particles that actually get into the plants. Second, because of the relatively small sample sizes and low statistical power, the tests presented above could have detected only large deviations. The results clearly show, however, precisely that the markers do not have large effects, if any, and that therefore recombination estimates would be affected only very slightly by any hypothetical selective effects of the markers. Because of this, along with the fact that the introduced markers provoke silent substitutions in the CaMV genome, we assumed that markers were effectively neutral in the rest of the analysis. The dataset used to estimate the recombination frequency consisted of the 500 full-genome-length viral clones (50 from each of ten co-infected plants) individually genotyped for each of the four markers. As discussed in detail in the Results, recombination was very frequent and concerned all four markers. Indeed, approximately half of the genotyped clones exhibited a recombinant genotype. It was therefore meaningful to try to obtain quantitative estimates of recombination from our data. Our aim was to analyze viral recombination in a live host. Consequently, we had to deal with the fact that more than one viral replication cycle occurred during the 21 d that infection lasted in our experiment (we had to wait that long for the disease to develop and to be able to recover sufficient amounts of viral DNA from each infection). Based on the kinetics of gene expression [40] , we postulate that each replication cycle lasts between 2 and 3 d, and that therefore seven to ten cycles occurred between infection and the sampling time. In case this assumption is incorrect, we did calculations assuming five, seven, ten, or 20 replication cycles during these 21 d. As shown, the results were not affected qualitatively, and only slightly quantitatively. It is important to note that we assumed that recombination occurred through a template-switching mechanism, and that therefore, from a recombination point of view, the CaMV genome is linear. The reverse transcription starts and finishes at the position 0 in Figure 1 , which is the point of circularization of the DNA genome. This implies that changes between contiguous markers a-b, b-c, and c-d can be considered as true recombination whereas those between a and d cannot, as they may simply stem from circularization of DNA, during the synthesis of which the polymerase has switched template once anywhere between a-b, b-c, or c-d. To estimate the recombination rate between markers, we wrote recurrence equations describing the change in frequency of each genotype over one generation, assuming random mating and no selection (i.e., the standard Wright-Fisher population genetics model). We then expressed the frequency of all possible genotypes n generations later as a function of their initial frequency and of the recombination parameters. Subsequently we calculated the maximum likelihood estimates of the recombination parameters and their asymptotic variances given initial frequencies (we assumed that the two ''parental'' genotypes, Mark-S and Cabb-S, had equal initial frequencies of 0.5 and that all other genotypes had initial frequencies of zero) and frequencies after n generations (the observed frequencies; as stated above we used different values of n). All algebraic and numerical calculations were carried out with the software Mathematica. The recombination parameters are the recombination rates between two adjacent loci, e.g., r 1 for the recombination rate between markers a and b, and the interference coefficients, e.g., i 12 for interference between recombination events in the segments between markers a and b and b and c. To define these parameters we followed Christiansen [48] , and in particular the recombination distributions for two, three, and four loci (respectively, Tables 2.7, 2.8, and 2.9 of [48] ). It is important to realize that given the definitions of these parameters, the estimator of the recombination rate between two loci is not affected by the number of loci considered. In other words, we obtain the same estimation of the recombination rate between markers a and b whether we consider genotypic frequencies at just these two loci, or the frequencies at these two loci plus a third locus, or the complete information to which we have access, the fourmarker genotypes. Information on additional loci only affects the estimates of the interference coefficients. It proved impossible to carry out the calculations for four loci algebraically. Instead, we used a computer program to calculate the expected genotypic frequencies at all four loci after n generations, given the above stated initial frequencies and specified recombination parameters. For each combination of recombination parameters we calculated a Euclidean distance between the vector of the expected genotypic frequencies and the observed genotypic frequencies, and considered that the estimated recombination parameters were those yielding the minimal Euclidean distance. In all cases, the estimated recombination rates between pairs of loci were equal to the second decimal to those estimated algebraically from data for three or two loci."
26
"Torsional restraint: a new twist on frameshifting pseudoknots"
"mRNA pseudoknots have a stimulatory function in programmed −1 ribosomal frameshifting (−1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for −1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168–174]. A separate ‘torsional restraint’ model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115–1127]. Here, experiments using a series of ‘pseudo-pseudoknots’ having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that −1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot."
"The structure of an RNA molecule is widely recognized to play a role in many processes, including structurally organizing complex RNAs, the assembly of ribonucleoprotein complexes, and in translational recoding and regulation [reviewed in (1) ]. One common RNA folding motifs is a pseudoknot, the folding back of a single-stranded RNA onto itself to form two helical structures with single-stranded loops joining them (2) . Many such structures can be inferred from RNA sequences and frameshifting function has been demonstrated for some of these [reviewed in (3) (4) (5) ]. However, though much theoretical progress has been made in understanding how mRNA pseudoknots promote efficient À1 ribosomal frameshifting (6), a complete understanding of this mechanism remains untested. Programmed À1 ribosomal frameshift signals are typically divided into three components. From 5 0 to 3 0 these are (i) a 'slippery site' in the form N NNW WWH, where N must be a stretch of any three identical nucleotides, where W is either three A or U residues, and H is A, C or U (spacing indicates the unshifted zero frame), (ii) a spacer region and (iii) an mRNA structural element, most often a pseudoknot. The general model posits that upon encountering the mRNA pseudoknot, an elongating ribosome is forced to pause such that the anticodons of its A-and P-site tRNAs are base-paired with the zero-frame codons of the slippery site. The nature of the tRNA-mRNA interactions is such that a relative slip of À1 nucleotide still allows base-pairing in the non-wobble positions. The slippage occurs during the ribosomal pause, and it has been shown that changes affecting ribosome pause times affect frameshift efficiencies [reviewed in (7) ]. An important observation is that even though mRNA pseudoknots and energetically equivalent stem-loop structures appear to promote ribosome pausing with equal effectiveness, mRNA pseudoknots are more efficient at promoting À1 PRF (8). Our '9 Å ' model (6) provided a refinement of the original 'simultaneous slippage' (9, 10) model of frameshifting by suggesting that rather than the entire ribosome having to slip one base in the 5 0 direction, slippage could be accomplished by moving the small section of mRNA in the downstream tunnel by one base in the 3 0 direction. We have proposed that this is accomplished by the bulky and difficult to unwind mRNA pseudoknot structures becoming wedged in the downstream entrance tunnel of the ribosome, preventing the downstream region of the mRNA from being pulled into the ribosome by the equivalent of one base during the accommodation step of elongation. This blockage would introduce tension into the spacer region, which could be resolved by unpairing the mRNA from the tRNAs, allowing the mRNA to slip 1 nt backwards, resulting in a net shift of reading frame by À1 base. Though the 9 Å model provides a partial explanation for why mRNA pseudoknots promote programmed À1 ribosomal frameshifting (À1 PRF) more efficiently than simple *To whom correspondence should be addressed. Tel: +1 301 405 0981; Fax: +1 301 314 9489; Email: dinman@umd.edu ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org stem-loop structures, it does not answer the question of how the mRNA pseudoknot directs the ribosome to pause at the correct position along the mRNA. A complementary 'torsional restraint' model addresses this issue (11) . When a stem-loop structure is unwound by an elongating ribosome, unwinding of the stem forces the loop to rotate. Since a simple stem-loop is not restrained, the loop can rotate freely and only the base pairs within the stem resist ribosomal movement, and thus the potential energy of unwinding should be distributed along the length of Stem 1 ( Figure 1A ). However, if the loop is anchored or restrained, as it is in a pseudoknot by Stem 2, since the intrinsic ribosomal helicase is processive (12) , Stem 1 cannot be fully unwound until Stem 2 is first denatured. Mechanically, as the ribosome begins to unwind the base of Stem 1, Stem 2 forces the supercoiling in the remainder of Stem 1, providing extra resistance to ribosome movement. At some specific point, the resistance to ribosome movement provided by the supercoiling counteracts the forward movement of the ribosome, increasing the likelihood that the ribosome will stop at a precise point along the mRNA. Energetically, since full unwinding of Stem 1 is dependent on complete denaturation of Stem 2, the potential energy of unwinding of the pseudoknot structure should similarly be directed toward one point. Viewed either mechanically or energetically, this point is where ribosomes will be directed to specifically pause on the mRNA. If it occurs with the tRNAs in ribosomal A-and P-sites positioned at the slippery site, then frameshifting is stimulated. This is summarized in Figure 1B . The efficiency of À1 PRF can thus be viewed as a function of (i) the fraction of ribosomes paused over the slippery site and (ii) the rate at which the structure can be denatured. There is increasing evidence from single molecule experiments that unfolding occurs in quick 'rips' at a particular force (13) , suggesting that in the case of unfolding pseudoknots, frameshifting efficiency is related to both the energy barriers to unfolding the pseudoknot structure and the resistance of the structure against the force of the ribosome. In the context of the torsional restraint model, this resistance is dependent on the ability of Stem 2 to remain intact while Stem 1 is being unwound. There is experimental data that indirectly support this model: (i) disruption of the first 3 bp of Stem 1, which would displace the ribosome's pause site to a point 3 0 of the slippery site, has been shown to eliminate frameshifting (14) ; (ii) destabilizing Stem 2, which would allow it to be unwound more readily, has been shown to result in decreased frameshifting efficiency (15) (16) (17) ; (iii) replacing bulges in Stem 1 with base pairs would increase the energy required to unwind the first three bases, and a longer ribosomal pause over the slippery site would follow, yielding increased efficiencies in À1 frameshifting (15, 18) ; (iv) destabilizing the base of Stem 1 by replacing G:C base pairs with A:U pairs decreases À1 frameshifting efficiencies (19, 20) ; (v) the model eliminates the need for a 'pseudoknot recognizing factor', the evidence of which has not been forthcoming in either competition assays in in vitro translation systems (21) or by gel retardation assays (J. D. Dinman, unpublished data); and (vi) elimination of a potential torsion-restraining Stem 2, but not of a non-torsion-restraining Stem 2 in HIV-1, resulted in decreased À1 PRF efficiencies (22) . Though all of the cited studies support the torsional restraint model, none has directly addressed it. In the experiments presented in this study, a series of 'pseudo-pseudoknot' containing reporter constructs were used to test the torsional restraint hypothesis. In vitro frameshifting assays show that frameshifting can be significantly stimulated by limiting the rotational freedom of the loop region of a stem-loop structure, and that the degree of rotational freedom of Stem 1 is important in determining the extent of À1 PRF. Furthermore, mRNA toeprint analyses reveal a pseudo-pseudoknot-specific strong stop 16 nt 3 0 of the slippery site, consistent with this structure being able to direct ribosomes to pause with their A-and P-sites positioned at the slippery site. All synthetic DNA oligonucleotides were purchased by IDT (Coralville, IA). The modified L-A viral À1 PRF signal containing the G GGU UUA slippery site followed by a simple stem-loop was amplified from pJD18 (23) using the primers luc5 0 b (5 0 -CCCCAAGCTTATGACTTCTAGGCAGGGTTT-AGG-3 0 ) and luc3 0 b (5 0 -CCCCCCATGGGACGTTGTAAA-AACGACGGGATC-3 0 ). These were digested with HindIII and NcoI (restriction sites are underlined) and cloned into the firefly luciferase reporter plasmid pT7-LUC minus 3 0untranslated region-A50 (24) . In the resulting reporter construct (pJD214-18), expression of firefly luciferase requires a À1 frameshift, and the 5 0 sequence of the Stem 2 is not able to base pair with the 3 0 sequence, so that only a stem-loop rather than a pseudoknot is able to form. The same primers were used to amplify DNA from pJDRC (23) to make pJD214-Ry. In this construct, complementary mutations (5 0 -GCUGGC-3 0 to 5 0 -CGACCG-3 0 ) in the 3 0 acceptor sequence of the pseudoknot-forming region of Stem 2 allow the formation of an mRNA pseudoknot that has previously been shown to promote frameshifting at the same frequency as the wild type (23) . The primer luc5 0 CON (5 0 -CCCCAAGCTTATGACTTC-TAGGCAAGGGTTTAGG-3 0 ) contains an additional A nucleotide upstream of the slippery site and was used to make pJD214-0, the zero-frame control. To eliminate the possibility of internal initiation occurring at the luciferase initiation codon downstream of the frameshift signals, the AUG codon was changed to AUA. The Stratagene Quik-Change kit was used to mutate pJD214-18 and pJD214-Ry into pJD336-18 and pJD366-Ry, respectively, using the oligonucleotides 5 0 -GGCGTTCTTCTATGGGACGTTGTA-AAAACGGATC-3 0 and 5 0 -GATCCGTCGTTTTTACAACG-TCCCATAGAAGACGCC-3 0 (the mutated codon is underlined). pJD366-18 was further mutated to make a zero-frame control by placing an A upstream of the slippery site using the oligonucleotides 5 0 -TGACTTCTAGGCAAGGGTTTAGGAG-TG and 5 0 -CACTCCTAAACCCTTGCCTAGAAGTCA (the inserted base is underlined). A series of synthetic DNA oligonucleotides were designed to join the loop acceptor region of mRNA transcribed from pJD366-18 to the downstream region that forms the pseudoknot in the wild-type L-A À1 PRF signal. In the J-oligos, the 3 0 sequences base pair with the loop of the mRNA transcribed from pJD366-18, and the 5 0 regions of these oligos base pair with the downstream sequence. This orientation is reversed for the R-oligos. These general orientations are shown in Figure 3B . The naming of the oligonucleotide names refers to the number of additional residues placed between the regions of complementarity. The bases complementary to the pJD366-18 sequence are underlined. Plasmid DNAs were prepared using the Qiagen mini-prep kits and were linearized with DraI in a total volume of 20 ml. Proteins were eliminated by the addition of 2 ml of 1 mg/ml proteinase K and SDS to a final concentration of 0.5% followed by digestion at 50 C for 30 min. Volumes were then increased to 100 ml, extracted twice with phenol/chloroform, and DNA was precipitated with 10 ml NH 4 Ac and 250 ml ethanol. The purified DNA was resuspended in DEPCtreated H 2 O. To prepare synthetic mRNAs, 2 ml of purified linear DNAs were used for in vitro transcription using the Ambion T7 mMachine mMessage kit. RNAs were precipitated using 30 ml DEPC H 2 O and 25 ml LiAc. The RNA was resuspended in 11 ml DEPC H 2 O (1 ml in 500 would give an OD 260 of 0.05-00.1; 1-2 mg/ml). To anneal the oligonucleotides with the mRNA, J-oligos, R-oligos or the equivalent volumes of dilution buffer alone (20 mM Tris, pH 7.4, 2 mM MgCl 2 and 50 mM EDTA final concentration) were added to synthetic mRNA (0.5 mg), and the mixtures were first incubated in a 70 C heating block for 10 min; the block was then removed and allowed to cool to 37 C (30 min), after which they were briefly spun down and incubated on ice. In all experiments, the molar ratios of J-and R-oligos to synthetic mRNAs were 100:1. In experiments using the competing oligonucleotide (C-oligo), this was added to either 0.5:1 or 1:1 molar ratios with either J-or R-oligonucleotides. Reticulocyte lysates were thawed on ice, 15 ml of Àmet and 15 ml of Àleu master mixes plus 20 ml of H 2 O were added to 400 ml of lysate, and 19 ml of this was added to each annealed reaction to start the in vitro translation reactions. These were incubated at 30 C for 60 min (the reaction reached a plateau after 30-35 min where the greatest difference was seen between the zero-frame controls and the frameshifting plasmids) (data not shown), and the reactions were then placed on ice. An aliquot of 7.5 ml from each in vitro translation reaction was added to 50 ml of the prewarmed luciferase reagent, and luminescence readings were taken after a 3 s delay for 15 s in triplicate using a Turner 20/20 Luminometer. Synthetic transcripts generated from DraI-digested pJD366-18 ($1.7 kb) were 5 0 end labeled using [g-32 P]CTP. These RNAs (4 ml) were incubated with 1 ml of annealing buffer and either 1 ml of H 2 O or 1 ml of an oligo (0.25 ng) at 70 C. The heating block was allowed to cool at room temperature for 40 min before 8 ml of RNaseH buffer was added (20 mM HEPES, 50 mM KCl, 10 mM MgCl 2 and 1 mM DTT). An aliquot of 1 ml of enzyme was added (mung bean nuclease, RNaseH or RNaseT1) and the reactions incubated at 37 C for 1 h. The reactions were stopped by adding 4 ml of stop solution, the products separated through a 6% polyacrylamide-urea denaturing gel and visualized by autoradiography. mRNA toeprinting JD366-18 mRNA (1 mg in 8 ml) was annealed with 2 ml of 3 0 end-labeled toeprinting primer (5 0 -CGTACGTGATCTTCA-CC-3 0 , complementary to sequence 240 bp 3 0 of the slippery site) as described above. This was added to 15 ml of lysate (200 ml Ambion retic lysate, 7.5 ml of each master mix [Àleu and Àmet] and 70 ml of 250 mM KCl), except for 2 ml, which was added to 15 ml of RT buffer [50 mM Tris-HCl (25), 40 mM KCl, 6 mM MgCl 2 , 5 mM DTT and 575 mM dNTPs] to be used as a no-ribosome control. In vitro translation reactions were incubated at room temperature for 10 min, which was empirically determined to provide the optimum amount of time to allow ribosomes to initiate translation and pause at the frameshift signal. Subsequently, 15 ml of RT buffer containing RNasin inhibitor and cycloheximide (to a final concentration of 100 ng/ml) was added to stop translation. To this, 2 ml of Superscript II (Invitrogen) was added and the reaction incubated at room temperature for 10 min. Reactions were terminated by phenol:chloroform extraction and 15 ml of stop solution added. The toeprinting primer was also used in conjunction with pJD366-18 to produce sequencing ladders by standard dideoxynucleotide chain termination methods using Sequenase (USB). Products were separated though 6% polyacrylamide-urea denaturing gels and visualized using a Storm phosphorImager (Pharmacia). Pseudo-pseudoknots stimulate frameshifting, and frameshifting efficiency changes with the degree of pseudo-pseudoknot rotational freedom We previously showed in intact yeast cells that the pseudoknot containing mRNA produced from pJDRC was able to promote efficient À1 PRF, whereas one in which only a stem-loop can form, transcribed from pJD18, could not (23) . As a first step in this study, we tested the ability of synthetic mRNAs produced from pJD366-RC and from pJD366-18, two plasmids derived from these parental constructs, to promote À1 PRF. Total luciferase activities produced from these synthetic mRNAs were divided by the luciferase activity produced from the zero-frame control plasmid, pJD366-0, and multiplied by 100% to determine À1 PRF efficiencies. The results show that the trends observed in yeast were replicated in vitro, i.e. JD366-RC mRNA promoted $8% efficiency of À1 PRF as compared with $1.1% promoted by JD366-18 mRNA (Figure 2 ). The 'torsional restraint' model predicts that conditions that would inhibit the rotational freedom of the loop region of the pJD366-18-derived mRNA should result in enhanced À1 PRF efficiency. The strategy used in this study was to anneal this mRNA with synthetic oligonucleotides complementary to both the loop region and to the sequence downstream that is normally involved in pseudoknot formation. These 'pseudopseudoknots' would be predicted to restore a pseudoknot-like structure to the mRNA. This is diagrammed in Figure 3A . Two different classes of oligonucleotides having different orientations relative to the mRNA were used to this end: 'joining' (J-) and 'reverse' (R-) oligos. The orientation of the J-oligos promotes the formation of a structure containing the equivalent of a Loop 2 region, while that of the R-oligos promotes a Loop 1 equivalent. The model also predicts that pseudo-pseudoknots having different degrees of rotational freedom should promote different frequencies of ribosome pausing over the slippery site, resulting in different efficiencies of À1 PRF. In order to control this parameter, increasing numbers of nucleotides were inserted between the mRNA hybridizing regions of the J-and R-oligos. The additional non-complementary bases in the J-oligos are 3 0 to the stem-loop residues involved in Stem 2, thus effectively increasing Loop 2. Similarly, the additional non-complementary bases in the R-oligos are 5 0 to the loop acceptor residues and correspond to an increased Loop 1. The structure of the stem-loop of pJD366-18 and its maximum base-paired interactions with representative J-and R-oligos are shown in Figure 3B . To demonstrate that an oligonucleotide-mRNA hybrid was capable of forming under the assay conditions, the J1-oligo was incubated with 5 0 [ 32 P]labeled JD366-18 mRNA and subjected to RNaseH digestion. Digestion of the RNA-DNA hybrid resulted in a labeled 110 nt fragment, demonstrating that the oligonucleotide bound to the mRNA at the position of the pseudoknot (Figure 4) . Having demonstrated the utility of the in vitro frameshifting assay and that the J-and R-series of oligonucleotides were able to hybridize with synthetic mRNA produced from pJD366-18, the next step was to monitor frameshifting efficiencies promoted by these hybrid species. Significant increases in frameshifting were observed with the incubation of pJD366-18 mRNA with oligonucleotides J1 ($10%) and J2 ($35%), while only modest increases were seen with J3 and J4 ( Figure 5 ). These findings are consistent with the notion that changes in the degree of rotational freedom of the structure would affect the distribution of paused ribosomes in the vicinity of the slippery site. One potential complication with the J-oligos is the possibility that they could interact with the Loop 2-Stem 1 region. In the R-oligos, the additional bases are distal to any possible Loop 2-Stem 1 interactions and would be more analogous to increasing Loop 1. The R-oligos stimulated À1 PRF to an even higher extent than the J-oligos ( Figure 5 ). Importantly, increasing the length of the bridging regions in these oligonucleotides (R1 to R3), which is predicted to increase the rotational freedom of the stem-loop, resulted in decreased frameshifting activity as predicted by the torsional resistance model. However, addition of three residues between the two binding regions of the R-oligo (R4) resulted in an unexpected increase in frameshifting with a very large amount of variation. In a series of control experiments, 8 nt oligos complementary to the 5 0 (Loop 1) and 3 0 Stem 2 forming regions of the pseudo-pseudoknot were hybridized to the SL mRNA and À1 PRF assays were performed. Neither of these were able to stimulate À1 PRF, even at concentrations in 100-fold molar excess to the mRNA template (data not shown). Though supportive of our central hypothesis, it is also possible that these results were due to the thermodynamic instability of the RNA:DNA duplexes through the course of the experimental protocol. To determine whether the stimulation of frameshifting was specifically due to the bridging of the stem-loop with downstream sequence (the pseudo-pseudoknot), as opposed to the nonspecific presence of an RNA:DNA hybrid, the competing oligonucleotide (C-oligo) was designed to form a 15 bp duplex with JD366-18 mRNA, including the 3 0 Stem 2 forming region, which was expected to significantly out compete either the J-or R-oligos from binding to this site, thus disrupting formation of the pseudo-pseudoknot (see Figure 3A ). Additionally, in the presence of the C-oligo, the J-and R-oligos were still predicted to hybridize with the 5 0 Stem 2 forming region, enabling us to address the question of whether this interaction alone was able to stimulate frameshifting. The results demonstrate that the addition of the C-oligo severely inhibited the abilities of both the J-and R-oligos to promote efficient frameshifting ( Figure 6 ). These findings demonstrate that (i) frameshifting was specifically stimulated by bridging of the 5 0 and 3 0 Stem 2 forming regions by the J-and R-oligos, and (ii) that the presence of an RNA:DNA hybrid at the 5 0 Stem 2 forming region was not sufficient to stimulate frameshifting by itself. The torsional restraint model predicts that pseudoknots should direct elongating ribosomes to pause at one specific location 1 2 3 4 5 6 7 8 . Efficient frameshifting is stimulated by pseudo-pseudoknots. In vitro translation assays were performed in retic lysates with mRNAs derived from pJD366-18 (SL) to which J-or R-oligos were annealed. Luciferase activities were divided by those obtained using mRNAs generated from pJD366-0, and the resulting ratios were multiplied by 100 to calculate percent frameshifting. The averages of three independent experiments performed in triplicate are shown. Error bars denote standard deviation. on the mRNA, rather than being distributed along Stem 1. We used mRNA toeprint assays to test this hypothesis. In mRNA toeprint reactions, the movement of reverse transcriptase is blocked by paused ribosomes, resulting in a strong stop positioned $16-18 nt 3 0 of the P-site of eukaryotic ribosomes (25) . Synthetic JD366-18 mRNAs were annealed with the sequencing oligonucleotide and either J1, R1 or no second oligo, and these were then used for in vitro translation reactions. After a period of time (10 min were empirically determined to be optimal), elongation reactions were stopped by the addition of cycloheximide, and reverse transcription reactions were initiated on the sequencing oligonucleotides. In parallel, control reverse transcription reactions were carried out using synthetic JD366-18 mRNA and oligonucleotides, but without in vitro translation. The results are consistent with the model, showing that the J1-and R1-oligos specifically promoted one strong reverse transcriptase stop 16 nt 3 0 of the P-site of the slippery site only the in the in vitro translation reactions ( Figure 7 ). As further predicted by the model, a broad distribution of stops of equal intensities was observed in this region with JD366-18 mRNA alone (Figure 7, lane 1) . Importantly, the +16 stop was not observed when toeprint reactions were carried in the absence of ribosomes. Additional strong stops were also of interest. One corresponding to the 3 0 end of the base of Stem 1 was observed in all samples, consistent with the presence of this structure. Both J-and R-oligo-specific pauses were also observed. The reason for the strong pause in the J-oligo is unknown. The R-oligo-specific pause is perhaps more revealing. It occurs at the 3 0 end of the RNA:DNA hybrid formed by this oligo and the mRNA, a structure that should also promote pausing of reverse transcriptase. The results presented in this study provide strong support for the torsional restraint model of programmed À1 frameshifting. Specifically, we demonstrated that RNA:DNA hybrids that mimic mRNA pseudoknots can significantly stimulate frameshifting. As predicted by the model, changing the rotational freedom of the structure by altering the lengths in the J1-and R1-oligos between the 5 0 and 3 0 mRNA hybridizing regions resulted in changes in their abilities to stimulate À1 frameshifting. The demonstration that these 'pseudopseudoknot' structures cause elongating ribosomes to specifically pause with their A-and P-sites positioned at the slippery site provides independent evidence in support of the model. In the case of the J-oligo series, frameshifting was best stimulated by J2, suggesting the structure created and the rotational freedom allowed by it was optimal for À1 PRF. The experimental design is such that we assume a similar rate of unfolding for each oligo as the predicted maximum base pairing is the same for them all. However, we do note that the type of nucleotides separating the two, separately paired regions of the oligos, and their presentation, may play a role in À1 PRF efficiency. The recent NMR structural solution of the SRV-1 pseudoknot revealed a highly structured Loop 2-Stem 1 interface including base triples involving an A residue at the 3 0 end of Loop 2 (26) . The additional base in the J2oligonucleotide is also an A. Mutagenesis experiments in this region by other groups showed, for example, that replacing the 3 0 base in Loop 2 of IBV with an A residue promoted a significant increase in frameshifting efficiency (27) , and mutation or removal of the A residue at the 3 0 base in Loop 2 of the BWYV pseudoknot reduced frameshifting levels (17) . This part of the pseudoknot has been proposed to be important in a frameshifting model where differential transition state energy barriers (due to small differences in local structure, stability or dynamics) are the primary determinant of frameshifting efficiency (3). Indeed, a Loop 2-Stem 1 triplex interaction seen in smaller frameshifting pseudoknots from luteoviruses has been shown to be critical for À1 PRF, and that similar pseudoknots lacking the triplex are less efficient at frameshifting [(28) and references therein]. This extra structural feature would limit the rate of unfolding and provide extra anchoring of Stem 2 as the ribosome attempts to unwind Stem 1, i.e. it too would help to provide additional torsional restraint. It is also possible that although the J3-and J4oligonucleotides also help to form a pseudo-pseudoknot, the additional bases may interfere with the stabilization of Stem 1. With the R-oligos, a general correlation was observed between minimization of rotational freedom and frameshifting efficiency, though this was not the case of the R4-oligo. Since the stability of the pseudo-pseudoknot generated with R4 should be similar to that of the other oligonucleotides based on the base-pairing, this result suggests that there are additional considerations to be uncovered with regard to the Frameshifting (% stimulated by R1) Figure 6 . Competition for J-or R-oligo binding sites inhibits its ability to promote efficient frameshifting. mRNA transcribed from pJD366-18 (SL) was annealed with either J-or R-oligos alone, or in combination with different concentrations of competing (C-) oligos (in ratios of 2:1 or equimolar as indicated). Sample marked SL is mRNA alone. Luciferase activities generated from in vitro translation reactions in rabbit reticulocyte lysates were divided by those obtained using mRNAs generated from pJD366-0, and the resulting ratios were multiplied by 100 to calculate percent frameshifting. pseudoknot structure influencing frameshifting. Addition of residues in the R-oligos was analogous to lengthening Loop 1, which is typically short in À1 frameshifting pseudoknots. Limited and conflicting data are available on the importance of Loop 1 in À1 frameshifting pseudoknots. In one study, addition of three A bases to Loop 1 did not affect frameshifting efficiency (15) , while in another all the mutations made in this region were detrimental to frameshifting efficiency (17) . Given the complex interactions occurring between the helices and loops in this region, we cannot yet account for why the R4-oligo stimulated frameshifting so efficiently and with such variable results. Examination of the RNA toeprint data presented here reveals that both of the pseudo-pseudoknot structures formed by the J1-and R1-oligos promoted strong stops of the reverse transcriptase $16 nt 3 0 of the P-site codon of the slippery site, consistent with the hypothesis that the presence of Stem 2 forces ribosomes to pause with their A-and P-sites positioned over the slippery site. Previous studies mapping the lagging edge of paused ribosomes, i.e. mRNA heelprint studies, did not reveal any striking differences between the effects of pseudoknots versus stem-loops (8, 16) . Interestingly, using this method, the ribosomal pauses appeared distributed over a broader stretch of mRNA ($4 nt) than observed here. It is possible that some critical level of resolution is lost in the requirement for many additional manipulations of substrates using the mRNA heelprint as compared with the toeprint methods. A remaining question centers on whether the role of the RNA pseudoknot in À1 PRF is passive or active. In the '9 Å solution' (6), the frameshift mechanism is activated by movement of the A-site codon-anticodon complex by 1 base in the 5 0 direction upon accommodation. As currently described, the mRNA pseudoknot merely passively blocks entry of the downstream message into the ribosome, resulting in stretching of the segment of mRNA located between the codon-anticodon complex and the pseudoknot. By this model, all of the energetic input for the frameshift is derived from hydrolysis of GTP by eEF1A. However, it is possible that the pseudoknot may also actively contribute to the frameshift mechanism. Specifically, pulling the downstream message into the ribosome at accommodation could result in unwinding of Stem 1 of the pseudoknot by one additional base pair. The energetic cost of so doing would be to introduce an equivalent amount of torsional resistance into Stem 2. If Stem 2 were to release this resistance by 'pulling back', the base pair in Stem 1 would be re-formed, which in turn would contribute to the energy required to dissociate the A-and P-site codonanticodon complexes from the zero-frame. This would be followed by slippage of the mRNA by 1 base in the 3 0 direction relative to the ribosome, followed by the formation of À1 frame codon-anticodon complexes. As such, the proposed active role for the mRNA pseudoknot would further reduce the energetic barrier to À1 PRF. In sum, we suggest that the 'torsional restraint model' can be combined with the '9 Å solution' to mechanistically explain the original 'simultaneous 3' mRNA + Ribos. mRNA Figure 7 . Pseudo-pseudoknots direct ribosomes to pause over the slippery site. mRNAs generated from pJD366-18 (SL) were annealed with the sequencing oligonucleotide and either J1-, R1-or no oligo (lanes 1, 2 and 3, respectively), and these were then used for in vitro translation reactions. Reactions were stopped after 10 min by the addition of cycloheximide, and reverse transcription reactions were initiated on the sequencing oligonucleotides. In parallel, control reverse transcription reactions were carried out using synthetic JD366-18 mRNA and oligonucleotides, but without in vitro translation (lanes 4-6). The positions of the slippery site, loops and stems of the pseudo-pseudoknots are indicated next to a sequencing reaction. Arrowheads indicate positions of reverse transcriptase strong stops and these are mapped to a representation of the stem-loop structure of pJD366- 18. slippage' model of À1 PRF (9, 10) . In other words, the 9 Å solution + torsional restraint = simultaneous slippage. Two recent publications have also shown that oligonucleotide:mRNA duplexes can stimulate efficient À1 ribosomal frameshifting (29, 30) . These studies differed from the present one in a number of ways, particularly insofar as they examined the effects duplex structures immediately 3 0 of the slippery site rather than addressing mRNA pseudoknot related questions. The findings support the notion that the specific location of ribosome pausing on the mRNA plays a critical role in determining frameshifting, though they do come with caveats, e.g. neither study directly mapped ribosomal pausing, and the use of different slippery sites and downstream contexts likely contributed to disparate findings for the optimal distances between the 3 0 ends of slippery sites and 5 0 ends of frameshift-stimulating oligonucleotides. Although potentially useful therapeutically there are no known natural examples of frameshifting stimulated in this manner, and thus these results do not affect the hypothesis presented here. However, these studies are important in that they raise the possibility for a new role for micro-RNAs in regulating gene expression, and for therapeutic approaches to correcting inborn errors of metabolism due to the presence of frameshift mutations."
27
"Correcting errors in synthetic DNA through consensus shuffling"
"Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ∼60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ∼1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences."
"Methods for the automated chemical synthesis of oligonucleotides (1, 2) and their assembly into long double-stranded DNA (dsDNA) sequences by PCR (3, 4) and LCR (5) have enabled the chemical synthesis of genes and even entire viral genomes (6, 7) . These technological advances have helped spur the formation of the new field of synthetic biology (8) , which aims at defining the functional units of living organisms through the modular engineering of synthetic organisms. In addition, the demand for fully synthetic gene length DNA fragments of defined sequence has dramatically increased in recent years for use in applications such as codon optimization (9), construction of DNA vaccines (10) , de novo synthesis of novel biopolymers (11) , or simply to gain access to known DNA sequences when original templates are unavailable. The future demand for long synthetic DNA is likely to dramatically increase when it becomes cheaper/faster to synthesize a desired sequence than to obtain it by other means. The assembly of DNA is currently limited by the presence of random sequence errors in synthetic oligonucleotides that arise from side reactions during synthesis (incomplete couplings, misincorporations, etc.) and resulting in 1-3 errors/kb (7, 12, 13) . The deleterious impact of these errors becomes more significant as the desired lengths of synthetic DNA increase. Indeed, in the remarkable assembly of the PhiX 174 bacteriophage genome (5386 bp) using gel-purified, synthetic oligonucleotides, the products contained an average of $2 lethal errors/kb resulting in 1 plaque-forming genomes per 20 000 clones (7) . A functional selection (plaque formation) was required in this study to identify a clone with the correct sequence. Thus, error reduction/correction is a requirement for the efficient production of long synthetic DNA of defined sequence. However, the process of sequencing multiple clones and manual correction of errors is both costly and time consuming. Several methods have been reported for the removal of error-containing sequences in populations of DNA. These methods rely upon the selective destruction (14, 15) or physical separation (16, 17) of mismatch-containing heteroduplexes. Smith and Modrich (14) reported the selective destruction of error-containing sequences in PCR products by generating dsDNA breaks upon overdigestion with the Escherichia coli mismatch-specific endonuclease MutHLS (18) . Gel purification and cloning of the remaining full-length DNA resulted in an apparent 10-fold reduction in the error rate for PCR products. However, the existing approaches are not well suited for error removal in long synthetic DNA sequences where virtually all members in the population contain multiple errors. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org Error correction with MutS is outlined in Figure 1 . The population of DNA molecules containing random errors is first re-hybridized to expose synthesis errors as mismatches ( Figure 1A ). Duplexes containing mismatches can then be removed from the population by affinity capture with immobilized MutS ( Figure 1B) , a process we term coincidence filtering, since both strands of the duplex must match to pass this filtering step. For long synthetic DNA sequences or for sequences with high error rates, coincidence filtering is ineffective, since the likelihood of both strands being perfectly matched after re-hybridization is very low. To generalize MutS error filtering for application on synthetic DNA, the synthetic DNA is cleaved into small overlapping fragments before MutS filtering. Fragments containing mismatches are selectively removed through absorption to an immobilized maltose-binding protein (MBP)-Thermus aquaticus (Taq) MutS-His 6 fusion protein (MBP-MutS-H6) (18) (19) (20) . The remaining mixture of fragments (enriched with fragments of the correct sequence) serves as a template for assembly PCR to produce the full-length product ( Figure 1C ). This process can be iterated until the consensus sequence emerges as the dominant species in the population. This approach is equivalent to DNA shuffling (21) with additional mismatch exposure and removal steps. In this report, we assemble GFPuv from synthetic oligonucleotides and apply both coincidence filtering and consensus shuffling protocols to reduce errors in the resultant DNA populations. The error rates are characterized by gene function (fluorescence) and by DNA sequencing. We also provide a mathematical model describing the error reduction protocols to aid predictions about parameters influencing their effectiveness. Chemicals were from Sigma. Restriction enzymes were from Promega and New England Biolabs. KOD Hot Start DNA Polymerase was from Novagen. Amylose resin was from NEB (catalog no. E8021S). Ni-NTA resin was from Novagen (catalog no. 70666). Ultrafiltration device from Millipore (catalog no. UFC900524). Slide-A-Lyzer dialysis membrane was from Pierce (catalog no. 66415). Full-length Taq MutS was amplified from template pETMutS (22) with primers 5 0 -AAA AAA CAT ATG GAA GGC ATG CTG AAG G-3 0 and 5 0 -AAA AAT AAG CTT CCC CTT CAT GGT ATC CAA GG-3 0 and cloned into the Nde1/HindIII sites of vector pIADL14 (23) to give plasmid pMBP-MutS-H6. E.coli strain BL21(DE3) transformed with pMBP-MutS-H6 was grown to OD 600 $1.0 and induced using 1 mM isopropylb-D-thiogalactopyranoside for 4 h at 37 C. Cells from 4 l of culture were pelleted and resuspended in 60 ml of buffer A (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM EDTA, 1 mM DTT and 1 mM phenylmethlysulfonyl fluoride). Cell suspension was sonicated on ice and insoluble material was removed by centrifugation at 50 000 g for 10 min at 4 C. Supernatant was applied to 5 ml amylose resin pre-equilibrated in buffer A. Bound MBP-MutS-H6 was washed three times using 20 ml buffer B (20 mM Tris-HCl, pH 7.4, 300 mM NaCl) and stored The re-hybridized gene synthesis products are fragmented, and error containing fragments are precipitated by MBP-MutS-H6 immobilized on amylose support. Error reduced fragments (orange, blue and red) are reassembled into the full-length gene followed by PCR amplification to generate error reduced products. Primers: black lines. overnight at 4 C. MBP-MutS-H6 was eluted using 20 ml buffer B + 10 mM maltose. Eluate was applied to $4 ml of Ni-NTA resin pre-equilibrated in buffer B. Bound MBP-MutS-H6 was washed four times using 20 ml buffer B + 25 mM imidazole. Bound MBP-MutS-H6 was eluted using buffer B + 1 M imidazole. Eluate was concentrated via ultrafiltration using Amicon Ultra 5 kDa MWCO at 4 C. Concentrated sample was dialyzed extensively against 2· storage buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.2 mM EDTA and 0.2 mM DTT) using a Slide-A-Lyzer 10 kDa MWCO cassette at 4 C. Protein concentration was determined using A 280 and a calculated extinction coefficient of 119 070 M À1 cm À1 . Dialyzed sample was diluted using an equal volume of glycerol and stored at À20 Oligonucleotides were purchased from Qiagen with 'salt-free' purification. Sequence 261-1020 of pGFPuv (GenBank accession no. U62636 with T357C, T811A and C812G base substitutions) was assembled using 40mer (37) and 20mer (2) oligonucleotides with 20 bp overlap (Supplementary Table 1 ). Assembly reactions contained the following components: 64 nM each oligonucleotide, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. Assembly was carried out using 25 cycles of 94 C for 30 s, 52 C for 30 s and 72 C for 2 min. PCR amplification of assembly products contained the following components: 10-fold dilution of assembly reaction, 25 mM of 20 bp outside primers, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. PCR was carried out using 35 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 1 min followed by a final extension at 72 C for 10 min. PCR products were purified using the Qiagen QIAquick PCR purification kit with elution in dH 2 O followed by speed-vac concentration. Assuming an error rate of 1 · 10 À6 /bp/duplication for KOD DNA polymerase (24) , 35 cycles of PCR would be expected to introduce $0.053 mutations per assembled GFPuv molecule. Assembled GFPuv was diluted to 250 ng/ml in 10 mM Tris-HCl, pH 7.8, 50 mM NaCl and heated to 95 C for 5 min followed by cooling 0.1 C/s to 25 C. Heteroduplex for consensus filtering was split into three pools and digested to completion with NlaIII (NEB), TaqI (NEB) or NcoI plus XhoI (Promega) for 2 h following the manufacturer's protocols. Digests were purified using the Qiagen QIAquick PCR purification kit with elution in dH 2 O. Samples were pooled and the concentration was determined by measuring A 260 . MBP-MutS-H6 binding reactions contained $11.5 ng/ml DNA and $950 nM MBP-MutS-H6 dimers in 1· binding buffer (20 mM Tris-HCl, pH 7.8, 10 mM NaCl, 5 mM MgCl 2 , 1 mM DTT and 5% glycerol). Reactions were allowed to incubate at room temperature for 10 min before incubation for 30 min with an equal volume of amylose resin pre-equilibrated in 1· binding buffer. Protein-DNA complexes were removed by low-speed centrifugation and aliquots of supernatant were removed for subsequent processing. Supernatant (50 ml) from consensus filtering experiments was desalted using Centri-Sep spin columns (Princeton Separations) and concentrated. Purified and concentrated DNA fragments were reassembled as above with aliquots removed at varying cycles. Aliquots of assembly reactions were resolved on 2% agarose gels to monitor the reassembly process. Aliquots showing predominantly reassembled fulllength GFPuv were PCR amplified as above. Aliquots of supernatant from coincidence filtering experiments were diluted 10-fold and PCR amplified as above. PCR products were digested with BamHI/EcoRI and ligated into the 2595 bp BamHI-EcoRI fragment of pGFPuv. Ligations were transformed into E.coli DH5 and fluorescent colonies were scored using a handheld 365 nm ultraviolet (UV) lamp. Preparation of substrate for consensus shuffling from 10 non-fluorescent GFPuv clones Ten non-fluorescent GFPuv clones were pooled in equal amounts. The nature and location of the mutations in these clones is shown in Figure 4 . The GFP coding region was PCR amplified from the mixture and submitted to the consensus shuffling protocol with and without the application of the MBP-MutS-H6 error filter. To create an error filter, we constructed a fusion protein between MBP (19) and the mismatch binding protein from T.aquaticus (22) with a C-terminal His 6 tag (MBP-MutS-H6). MBP-MutS-H6 was overexpressed and purified from E.coli to >95% purity (Supplementary Figure 1) . MBP-MutS-H6 immobilized on amylose resin was shown to selectively retain a 40mer heteroduplex containing a deletion mutation over wild-type homoduplex (Supplementary Figure 2) . To demonstrate error correction, unpurified 40mer oligonucleotides were assembled by PCR (3) to produce a 760 bp gene encoding green fluorescent protein (25) (GFPuv). Two independent preparations of GFPuv containing typical gene synthesis errors (Figure 3 and Table 1 ) were re-hybridized and subjected to two iterations of coincidence filtering or consensus shuffling. For consensus shuffling, the GFPuv assembly product was split into three pools and digested into sets of overlapping fragments using distinct Type II restriction enzymes ( Figure 2 ). The digests were pooled and subjected to error filtering with or without added MBP-MutS-H6. The unbound fragments were reassembled into full-length products and PCR amplified. For coincidence filtering, unbound fulllength GFPuv was PCR amplified following treatment with the error filter. After cloning in E.coli, error rates were estimated by scoring colonies for fluorescence under a handheld UV lamp (Figure 3) . The actual error rates of the input and consensus shuffled populations were determined by sequencing plasmid DNA from randomly selected colonies (Figure 3) . The results show that two rounds of consensus shuffling increased the percentage of fluorescent colonies from $60 to >90% and Table 1 . Sequence errors in input and consensus shuffled DNA Table 1 . Although DNA shuffling has traditionally been used to create diversity through the combinatorial shuffling of mutations in a population, DNA shuffling also creates a sub-population of sequences with a reduction in diversity, as correct fragments can recombine to produce error-free sequences. Indeed, with consensus shuffling, it is possible to start with a population of DNA molecules wherein every individual in the population contains errors and create a new population where the dominant sequence is error free. To demonstrate this, 10 nonfluorescent GFPuv clones, each containing a deletion mutation (Figure 4) , were pooled and subjected to either DNA shuffling alone or two iterations of consensus shuffling. Products were cloned in E.coli, and the percentage of fluorescent colonies was monitored as an indication of progress toward the consensus sequence. DNA shuffling alone (no MBP-MutS-H6) increased the percentage of fluorescent colonies to 30% (387 colonies total) similar to a previous report (26) . Two rounds of consensus shuffling gave a new population that was 82% fluorescent (551 colonies total), indicating that the dominant species was likely the consensus sequence of the input population. Both consensus shuffling and coincidence filtering protocols were effective in reducing errors in synthetic GFPuv populations ( Figure 3 ). In both cases, two iterations of either consensus shuffling or coincidence filtering increased fluorescent colonies from average values of $60 to >90%. Sequencing data from two independent experiments showed a 4.3-and 3.5-fold reduction in the error rate for the consensus shuffled populations compared with the input populations giving final error rates of 0.3 and 0.28 errors/kb, respectively. These results demonstrate the usefulness of the MBP-MutS-H6 error filter in both consensus shuffling and coincidence filtering protocols. Taq MutS has previously been shown to bind to deletion mutations with high affinity (27) , a mutation common in synthetic DNA. However, it is important to note that Taq MutS has lower affinity for specific point mutations and binds weakly to homoduplex DNA (27) . These factors may limit the stepwise efficiency of the error filter. Moreover, specific point mutations may be refractory to removal even after multiple rounds of consensus shuffling. Two rounds of consensus shuffling using the MBP-MutS-H6 error filter proved most effective in reducing deletions and G/C to A/T transitions, consistent with previous reports for the selectivity of Taq MutS (27) . However, it must be emphasized that each synthetic oligonucleotide point mutation would generate two heteroduplex DNA molecules containing unique mismatches after PCR amplification and re-hybridization ( Figure 1A and Table 1 ). For example, a G to A transition mutation in a synthetic oligonucleotide would generate heteroduplexes with G-T or A-C mismatches after PCR amplification and re-hybridization. For consensus shuffling, either of these mismatch containing heteroduplexes could evade precipitation by the MBP-MutS-H6 error filter and participate in the reassembly of full-length GFPuv. Therefore, Table 1 lists the pair of mismatches that could give rise to the observed transition or transversion mutation. These results show that the MBP-MutS-H6 error filter was most effective at removing insertion/deletion loops and G-T/A-C mismatches from the population. It should be possible to generalize the consensus shuffling protocol to a large number of synthetic DNA constructs. GFPuv was chosen as the synthetic construct in this study for its advantages as a fluorescent reporter gene. This allowed easy optimization of our protocol without the need to sequence thousands of base pairs of DNA. We expect the results reported here for consensus shuffling to readily translate to synthetic DNA constructs of varied sequence, greater overall length and/ or higher initial errors/kb. Synthetic DNA constructs of varied sequence can be digested into a defined set of fragments using Type II restriction enzymes or fragmented into any desired size range using controlled DNase I digestion (26) . Digestion and reassembly of a large number of different genes is expected to be as robust as the protocol of DNA shuffling (28) , which has been broadly applied to a variety of gene sequences. Synthetic DNA constructs larger than GFPuv are expected to be amenable to error correction by consensus shuffling, as the error filtering is conducted on gene fragments before reassembly of the full-length gene. Thus, the errors/kb data presented in this study are expected to translate to larger genes with similar initial errors/kb (excepting mutations introduced by PCR amplification following the final application of the error filter). Synthetic DNA constructs of higher initial errors/kb are expected to be amenable for error correction by consensus shuffling. However, these constructs will require digestion into smaller sized gene fragments that may affect the efficiency of error correction. In contrast to consensus shuffling, an increase in the size of the synthetic DNA product or an increase in errors/kb would preclude the use of the coincidence filtering protocol, as every molecule in the population would contain one or more errors. As proof of the utility of the consensus shuffling protocol, 10 non-fluorescent GFPuv clones containing one or more errors (Figure 4 ) were converted into a population where 82% of the clones were fluorescent. It is important to note that DNA shuffling alone shows an improvement in percent fluorescent colonies in this example (from 0 to 30%). For synthetic DNA populations, DNA shuffling alone shows no improvement in percent fluorescent colonies (see Figure 3 'no MutS' treatments). DNA shuffling alone improves the overall number of correct sequences only for small DNA populations with low error rates. For example, when shuffling 10 clones with a unique mutation in each clone, one would expect the fraction of correct products to be (9/10) 10 = 35% (26), very close to the value of 30% that we observed. A mathematical model describing the error rates for shuffling and error filtering of synthetic DNA populations is presented below. To estimate some parameters of consensus shuffling and coincidence filtering, a simple mathematical model (Equations 1-6) was constructed. An input population of dsDNA molecules of length N, containing E errors/base is re-hybridized, fragmented into shorter dsDNA fragments of average length S, error filtered and reassembled. P(F) is the probability a fragment of length S will have a correct sequence. We determine the probability that re-hybridized duplexes will have zero (C), one (H ) or both (I ) strands with errors. Equation 5 estimates the probability that a fragment will be correct after a cycle of MutS filtering, P(F 0 ), by applying a MutS selectivity factor (M ) to adjust the relative amounts of mismatch containing duplexes (I, H ) while accounting for the total fraction of correct strands in the re-hybridized duplexes. The probability of obtaining an error free assembly product, P(A), is then given by Equation 6 . From our consensus shuffling error rate data (Figure 3 ), we estimate the MutS selectivity factor M to be $2.2. Figure 5 shows some predictions that emerge from this model assuming typical length (2 kb), fragment sizes (200 bp) and error rates (1.8 errors/kb). Consensus shuffling is predicted to be most effective with smaller fragment sizes ( Figure 5A ). As mentioned above, smaller fragment sizes could be obtained by controlled digestion with DNase I (21) . In addition, multiple iterations of MutS filtering can have dramatic results on populations with few correct sequences ( Figure 5B ), although the model does not account for the differing specificity of MutS toward the various types of mismatches. The model also predicts that even modest improvements in the MutS selectivity factor through optimization of the MutS-DNA binding conditions and/or the use of a combination of MutS homologs with varying mismatch specificity (29) could dramatically improve the consensus shuffling protocol ( Figure 5C ). Coincidence filtering (N = S) is predicted to be effective for populations with low error rates per clone ( Figure 5D ) but becomes ineffective when the majority of re-hybridized duplexes contain mismatches. We have demonstrated consensus shuffling and coincidence filtering as experimental methods to significantly reduce errors in synthetic DNA. Consensus shuffling should be generally applicable for error correction on synthetic genes of typical lengths and error rates. Two iterations of consensus shuffling ($6 h/iteration) generated a population with $1 error/3500 bp. This reduction in error rate will allow the identification of a correct clone after sequencing DNA from a reduced number of colonies. Coincidence filtering is a simple and effective procedure to reduce errors in synthetic DNA populations with low error rates per clone. These methods should significantly increase the speed and decrease the cost of production of synthetic genes. Note: While this manuscript was under review, Carr et al. (30) independently reported the application of Taq MutS in protocols for error reduction on synthetic DNA."
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"Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces"
"While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data."
"Purity and integrity of RNA are critical elements for the overall success of RNA-based analyses, including gene expression profiling methods to assess the expression levels of thousands of genes in a single assay. Starting with low quality RNA may strongly compromise the results of downstream applications which are often labor-intensive, time-consuming and highly expensive. However, in spite of the need for standardization of RNA sample quality control, presently there is no real consensus on the best classification criteria. Conventional methods are often not sensitive enough, not specific for single-stranded RNA, and susceptible to interferences from contaminants present in the sample. For instance, when using a spectrophotometer, a ratio of absorbances at 260 and 280 nm (A 260 :A 280 ) greater than 1.8 is usually considered an acceptable indicator of RNA purity (1, 2) . However, the A 260 measurement can be compromised by the presence of genomic DNA leading to over-estimation of the actual RNA concentration. On the other hand, the A 280 measurement will estimate the presence of protein but provide no hint on possible residual organic contaminants, considered at 230 nm (3) (4) (5) . Pure RNA will have A 260 :A 230 equal to A 260 :A 280 and >1.8 (1) . A second check involves electrophoresis analysis, routinely performed using agarose gel electrophoresis, with RNA either stained with ethidium bromide (EtBr) (6) (7) (8) (9) , or the more sensitive SYBR Green dye (10) . The proportion of the ribosomal bands (28S:18S) has conventionally been viewed as the primary indicator of RNA integrity, with a ratio of 2.0 considered to be typical of 'high quality' intact RNA (1) . However, these methods are highly sample-consuming, using 0.5-2 mg total RNA and often not sensitive enough to detect slight RNA degradation. Today, microfluidic capillary electrophoresis with the Agilent 2100 bioanalyzer (Agilent Technologies, USA) has become widely used, particularly in the gene expression profiling platforms (11, 12) . It requires only a very small amount of RNA sample (as low as 200 pg), the use of a size standard during electrophoresis allows the estimation of sizes of RNA bands and the measurement appears relatively unaffected by contaminants. Integrity of *To whom correspondence should be addressed. Tel: The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org the RNA may be assessed by visualization of the 28S and 18S ribosomal RNA bands ( Figure 1A and B); an elevated threshold baseline and a decreased 28S:18S ratio, both are indicative of degradation. A broad band shows DNA contamination ( Figure 1C ). As it is apparent from a review of the literature, the standard of a 2.0 rRNA ratio is difficult to meet, especially for RNA derived from clinical samples, and it now appears that the relationship between the rRNA profile and mRNA integrity is somewhat unclear (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) . On the one hand, this may reflect unspecific damage to the RNA, including sample mishandling, postmortem degradation, massive apoptosis or necrosis, but it can reflect specific regulatory processes or external factors within the living cells. Altogether, it appears that total RNA with lower rRNA ratios is not necessarily of poor quality especially if no degradation products can be observed in the electrophoretic trace ( Figure 1D ). For all these reasons, the development of a reliable, fully integrated and automated system appropriate for numeric evaluation of RNA integrity is highly desirable. Standardized RNA quality assessment would allow a more reliable comparison of experiments and facilitate exchange of biological information within the scientific community. With that prospect in mind, and with the aim of anticipating future standards by pre-normative research, we identified and tested two software packages recently developed to gauge the integrity of RNA samples with a user-independent strategy: one open source, the degradometer software for calculation of the degradation factor and 'true' 28S:18S ratio based on peak heights (24) and the freely available RIN algorithm of the Agilent 2100 expert software, based on computation of a 'RNA Integrity Number' (RIN) (25) . Both tools were developed separately to extract information about RNA integrity from microcapillary electrophoretic traces and produce a userindependent metrics. Using these tools, we assessed the purity and integrity of 414 RNA samples, derived from 14 different human adult tissues and cell lines, many of which representing tumors. Those results were compared with conventional RNA quality measurement approaches as well as with highly expert human interpretation. We evaluated the simplicity for users and examined the potential, accuracy and efficiency of each method to contribute to standardization of RNA integrity assessment upstream of biological assays. These procedures were further validated by real-time RT-PCR quantitation of the expression levels of three housekeeping genes, using the same RNA samples, at different levels of degradation. Total RNA was prepared from human cell lines (especially from the ATCC bio-resource center, N = 50) and tissue samples (clinical samples, N = 285) from 13 different human adult tissue types, i.e. blood, brain, breast, colon, epithelium, kidney, lymphoma, lung, liver, muscle, prostate, rectum and thyroid. RNA purification was performed by cesium chloride ultracentrifugation according to Chomczynski and Sacchi (26) , by phenol-based extraction methods (TRIzol reagent, Invitrogen, USA), or silica gel-based purification methods (RNeasy Mini Kit, Qiagen, Germany; Strataprep kit, Stratagene, USA or SV RNA isolation kit, Promega, USA) according to the manufacturer's instructions with some modifications. Material was maintained at À80 C with minimal handling. RNA extraction was carried out in an RNase-free environment (see Supplementary Table 1 online) . The commercially available RNA samples were the 'Universal Human Reference' (N = 75) distributed by Stratagene (USA), and human brain (N = 2) and muscle (N = 2) RNAs supplied by Clontech (USA). Once extracted, RNA concentration and purity was first verified by UV measurement, using the Ultrospec3100 pro (Amersham Biosciences, USA) and 5 mm cuvettes. The absorbance (A) spectra were measured from 200 to 340 nm. A 230 , A 260 and A 280 were determined. A 260 :A 280 and A 260 :A 230 ratios were calculated. For microcapillary electrophoresis measurements, the Agilent 2100 bioanalyzer (Agilent Technologies, USA) was used in conjunction with the RNA 6000 Nano and the RNA 6000 Pico LabChip kits. In total, 39 assays were run in accordance with the manufacturer's instructions (see Supplementary Notes online). To evaluate the reliability of the classifier systems described in this study, replicate runs were done on a set of 56 RNA samples loaded on different chips, resulting in 2 (N = 41), 3 (N = 12), 7 (N = 2) and 50 (N = 1) data points per sample. Human RNA integrity categorization RNA integrity checking was performed by expert operators who classified each total RNA sample within a predefined discrete category from 1 to 5, examining the integrity of the RNA from electropherograms (see Supplementary Table 2 online). A low number indicates high integrity. Reference criteria parameters include ribosomal peaks definition, baseline flatness, existence of additional or noise peaks between ribosomal peaks, low molecular weight species contamination and genomic DNA presence suspicion. A smearing of either 28S and 18S peaks, or a decrease in their intensity ratio indicate degradation of the RNA sample and results in the classification into the higher categories. To evaluate the robustness of this human interpretation, five highly experienced operators, trained in these cataloging steps, separately classified a subset of 33 samples from breast cancers. It included samples with varying levels of integrity: intact RNA (33%), low quality samples (20%) and a wide range of degradation (47%). Bioanalyzer electrophoretic data were exported in the degradometer software folder (.cld format). For comparison of samples, the original data were re-scaled by the classifier system, first along the time-axis to compensate for differences in migration time, then along the fluorescence intensity-axis to compensate for variation in total RNA amount. As a result, fluorescence curves that have the same shape will have the same peak heights after re-scaling. Then, Degradation Factors (DegFact) and corrected 28S:18S ratios were calculated (see Supplementary Table 3 online) using the mathematical model developed by Auer et al. (24) , examining additional 'degradation peak signals' appearing in the lower molecular weight range and comparing them to ribosomal peak heights. Calculation of the DegFact is based on a numbering of continuous metrics, ranging from 1 to ¥; increasing DegFact values correspond to more degradation, and a new group of integrity is defined after 8 graduation steps. Once the classification of the RNA samples is completed, 4 groups of integrity are displayed, 3 showing an alert warning indicative of some measurable degradation (Yellow: 8-16, Orange: 16-24 and Red: >24), while all non-reliable data come together and form the fourth group (Black). We introduced a fifth class labeled White (<8), when no alert was produced by the software. Software and manual are freely available at http://www. dnaarrays.org/downloads.php. Degradometer version 1.4.1 (released in May 2004) of the software was used. Bioanalyzer electrophoretic sizing files (.cld format) collected with biosizing software version A.02.12.SI292 (released in March 2003) were imported in the Agilent 2100 expert software (RIN beta release). The RIN algorithm allows calculation of RNA integrity using a trained artificial neural network based on the determination of the most informative features that can be extracted from the electrophoretic traces out of 100 features identified through signal analysis. The selected features which collectively catch the most information about the integrity levels include the total RNA ratio (ratio of area of ribosomal bands to total area of the electropherogram), the height of the 18S peak, the fast area ratio (ratio of the area in the fast region to the total area of the electropherogram) and the height of the lower marker. A total of 1300 electropherograms of RNA samples from various tissues of three mammalian species (human, mouse and rat), showing varying levels of degradation and an adaptive learning approach were used in order to assign a weight factor to the relevant features that describe the RNA integrity. A RIN number is computed for each RNA profile (see Supplementary Table 4 online) resulting in the classification of RNA samples in 10 numerically predefined categories of integrity. The output RIN is a decimal or integer number in the range of 1-10: a RIN of 1 is returned for a completely degraded RNA samples whereas a RIN of 10 is achieved for intact RNA sample. In some cases, the measured electropherogram signals are of an unusual shape, showing for example peaks at unexpected migration times, spikes or abnormal fluctuation of the baseline. In such cases, a reliable RIN computation is not possible. Several separate neural networks were trained to recognize such anomalies and display a warning to the user or even suppress the display of a RIN number. Combining the results of the neural network for the RIN computation and the neural networks to detect anomalies, the RIN algorithm achieves a mean square error of 0.1 and a mean absolute error of 0.25 on an independent test set. The beta release of the software and manual are freely available at http://www.agilent.com/chem/RIN. Agilent 2100 expert version B.01.03.SI144 (released in November 2003) of the software was used. Expression levels of three housekeeping genes (HKG)-GAPD, GUSB and TFRC-were measured by quantitative PCR using the TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems, USA). Sixteen aliquots of a unique batch of RNA sample (Universal Human Reference RNA, Stratagene, USA) of various levels of integrity (cf. Table 1 ) were used to test the influence of RNA quality on the relative expression of those three genes. In parallel, a 5 0 to 3 0 comparison was done using two separate GUSB and TFRC TaqMan probes. An homogeneous quantity (0.8-1 mg) of the RNA samples was subjected to a reverse transcription step using the highcapacity cDNA archive kit (Applied Biosystems, USA) as described by the manufacturer. Single-stranded cDNA products were then analyzed by real-time PCR using the TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems, USA). Single-stranded cDNA products were analyzed using the ABI PRISM 7700 Sequence Detector (Applied Biosystems, USA). The efficiency and reproducibility of the reverse transcription were tested using 18S rRNA TaqMan probes. Five assays were used, GAPDH-5 0 (Hs99999905_m1), GUSB-5 0 (Hs00388632_gH), GUSB-3 0 (Hs99999908_m1), TFRC-5 0 (Hs00951086_m1) and TFRC-3 0 (Hs00951085_m1). In each case, duplicate threshold cycle (Ct) values were obtained and averaged; then expression levels were evaluated by a relative quantification method (27) . The fold change in one tested HKG (target gene) was normalized to the 18S rRNA (reference gene) and compared to the highest quality sample (calibrator sample), using the following formula: Fold change = 2 ÀDDCt , where DDCt = (C t-target À C t-reference ) sample-n À (C t-target À C t-reference ) calibrator-sample . Sample-n corresponds to any sample for the target gene normalized to the reference gene and calibrator-sample represents the expression level (1·) of the target gene normalized to the reference gene considering the highest quality sample. Mean 2 ÀDDCt and SD were calculated, considering the samples either individually or grouped by quality metrics categories, based on RIN metrics or DegFact values, together with the lower and upper bound mean of 95% Intervals of Confidence (IC). Using this analysis, if the expression levels of the HKG are not affected by the RNA degradation, the values of the mean fold change at each condition should be very close to 1 (since 2 0 = 1) (27) . Descriptive statistics were executed using the XLSTAT software, version 7.1 (Addinsoft, USA), P = 0.05. Mean, SD and coefficient of variation (variation or CV) between and within groups of samples were calculated, together with a measure of the dispersion (range), inter-quartile range (1st and 3rd quartiles, Q1-Q3) and evaluation of the lower and upper bound mean of 95% Interval of Confidence (IC). Comparative statistical analyses between groups were completed, P = 0.05, using non-parametric statistical tests: two-independent Mann-Whitney U-test and k-independent Kruskal-Wallis test. We analyzed 414 total RNA sample profiles from various human tissues (69%) and cell lines (31%) of either tumoral (85%) or normal (15%) origin, with varying levels of RNA integrity. Supplementary Table 1 online for details). Significant differences in A 260 :A 280 ratios were observed between specific groups of samples (i.e. tumoral versus normal or tissues versus cell lines). For instance, RNA extracted from normal samples displayed an improved ratio of 1.97, with 97% falling within the desired range ( Figure 2A ). In contrast, the distribution of A 260 :A 280 ratios was not found to correlate with either purification methods or tissues of origin. RNA integrity was further assessed by resolving the 28S and 18S ribosomal RNA bands using the Agilent 2100 bioanalyzer and the RNA 6000 protocol. The analysis was done on 399 RNA profiles; data from 15 samples was not obtained due to device problems during the runs. The system automatically provided 28S:18S ratios for 348 (87%) of the 399 profiles. Figure 2B shows the distribution of the 28S:18S computed values, with a median ratio around 1.7 and a variation of 54% from the mean (IC 1.9-2.1 and Q1-Q3 1.4-2.5). In addition, a significant degree of variability of the 28S:18S ratio (19-24%) was found for identical samples from replicate runs (2-50 times). Among those RNA samples, 28S:18S ratios of 2.0 or greater were rare, less than 44% of the values measured being within the theoretically desired range, except for the samples prepared from cultured cells ( Figure 2B ). The integration failed in the remaining 51 cases, displaying an atypical migration, with no clear 28S and 18S rRNA bands, and no 28S:18S ratio was computed (data not shown). Expert operators categorized the set of RNA samples by inspecting the electrophoretic traces of successful assays. Over the 399 RNA profiles checked, 379 (95%) were scored within predefined categories ( Figure 2C ), namely good [Human Categorization (HC)-level 1], regular (HC-level 2), moderate (HC-level 3), low (HC-level 4) and degraded (HC-level 5). The remaining 20 (5%) were flagged as displaying a temperature-sensitive profile: RNA samples initially found intact became highly degraded when heated, although no RNase contamination was observed (data not shown). Estimation of the robustness of this cataloging was done through comparison of qualifying criteria using a set of 33 breast cancer samples (see Materials and Methods). Integrity of the samples was evaluated independently by five expert operators, and categorization was found highly reliable with a coefficient variation (CV) $16%. This is low considering that individual interpretation is involved, but can be explained by the fact that very experienced operators accomplished the scoring based on a clearly defined set of instructions, thus limiting frequently observed subjective visual interpretation and inconsistency of human categorization. Predictably, a 28S:18S ratio of 2.0 denoted high quality for a majority of RNA samples, 91% being classified in HC-levels 1 to 3. However, 83% of total RNAs with 28S:18S > 1.0 but a low baseline between the 18S and 5S rRNA or front marker were also classified in HC-levels 1-3 (see Figure 1D ) and could be considered suitable for most downstream applications. RNA degradation was first assessed using the degradometer software (see Materials and Methods). Over the 399 RNA profiles checked, all were scored in one of the five predefined classes ( Figure 3A) . Altogether, 334 (84%) Degradation Factors (DegFact) values were computed, the remaining 65 RNA samples (16%) displaying profiles that could not be interpreted reliably; no DegFact values could be scored, and samples were flagged in the Black category ( Figure 3A ). Most of them (80%) correspond to samples previously classified by our operators as degraded (HC-level 5). The remaining cases had an average degradation factor of 7.5 (IC 6.7-8.3) with large variations over the entire set of samples (over 103% from the mean, range 1-52). A lower variability was persistently found when identical samples from replicate runs were considered, resulting in observed DegFact values with a 26-32% CV. In addition, statistically significant differences were found between DegFact values of samples sorted by types. The highest DegFact values were found characteristic of tissue samples, 41% of them displaying a DegFact > 8, as compared with 6% for the cell lines (data not shown). Remarkably, we found a significant linear relationship between the DegFact values distribution and the explicit human categorization. Most HC classes corresponded to an unambiguous DegFact distribution ( Figure 3B ), while HClevels 2 and 3 form a single class: HC-level 1, mean DegFact of 3.3, SD of 2.8 (IC 2.8-3.7); HC-level 2 and 3, mean Deg-Fact of 8.8, SD of 6.8 (IC 7.5-10.2); HC-level 4, mean DegFact of 15.9, SD of 7.8 (IC 12.7-19.1); HC-level 5, mean DegFact of 26.0, SD of 7.5 (IC 21.9-30.1). It is worth mentioning that the normalized heights of 18S and 28S peaks, and the interval between them after rescaling gradually decrease and then reverse with increasing degradation ( Figure 3B ). Integrity of RNA samples was measured in parallel based on the RNA Integrity Number metrics using an artificial neural network trained to distinguish between different RNA integrity levels by examining the shape of the microcapillary electrophoretic traces (see Materials and Methods). Over the 399 RNA profiles checked, 363 (91%) were scored successfully ( Figure 4A) , with an average RIN of 7.7 (IC 7.4-8.0). The remaining 36 (9%) samples were associated with various unexpected signals, disturbing computation of the RIN using default anomaly detection parameters. In each case, a flag alert was added corresponding to critical anomalies including unexpected data in sample type, (or) ribosomal ratio, (or) baseline and signal in the 5S region (data not shown). RIN categorization was found regular, variability between replicate runs, compared to the other methods, being consistently very small (CV 8-12%). As expected, the highest RIN were characteristic of cell line samples, 72% of them displaying a RIN > 9, as compared with 47% for the tissue samples (data not shown). A first group, corresponding to 295 (82%) of the 363 RNA profiles, was analyzed using the default settings of the RIN system, but with a lower threshold of RNA quantity loaded (20 ng) for reliable detection of anomalies than that recommended by the manufacturer (50 ng). A significant linear relationship was found between the RIN number and both the explicit human classification provided by our operators, Figure 3 . RNA degradation characterization. Integrity of 399 RNA sample profiles was scored using the degradometer software. (A) A total of 334 RNA profiles were successfully categorized into 5 predefined alert classes using a mathematical model that quantifies RNA degradation and computes a degradation factor (DegFact). Four classes (White, Yellow, Orange and Red) are associated with different levels of degradation. A fifth class, Black alert corresponds to samples that the system was not able to qualify with accuracy (n.d.). The distribution is represented by the number of records in each class. (B) Comparative analysis was done using human evaluation (x-axis) based on electrophoresis analysis as a reference for RNA integrity classification; observations of rRNA peak heights and DegFact values were taken at each of the 5 HC levels. Histograms refer to the mean 28S and 18S rRNA peak heights and 95% confidence intervals (fluorescence intensities; left scale). Mean DegFact values and 95% confidence intervals (arbitrary unit, right scale) are plotted with the means joined. and the DegFact values calculated by the degradometer software ( Figure 4B ). Each distinct HC class corresponds to an explicit RIN number, with HC-levels 2 and 3 forming once again a single class: HC-level 1, mean RIN of 9.6, SD of 0.7 (IC 9.5-9.7); HC-level 2 and 3, mean RIN of 8.6, SD of 0.9 (IC 8.4-8.9); HC-level 4, mean RIN of 6.1, SD of 1.5 (IC 5.2-7.1); HC-level 5, mean RIN of 3.7, SD of 2.0 (IC 2.9-4.5). For the remaining 68 samples (assay done with <20 ng of RNA), two separate groups were considered: 41 samples with a computed RIN below 5.0, and 27 above 7.0. All samples in the first group were derived from RNA 6000 Nano assays, with mean RNA quantities loaded below 10 ng (Q1-Q3, 5-12 ng), i.e. below the lower limit of quantitation indicated by the manufacturer. All but 8 of these samples were estimated by our operators to be of poor quality (HC-level 4; N = 3) or degraded (HC-level 5; N = 30), and all but 4 were flagged Black by the degradometer software and no DegFact values were scored. These RNA profiles could not be interpreted reliably, possibly due to either the low RNA concentration or the unusual migration behavior and shifted baseline values of degraded samples. Thus, the two automated systems were in disagreement for these samples; while human interpretation was in most cases in agreement with the RIN system, with less than 20% of inconsistency. In the second group of 27 samples, 20 of the profiles were derived from RNA 6000 Pico assays with RNA quantities loaded being on average below 4 ng (Q1-Q3, 0.5-0.8 ng), which is within the manufacturer specifications. All but 3 of them were estimated by our operators to range from high (HC-level 1; N = 12) to correct (HC-level 2 and 3; N = 12) quality levels. In addition, all RNA profiles except 1 were scored by the degradometer software, most of them displaying an alert flag (N = 20); some slight degradation was detected, associated to a low mean DegFact value of 9.7 (IC 8.1-11.3; Q1-Q3, 6.2-12.6). Thus, both automated systems and human interpretations agreed in most of these cases, with <11% of inconsistency. The influence of RNA quality categorization obtained with both user-independent classifiers on gene expression profiling was explored using real-time RT-PCR. The expression levels of three housekeeping genes (HKG)-GAPDH, GUSB and TFRC-were measured in 16 aliquots of a unique RNA displaying various integrity metrics ( Table 1 ). The mean correlation coefficient (r) between the threshold cycle (Ct) among the 16 samples and both quality metrics was found high: r = À0.87 considering the RIN metrics and r = 0.85 considering the DegFact values. The values of the mean fold changes, calculated according to the 2 ÀDDCt quantification method (see Materials and Methods), were found lower than 1.0, corresponding to the expression level (1·) in the sample exhibiting the highest RNA quality (Table 2 and Figure 5 ). Considering that HKG expression was measured relative to the reference sample, an obvious decline of the relative expression levels was observed, up to 24, 70 and 82%, in samples categorized according to the RIN metrics ( Figure 5A) and DegFact values ( Figure 5B ). These results indicate that 2-to 7-fold differences may be expected in the relative expression levels of genes in samples that differ only by their quality (Table 2 ). These fold differences are much larger than those measured for RNA samples of comparable integrity, consistently lower than 1.6 (Table 2 and Figure 5 ). In addition, an unambiguous gap in the distribution may be defined ( Figure 5A and B) , distinguishing the RNA samples of the higher quality categories (RIN > 8 and DegFact values < 7) from those of the lower categories (RIN < 8 and DegFact values > 12). It would be expected that measuring expression of an intact mRNA would yield approximately equal results regardless of the region being probed, and if mRNA fragmentation had occurred, then some sequences may be more abundant than others. We thus tested the effect of PCR probe location on the RNAs. The 5 0 and 3 0 GUSB probes, separated by 1209 nt, were associated with highly similar threshold cycle (Ct) measures (r = 0.98, b parameter = 0.88) ( Figure 5C ). Similar results were obtained for TFRC, with probes separated by 2066 nt (r = 0.84, b parameter = 0.92, data not shown). It seems therefore that the region being probed is not a source of variation in our results. It is universally accepted that RNA purity and integrity are of foremost importance to ensure reliability and reproducibility of downstream applications. In the biomedical literature (PubMed, November 2004), from the 485 090 articles that relate to RNA, and the 287 515 or 40 395 including respectively the 'quality' or 'integrity' term, less than 100 were found to contain 'RNA quality' or 'RNA integrity' terms. Interestingly, half of them were published between 2001 and 2004; but none is proposing a standard operational procedure for RNA quality assessment to the scientific community. Except for two studies (24, 25) , those reports are based on 10 to 15 years old methods (1), indicating that they represent the established and currently mostly used methods. Our results strongly challenge the reliability and usefulness of those conventional methods, demonstrating their inconsistency to evaluate RNA quality. First, the A 260 :A 280 and A 260 :A 230 ratios are reflecting RNA purity, but are not informative regarding the integrity of the RNA. Available RNA extraction and purification methods yield highly pure RNA with very little DNA or other contaminations, resulting most often in both ratios )1.8, although 18% of the samples were found degraded and 7% more of poor quality. The high A 260 :A 280 ratios are indicative of limited protein contaminations, whereas high A 260 :A 230 ratios are indicative of an absence of residual contamination by organic compounds such as phenol, sugar or alcohol, which could be highly detrimental to downstream applications. Nonetheless, samples displaying low A 260 :A 230 ratios ((1.8) did not exhibit any inhibition during downstream applications, such as cDNA synthesis and labeling or in vitro transcription (data not shown). Second, due to a lack of reliability, the 28S:18S rRNA ratios may not be used as a gold standard for assessing RNA integrity. When ribosomal ratios were calculated from identical samples but through independent runs, a large degree of variability (CV 19-24%) was observed. Moreover, using the biosizing software, we found 28S:18S rRNA ratios evaluation compromised by the fact that their calculation is based on area measurements and therefore heavily dependent on definition of start and end points of peaks. In 13% of the cases, the system was unable to localize the ribosomal peaks, and therefore no 28S:18S ratios were computed. For the remaining samples, no clear correlation between 28S:18S ratios and RNA integrity was found although RNAs with 28S:18S >2.0 were usually of high quality. Most of the RNAs we studied (83%), displaying a 28S:18S > 1.0, could be considered of good quality. Interestingly, Auer et al. (24) in a study on 19 tissues from seven organisms, reported that an objective measurement of the RNA integrity may possibly be done through comparison of re-scaled 28S and 18S peak heights, but not of the corresponding areas. Actually, we observed a linear relationship between RNA integrity and differences in normalized 28S and 18S peak heights. Increased degradation resulted in a significant decrease in the scaled corrected heights of the ribosomal peaks, with inversion of the ratio at the highly degraded stages (cf. Figure 3B ). In comparison to the area computation, 28S:18S rRNA re-scaled peak height measurement produced more consistent values, with a CV reduced to 12-14%, and displayed clear concentration-independent values (see Supplementary Tables 1 and 3 online) . Human evaluation of the integrity of RNA through visual inspection of the electrophoresis profiles provided very consistent data. Variability between classifications produced by five independent expert operators (CV 16%) was lower than with automated management of more conventional control 28S:18S area values (CV 19-24%). It is, however, very time-consuming and strongly dependent on individual competence. Even with highly trained specialists, 5% of the set of RNA samples could not be allocated to any of the five predefined categories; their corresponding profiles were considered by our experts as atypical, displaying a temperature-sensitive shape (data not shown). These strategies appear unsuitable for standardization and quality control of RNA integrity assessment, which require simple but consistent expert-independent classification, facilitating information exchanges between laboratories. The N-value corresponds to the number of samples by category. The mean quality metrics, i.e. RIN and DegFact and the mean fold change (2 ÀDDCt ) relative to the reference sample are indicated, together with the 95% confidence intervals. Observed technical variation (IC-rep, P = 0.05) is also specified, considering duplicate (two per gene per target sample) and replicate (six per gene per calibrator sample) measures. The reference sample exhibits a RIN of 9, a DegFact value of 4.9 and by default mean fold change set to 1. The observed decrease in the expression (relative expression, %) relative to the reference sample is calculated. The fold differences refer to the fold-ratios that are expected in the expression levels for a gene, across categories (between categories), given that the samples only differ by their quality, and within each category (within categories), considering RNA of comparable integrity. The fold-ratios (technical variation) that may be expected by chance in the gene expression levels, P = 0.05, from some technical reasons, are also considered. We therefore investigated the performance of two recently developed user-independent software algorithms (24, 25) . The degradometer software provided a reliable evaluation of RNA integrity based on the identification of additional 'degradation peak signals' and their integration in a mathematical calculation together with the ribosomal peak heights. It allowed characterization of the integrity of 84% of the samples tested, one-third with an alert flag, which was first found to be fairly informative, as it strongly reduces the complexity of the metrics by introducing three distinct classes labeled Yellow, Orange and Red, and can be used as a first straightforward simple filtering step. However, degradation factors (DegFact) metrics yield precise measures with less than 32% CV and are much more valuable than flag alerts for the purpose of standardization. The same is true for the RNA Integrity Number 'RIN' software which allowed the characterization of the integrity of 91% of the RNA samples tested, with a RIN value for 363 RNA sample profiles with less than 12% CV. In general, there was a good agreement between the human classification, the degradation factor and the RIN (see Figure 4B ). This provided a cross-validation of the user-independent qualification systems tested. Both resulted in the refinement of human interpretations, validating four statistically relevant classes of samples, namely good (HC-level 1), regular/ moderate (HC-level 2 and 3), poor (HC-level 4) and degraded (HC-level 5). Moreover, the 5% RNA samples previously flagged by the operators as displaying an atypical temperature-sensitive shape were unambiguously assigned to one or the other category of samples [RIN = 7.3 (IC 6.8-7.8); DegFact = 11.9 (IC 9.5-14.2); data not shown]. Altogether, we found the degradometer and RIN algorithms to be highly reliable user-independent methods for automated assessment of RNA degradation and integrity. The RIN system is a slightly more informative tool, able to compute assessment metrics for 91% of the RNA profiles, compared to 84% with the degradometer software; the remaining being flagged respectively as N/A or Black alert. For samples available below a low limit of 20 ng (N = 80) the RIN system provided Figure 6 . Workflow of operational procedure for RNA quality assessment. Integrity of the RNA, once extracted and purified from cell lines, clinical or biological tissues samples, is controlled from the widely used bioanalyzer electrophoretic traces. As standard part of the Agilent analysis software (25), a RIN metrics is first calculated, scoring each RNA sample into 10 numerically predefined categories of integrity (RIN, from 1 to 10; N is a threshold value). As an independent control, a degradation factor metrics (DegFact, from 1 to ¥; N 0 is a threshold value) may optionally be allocated to each RNA sample using the bioanalyzer-independent degradometer software (24) . In a standard operating procedure, RIN and/or DegFact metrics will first be used as a standard exchange language to document RNA integrity and degradation, second to classify the RNA in homogeneous groups, and finally to select samples of comparable RNA integrity to improve the scheme of meaningful downstream experiments. The standard operating procedure will benefit from feedback information that will help users to define threshold integrity metrics values based on the results of RNA-based analyses. metric values for 85% of them, compared to only 46% with the degradometer software. Similarly, the RIN system was able to provide metric values for 81% of poor quality samples (including low quality and degraded samples; N = 96), whereas the degradometer software could classify only 44% of them. Another advantage with the RIN classifier is that, if there are critical anomalies detected (including genomic DNA contamination, wavy baseline, etc.), threshold settings may be changed and a reliable RIN value computed. This was the case for 25 of the 363 RNA sample profiles successfully classified by the system. While intact RNA obviously constitutes the best representation of the natural state of the transcriptome, there are situations in which gene expression analysis may be desirable even on partially degraded RNA. Some studies report collection of reasonable microarray data from RNA samples of impaired quality (28) , leading to meaningful results if used carefully. Moreover, Auer et al. (24) recently concluded that degradation does not preclude microarray analysis if comparison is done using samples of comparable RNA integrity. We confirmed the direct influence of the RNA quality on the distribution of gene expression levels, by detecting using Q-PCR a significant (up to 7-fold) difference in the relative expression of genes in samples of slightly decreased RNA integrity, which is much larger than the variation within comparable RNA quality categories (cf. Figure 5 and Table 2 ). This may correlate with ratio discrepancies in gene expression experiments, and therefore with false positive and false negative rates of differential gene expression when comparing two samples. Therefore, computing reliable metrics of RNA integrity, even if the RNA is found to be partially degraded, may be highly valuable. The straight and unambiguous relationships established between human interpretations and both RIN and DegFact distributions indicates that, using these metrics, it should be possible to distinguish specific samples that are too disparate to be included in comparative gene expression analyses without compromising the results. Although the information provided by these user-independent classifiers is not a guarantee for successful downstream experiments, it gives a more comprehensive picture of the samples and can be used as a safeguard against performing useless and costly experiments. Thus, the RIN system may be used as simple metrics that can be easily integrated in any sample tracking information system for definition of standard operating procedures under quality assurance following a scheme such as the one described in Figure 6 . In this context, we suggest that the growing number of laboratories performing RNA Quality Control by microcapillary electrophoresis should be offered the option to report objective RNA quality metrics as part of the 'Minimum Information About a Microarray Experiment' MIAME standards (29) . Through registration of RNA profiles in a public electronic repository, such standardized information should enable and facilitate comparisons of RNA-based bioassays performed across laboratories with RNA samples of similar quality, in much the same way as sequencing traces are compared."
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"Factors affecting translation at the programmed −1 ribosomal frameshifting site of Cocksfoot mottle virus RNA in vivo"
"The ratio between proteins P27 and replicase of Cocksfoot mottle virus (CfMV) is regulated via a −1 programmed ribosomal frameshift (−1 PRF). A minimal frameshift signal with a slippery U UUA AAC heptamer and a downstream stem–loop structure was inserted into a dual reporter vector and directed −1 PRF with an efficiency of 14.4 ± 1.9% in yeast and 2.4 ± 0.7% in bacteria. P27-encoding CfMV sequence flanking the minimal frameshift signal caused ∼2-fold increase in the −1 PRF efficiencies both in yeast and in bacteria. In addition to the expected fusion proteins, termination products ending putatively at the frameshift site were found in yeast cells. We propose that the amount of premature translation termination from control mRNAs played a role in determining the calculated −1PRF efficiency. Co-expression of CfMV P27 with the dual reporter vector containing the minimal frameshift signal reduced the production of the downstream reporter, whereas replicase co-expression had no pronounced effect. This finding allows us to propose that CfMV protein P27 may influence translation at the frameshift site but the mechanism needs to be elucidated."
"The principal mechanism of translation is the accurate decoding of the triplet codon sequences in one reading frame of mRNA. Specific signals built into the mRNA sequences can cause deviations from this rule. Viruses exploit several translational 'recoding' mechanisms, including translational hopping, stop codon readthrough and programmed ribosomal frameshifting (PRF) [reviewed in (1, 2) ], for regulating the amount of proteins produced from their polyproteins. For positive-stranded RNA viruses, À1 PRF is the prevailing recoding mechanism and an essential determinant of the stoichiometry of synthesized viral proteins. Most viral À1 PRF signals are regulating the production of replication-associated proteins. Depending on the virus, the efficiency of À1 PRF can vary between 1 and 40% (3) , and changes in the efficiency can inhibit virus assembly and replication (4) (5) (6) . Therefore, À1 PRF can be regarded as a potential target for antiviral agents (4, 7) . However, the development of efficient antiviral drugs is still hindered, since little is known about the trans-acting factors and the biophysical parameters affecting the À1 PRF efficiencies. Database searches have identified putative frameshift signals from a substantial number of chromosomally encoded eukaryotic mRNAs (8) . Thus, À1 PRF may also have an impact on the complexity of the proteome of several eukaryotic organisms. Two cis-acting signals, a slippery heptamer X XXY YYZ (the incoming reading frame indicated) and a downstream secondary structure, direct the slippage and are therefore essential for this event (9) . À1 PRF takes place after the accommodation step in the slippery sequence by simultaneous slippage of both tRNAs into the overlapping À1 frame XXX YYY (9, 10) . The sequence of the heptamer allows postslippage base-pairing between the non-wobble bases of the tRNAs and the new À1 frame codons of the mRNA. Downstream RNA secondary structures [reviewed in (11) ] force the ribosomes to pause, and place the ribosomal A-and P-sites correctly over the slippery sequence (12) . However, the pausing of the ribosomes is not sufficient for À1 PRF to occur (13) ; in fact, the duration of the halt does not necessarily correlate with the level of the À1 PRF observed (12) . Crystallographic, molecular, biochemical and genetic studies suggest that a pseudoknot restricts the movement of the mRNA during the tRNA accommodation step of elongation by filling the entrance of the ribosomal mRNA tunnel (14) . This restriction can be eased either by unwinding the pseudoknot, which allows the mRNA to move forward, or by a slippage of the mRNA one nucleotide backwards. Chemical agents such as *To whom correspondence should be addressed. Tel: +358 9 19158342 ; Fax: +358 9 19158633; Email: kristiina.makinen@helsinki.fi ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org antibiotics, certain mutations in the translation apparatus, and in translation elongation factors that change the translation fidelity and kinetics, have been shown to influence À1 PRF efficiency [(10,15) ; reviewed in (16) ]. The parameters known to contribute to the efficiency of À1 PRF are the sequence of the slippery heptamer, the downstream secondary structure, and the length and sequence of the spacer between the two cis-acting signals. Up-and downstream sequences such as termination codons in the vicinity of the À1 PRF signals, or even several kilobases away from them, can affect the À1 PRF efficiencies (3, (17) (18) (19) (20) (21) (22) . A specific sequence in the Barley yellow dwarf virus (BYDV) 3 0 untranslated region (UTR), 4 kb downstream from the slippage site, is vital for À1 PRF (6, 19) . A stimulating effect is achieved through the formation of a tertiary structure, where complementary nucleotides from the 3 0 UTR base pair with a single-stranded bulge in the cis-acting stem-loop (6). Human immunodeficiency virus (HIV) was also shown to require a more complex secondary structure instead of a simple stemloop for optimal À1 PRF in vivo (21, 22) . These investigations suggest that À1 PRF studies carried out with minimal frameshift signals may lead to inaccurate estimates of the stoichiometry of synthesized viral protein products during infection. Cocksfoot mottle virus (CfMV; genus Sobemovirus) infects a few monocotyledonous plant species such as barley, oats and wheat. It has a monopartite, single-stranded, 4082 nt long, positive-sense RNA genome (23, 24) . The polyprotein of CfMV is translated from two overlapping open reading frames (ORFs) 2A and 2B by a À1 PRF mechanism (25) . In this study, we wanted to determine the in vivo À1 PRF efficiency guided by the CfMV U UUA AAC heptamer and the stem-loop structure. In addition to the minimal signal (18) , we decided to test the effect of flanking CfMV sequences for their ability to contribute to À1 PRF. We found that the surrounding viral sequences promoted more efficient À1 PRF than the minimal signal sequence in vivo when measured with the dual reporter vector system developed by Stahl et al. (26) . Therefore, we carried out an expression pattern and deletion analysis to understand the molecular basis of the observed upregulation. In addition, we critically analysed the suitability of the implemented experimental system for this type of a recoding study. An interesting possibility is that the viral proteins produced via À1 PRF could regulate À1 PRF. This hypothesis was tested by co-expressing the CfMV proteins P27 and replicase together with the dual reporter vectors. Three regions from the CfMV polyprotein ORFs ( Figure 1A) were cloned into the NheI and BclI sites between the lacZ and the luc ORFs in pAC74 (26) . This dual reporter vector was a generous gift from Dr J. Rousset of the Universite Paris-Sud, France. The inserted sequences 1602-1720 (A region), 1386-2137 (B region) and 1551-1900 (C region), were amplified by PCR using pAB-21 as a template (18) . Primers were used to introduce NheI and BglII sites to the flanking ends of the inserts. Since NheI digestion removed lacZ ORF, it was reintroduced into the plasmids as a final cloning step. The resulting plasmids were named pAC-A, pAC-B and pAC-C. Corresponding inframe controls, where one nucleotide was added in front of the slippery heptamer, were generated by PCR-based mutagenesis (Exsite, Stratagene) and named as pAC-Am, pAC-Bm and pAC-Cm, respectively. Deletion plasmids pAC-AB/ABm (1602-2137), pAC-AC/ACm (1602-1900), pAC-BA/BAm (1386-1720) and pAC-CA/ CAm (1551-1720) were also generated. The target sequences are shown in Figure 1B . The base numbering refers to the CfMV genome as in (23) . Transcription was driven from SV40 promoter. Plasmids encoded leucine (LEU2) and b-lactamase (ampicillin resistance) as selective markers. Plasmids were transformed into Saccharomyces cerevisiae H23 [MATa hsp150::URA3 ura3-1 his3-11 15leu2-3 112trp1-1 ade2-1 can100]. Dual reporter plasmid pAC1789 and the inframe control pAC1790 containing a 53 bp sequence from the HIV-1 frameshift region (26) were used as a positive control for monitoring the À1 PRF efficiency. To analyse the proteins produced during À1 PRF, lacZ-A/ Am/B/Bm/C/Cm-Fluc fragments were cloned inframe with the N-terminal 6xhistidine-tag in pYES2/NT KpnI and XhoI sites (Invitrogen). Reporter fusions were amplified by PCR using pAC-A/Am, pAC-B/Bm or pAC-C/Cm as templates. The resulting plasmids were named pYES2/NT-A/ Am, B/Bm and C/Cm. Protein expression was regulated from GAL1 promoter. Two CfMV encoded proteins, P27 (C-terminal end of ORF2A) and replicase (ORF2B), were cloned into pYES2 (Invitrogen). Translation initiation codons were introduced within the oligonucleotides during PCR. The resulting plasmids were named pYES-P27 and pYES-Rep. Control plasmids, which lacked the translation initiation codons were prepared by PCR-based mutagenesis (pYES-P27DAUG and pYES-RepDAUG) and the resulting plasmids were verified by sequencing. Plasmids encoded auxotrophic marker for uracil (URA3). All cloning steps were performed using standard protocols. Plasmids were amplified either in Escherichia coli DH5a or JM110, and purified with Qiagen columns. Inserts were verified by sequencing. Yeast transformations were done using the LiAc method (27) , and transformants were selected on a synthetic minimal defined medium (SC) lacking the corresponding auxotrophic marker(s) encoded by the used plasmid(s). Bacteria (E.coli DH5a) were grown in LB-medium containing ampicillin, whereas yeast cells were grown either in YPD, or in an SC medium. Protein expression from GAL1 promoter was repressed during growth at SC medium containing 2% glucose. Expression was induced by replacing glucose with 2% galactose and 1% raffinose. Reporter fusions were expressed in S.cerevisiae INVSc1 (his3D1/his3D1, leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52) (Invitrogen) overnight. Protein fusions were purified in denaturing conditions using Ni-NTA agarose (Qiagen), and analysed in 6% SDS-PAGE gels. Proteins were visualized either by Coomassie staining, or by using antisera raised against the CfMV polyprotein region 1386-1724 encoding CfMV VPg (28) . Protein antibody complexes were visualized with horseradish peroxidase-conjugated anti-rabbit antibodies (Sigma) and ECL chemiluminescent reagents (Amersham). Plasmids pYES-P27, pYES-Rep, pYES-P27DAUG, pYES-RepDAUG, or empty pYES2 were co-expressed with pAC-A or with the corresponding pAC-Am inframe control in S.cerevisiae EGY48 strain (MATa, ura3, trp1, his3, 6lexAop-LEU2) (Invitrogen). Transformants were grown overnight in SC-Leu-Ura media in non-inducing conditions, and used to inoculate induction medium. Cells were harvested at late logarithmic phase. Expression of the CfMV proteins was confirmed by western blotting using polyclonal antisera against the CfMV ORF 2a and 2b proteins (28) . Determining the enzymatic activities as described below monitored the effect of CfMV P27 and replicase on À1 PRF. For the in vitro analysis, the lacZ-gene of pAC-A/Am, -B/Bm and -C/Cm vectors was replaced with PCR-amplified Renilla luciferase (Rluc) gene from pRLnull vector (Promega). The resulting pACRF plasmids were used as templates for PCR in order to add T7 promoter upstream of the Rluc gene. These PCR products were used for RNA synthesis with RiboMax kit (Promega). Transcripts were treated with RQ1-DNase (Promega), purified with Qiagen RNeasy columns, and quantified spectrophotometrically. The integrity of the transcripts was checked in agarose gels. In vitro translations were carried CfMV À1 PRF test and control sequences were cloned between the b-galactosidase (LacZ) and firefly luciferase (Luc) genes into a dual reporter vector pAC74 (26) . Inframe control constructs had one extra nucleotide inserted in front of the slippery heptamer, which fused the reporters into the same reading frame. Thus, translation of the inframe control results in the production of a b-galactosidase-CfMV-firefly luciferase fusion. Translation of the test constructs in the incoming 0-frame yields a b-galactosidase-CfMV fusion, whereas À1 PRF produces a b-galactosidase-CfMV-firefly luciferase fusions identical to those produced from the inframe controls. À1 PRF efficiencies were calculated from the firefly luciferase activities after b-galactosidase normalization with the given formula. (B) CfMV polyprotein is encoded by two overlapping ORFs, 2A and 2B via À1 PRF. Sequence regions tested in the dual reporter vectors for their activity to promote À1 PRF are indicated. The numbering refers to the CfMV RNA sequence as published in (23) . out in wheat germ extract (WGE) according to the manufacturer's protocols (Promega). Reactions were incubated in room temperature for 60 min, and stopped on ice prior to enzymatic measurements. Cell cultures were started from at least three independent clones and grown until the late exponential phase. Cells were collected by centrifugation, frozen in liquid nitrogen and stored at À70 C. Bacterial cells were lysed by sonication (3 · 15 s), and yeasts by vortexing with glass beads (0.5 vol) in +4 C for 30 min. Lysates were cleared by centrifugation, and enzymatic activities were determined immediately. Total protein concentrations were measured by using a Bradford protein assay reagent (Bio-Rad). b-Galactosidase (LacZ) and firefly or Renilla luciferase (LUC or RUC) activities were measured with commercial kits from Promega according to the manufacturer's instructions. LacZ activity was determined as the colour intensity at A414 nm. Luciferase activities were measured as relative light units (RLUs) with luminometer (Biohit or ThermoLabsystems). À1 PRF efficiencies were calculated from normalized firefly luciferase activities with the following formula: [(LUC activity from the test construct)/(LacZ or RUC activity from the test construct)]/[(LUC activity from the inframe control)/(LacZ or RUC activity from the inframe control)] · 100%. In CfMV, the motif for À1 PRF is the slippery heptamer U UUA AAC and a stem-loop structure 7 nt downstream (25) . The efficiency of À1 PRF directed by CfMV cis-acting signals was assayed in vivo using a dual reporter vector system ( Figure 1A ). Since reporters are produced from one single mRNA, factors that affect the stability of the mRNA as well as the rate of translation initiation have a similar influence on the expression of both reporters, and these variations can be monitored as changes in the activity of the upstream reporter. We quantified À1 PRF by comparing the b-galactosidase normalized firefly luciferase activities derived from the test constructs via À1 PRF to those obtained from the inframe controls, in which identical b-galactosidase-CfMV-firefly luciferase fusions are produced without À1 PRF due to the added nucleotide in front of the slippery heptamer (see Figure 1A ). Similar vectors have been shown to detect even small changes in the recoding efficiencies resulting from alterations in the cis-or trans-acting factors (26, (29) (30) (31) (32) . Three inserts of varied lengths from the CfMV polyproteinencoding region (ORF2A/2B) were introduced between the two reporters ( Figure 1B) . The A-region, which at 119 bp was the shortest, represented approximately the minimal frameshift signal proven to be functional in vitro (18) . The longest region was the B-insert. At 752 bp, it started from the 5 0 -terminus of the 12 kDa viral genome-linked protein (VPg) gene and continued to the end of ORF2A. This region encodes CfMV protein P27 with an unknown function (28) . Since the minimal requirements for the functional frameshift signal in vivo were not known, an intermediate 349 bp C-sequence was also selected for the analysis. A well-characterized 53 bp frameshift cassette derived from HIV-1 RNA was used as a positive control. Our results regarding the HIV À1 PRF efficiency, 0.7 -0.1% in bacteria, and 4.5 -1.1% in yeast (Figure 2) , are corroborated by those published earlier (26, 33, 34) indicating that our dual reporter system was fully functional. b-Galactosidase has been shown to retain its specific activity well, irrespective of the C-terminal fusions (35) . This is important, since the first reporter serves to control the variations among the abundance and translation rates of the studied mRNAs (26, 30) . In addition to changes in specific activities, heterologous fusions can cause alterations in the solubility and conformation, which can expose cryptic protease target sites and reduce the stability of the proteins (36) . Therefore, for a reliable quantification of À1 PRF, it was important to test that equimolar amounts of fusions produced from the corresponding test and control constructs had similar enzymatic activities. Most inframe controls and the analogous test constructs had equal absolute b-galactosidase activities ( Table 1) . Comparable results were obtained, if activities were normalized with total protein concentration (data not shown). These results indicated that the length of the fusion as such did not affect the specific activities. The b-galactosidase activity from pAC-Am inframe control was also comparable to activity obtained from an empty pAC74, where this enzyme has no fusion (data not shown). This further supported the view that the few observed variations in the b-galactosidase activities more likely resulted from the changes in translatability or stability of the transcripts. In addition to pAC-Cm, two inframe controls pAC-Am and pAC-ACm showed $25% lower b-galactosidase activities when compared to the equivalent test constructs ( Table 1 ), indicating that the productivity from these constructs was reduced. Taken together, b-galactosidase seemed to fit well to be used as the first reporter and thus normalization factor in the in vivo experiments of this study. CfMV frameshift signals generated significant À1 PRF in yeast. À1 PRF level measured from pAC-A was 3-fold higher than from HIV RNA (Figure 2A and B) . The extent of À1 PRF directed by the minimal region A in yeast, 14.4 -1.9%, was at the same level as that reported for the CfMV minimal frameshift signal in vitro (12.7%) (18) . In contrast to our earlier in vitro observations (18) , the longer CfMV sequences upregulated À1 PRF in vivo. In yeast, the level of upregulation was 2-fold for pAC-B, the À1 PRF frequency being 26.3%, and almost 5-fold for pAC-C resulting in efficiency close to 70% (Figure 2A) , which is an extremely high value, if compared to the other values published earlier (3). CfMV frameshift signals directed À1 PRF at a lower level in bacteria than in yeast ( Figure 2B ). The extent of À1 PRF directed by region A in bacteria was 2.4 -0.7%. As in yeast, the longest B region stimulated À1 PRF 2-fold in bacteria when compared with pAC-A. However, region C did not further improve À1 PRF, but programmed À1 PRF to similar levels as pAC-B, the percentages being 4.7 -1.6% for pAC-C and 5.5 -1.5% for pAC-B. To identify the sequence(s) responsible for the enhancement of À1 PRF in vivo, a deletion analysis was carried out. The 5 0 -or the 3 0 -sequences flanking the A-region were deleted from pAC-B/Bm or pAC-C/Cm as indicated in Figure 1B , which generated vectors pAC-AB/ABm, pAC-BA/BAm, pAC-AC/ ACm and pAC-CA/CAm. À1 PRF frequencies were determined in yeast ( Figure 2C ). Increased À1 PRF was observed in all deletion constructs in comparison to the À1 PRF directed by the A region. The BA and AB regions promoted À1 PRF as efficiently as the B region, whereas regions CA and AC were better than region A, but not as good as region B. In other words, the presence of nucleotides 1386-1720, or downstream nucleotides 1602-2137, was sufficient to increase À1 PRF to the level directed by the region B. Thus, the deletion analysis did not identify single specific sequence region as being responsible for the increased À1 PRF frequencies. The expression pattern of the test and control constructs was analysed to understand the basis for the observed upregulation in yeast. Cassettes containing the reporters and the studied intercistronic sequences were expressed and purified as N-terminal histidine fusions. This allowed us to capture all the N-terminally intact products. The affinity-purified proteins were separated in SDS-PAGE gels, and visualized either by Coomassie staining (data not shown), or by western blotting with the CfMV-specific anti-VPg antibodies. The expected b-galactosidase-CfMV fusions terminating at the end of the 0-frame in the test constructs were detected. Also, the longer transframe b-galactosidase-CfMV-firefly luciferase fusion proteins were present in both the test and the inframe constructs ( Figure 3) . Comparison of the Coomassie-stained gels with the western blots revealed that the antisera recognized the products terminating at the CfMV-encoding regions better than the transframe products. Furthermore, the small size of the CfMV-specific region in the pYES2/NT-Am decreased the binding of the antibodies to these inframe control fusions. Thus, this data were not suitable for quantitative analysis of À1 PRF. Interestingly, an additional protein, which reacted with CfMV-specific antisera, was co-purified from the cells expressing pYES2/NT-Bm and pYES2/NT-Cm inframe controls ( Figure 3 ). The size of these fusions suggested that translation had terminated approximately at the site for À1 PRF signals. If such putative termination products were also present in cells expressing the test constructs, the correctly terminated 0-frame products in the western blots masked these products. A closer look at the absolute b-galactosidase and firefly luciferase activities revealed that firefly luciferase expression from pAC-Cm was clearly reduced (data not shown). In fact, expression from the inframe control was comparable to the corresponding pAC-C test construct. This was also obvious when the firefly luciferase activities were normalized with the total protein amount. After setting the activity from pAC-Am to a relative value of one, the corresponding values from pAC-Bm and pAC-Cm were 0.80 and 0.28. Although the b-galactosidase measurements (Table 1) suggested that the overall translatability of the pAC-Cm mRNA was also reduced to some extent, it explained the decrease in firefly luciferase expression only partially. In the light of these findings, the extremely high À1 PRF frequency estimate calculated for the C-region could be explained with more frequent translation termination at the frameshift signals of the pAC-Cm mRNA, which reduced firefly luciferase activity in relation to b-galactosidase. À1 PRF was also assayed in vitro in WGE. Although LacZ-encoding gene is suitable for the in vivo studies, it is an unsuitable first reporter for the in vitro determination of À1 PRF efficiencies due to its big size (30) . In good agreement with this, we observed several unexpected products in the in vitro translations programmed with LacZ-CfMV-luc mRNAs (data not shown). Renilla luciferase has been shown to retain its specific activity irrespective of the C-terminal fusions (30) . Therefore, we decided to use Rluc-CfMV-luc transcripts to determine the À1 PRF efficiencies in the cellfree system. First, we verified the suitability of Renilla luciferase for the intended in vitro experiments as described in (30) . Transcripts encoding monocistronic Renilla luciferase and Renilla luciferase fused to firefly luciferase (Rluc-Am/ Cm-luc) were mixed in different ratios and used to program the in vitro translations. Increasing concentrations of transcripts encoding the Rluc-Am-luc fusion resulted in linearly growing firefly luciferase activities. At the same time Renilla luciferase activities remained constant, which showed that its enzymatic activity was not sensitive to the C-terminal fusions ( Figure 4A ). Similar results were obtained with Rluc-Cm-luc mRNA (data not shown). À1 PRF efficiencies were then determined with transcripts that contained CfMV regions A, B and C, and their corresponding inframe controls. In all cases, slightly higher À1 PRF frequencies were obtained than in vivo. In nice correlation with the in vivo results, enhanced À1 PRF was observed with the region B, although the effect was weaker than in vivo. In this context, region C did not differ from the minimal region A in its capacity to program À1 PRF ( Figure 4B) . The ratio between the CfMV P27 and replicase is regulated by À1 PRF during CfMV infection (28) . We studied whether these proteins could regulate the À1 PRF process. P27, replicase, or an empty expression vector was co-expressed in yeast together with the dual reporter vectors containing the minimal À1 PRF test and inframe control regions as intergenic sequences (pAC-A and -Am). P27 and replicase expression was verified by a western blot analysis ( Figure 5) . A faint band having nearly the same mobility as the replicase was detected in cells grown under repressing conditions. However, due to the small size difference, this protein was not regarded as replicase. Enzymatic activities were measured from yeast lysates prepared from induced cultures. Measurements showed comparable levels of b-galactosidase in all the samples, indicating that P27 or replicase expression did not affect the stability of the dual reporter mRNA or the translatability of the first reporter ( Table 2 ). The effect of P27 or replicase expression was monitored by comparing the reporter activity ratios to those measured from cells harbouring the empty expression plasmids (Table 2) . Co-expression of CfMV replicase did not affect the normalized firefly luciferase expression (LUC/LacZ) from the inframe control, whereas slightly increased luciferase expression from the test construct was observed. In contrast, P27 expression reduced firefly luciferase expression both from the test and the inframe constructs. The effect was stronger in the presence of inframe control as normalized firefly luciferase levels reached only 54% of expression measured from the empty vector control. To verify that the observed differences in firefly luciferase production depended on the studied CfMV proteins, we co-expressed the dual reporter vectors with plasmids having the first translation initiation codons of P27 and replicase deleted (pYES-P27DAUG and pYES-RepDAUG). Western blot analysis with antisera against ORF2A or 2B did not detect any proteins produced from these vectors (data not shown). The obtained LUC/LacZ ratios were compared to those measured from cells expressing the CfMV proteins (pYES-P27 or pYES-Rep). LUC/LacZ ratios measured from cells expressing replicase were slightly lower than the ratios calculated from cells harbouring pYES-RepDAUG plasmids, being $90% when co-expressed with pAC-A and $84% when co-expressed with pAC-Am. In the presence of P27, LUC/ LacZ ratio of pAC-A reached $81% of expression measured from cells transformed with pYES-P27DAUG. Again the effect of P27 expression was more evident with pAC-Am inframe control as P27 expression reduced LUC/LacZ ratio to half ($48%) when compared to the corresponding value measured from the cells harbouring pYES-P27DAUG. This verified that CfMV P27 was able to reduce the downstream reporter expression from dual reporter mRNAs. Since CfMV P27 had a proportionally stronger effect to firefly luciferase production from the inframe control mRNAs in comparison to the test mRNAs (Table 2) , the calculated À1 PRF efficiency increased from 14.7 to 22.4%. Since À1 PRF studies are affected by a huge number of different parameters, it is not an easy task to determine the real ratio between the proteins produced via this mechanism in vivo. However, in viral systems, the efficiency of À1 PRF is an essential determinant of the stoichiometry of synthesized viral protein products, which must be rigidly maintained for efficient propagation of the virus. For example, frameshifting in retroviruses determines the ratio of structural (Gag) to enzymatic (Gag-Pol) proteins, and plays a critical role in viral particle assembly (5) . In this study, the capacity of CfMV frameshift signals to direct efficient À1 PRF was analysed in vivo by using dual reporter vectors. The length of the CfMV sequence clearly affected the actual efficiency percent in vivo. The PRF efficiency was elevated when longer viral sequences were directing the À1 PRF, but the deletion analysis did not identify any specific region as being solely responsible for the enhancement. Up-and downstream sequences nearby or far away from the cis-acting signals have been reported to enhance À1 PRF in other viruses, such as HIV, human T-cell leukaemia virus and BYDV (6, 19, 20) . Also out-of-frame stop codons have been shown to influence À1 PRF frequency in vitro in retroviruses (17) and in CfMV (18) . A study on the spacer sequences located between the cis-acting signals showed that high slippage frequencies were obtained when the first three nucleotides were G/U, G/A and G/A, the first two being the most important (37) . In CfMV, the spacer starts with UAC, which partially explains the capacity of the CfMV sequence to promote high slippage levels. In this study, the observed enhancement of À1 PRF was, however, caused by sequences that were not in the immediate vicinity of the Figure 5 . Co-expression of CfMV P27 or replicase simultaneously with the minimal frameshift signal construct pAC-A or the corresponding inframe control pAC-Am in yeast. Yeast total protein samples were separated in 12% SDS-PAGE gels, transferred onto PVDF membranes, and immunocomplexes detected by ECL chemiluminescent system. CfMV P27 expression was verified by western blotting with antisera raised against ORF2A (A), and CfMV replicase expression was detected with antisera raised against ORF2B (B). Abbreviations: À, repressed; +, induced; C1, pMAL-VPg $53 kDa; and C2, baculovirus expressed CfMV replicase. slippery sequence thus indicating that CfMV sequences further away also have an influence on the level of frameshifting in vivo. We conclude that the most reliable estimates for À1 PRF and consequently for the amount of replicase versus the 0-frame translation product P27 can be obtained only by using the full-length viral sequences. In reality, such a study would however be hampered by the non-quantitative nature of the western blot analysis, the presence of different polyprotein processing intermediates, and the differences in the stabilities of the end products in the infected cells. The overall competence of CfMV signals to direct À1 PRF was high, when compared to related plant viruses, such as Potato leaf roll virus and BYDV. À1 PRF values of $1% have been reported for these viruses when measured with reporter-based assays (6, 38) . We can hypothesize that one reason for the high efficiency is the slippery tRNA Asn encoding the AAC triplet of the CfMV heptamer. Equal U UUA AAC slippery heptamer has been measured to induce 20-40% of À1 PRF in a diversity of animal viruses [(39); reviewed in (3)]. The low fitness of CfMV À1 PRF signals in bacteria is in agreement with the poor functioning of the eukaryotic slippery heptamers of the order X XXA AAC in prokaryotes (40) (41) (42) . IBV RNA, having an identical shifty heptamer, has been shown to direct À1 PRF at similar 2-3% level in bacteria (41) . A recent study reported that XXXAAAC heptamers dictate À1 PRF to occur via the slippage of two adjacent tRNAs placed over the heptamer, irrespective of whether the host is an eukaryote or a prokaryote (42) . Therefore, the inability of prokaryotic translation systems to direct efficient À1 PRF from this heptamer is not an inherited property of prokaryotic tRNA Asn , but results from differences in the ribosomes (42) . Paused ribosomes can pass the À1 PRF site by À1 frameshifting, resumption of 0-frame translation, or termination (43) . Transient polypeptide intermediates that result from the pausing of ribosomes in the slippery sequences have been observed during IBV and S.cerevisiae L-A virus polyprotein synthesis (12, 13, 43, 44) . A pseudoknot structure formed by IBV mRNA causes a translational pause at fixed position upstream the secondary structure regardless of whether the slippery heptamer is present or absent (12) . Based on the findings of this study, we propose that also here a certain percent of ribosomes stalled at the secondary structure of the frameshift site in our inframe control and test mRNAs in yeast, and this led to the prematurely terminated products observed with the inframe control constructs pAC-Bm and -Cm. Although not unambiguously proven by this study, high frequency of termination of translation especially at the frameshift site of the pAC-Cm mRNA would nicely explain the extremely high calculated À1 PRF efficiency. Factors that change the translation fidelity and kinetics have been shown to influence À1 PRF efficiency [ (10, 15) ; reviewed in (16) ]. Autoregulation of +1 frameshifting by mammalian ornithine decarboxylase antizyme has been reported (45) . This mechanism allows modulation of frameshifting frequency according to the cellular concentration of polyamines. One could speculate that such a regulation mechanism could also be useful to adjust the amounts of the replicationassociated proteins to match the requirements of different phases in viral replication cycle. This hypothesis was studied by expressing CfMV proteins P27 and replicase together with pAC-A and pAC-Am in yeast cells. Since b-galactosidase production remained constant regardless of the presence or absence of CfMV proteins, they did not interfere with translation initiation from pAC-A/Am mRNAs per se. However, P27 expression caused a reduction in the firefly luciferase production especially from the inframe control, whereas replicase production only slightly increased the firefly luciferase production from pAC-A, but not from pAC-Am. Since replicase expression had only a faint effect on the normalized firefly luciferase production via À1 PRF, our conclusion is that CfMV replicase had no pronounced effect on translation at the frameshift site. Co-expression of the non-translatable form of P27 with the dual reporter vectors verified that P27 truly affected firefly luciferase expression on the protein level. Therefore, we propose that CfMV protein P27 may influence translation at the frameshift site. If CfMV P27 indeed interferes with viral protein synthesis during CfMV infection, the mechanism, its specificity and the possible biological role needs to be elucidated in the future."
30
"Australian public health policy in 2003 – 2004"
"In Australia, compared with other developed countries the many and varied programs which comprise public health have continued to be funded poorly and unsystematically, particularly given the amount of publicly voiced political support. In 2003, the major public health policy developments in communicable disease control were in the fields of SARS, and vaccine funding, whilst the TGA was focused on the Pan Pharmaceutical crisis. Programs directed to health maintenance and healthy ageing were approved. The tertiary education sector was involved in the development of programs for training the public health workforce and new professional qualifications and competencies. The Abelson Report received support from overseas experts, providing a potential platform for calls to improve national funding for future Australian preventive programs; however, inconsistencies continued across all jurisdictions in their approaches to tackling national health priorities. Despite 2004 being an election year, public health policy was not visible, with the bulk of the public health funding available in the 2004/05 federal budget allocated to managing such emerging risks as avian flu. We conclude by suggesting several implications for the future."
"Public health is a small component of the health system, both in terms of budgetary allocation at either state or national level and in terms of the number of practitioners. It incorporates a myriad of activities; legislation and regulation for health protection, preventive services directed at specific diseases and populations, and health promotion programs geared towards particular risk factors and vulnerable groups in the community. As such, it looks like a disparate collection of programs and investments. In Australia, there is also confusion about the very terminology of 'public health'. Despite its extensive history and global understanding, in Australia the term is used variously; to refer to publicly funded health services, and interventions (regardless of the funding source) which are aimed at primary prevention and the promotion and protection of the public health ('rats and drains'). This has led to an increasing number of jurisdictions adopting the label 'population health'. Renovation of the public health system has been on the international agenda for some years. In the US, the Institute of Medicine released reports during 2003 about the public health workforce required for 21 st century challenges [1], as well as re-visited and updated its landmark report, The Future of Public Health in the 21st Century [2] . In the UK, following the path-breaking review of the NHS by Derek Wanless [3] the Treasury commissioned him, in 2003, to undertake a review of whole-of-government effort in public health. Arising in part from the challenges that confronted Canada during the outbreak of sudden acute respiratory syndrome (SARS) in 2003, a new public health agency, at arms length from government, is being created. Public health in Australia, meanwhile, remained fragmented -by programs, across jurisdictions (particularly the states and territories) -and without a systematic approach to funding, organisation, or conceptualisation. In 2003/04, the gap between rhetoric and funding continued to be noticeable, along with the tension between framing priorities for popular appeal versus the technical language of the evidence base. This article will examine some of the indicative developments of public health in Australia in 2003/04. The key developments are identified, and a number of them are selected for in-depth analysis. In this article, we use the traditional meaning of the term 'public health' and focus on activities which are usually designed to promote and protect the health of the population. The drivers for these developments, their short term implications and some signposts for the future are suggested. While early global anxiety over SARS occupied headlines between February and May, the more persistent popular headline in 2003 focused on obesity. Summits were held in NSW and Victoria, while the National Obesity Taskforce was convened under the auspice of the Australian Health Ministers Council (AHMC). When Kay Patterson was the Federal Health Minister, she declared that prevention was the fourth pillar of Medicare and she wanted to be 'Minister for Prevention'. Indeed, the 2003/04 federal budget, although limited, contained a bundle of initiatives entitled "Prevention on the Health Agenda". In particular, a number of immunisation and health promotion programs were included. Significant amongst the funding initiatives for public health announced in 2003/04 was government support for the meningococcal vaccine. Although this was the culmination of many months of careful planning, a perception existed that this only occurred after considerable public interest in and anxiety about deaths from outbreaks of this disease. Further changes to the recommended schedule in 2003 were made by the Australian Technical Advisory Group on Immunisation (ATAGI), in particular the inclusion of pneumococcal and varicella vaccines; however, these did not result in similar prescribed vaccine programs or in similar funding. These three developments are reviewed in greater detail in the next section. The National Public Health Partnership (NPHP) and the AHMC adopted the influenza pandemic plan in October 2003, and with the advent of the newly-identified disease SARS, as well as outbreaks of meningococcal disease, management and prevention of communicable diseases was prominent. Following on from the significant funding boost for bioterrorism preparedness in 2002/03, public health preparedness became a more generic theme. The arrival of SARS occupied the national popular and political imagination as well as tested the infrastructure capacity of public health. Australia fared well during the outbreak. Apart from escaping with only six Australian cases, it provided an opportunity to establish a coordinated approach between the Commonwealth and the states/territories and also contributed to the global epidemiological investigation and prevention effort. SARS also prompted amendments to the Quarantine Act [4] . While the recall following the Pan Pharmaceutical crisis put the Therapeutic Goods Administration (TGA) under the spotlight, it also managed to conclude negotiations that had been in train for several years on a Trans-Tasman regulatory regime and authority. Also on the regulatory front, the Australian New Zealand Food Regulation Ministerial Council endorsed a nutrition, health and related claims policy guidelines and established a review of genetically modified (GM) labeling of foods [5] . All these developments pointed to the global nature of public health, and the intersection between public health activities and the economy. Policy development in public health has never been confined to a set of health programs, and in 2003/04, the lead was often taken from outside the health sector. Most significant was the adoption of the National Agenda for Early Childhood [6] , pushed by public health advocates for child health since the mid 1990s. The National Public Health Partnership responded by coordinating a scoping of child health strategies across Australia. Elsewhere in Government, "Promoting and Maintaining Good Health" was adopted as one of the National Research Priorities [7] . Healthy ageing also emerged as a policy theme in Ageing Research. Public health workforce development was pursued outside the mainstream education and training arrangements for public health in universities. The Community Services and Health Training Board commissioned a consultative process to develop population health competencies for the Vocational Education and Training (VET) sector [8] . New population health qualifications and competencies were proposed for incorporation into the Health Training Package -including certificates in population health and in environmental health, and diplomas in population health and in indigenous environmental health. The release in 2003 of the report "Returns on Investments in Public Health: an epidemiological and economic analysis" [9] (often referred to as the Abelson report), may have a significant impact in subsequent years. Commissioned several years earlier by the Population Health Division of the Department of Health and Ageing (DoHA), the report experienced a relatively low profile until Derek Wanless visited from the UK. Having chaired a review that contributed to a significant budgetary increase for the NHS, Wanless had been commissioned by the British Treasury to examine prevention across government. In September 2003, at a meeting in Canberra with senior officials across key agencies, Wanless marveled at the value of the Abelson report, described in more detail below. Although 2004 was an election year, public health policy was neither visible during the campaign or in policy development more generally. The Federal Government's initiative to wind up the National Occupational Health and Safety Commission received little publicity and comment, even though it indicated the Commonwealth's increasing tendency to pursue its own pathway, separate from states and territories, and to bring the functions of statutory bodies into departments. Jurisdictional and annual reports show that across the states and territories, there were multiple plans, draft guidelines, meetings, episodic training and programs across a broad range of areas. Some health issues are being taken up across jurisdictions -particularly tobacco control, sexually transmitted infections, Aboriginal health, and vaccination. Innovative activities were reported in some jurisdictions, such as a new Health Impact Assessment Branch and a new public health training program in Western Australia. There was, however, no apparent consistency in health priorities across the nation, and an apparent divergence in the interests of the states/territories and the federal government. While the "prevention and management of overweight and obesity" agenda may have appeared to many observers as a new issue in 2003, its arrival was preceded by several years of intensive work. The NHMRC had released Acting on Australia's Weight: Strategic plan for the prevention of overweight and obesity in 1997 [10] , the same year the ABS published the findings from the 1995 National Nutrition Survey, revealing that 45% of men and 29% of women in Australia were overweight, with an additional 18% of men and women classified as obese [11] . Furthermore, overweight and obesity were more common in lower socio-economic groups, in rural populations, in some immigrant groups, and in Aboriginal and Torres Strait Islander (ATSI) peoples. Despite longstanding national cooperation on nutrition (since the days of the National Better Health Program in the late 1980s), and even more recent national cooperation on physical activity, public and political imagination was not captured until the same issues were recast as 'obesity', with a focus in particular on childhood obesity. Following from the NSW Childhood Obesity Summit in late 2002, the Australian Health Ministers agreed that a national approach was required and established a National Obesity Taskforce [12] . In 2003, NSW Health released it's response to the Summit recommendations and supported the vast majority of the 145 resolutions [13] . The Victorian Department of Human Services also held a summit [14] , while Healthy Weight 2008 -Australia's Future was released by the Commonwealth [15] . The NHMRC joined in with release in late 2003 of clinical practice guidelines for general practitioners and other health professionals [16] . While the specifics vary, the major themes and strategies are captured in Healthy Weight 2008. These are summarised in the Table 1. The Commonwealth strategy is, however, relatively weak on intersectoral policy and regulatory measures. As an illustrative example of the contrast at the state level, implementation in NSW now ranges from school physical activity and nutrition survey, to a school canteen strategy, to negotiating with Commercial Television Australia about their code of practice on advertising in peak children's viewing hours. The Commonwealth apparently chose not to consider how it might exercise its relevant taxation or legislative powers, despite the history of health promotion pointing to the importance of public policy measures beyond the health system. An examination of the manner in which the obesity issue was framed, and the details contained in the national strategy, raises a number of issues and questions: -Why was framing the issues as 'obesity' more successful than the focus on 'nutrition' and 'physical activity'? Why did 'obesity' gain traction while the other terms did not? -Why did the Commonwealth opt for the softer programmatic approach, rather than tackle obesity with stronger public policy measures (such as taxation and regulation), and demonstrate its national leadership capacity? -Was the absence of stronger public policy measures because 'obesity' is regarded as largely a health issue, rather than a whole-of-government issue? Or was the Government waiting to see if the US opposed the WHO Global Strategy on account of the strength of the industry lobby? -After a number of years of public concern about eating disorders and whether they arise in part because of promotion of certain types of body image, was the 'obesity' label a backward step for mental health and a return to traditional images of beauty? -Is there a risk that people, including children, who are labeled as 'overweight and obese' will be stigmatised? To what extent have the voices of affected communities been incorporated into the development of national strategies, if at all? -Given the correlation between obesity and socioeconomic disadvantage, how would the proposed strategy not exacerbate those inequalities? -Were children targeted because they are a "captive audience" and therefore easy targets or did the evidence suggest the best return on investment (in terms of health gain and managing demand on the health care system) would come from a focus on children? -Was the move to appeal to a populist agenda, while simultaneously progressing the longer-term agenda of tackling health inequalities through multi-sectoral partnerships, a triumph for public health advocates? These complex threads are interwoven. For the moment, the publicly enunciated agenda represents a confluence of a number of rationales. During 2003-4 three new vaccines were added to the schedule of recommended vaccines for Australians (an additional change to the schedule, recommending that polio immunisation be changed from oral to injected (IPD) vaccine, will not be discussed here). These vaccines protect against serogroup C meningococcal disease, some strains of Pneumococcal disease, and chicken pox [17] . For the first time, not all of these recommended vaccines will be funded by Government. Prior to the introduction of these vaccines, the quality of information about the epidemiology and burden of disease caused by these three infections was extremely variable. Meningococcal disease has been notifiable for many years, and in Australia almost all is caused by serogroups B and C. Whilst serogroup B predominantly occurs in young children, a new strain of serogroup C [18] was causing increasing anxiety amongst public health professionals, microbiologists, staff of accident and emergency departments, intensive care units and of course the public and media. The cause of anxiety amongst health professionals was based on the fact that this new strain carried a high fatality rate with severe after-effects in a high proportion of survivors. The attack rate, although still small, was increasing exponentially each year and reaching an important trigger point, and the majority of cases were now healthy teenagers and young adults. Although an initial accelerated catch-up programme was introduced for teenagers (the major risk group), the new conjugated vaccine was also introduced to the childhood schedule at age one, as from that age, only one dose (at a cost of $30-$60) was considered necessary for full protection from serogroup C disease. Pneumococcal disease became notifiable in 2001, however, with such a short surveillance history, not much is certain locally, epidemiologically speaking, about risk groups and effects (although there is no reason to suppose that it has a different epidemiological pattern from other developed countries). Pneumococcal disease is thought to occur at least four times as often as meningococcal disease, is known to carry major sequelae and has a high case fatality rate. For some time it has been known to be even more common amongst the indigenous Australian population with attack rates of up to 1 in 500 each year, knowledge which underpinned the 1999 decision to target Aboriginal people for free vaccination as soon as the new vaccines became available. Unfortunately at about $120 per dose, conjugate pneumococcal vaccine is very expensive and, for the protection of the very young children who bear the brunt of this disease, it is licensed only to be given as a three dose course, making provision of this vaccine to all Australian children prohibitively expensive. Varicella, predominantly a childhood disease, is caused by a Herpes virus known as herpes virus 3 or varicella-zoster virus or VZV. It is not notifiable in Australia; therefore no epidemiological population data are available. A reliable varicella vaccine has been available since the mid 1990s in the USA and is part of American routine immunisation schedule. This vaccine became available in Australia in 2000, at a cost of about $75-$90 per dose, with two doses being required for full protection. In 2003 the Commonwealth provided its periodic update on the Australian Standard Vaccination Schedule, the list of vaccines it provides as appropriate at no cost to all Australians [19] . For the first time it differed from the National Immunisation Program recommendations in that besides meningococcal serogroup C conjugate vaccines, pneumococcal vaccine, varicella vaccine and also inactivated polio (injected) vaccine were also recommended: however, funding was only secured for meningococcal conjugate vaccines, with a continuation of the provision of pneumococcal vaccines for indigenous children. As a result, although recommended, pneumococcal and varicella vaccines were not funded and parents would have to decide whether or not to pay for them. These funding decisions had important implications. Vaccines protect most of their recipients from unpleasant and sometimes life-threatening disease. One view, subscribed to in the UK, is that ethically, children should not be denied access because of their parents' inability to pay. These vaccines have been the subject of several cost-benefit studies, with generally favourable to extremely favourable pro-vaccination results. Table 2 summarises the various models for framing policy. The policy of funding meningococcal serogroup C vaccine was built on a sustained program of epidemiological evidence, ethical decision-making and public support (and was arguably honed by public pressure). Pneumococcal disease and varicella vaccination programs however, were neither supported by good local epidemiological evidence nor respectable levels of public awareness about these diseases. There had not been a similar program of sustained policy building to support or drive a decision to fund these vaccines. As a funding policy, this was noteworthy in that it marked a departure from previous policies where all recommended vaccines were fully funded by governments. National vaccination policy is designed to advise vaccination policy makers and practitioners of the most up-to-date thinking about optimal vaccination schedules for Australian children, and is not therefore proscriptive, unlike the United Kingdom (UK). Changing or adding vaccines to the recommended schedule is therefore an advisory matter, and the question of funding the vaccination program is decided separately. Cost benefit studies indicate pneumococcal polysaccharide and conjugate vaccines can be cost-effective although vaccine costs clearly affect ratios of cost to benefit greatly [20, 21] . Varicella vaccine is more contentious, because this disease is more severe in older cases, and it is possible that one result of a vaccination program could be an increase in older cases (and therefore severe disease). Whilst the vaccine undoubtedly works, there is no consensus about precisely who should be vaccinated for maximum population health as well as cost benefit, and again potential financial savings are highly dependant upon vaccine costs [22, 23] . The costs of preventive vaccine programs and curative medicine are funded from different sources. Vaccines are currently funded by the Commonwealth and subsidised through the states according to local vaccination policies, whilst the costs of curing cases of these diseases is broadly funded through the Medicare and private health insurance systems. Savings to Medicare and health insurance funds, as a result of successful vaccination programs, are not automatically transferred to the Commonwealth to fund the vaccine programs. Savings -or costs -in one area are of little interest or importance to other program areas. In 2004 the Government revised this funding policy, providing funding for conjugate pneumococcal vaccines population immunisation program for all children under seven years of age (as well as specific people in other risk groups) to commence in January 2005. The Australian Technical Advisory Group on Immunisation (ATAGI) completed Ministerial reports on both varicella and polio (injected as well as oral) vaccination late in 2004, and it is possible that programs for these vaccines will also be funded in the future. The 2002/2003 Federal Budget papers stated that "the Government is committed to making disease prevention and health promotion a fundamental pillar of the health system": however, this was not evident in the subsequent 2003/2004 budget. The Government's Focus on Prevention Package in 2002/03 aimed to incorporate disease prevention into the core business of the primary health care system and was reflective of how the public health agenda was evolving at the national level [24] . The package was comprised largely of a range of measures directed at specific diseases, plus a bundle of initiatives for general practitioners, also referred to as the "primary health care system". Amongst health conditions affecting Australians, breast cancer received the most attention, with the National Breast Cancer Centre being funded to develop a partnership approach to the review and dissemination of new information, along with information, support and management initiatives for rural women diagnosed with breast cancer. Hepatitis C also received some attention, with funding for national education and prevention projects. Financial support was offered for the SARS efforts that had been undertaken by states and territories, in particular for providing medical personnel at international airports. A clear process for assessing priorities under the broad banded National Public Health Program was also flagged. For purposes of the budget, primary health care was defined as general practitioners, and the measures funded included: • "Lifestyle prescriptions" to help GPs "raise community awareness and understanding of benefits of preventive health"; • Collaborative approach to learning, training education and support systems; • Coordinated care plans for people with chronic or terminal conditions; and • Involvement in multidisciplinary case conferencing. The budget did not adopt a comprehensive approach to the primary health care system, perhaps because many community health services, which represent the other important arm for delivery of public health services, are the responsibility of states. The timetable for renewing Public Health Outcome Funding Agreements (PHOFAs) between the Commonwealth and states and territories in 2004 raised in the minds of some stakeholders, the possibility that the Commonwealth might adopt a more comprehensive and strategic approach, linking public health and primary health care funding streams. Judging by the actual quantum of funds made available in the 2003/2004 budget, it would seem that most elements from the package did not actually receive additional funding, as shown in Table 3 . Indeed, many of the GP initiatives, previously cast as improving primary health care, were subsequently packaged as 'prevention'. The combination of these measures reflected a tight fiscal climate, with little growth in the overall health budget, as well as that of other portfolios. It was also a package that demonstrated relatively limited imagination, with support for established issues (such as breast cancer) and repackaging general practice measures that were already in train. With Medicare spending "uncapped" (and targeted public health programs "capped"), attaining more prevention dollars through the GP sector may appear to be one of the few ways to 'grow' dollars for prevention. Although this could be considered to be consistent with the Ottawa Charter of "reorienting health services", many GPs are not trained in a population-based approach to practice, and simply providing new for payments to all represents an undifferentiated, uncoordinated and untargeted approach to prevention. If there is limited support to GPs, and little monitoring, then these measures are unlikely to translate into improved health outcomes. Funding for the Tough on Drugs strategy was announced outside the Focus on Prevention package; perhaps due to Source: [28] the fact that the Tough on Drugs was the responsibility of the Parliamentary Secretary therefore requiring a separate communications strategy, or because the Prime Minister has a strong personal interest in the illicit drug strategy. The range of measures funded (which included introduction of retractable needle and syringe technology, addressing problems related to increased availability and use of psycho stimulants, establishing a research fund, supporting alcohol and drug workforce development needs, promoting access to drug treatment in rural areas, and tackling problems faced by drug users with concurrent mental health problems) certainly suggested more serious government interest and commitment to illicit drugs. During the course of the Howard Government, there has been a gradual process of re-casting the "landscape" of interest groups and policy constituencies. Strong support for breast cancer and zero-tolerance on illicit drugs contrasts sharply with the delays experienced in renewal of the National HIV/Hepatitis C Strategy. The new prominence given to meningococcal vaccine, child health and obesity creates space for other interest groups: even if the re-framing was shaped by nutrition and physical activity lobbies, other clinical interests have been brought into the picture. These developments illustrate how 'political' considerations are important in determining 'public health policy'. It was interesting however, to observe the interest in prevention from outside the health portfolio, particularly from Treasury. This was motivated in part by the Intergenerational Report and concerns about both the sustainability of Medicare as well as the social and economic cost burden arising from an ageing society. This helped to ensure interest in the Abelson Report [9] . Few countries have conducted research on return of investment from prevention efforts. Australia was praised by Derek Wanless at a high-level consultation for completing such an analysis, during his visit to Canberra while conducting a review for the UK Treasury, "Securing Good Health for the Whole Population" [26] . His final report pointed to Australia and Netherlands as two countries that were increasingly using economic evaluation in public health programs. It will be interesting to see if public health policy analysts and Treasury officials draw on this report in future years. In the future it will be interesting to see if the focus on high-visibility programs can demonstrate short-term economic returns. Given 2004 was an election year, the "political economy" of prevention programs could arguably have become a focus of future public health policy, with the 2003/4 agenda providing the Government with the opportunity to gauge public reaction to this new positioning and design their election campaign appropriately. This was, however, not the case. The American emphasis on 'preparedness' appears not to resonate with the Australian public in the same way. From the perspective of public health policy advocates, some lessons that can be drawn from 2003/04 are: • Government's response to public health proposals are shaped by its understanding of the popular interest and desire to communicate directly with the general public; • Longer term public health issues which have struggled to gain support can be progressed if they are cleverly shaped to fit the Government's "formula"; • Develop and nurture new advocates, particularly in seeking to engage with the broader health system; and • Work with the media as partners rather than adversaries These lessons need to be learned well and quickly, to assist with moving the forum for public health policy debate more into the public domain; beyond an essentially "in house" discourse between politicians, researchers and public health advocates. If a more engaged and informed community takes up a public health issue, government will be more likely to respond. "
31
"GIDEON: a comprehensive Web-based resource for geographic medicine"
"GIDEON (Global Infectious Diseases and Epidemiology Network) is a web-based computer program designed for decision support and informatics in the field of Geographic Medicine. The first of four interactive modules generates a ranked differential diagnosis based on patient signs, symptoms, exposure history and country of disease acquisition. Additional options include syndromic disease surveillance capability and simulation of bioterrorism scenarios. The second module accesses detailed and current information regarding the status of 338 individual diseases in each of 220 countries. Over 50,000 disease images, maps and user-designed graphs may be downloaded for use in teaching and preparation of written materials. The third module is a comprehensive source on the use of 328 anti-infective drugs and vaccines, including a listing of over 9,500 international trade names. The fourth module can be used to characterize or identify any bacterium or yeast, based on laboratory phenotype. GIDEON is an up-to-date and comprehensive resource for Geographic Medicine."
"As of 2005, the world is confronted by 338 generic infectious diseases, scattered in a complex fashion across over 220 countries and regions. Each new day confronts health care workers with unexpected outbreaks, epidemics and heretofore unknown pathogens. Over 2,000 named bacteria, viruses, fungi and parasites are known to cause human disease; and are confronted by 328 anti-infective agents and vaccines. Experts working in Health Geographics share an obvious and immediate need for comprehensive and timely data on the status of infection around the globe. A recent outline of GIDEON addressed uses for the Infectious Diseases clinician [1] . This review will focus on the Global Health aspect of the program. In 1990, we initiated a project to design computer systems to follow all diseases, outbreaks, pathogens and drugs. The initial DOS-based program was written in Paradox for floppy disks, later evolving through a compact disk-based program in Windows. A commercial web-based program was eventually released under the name, GIDEON (Global Infectious Diseases and Epidemiology ON-line, Gideon Informatics, Inc, Los Angeles, California) at http:/ /www.GideonOnline.com. The current version is available on CD (updated every three months) or web subscription (updated every week). The program consists of four modules: Diagnosis, Epidemiology, Therapy and Microbiology. Program modules of peripheral interest in Health Geographics (Therapy and Microbiology) will be discussed only briefly. The Diagnosis module is designed to generate a ranked differential diagnosis based on signs, symptoms, laboratory tests, incubation period, nature of exposure and country of disease origin. Figure 1 depicts the data entry screen for a patient suffering from fever and joint pain following a trip to Indonesia. The lower 'Personal notes' box is used to record additional case data, and can be written in the user's own language. The differential diagnosis list for this case (figure 2) indicates that this patient may be suffering from Chikungunya. The appearance of many diseases on the list indicates that the user failed to enter all positive, and negative findings. For example, the fact that cough was absent would have reduced the likelihood of the second disease listed (Mycoplasma infection) and increased the statistical probability of Chikungunya. At this point, the user can generate a hard copy or e-mail report, access a table comparing the clinical features of the diseases listed, or examine the ranking or omission of specific diseases. If the user clicks on a specific disease name, clinical and epidemiological data on the disease in question are depicted (figure 3). The differential diagnosis list is generated by a Bayesian formula which compares the product of disease-incidence and symptom incidence, for all compatible infectious diseases. In the above example, a number of diseases known to occur in Indonesia were capable of producing fever, and joint pain. The statistical likelihood of Chikungunya in this case can be computed by a simple Bayesian formula, as follows: Two spreadsheets in the GIDEON database respectively follow the incidence of all symptoms for every disease, and the incidence of all diseases for every country. When a clinical case is "entered" into GIDEON, the program identifies all compatible diseases and ranks their relative likelihoods as determined by the above formula, ie: P-(C/ S) vs. P-(D2/S) vs. P-(D3/S) ... vs. P-(Dn/S). A blinded study of 500 cases conducted by this author found that the correct diagnosis was listed in the differential list in 94.7% of cases, and was ranked first in 75% [2] . A second study of hospitalized patients in Boston found that the correct diagnosis was listed in only 69%, and was ranked first in 60% [3] . It is likely that inclusion in the differential diagnosis list may be more important than disease ranking in such systems [4] . A "Bioterrorism" option generates the differential diagnosis for diseases associated with suspected bioterror scenarios. In Figure 4 , "<bioterrorism simulator>" has been substituted for Indonesia, given the above constellation of fever, joint pain, etc. The resulting differential diagnosis lists Ebola (42.9% probability), followed by Crimean-Data entry screen for a bioterrorism scenario Figure 4 Data entry screen for a bioterrorism scenario. Congo hemorrhagic fever (12.6% probability). A similar "Worldwide" option can be used to explore all of the worlds diseases consistent with given clinical features, and access text on the global status for individual diseases. In theory, data entry by users can be monitored at the server level for purposes of surveillance. For example, if one or more users in China were to enter cases of fatal pneumonia, a "red-flag" at any monitoring agency (i.e., the World Health Organization) could indicate the possible appearance of SARS -long before submission of specimens or reporting of the case to local authorities. Similarly, the appearance of multiple cases of "dysentery" by users in a given community could indicate a possible outbreak of shigellosis. The Epidemiology module presents detailed country-specific information on the status of each disease, both globally and within each relevant country. The current version contains over two million words in 12,000 notes. All data are derived from Health Ministry publications, peer-review journals, standard textbooks, WHO and CDC websites and data presented at conferences. The user may also access over 30,000 graphs which follow disease incidence, rates and other numerical data. The main Epidemiology screen is shown in Figure 5 . Note that the user can append custom "personal notes" -in any national language or font-regarding the status of every disease in their own institution. Such notes would be accessible by all colleagues using GIDEON on the local network. Maps which depict the global distribution of each disease can be accessed through the 'Distribution' tab ( Figure 6 ). Epidemiology module, main screen Figure 5 Epidemiology module, main screen. The 'images' tab has been pressed, to access thumbnail images of Plague. These can be maximized and copied to PowerPoint, etc. Note addition of 'Personal notes' by the user, at lower right. Text outlining country-specific data for the disease ( Figure 7) is available through either a list of countries displayed in this module, or by clicking the relevant 'red dot' on the map. These text boxes also include data sets which automatically generate incidence / rate graphs (Figure 8) , a chronological account of all disease outbreaks, and numbered reference links to relevant journal publications and reports of ongoing outbreaks from ProMed http:// www.promedmail.org. A separate 'Graphs' option allows the user to generate custom-made graphs comparing multiple disease rates, or rates in multiple countries. (Figure 9 ). Additional tabs access the descriptive epidemiology and clinical background of each disease. Synonym tabs generate lists of alternative terms for diseases and countries in Spanish, German, Norwegian, etc. Historical data record the incidence of individual diseases and significant outbreaks spanning decades. An additional "Fingerprint" option generates a list of diseases compatible with any set of epidemiological parameters. For example, in Figure 10 we see that ten parasitic diseases are transmitted by fish in Japan. The Therapy module follows the pharmacology and application of all drugs and vaccines used in Infectious Diseases. The current version contains 264 generic drugs and 64 vaccines. Various sub-modules present the mechanism of action; pharmacology, dosages, drug-drug interactions, contraindications, spectrum, and susceptibility testing standards. An international synonym lists contains over 9,500 trade names. As in other modules, users may add Epidemiology module Figure 6 Epidemiology module. Map depicting the global distribution of plague. Specific map areas can be expanded, and all elements can be copied for reproduction as necessary. Country-specific notes regarding plague appear when corresponding red dots are clicked. custom notes in their own language for each drug or vaccine: prices, resistance patterns, local trade names, etc. The Microbiology option is similar to the Diagnosis module. Users may enter any combination of phenotypic tests, and obtain a ranked probability list of compatible bacteria. The current version incorporates more than 1,300 taxa. The Microbiology module is also designed to list the phenotype, prior names, ecology and disease association for any organism, or compare the phenotypes of any combination of organisms selected by the user. Since the graphic and mapping functions of GIDEON treat individual countries as whole units, data presentations lack a certain degree of "granularity." Thus, the dif-ferential diagnosis of fever in Venezuela will include malaria, even if the patient is living outside of the endemic, southern region. This problem is corrected to a large extent by text in the associated country-specific notes and the general knowledge base of the treating physician. In theory, the manufacturer could follow the incidence of each disease for every state, district, province and oblast; but variability would still exist according to occupation, rural vs. urban setting, season, etc. An additional problem relates to the availability and quality of valid epidemiological data. Disease reporting varies widely from country to country. For example, AIDS reporting statistics from sub-Saharan Africa are generally inadequate. Where necessary, the spreadsheets used by GIDEON record published true estimates rather than questionable reports. In other instances, Health Ministry Figure 7 Plague in Tanzania. Clicking on relevant data sets will generate incidence and rates graphs. Note several numbered links to journal publications. Plague -Worldwide incidence and rates per 100,000 Figure 8 Plague -Worldwide incidence and rates per 100,000. data conflict with reports of the World Health Organisation, a fact which is recorded in relevant GIDEON country notes. Occasionally, major diseases are not reported at all. For example, several recent cases of cholera in Japan originated from Thailand; but Thailand has not officially reported a single case in many years. Where possible, the GIDEON data base relies on published best estimates, and at times 'educated guesses' when data are entirely lacking. Thus, there are few published data for disease incidence in Togo, and the program is forced to rely on publications for neighboring Ghana. The reader is referred to the GIDEON website http:// www.GideonOnline.com for an extensive listing of data sources, published reviews, technical background and pricing information. Graph contrasting AIDS rates among user-selected countries Figure 9 Graph contrasting AIDS rates among user-selected countries. Publish with Bio Med Central and every scientist can read your work free of charge "
32
"Globalization and Health"
"This debut editorial of Globalization and Health introduces the journal, briefly delineating its goals and objectives and outlines its scope of subject matter. 'Open Access' publishing is expected to become an increasingly important format for peer reviewed academic journals and that Globalization and Health is 'Open Access' is appropriate. The rationale behind starting a journal dedicated to globalization and health is three fold: Firstly: Globalization is reshaping the social geography within which we might strive to create health or prevent disease. The determinants of health – be they a SARS virus or a predilection for fatty foods – have joined us in our global mobility. Driven by economic liberalization and changing technologies, the phenomenon of 'access' is likely to dominate to an increasing extent the unfolding experience of human disease and wellbeing. Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve. And lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic. A balanced view would propose that the effects of globalization on health (and health systems) are neither universally good nor bad, but rather context specific. If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first."
"Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve. And lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic. A balanced view would propose that the effects of globalization on health (and health systems) are neither universally good nor bad, but rather context specific. If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first. I am pleased to introduce 'Globalization and Health', a peer reviewed, open access (free to the end user) journal. In this, the début editorial, I will briefly outline the purpose and scope of this journal highlighting our intention to publish a balanced mixture of opinion on the subject. That the journal be 'Open Access' is entirely appropriate. Knowledge, at its best utility, is a 'public good' i.e. nonrival, non-excludable. While this journal will deal with the subject matter of creating 'global public goods for health', it will also by virtue of its very existence, contribute toward that process. Globalization and Health's 'Open Access' policy changes the way in which articles are pub-lished. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [1] . Third, a copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal. Globalization and Health's articles are archived in PubMed Central [2], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [3] in Germany, at INIST [4] in France and in e-Depot [5], the National Library of the Netherlands' digital archive of all electronic publications. Importantly, the results of publicly funded research will be accessible to all taxpayers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [6]. Added to this, a country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones (although creating access to the internet is another matter [7] ). The rationale behind starting a journal dedicated to globalization and health is three fold: Firstly: Globalization is reshaping the social geography within which we might strive to create health or prevent disease. The determinants of health -be they a SARS virus or a predilection for fatty foods -have joined us in our global mobility. Driven by economic liberalization and changing technologies, the phenomenon of 'access' is likely to dominate to an increasing extent the unfolding experience of human disease and wellbeing. Secondly: Understanding globalization as a subject matter itself needs certain benchmarks and barometers of its successes and failings. Health is one such barometer. It is a marker of social infrastructure and social welfare and as such can be used to either sound an alarm or give a victory cheer as our interconnectedness hurts and heals the populations we serve. And lastly: In as much as globalization can have an effect on health, it is also true that health and disease has an effect on globalization as exemplified by the existence of quarantine laws and the devastating economic effects of the AIDS pandemic. A balanced view would propose that the effects of globalization on health (and health systems) are neither univer-sally good nor bad, but rather context specific. The extent to which individual states are able to engage the process of globalization on their own terms differs widely from one country to the next. Child mortality, for example, changes quickly in response to subtle changes in purchasing power in impoverished communities. In affluent communities however, a small change in income has little effect on utility in either direction. As we consider the effects of globalization on wellbeing it becomes apparent that we need to consider both the long term scenarios for populations as a whole, and the immediate effects for the more vulnerable within those populations who are dependent on fragile local economies. If the dialogue pertaining to globalization is to be directed or biased in any direction, then it must be this: that we consider the poor first."
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Subset of CORD-19 for rapid prototyping of ideas in vector encodings and Weaviate.

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