In the long-established model of tumorigenesis, cells acquire specific mutations that disrupt the normal regulatory constraints on unlimited cell division. These mutations allow cells to proliferate at the expense of healthy tissues and develop into tumors. As a result, most cancer treatments use agents that curb tumor growth by inhibiting cell divisions. Surprisingly, a few conditions known to reduce a cell's ability to divide—including genetic traits, some diets, and chemotherapies—seem to increase the chances of tumor occurrence. In a new study, Ganna Bilousova and her colleagues shed new light on this troubling paradox by examining the development of mouse blood cell tumors (leukemias) in contexts that reduce cell division rates.All circulating blood cells are descendents of progenitor cells that reside in the bone marrow. The ultimate progenitors, called hematopoietic stem cells, give rise to all blood cells by producing more specialized progenitors that initiate distinct lineages, such as the red and white blood cells. Progenitors divide to ensure the constant renewal of blood cells, but their cell divisions must be tightly controlled to avoid generating too many blood cells and increasing the risk of tumors.Overproliferation of blood cell progenitors seems to underlie chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). The blood progenitors of both leukemias harbor a genetic anomaly known as the Philadelphia chromosome. This aberrant joining of Chromosomes 9 and 22 produces an aberrant protein called Bcr-abl, which promotes both cell proliferation and mutations. Either property could account for Bcr-abl's cancer-promoting, or oncogenic, potential. Yet, as the authors show, simply introducing a few blood progenitors carrying the Bcr-abl protein is not enough to produce leukemias in mice. Only when cell divisions are impaired in the mice blood progenitors does the proliferative advantage, and oncogenic potential, of Bcr-abl become obvious.Their experimental setup mimics the conditions of leukemia onset—in which an oncogenic mutation presumably arises in a single (or a few) blood progenitors—yet eventually allows the cell to supersede its nonmutant counterparts. The authors transplant a mixture of two types of progenitors—some express Bcr-abl and some don't—into mice, and examine their relative contribution to the recipients' blood a few weeks later. (The rodents' own blood progenitors had been previously destroyed by irradiation.) The authors find that whether they slow cell divisions genetically (using stem cells with mutations in key regulators of cell divisions) or chemically , the progenitor cells that carry Bcr-abl can overcome the hurdle and produce mature blood cells and, eventually, tumors. In the absence of this challenge, however, Bcr-abl cells barely contribute to the recipients' blood, and far fewer cancers arise. What's more, the authors can quench the oncogenicity of Bcr-abl progenitor cells simply by cotransplanting normal progenitor cells (free of Bcr-abl and able to divide). p53, an oncogene linked to a wide variety of cancers, confer the same advantage as Bcr-abl in this experimental setting, which suggests that the authors' model might apply to other tumors as well. While much more work needs to be done for the implications of this study to affect people, Bilousova and colleagues speculate that preventing cancer not only involves avoiding exposure to mutation-causing substances, but also a lifestyle that promotes healthy cell division behavior among tissue progenitors. —Francoise ChanutThe implication of these observations is that nonmutant progenitor cells normally outcompete a Bcr-abl cell arising in a bone marrow niche. But under conditions that impair cell divisions, whether genetic, environmental, or therapeutic, the mutated cell has the advantage and might eventually take over. Overcoming cell division blockers is a tall order that not all oncogenes are expected to fill. Yet mutations in


Information on the metastasis process in breast cancer patients undergoing primary tumour removal may be extracted from an analysis of the timing of clinical recurrence.The hazard rate for local-regional and/or distant recurrence as the first event during the first 4 years after surgery was studied in 1173 patients undergoing mastectomy alone as primary treatment for operable breast cancer. Subset analyses were performed according to tumour size, axillary nodal status and menopausal status.A sharp two-peaked hazard function was observed for node-positive pre-menopausal patients, whereas results from node-positive post-menopausal women always displayed a single broad peak. The first narrow peak among pre-menopausal women showed a very steep rise to a maximum about 8–10 months after mastectomy. The second peak was considerably broader, reaching its maximum at 28–30 months. Post-menopausal patients displayed a wide, nearly symmetrical peak with maximum risk at about 18–20 months. Peaks displayed increasing height with increasing axillary lymph node involvement. No multi-peaked pattern was evident for either pre-menopausal or post-menopausal node-negative patients; however, this finding should be considered cautiously because of the limited number of events. Tumour size influenced recurrence risk but not its timing. Findings resulting from the different subsets of patients were remarkably coherent and each observed peak maintained the same position on the time axis in all analysed subsets.The risk of early recurrence for node positive patients is dependent on menopausal status. The amount of axillary nodal involvement and the tumour size modulate the risk value at any given time. For pre-menopausal node-positive patients, the abrupt increase of the first narrow peak of the recurrence risk suggests a triggering event that synchronises early risk. We suggest that this event is the surgical removal of the primary tumour. The later, broader, more symmetrical risk peaks indicate that some features of the corresponding metastatic development may present stochastic traits. A metastasis development model incorporating tumour dormancy in specific micro-metastatic phases, stochastic transitions between them and sudden acceleration of the metastatic process by surgery can explain these risk dynamics. The 1970s and 1980s witnessed a revolution in the conventional approach to the treatment of primary breast cancer. Early in the 1970s, the favourable results of postoperative systemic adjuvant therapy in women with positive axillary lymph nodes  started Surgical resection of primary tumour removal may either 'cure' a significant fraction of patients, or it may even change the 'natural' recurrence and death timing for some others, by accelerating the metastatic development ,8. Some Careful inspection of the timing of tumour recurrence after resection can be of considerable interest. The recurrence risk pattern in a given follow-up span, a useful estimate of which is the hazard function , provideThe model and the results of its computer simulation suggest that the first peak may result from the superimposition of metastatic recurrences with different dynamics. Here we report a detailed analysis of the first peak of the hazard rate curve, that is, of the recurrence timing during the first 4 years, for patients undergoing mastectomy without any adjuvant chemotherapy or radiotherapy. Our findings suggest that metastatic growth dynamics after primary tumour removal is markedly different depending on menopausal status and, perhaps, axillary nodal involvement. These results support the concept that surgery may have differential perturbing effects on tumour growth kinetics depending on both tumour and host traits. These results strengthen the leading lines of the proposed model for metastasis development and may be relevant to adjuvant treatment strategies.All patients with mastectomy alone as primary treatment who entered into three different clinical trials for operable breast cancer were retrospectively evaluated for this analysis. The trials were performed at the Milan Cancer Institute between 1964 and 1980 to compare mastectomy with other surgical or combined therapeutic approaches. Before surgery, all patients underwent standard staging: complete physical examination, X-ray study of chest, skull, spine and pelvis, bilateral mammography, electrocardiogram, complete haemogram and routine biochemical tests. The primary tumour was treated by radical or modified radical mastectomy. No patient received postoperative radiotherapy or chemotherapy. After surgery, follow-up was performed as follows: physical examination, biochemical tests and chest X-ray every 6–8 months during the first 3 years and once a year thereafter; skeletal survey and mammography once a year. In the presence of controversial clinical findings, examinations were performed more often than originally planned. Appropriate radiological, radioisotopic and surgical investigations were performed whenever recurrence was suspected or clinically evident. Particular attention was paid to assessing the recurrence time by carefully reviewing clinical, radiological and laboratory documentation. More detailed characteristics of the studied series are reported in reference 12.Treatment failure (recurrence) was defined as the first clinically documented evidence of new disease manifestation(s) in either local-regional area(s)  or distant site(s) or any combination of these sites (classified as distant). Contra-lateral breast lesions may be either second primaries (confounding factors in the present investigation) or true metastases. Their annual incidence, together with the absence of a link with clinical prognostic factors of the primary breast cancer, suggest that most of them can be considered as second primary breast cancers ,19. ContRecurrence-free survival was considered as the time elapsed from the date of surgery to the first documented evidence of treatment failure; clinical evaluation without recurrence, death without recurrence, and second primary cancer  were considered censored events.Our application involved dividing the time axis into discrete intervals; the timing of the recurrence risk was studied by estimating the recurrence hazard rate, namely the conditional probability of manifesting recurrence in a time interval, given that the patient is clinically free of any recurrence at the beginning of the interval. The discrete hazards of recurrence and their standard deviations were calculated by means of the life-table method over 4 years. Because the event-specific rates display some instability arising from random variation, a kernel-like smoothing procedure  was adopMenopausal status, which was collected and recorded at the time of primary tumour diagnosis, was defined as 'post-menopausal' if 1 year had elapsed since the last menstrual period. Cumulative survival curves were compared by two-sided log-rank test.The main clinical characteristics of the 1173 analysed patients, all undergoing regular follow-up examinations in accordance with protocol requirements, are reported in Table As reported and discussed elsewhere , the hazAs a first step, the hazard rate pattern by type of recurrence  was studied and, because their timing proved to be remarkably similar, in the further analysis both recurrences were combined. Patients were then allocated to different subsets according to tumour size (T1 versus T2–T3) or to axillary nodal status (positive versus negative) or to menopausal status . The resulting hazard rate curves indicated that the recurrence risk pattern is definitely correlated to menopausal status, with a double-peaked curve for pre-menopausal patients Fig.  and a siPre-menopausal patients were allocated to subsets of node-negative, node-positive, one to three nodes positive and more than three nodes positive, and the hazard rates were estimated Fig. . The resA similar analysis was performed in post-menopausal patients. All subsets of patients displayed a wide, nearly symmetrical recurrence risk peak, with a maximum at 18–24 months and a height similar to the corresponding estimates of pre-menopausal patients  very few cases prove to be metastatic at standard staging today (less than 5%)  and (2) The first sharp peak, in our opinion, has a biological explanation and may result from some triggering event changing the unperturbed history of the disease. We suggest that primary tumour surgical removal is probably this event, selectively operating on some micro-metastases within similar biological conditions. This triggering effect could be modulated by the surgery extent . HoweverAssuming tumour dormancy release and the estimate of 8–10 months or less for tumour development from micro-metastasis to clinical metastasis, the hazard function should be considered as a description of the dormancy escape kinetics. A symmetrical, bell-shaped peak in the curve of hazard rate against time provides information about both the mean value (the peak position) and the variance (the peak width) of the timing of the constituent recurrences. The temporally stable position of the recurrence peak therefore suggests a common cause for recurrence development, a narrow peak indicates that the underlying event is most probably deterministic in nature, and a considerably wider peak suggests that some features of the transition from dormancy to growth may also present stochastic traits.We recently proposed a model ,18,31 inThe proposed model both reasonably explains and takes support from the findings of this study. The early sharp peak of the recurrence risk for pre-menopausal patients can be ascribed to a considerable switching of micro-metastatic foci to the angiogenic phenotype via the withdrawal of angiogenesis inhibitor(s) , and theAs regards differences between pre-menopausal and post-menopausal patients, it should be taken into account that peculiar conditions relevant to breast cancer development, in particular conditions acting on angiogenetic traits, may be dependent on the host hormone milieu. Angiogenesis in invasive breast carcinoma, as evaluated by micro-vessel counts, is associated inversely with patient age, and higher vascular intensity is observed in younger patients ,33. VascThe findings of this investigation would be much more complete if other prognostic and predictive factors could be analysed. However, this database, like most databases of patients undergoing surgery alone without any adjuvant treatment, goes back to years when factors other than TNM  staging and menopausal status were poorly known or completely unknown.Early metastatic growth dynamics shows a structured pattern that is correlated to menopausal status. Indeed, during the first 4 years, node-positive pre-menopausal patients display a split of the hazard function surge into two peaks, while post-menopausal patients always display a single peak. The timing and shape of peaks suggest the occurrence of some triggering event, correlated with primary tumour surgical removal, resulting in recurrence synchronisation, which seems to be especially important for pre-menopausal node-positive patients. In addition, findings support the hypothesis that some features of metastatic development may present stochastic traits. A metastasis development model incorporating tumour dormancy, stochastic transitions between specific micro-metastatic phases and sudden acceleration of the metastatic process due to surgery may be considered appropriate and to fit these findings well.The different post-resection relapse dynamics between pre-menopausal and post-menopausal patients may have important implications for adjuvant systemic treatments. For adjuvant chemotherapy the proposed picture makes it easier to understand retrospectively why adjuvant chemotherapy was found to be most effective in pre-menopausal patients, who during the first months after primary tumour removal would display an enhancement of actively proliferating, and hence more chemo-sensitive, micro-metastases . Most imThe authors declare that they have no competing interests.


Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. Neither the natural history of AF nor its response to therapy are sufficiently predictable by clinical and echocardiographic parameters.Atrial fibrillatory frequency (or rate) can reliably be assessed from the surface electrocardiogram (ECG) using digital signal processing  and shows large inter-individual variability. This measurement correlates well with intraatrial cycle length, a parameter which appears to have primary importance in AF domestication and response to therapy. AF with a low fibrillatory rate is more likely to terminate spontaneously, and responds better to antiarrhythmic drugs or cardioversion while high rate AF is more often persistent and refractory to therapy.In conclusion, frequency analysis of AF seems to be useful for non-invasive assessment of electrical remodeling in AF and may subsequently be helpful for guiding AF therapy. In recent years, mechanisms leading to atrial fibrillation (AF) induction and maintenance have begun to be explored and rapidly evolving therapies including new antiarrhythmic drugs, antitachycardia pacing algorithms and catheter ablation techniques have been introduced in clinical practice. Nevertheless, outcome of AF patients is still unsatisfactory which - to the greatest extent - can be attributed to the progressing nature of this arrhythmia and high AF recurrence rates following restoration of sinus rhythm. Moreover, AF treatment  may be viewed as being based on trial and error, since no test is able to predict the natural history of this arrhythmia or its response to treatment. Subsequently, current AF management guidelines  provide Virtually in every patient with AF, a standard surface electrocardiogram (ECG) is acquired, the main purposes being confirmation of arrhythmia presence and determination of ventricular rate. Fibrillation waves have, moreover, various appearances ranging from fine to coarse and from disorganized to organized, flutter-like. Interestingly, the mechanisms behind the various appearances of the fibrillatory process on the ECG and the possible prognostic information contained within fibrillation waves have just very recently begun to be explored.The aim of this article is twofold; (1) to describe novel frequency analysis techniques of the surface ECG for characterization of the fibrillatory process, and (2) to present possible applications of these methods, namely for (a) exploring autonomic modulation of AF, (b) monitoring and predicting antiarrhythmic drug effects, and (c) predicting AF recurrence following restoration of sinus rhythm. For this purpose, Medline-listed, peer-reviewed studies are included in this review.Shortening of atrial refractoriness and maladaptaion to rate are hallmarks of atrial electrical remodeling in AF .. DuringFirstly, spontaneous arrhythmia behavior is related to baseline fibrillatory cycle length. Asano et al induced AF with rapid pacing in 30 patients undergoing electrophysiologic study . PatientSecondly, response to antiarrhythmic drug therapy has been shown to be associated with baseline cycle length and drug-induced cycle length changes. Stambler and colleagues identified a mean right atrial cycle length of 160 ms (375 fpm) as a valuable cutoff-point for conversion to sinus rhythm with ibutilide . No patiFinally, there exists a close relation between the degree of electrical remodeling expressed as refractoriness changes and AF recurrence following cardioversion. Manios et al found that patients who failed to shorten the monophasic action potential duration to less then 195 ms at a pacing cycle length of 350 ms were more likely to have AF recurrence . This fiPrompted by the likely usefulness of non-invasive test that assesses the average fibrillatory rate from the surface ECG for exploration of AF pathophysiology and AF managment, frequency analysis techniques were independently introduced by our groups ,16, whilUsing digital recording techniques, traditional ECG signals are recorded. In most studies atrial fibrillatory rate has been obtained by spectral analysis techniques of resting ECG recordings such as standard 12-lead ,16 or  determine the appearance of the frequency power spectrum . VariantMore recently, a time-frequency analysis approach has been developed by which the time-frequency distribution is decomposed into a number of descriptive functions reflecting second-to-second variations in fundamental frequency and waveform morphology ,32. The To date, the dominant spectral peak has been presented as either dominating atrial cycle length (in ms), fibrillatory rate (in fpm) or fibrillatory frequency (in Hz). Since it has become evident that the results of this method might be of practical clinical importance, we believe that the results should be expressed in a way which is closest to the nomenclature of other surface ECG rate-variables  . The expA direct comparison between endocardially recorded electrograms and body surface recordings clearly evidences the validity of fibrillatory rate obtained from surface ECG as an index of the average atrial fibrillatory cycle and subsequently atrial refractoriness. Fibrillatory rates calculated from lead V1 substitute the right atrial free wall ,16 whilsAlthough the gross atrial fibrillatory pattern, studied by multiple simultaneous epicardial recordings, is reproducible already in repeated measurements of only 8 seconds duration , calculaIn persistent AF, there is minor short-term rate variability ,16,27 anVagal as well as sympathetic stimulation have been shown to reduce atrial refractory periods and increase their heterogeneity -39. In tSubsequently, atrial fibrillatory rate seems to be ideal for monitoring the effect of autonomic tone changes - either spontanenous (circadian) or by autonomic maneuvers provoked - on atrial electrophysiology. Indeed, the circadian variability of atrial fibrillatory rate has been explored in independent studies ,24. FibrThe second area of investigation concerns effects on atrial fibrillatory rate following vagal or sympathetic stimulation during experimental conditions. Carotid sinus massage, supposed to mainly induce vagal stimulation, resulted in a variable effect on fibrillatory rate in 19 patients studies . A reproWith the availability of instantaneous fibrillatory rates obtained from time-frequency analysis, their second-to-second variation can be explored by spectral analysis techniques, similar to those used for analyzing heart rate variability. By applying this approach, Stridh et al studies  noted a All these findings together clearly highlight the complexity of autonomous nervous system effects on atrial electrophysiology and justify further exploration.Class I and III antiarrhythmic drugs have been shown to increase atrial cycle length (decrease fibrillatory rate) which coincides with increased refractoriness and decreased conduction velocity . These mTwo examples of drug monitoring are presented in Besides direct monitoring of antiarrhythmic drug effects, it seems also possible to identify suitable patients for pharmacological cardioversion. A baseline fibrillatory rate of 360 fpm was highly sensitive and specific for prediction of AF termination following intravenous ibutilde  or oral Patients with a low fibrillatory rate may have a small number of wavelets (long wavelength), whereas those with higher rates have multiple wavelets (short wavelength) . In the One previous study suggested that a stepped conversion regimen of first-line ibutilide followed by electrical cardioversion for patients who fail to convert is less expensive and has a higher conversion rate than first-line electrical cardioversion . Given tPrevious investigations ,50 have Atrial premature beats with short coupling intervals have been shown to promote early AF reinitiation following cardioversion ,55. AF rTwo previous studies ,59 have These parameters seem therefore well suited to describe the individual atrial remodeling. The concept of ECG-guided cardioversion may gain even greater importance in the light of previous findings from two studies comparing rate- with rhythm-control strategies ,61. In tAtrial fibrillatory rate and its variability can be reliable obtained from the surface ECG in AF patients using spectral analysis techniques. These parameters exhibit a significant interindividual variability allowing individual quantification of the atrial electrical remodeling process. Frequency analysis of AF might prove useful in identification of underlying AF pathomechanisms, and prediction of therapy efficacy . Further larger studies are necessary to determine the role of these techniques in different AF management strategies in order to select and time the appropriate therapy for the individual patient.


Cope discusses a new study in rats suggesting that oxidative burst inducers might have a role to play in treating inflammatory arthritis. Activation requires association with a phosphorylated form of p47phox , p67phox, and the small GTPase Rac (phox deficiency in humans leads to neutrophil dysfunction and chronic granulomatous disease (CGD), a primary immunodeficiency disorder characterised by the inability to eradicate bacterial infections [For many years, reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and hydroxyl radicals, and their reaction products, were classically described as harmful by-products of aerobic metabolism capable of causing DNA mutations, lipid peroxidation, and protein oxidation . The idePase Rac . p47phoxfections . Since tBesides a role in phagocyte function and host defence, a large amount of evidence points to important roles for ROS in cell proliferation, apoptosis, angiogenesis, endocrine-related functions, and oxidative modification of the extracellular matrix. Indeed, increased ROS have been documented at sites of inflammation, such as synovial joints of patients with inflammatory arthritis , and circulating neutrophils and monocytes from patients with RA have increased NADPH oxidase activity ,5.phox subunit of NADPH oxidase was shown to induce granulomatous synovitis and exaggerated matrix destruction associated with enhanced expression of inflammatory mediators [Interestingly, polyarthritis has also been described in patients with CGD. More recently, induction of arthritis in mice deficient for the p47ediators . AlthougChronic inflammatory syndromes such as RA are complex polygenic diseases in which it is proposed that inheritance of gene polymorphism predisposes to distinct phenotypes such as the magnitude of the inflammatory response, the development of autoantibodies, or destruction of cartilage and bone. One productive approach for screening for such gene polymorphism has been to define in relevant rodent models chromosomal segments associated with each disease phenotype. Multiple backcrossing of these chromosomal segments to the disease-prone strain generates congenic animals, which are invaluable tools for further study. For example, linkage analysis in rats susceptible or resistant to pristane-induced arthritis  has defined quantitative trait loci (QTL) that associate with disease severity and cartilage destruction.pia4, which is implicated not only with arthritis, but also in rodent models of multiple sclerosis and uveitis [pia4 fragment from the arthritis-resistant E3 strain into arthritis-prone DA rats by serial backcrosses substantially reduced arthritis severity. By positional cloning, two structural polymorphisms of the Ncf1 gene (encoding p47phox) were identified in arthritis-susceptible DA rats characterised at the amino acid level by M106V and M153T substitutions. While the allelic variant had no effect on Ncf1 gene expression, the disease-associated allele conferred an unexpected reduction in oxidative burst upon stimulation of rat peritoneal cells with phorbol ester, presumably through the effects of the mutations on the function of the NADPH oxidase complex. Strikingly, this defective respiratory burst could be reversed in vivo by treating arthritis-prone DA rats with phytol, an alkane similar to pristane but that induces a robust reactive oxygen burst. Treatment not only restored the respiratory burst but also protected rats from developing arthritis.In 2003, Holmdahl and colleagues reported the results of a detailed analysis of one such QTL,  uveitis . IntroduPLoS Medicine, Holmdahl and colleagues explore in more detail the relationship between oxidative burst capacity and predisposition to inflammatory arthritis in an approach that has implications for better understanding this paradoxical relationship in humans [DA rats to levels observed in splenic granulocytes carrying the Ncf1E3 protective allele, whilst phytol induced only very modest increases in ROS production in T cells.In a new rodent study in n humans . First, Adoptive transfer: Transfer of cells from donor to recipient to induce or modify diseasephox:gp91 91-kD glycoprotein subunit of NADPH oxidase, a phagocyte oxidase LAT: Linker for activation of T cells, an adaptor protein involved in transmitting receptor proximal signals from the TCR to downstream intracellular signal transduction pathwaysMultiple backcrossing: Sequential breeding of F1 offspring with an animal derived from a particular inbred strain of mouse or rat; usually used to introduce a genetic fragment from one strain to anotherphox:p22 22-kD subunit of NADPH oxidasephox (Ncf1):p47 47-kD subunit of NADPH oxidase phox:p67 67-kD subunit of NADPH oxidase Positional cloning: Sequencing genes in the vicinity of phenotype-linked microsatellites  to identify the polymorphisms associated with the particular characteristic of interest Pristane-induced arthritis: Induction of arthritis using the alkane oil pristane, commonly used as a model of RARac: A small GTP-binding protein involved in cell signallingTCR ζ An invariant subunit of the T cell antigen receptor complex, also known as CD3ζThiols: Sulphhydryl groups Second, the authors explored the effects of phytol in different arthritis models, including collagen-induced arthritis, anti-collagen II antibody-induced arthritis, and non-oil collagen-induced arthritis, demonstrating both protective and therapeutic effects in all models; similar effects were observed in rats with normal oxidative burst capacity. Treatment resulted in reduction of the inflammatory response, attenuation of cell-mediated immunity, and downregulation of markers of cartilage destruction, modifying clinical disease to an extent at least as good as methotrexate or TNF blockade (now considered gold-standard therapy for RA). Histological analysis of joint sections confirmed the joint-protecting effects of phytol.Finally, an elegant series of experiments involving the transfer of T cells from pristane-inoculated rats to unmanipulated donor animals demonstrated, among other things, that the effects of phytol on arthritogenic T cells were rapid, since treatment of the donor T cells with phytol for as little as three hours was sufficient to prevent the development of arthritis in recipient rats. By contrast, adoptive transfer of a mixture of T cells from control and phytol-treated animals failed to inhibit arthritis, suggesting that the disease-modifying effects of phytol on T cells are cell intrinsic, affecting only those cells directly exposed to the therapeutic agent.Ncf1 in mice and humans predisposes to repeated insult with infectious pathogens [DA rats could explain predisposition to disease through persistent activation of innate immune responses. However, the authors argue that arthritis occurs in both conventional and specific pathogen-free facilities with equivalent severity, implying that the impact of deficient versus mutant Ncf1 gene products on host immunity might be distinct. Detailed analysis of the response of Ncf1 congenic rat strains to challenge with bacterial pathogens should now be undertaken to rule this out.Why should enhancing oxidative burst and ROS production paradoxically ameliorate arthritis? There are several possible explanations, none of which would necessarily be mutually exclusive. We know, for example, that deficiency of athogens ,9. AccorAnother conundrum arising from this work is the strikingly different levels of ROS induced in granulocytes and T cells after treatment with phytol in vitro; effects on T cell ROS levels were modest at best. So how could phytol regulate T cell reactivity? There is literature pointing to indirect effects of granulocyte- or macrophage-derived ROS on T cell phenotype and function. For example, coculture of T cells with ROS-producing neutrophils reduces T-cell reactivity as well as cytokine and proliferative responses . At the Ncf1 mutations), increased surface thiols would lower thresholds of T cell activation, permitting uncontrolled expansion of effector T cells in vivo. This would presumably influence thymic development as well, since lowering TCR-signalling thresholds selects a repertoire of T cells that might otherwise die by neglect when failing to receive appropriate survival signals.One intriguing possibility may relate to recent findings suggesting that cell surface thiols (–SH) are targets of redox regulation , which cThese intriguing results are important because they illustrate another means whereby redox reactions might be manipulated in the clinic. This would be especially attractive if, as this study demonstrates, the effects are rapid and reversible, because robust immunological and molecular tools are already available to monitor such interventions. This approach would be all the more appealing if genetic variation of subunits of the NADPH oxidase complex is associated with susceptibility to autoimmunity in humans. While the data provide unambiguous evidence for a dual role for the oxidative burst in innate and adaptive immune responses, it is now abundantly clear that waste products are not all bad.


There is growing evidence of the public health and community harms associated with crack cocaine smoking, particularly the risk of blood-borne transmission through non-parenteral routes. In response, community advocates and policy makers in Vancouver, Canada are calling for an exemption from Health Canada to pilot a medically supervised safer smoking facility (SSF) for non-injection drug users (NIDU). Current reluctance on the part of health authorities is likely due to the lack of existing evidence surrounding the extent of related harm and potential uptake of such a facility among NIDUs in this setting. In November 2004, a feasibility study was conducted among 437 crack cocaine smokers. Univariate analyses were conducted to determine associations with willingness to use a SSF and logistic regression was used to adjust for potentially confounding variables (p < 0.05). Variables found to be independently associated with willingness to use a SSF included recent injection drug use , having equipment confiscated or broken by police , crack bingeing , smoking crack in public places , borrowing crack pipes , and burns/ inhaled brillo due to rushing smoke in public places . The results suggest a strong potential for a SSF to reduce the health related harms and address concerns of public order and open drug use among crack cocaine smokers should a facility be implemented in this setting. Vancouver's Downtown Eastside (DTES) has been the site of an explosive HIV and HCV epidemic associated with a large open illicit drug use scene,2. The hTo date, the scientific evaluation of the supervised injection facility has documented several successes including high uptake of the facility, improved public order , and a pIn Europe, several safer smoking facilities (SSF) for non-injection drug users have been opened in addition to supervised injection facilities , and yetCurrently advocates and policy makers in Vancouver, Canada are calling for a medically supervised safer smoking facility to smoke pre-obtained non-injection drugs, particularly crack cocaine, and consideration is being given to applying for a federally-administered exemption from Health Canada (under exemption 56) to pilot a safe inhalation room ,26. AlthThe Vancouver Area Network of Drug Users (VANDU) is a drug user organization that formed in 1997 in response to a growing HIV epidemic and health emergency in the Downtown Eastside of Vancouver associated with illicit drug use, and perceived government inaction. The mission of VANDU is "to improve the lives of people who use illicit drugs through user-based peer support and education". Today, The Rock Users' Group (RUG) was formed through VANDU in response to a growing need to address the health needs of crack cocaine smokers. The RUG group meets weekly to educate members on the related concerns of crack cocaine use and discuss ways to expand harm reduction initiatives to include crack smokers. Through private donations, RUG has recently begun distributing safer smoking kits to users in the community at a cost of Can$1, including a mouthpiece, durex pipe, brass screens, lubricant, and condoms.The Community Health and Safety Evaluation (CHASE) Project is a prospective open cohort that was established to evaluate the impacts of recently implemented health initiatives on residents of the DTES; to identify priority health issues; shortfalls; and populations at greatest risk. All community residents are eligible to participate, and are enrolled through various recruitment strategies including community-based organizations, several storefront locations and door-to-door initiatives in a single room occupancy hotel (SRO) and subsidised housing buildings. The goal is to enrol a large representative sample of people residing in and having access to services in the DTES community. A short baseline questionnaire is administered by a trained peer interviewer, and elicits questions related to sociodemographic characteristics, health status, service utilization, barriers to healthcare access, and patterns of illicit drug use. In addition, permission is requested to link personal identifiers with a number of health related databases in the province. Participants are followed prospectively through these data linkages on a bi-annual bases. Upon completion of the survey, study participants receive an honorarium of $10 as compensation for their time. University of British Columbia / Providence Health Care Research Ethics Board provided ethical approval for this study.Through a community-based partnership between the Rock Users' Group (RUG) of Vancouver Area Network of Drug Users (VANDU) and the Community Health and Safety Evaluation (CHASE) Project of the BC Centre for Excellence in HIV/AIDS, the following assessment of health related harms of crack cocaine use was conducted in November of 2004. A total of 437 crack cocaine smokers participated in peer-administered interviews. To be eligible, individuals had to be current crack cocaine smokers . Participants were recruited through targeted recruitment strategy that included allocation of referral cards at staggered times and locations over a three-week period. Referral cards were handed-out by CHASE peers through street recruitment and VANDU members on alley patrol and outreach with active crack smokers, as well as through community-based organisations and service providers. Questionnaires were conducted at various storefront locations, and included women's only days to ensure inclusion of multi-risk women. Although participants were asked about former and current injection drug use practices, a history of injection drug use was not considered an eligibility criteria and thus both IDU and NIDU crack smokers were eligible to participate.Socio-demographic variables that were considered in this analysis included gender, age, ethnicity, housing status, education level, health status, health and addiction service uptake, recent incarceration, and drug use patterns. For the purpose of this analysis, unstable housing was defined as living arrangements that included SRO hotels, transitional housing, and no fixed address/ homelessness. Drug use behaviours included: frequency of cocaine injection, heroin injection, crystal methamphetamine injection, and crack cocaine smoking. As previously , "any dr2, normally distributed continuous variables were analyzed using t-tests for independent variables, and skewed continuous variables were analyzed using Mann-Whitney U tests. In order to identify factors independently associated with willingness to use a SSF, a logistic regression was performed. Variables found to be associated with willingness to use a SSF at the univariate level (p < 0.05) were entered into the logistic model. All reported p-values are two-sided.Descriptive and univariate analysis were used to determine bivariate associations between willingness to use a SSF and sociodemographic characteristics, selected drug use patterns, crack use behaviours and related risks. Mean averages were used to describe normally distributed variables, and median averages were used to describe skewed variables. Categorical and explanatory variables were analyzed using Pearson XA total of 437 participants were recruited over a three-week period in November 2004, and thus were eligible for the present analysis. Of the total, 289 (66%) were male and 145 (33%) were female. The median age was 41 years (interquartile range [IQR] = 35 – 45). One-hundred and eighty-four (42%) individuals self identified as Aboriginal, and 335 (77%) were living in unstable housing. Two-hundred and forty-six (56%) reported a history of injection drug use (either former of current IDUs), while 191 (44%) were NIDU crack smokers with no history of injection drug use.Of the 437 participants, 303 (69%) expressed a willingness to use a medically supervised safer smoking facility (SSF) if one was made available. The univariate analyses of associations between willingness to use a safer smoking facility and sociodemographic characteristics and selected drug use patterns are shown in Table The univariate analyses of associations between willingness to use a SSF and crack use behaviours and related risks are shown in Table Results of the multivariate logistic regression analysis of factors independently associated with willingness to use a SSF are presented in Table Of a total of 437 crack cocaine smokers, 303 (69%) reported a willingness to use a safer smoking site (SSS) should one be made available. A willingness to use a SSF was associated with recent injection drug use, having equipment confiscated or broken by police, crack binge use, smoking crack in public places, borrowing crack pipes, and burns/ inhaled brillo due to rushed smoking.The association between crack bingeing and willingness to use a SSF shows a potential for such a facility to intervene in risky drug use behaviours through increased contact and referral with primary care, education and addiction services . Drug biIn this context, the observed association between borrowing crack pipes and willingness to use a SSF is particularly noteworthy given the increasing evidence of blood borne transmission through the sharing of non-injection drug use implements and the potential of a SSF to reach this high-risk group. Oral sores, cuts, and burns are common among crack cocaine smokers and have been shown to facilitate HCV transmission, as well as increased potential risk for HIV transmission, through the sharing of contaminated equipment, such as crack pipes ,10,32. GThe observed association between willingness to use a SSF and recent injection use is also particularly relevant, given that this feasibility study was conducted a year following the implementation of the SIF in this community. As mentioned above, the SIF has had positive impacts by improving public order, minimisGiven the objective of drug consumption rooms to reduce public nuisance and a visible drug scene, the observed association between public crack use and increased willingness to use a SSF is also noteworthy. Although there is currently limited information available exclusively on SSFs, preliminary findings in Switzerland show an increase in public order and increased contact between NIDUs and health and social services. As wellWithin the context of public drug use, the associations between willingness to use a SSF, and burns or/ inhaled brillo due to rushing smoke and having equipment confiscated or broken by police, highlight a strong potential to reduce the community harms of public crack use and concerns of public order. Common modes of crack cocaine smoking such as metal pipes are known to cause frequent burns and blisters through excessive heat, while glass and durex pipes frequently splinter causing a smoker's lips to cut ,33. In aSeveral limitations should be considered. First, this study relied on self-reported information and thus is subject to socially desirable reporting. However previous studies have reported the validity of self-reported information among drug user populations. Second,The high reported rate of willingness to use a safer smoking facility (SSF) in this study highlights an important opportunity to connect with a known high-risk drug user population. Given the observed associations between willingness to use a SSF and public drug use and related harms, borrowing of crack pipes, and other risky drug use behaviours, this study identifies a strong potential to reduce community health risks, including infectious disease transmission, and address issues of open drug use and concerns of public order, if a facility was implemented in this setting.


One can no longer ignore mitochondria in cancer biology, argue Zanssen and Schon. In 1956, the German biochemist and Nobel Laureate Otto Warburg proposed that cancer is caused by altered metabolism and by deranged energy processing in mitochondria , mis, mis6], Chain-terminating point mutation: A point mutation that leads to a premature stop codonD-loop analysis: Analysis of polymorphisms in the mtDNA control region, known as the D-loopEthnic-specific haplotype-defining polymorphism: A polymorphism representing an mtDNA variant found exclusively in a specific population. Haplotypes H, K, J, and U are found in European populationsHomopolymeric sequences: Consecutive runs of the same nucleotideIntragenic deletion: Loss of a contiguous segment of DNA from a geneMissense mutation: A point mutation in a codon resulting in the alteration of one encoded amino acid to anotherMitomap database: An online human mitochondrial genome database (http://www.mitomap.org/)Nuclear mitochondrial pseudogenes: Nuclear DNA sequences homologous to mtDNA sequences, but without any functionROS: Reactive oxygen speciesrRNA: Ribosomal RNAtRNA: Transfer RNAThese findings pose a key conceptual question: how do these mutations arise and then establish themselves so quickly in the tumor? Many of the (noninherited) mtDNA mutations lead to the mtDNA of the tumor becoming heteroplasmic; i.e., they are a mixture of normal germline mtDNAs found in the noncancerous tissue and mtDNAs containing one or more mutations. Even more surprising than this heteroplasmy is the observation that most mutations are apparently homoplasmic , and further, some of those genomes contain not one, but two, three, and even four mutations, all on the same mtDNA circle. How can a cell with mitochondrial haplotype A in noncancerous tissue shift its mitochondrial genotype to haplotype B so rapidly in the adjacent tumor? Mathematical models of heteroplasmic shifting in rapidly dividing tumor cells  have beePLoS Medicine, Salas et al. question the very foundation of the observation of haplotype shifting in tumors [In this issue of n tumors . They prSalas et al. rightly emphasize the importance of the highest standards in mtDNA analysis to produce reliable results in the many investigations on this subject . In summHowever, although technical errors probably do account for some of the positives found in some of the literature, they cannot account for all of them. Putting aside the issue of mechanism, the question that remains—can mtDNA haplotypes shift rapidly in tumors—is not only still with us, but is almost certainly answered affirmatively.We would interpret the mtDNA sequencing data from the phylogenetic point of view differently than Salas and colleagues. mtDNA haplotype-specifying polymorphisms  arising Although the mechanism of rapid shifting is still unclear, random processes, extensively modeled in computer systems , may welWarburg was right. One can no longer ignore mitochondria in cancer biology. Mutations of mtDNA, even driven by random processes during malignant transformation, present an excellent possibility for early tumor detection using, e.g., D-loop analysis of bodily fluids from patients with tumors . –mediated pathways is challenging, but urgently needs experimental proof. Questions regarding whether the HIF-mediated pathway is also initiated in hypoxia and mitochondrial deficiency , both ch


Diabetes is an important predictor of mortality patients with ACS. However, little is known about the association between diabetes and health status after ACS. The objective of this study was to examine the association between diabetes and patients' health status outcomes one year after an acute coronary syndrome (ACS).This was a prospective cohort study of patients hospitalized with ACS. Patients were evaluated at baseline and one year with the Seattle Angina Questionnaire (SAQ). Socio-demographic and clinical characteristics were ascertained during index ACS hospitalization. One year SAQ Angina Frequency, Physical Limitation, and Health-Related Quality of Life (HRQoL) scales were the primary outcomes of the study.Of 1199 patients, 326 (37%) had diabetes. Patients with diabetes were more likely to present with unstable angina , less likely to present with STEMI , and less likely to undergo coronary angiography . In multivariable analyses, the presence of diabetes was associated with significantly more angina , cardiac-related physical limitation  and HRQoL deficits  at one year.Diabetes is associated with more angina, worse physical limitation, and worse HRQoL one year after an ACS. Future studies should assess whether health status outcomes of patients with diabetes could be improved through more aggressive ACS treatment or post-discharge surveillance and angina management. Acute coronary syndromes (ACS), including unstable angina, non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI), affect approximately one million Americans each year . As imprDiabetes is an established and highly prevalent risk factor for cardiac morbidity and mortality, and therefore is an important comorbidity in patients with ischemic heart disease (IHD). However, little is known about the association between diabetes and health status outcomes after ACS. It is important to assess the impact of diabetes on health status outcomes in cardiovascular populations. First, there is a general perception that patients with diabetes are less likely to experience angina than patients without diabetes. Yet, paThe goal of this study was to prospectively evaluate the association between diabetes and cardiac-specific health status  in patients one year after an ACS, independent of other known predictors. The results of this study are intended to inform clinicians about the importance of diabetes as a risk factor for health status outcomes after ACS, and potentially lead to interventions to improve health status outcomes in patients with diabetes.The INvestigation oF Outcomes from acute coRonary syndroMes (INFORM) study was a prospective cohort study designed to describe the health status of patients with ACS, to determine predictors of health status at one year following ACS, and to define the impact of ACS treatment on patients' health status. Patients were enrolled from two Kansas City, Missouri hospitals  between March 1, 2001 and October 31, 2002. In total, 10,911 consecutive hospitalized patients who had a troponin blood test performed were prospectively screened for a possible ACS. Standard definitions were used to diagnose ACS patients with either myocardial infarction (MI) or unstable angina. MI patients were defined by a positive troponin blood test in the setting of symptoms or electrocardiographic changes  consistent with an MI[Each participating patient was prospectively interviewed as early as possible during their index ACS admission to ascertain their socio-demographic and baseline health status characteristics. Detailed chart abstractions were performed to ascertain patients' medical history, laboratory results, disease severity and the processes of inpatient care. The Social Security Administration Death Master File was queried to determine patients' vital status in the year after enrollment. Surviving patients were contacted by telephone one year after ACS for a follow-up interview to reassess their clinical and health status. A minimum of 12 attempts to contact patients were made, including contacting up to 2 additional individuals designated by patients at the time of their baseline interview as people who would know their whereabouts.Disease-specific health status was measured at baseline (during index ACS admission) and at one year post-ACS by the Seattle Angina Questionnaire (SAQ). The SAQ is a 19-item disease-specific measure for patients with coronary artery disease that has well-established validity, reproducibility, sensitivity to clinical change, and prognostic value,11,12. Tcardiac-related physical limitation). A sensitivity analysis was done excluding those indicated limitations for non-cardiac related reasons, which did not significantly alter the results. Furthermore, secondary analyses were performed using the Physical Component Summary (PCS) score from the SF-12 to evaluate the association between diabetes and overall physical health status one-year after ACS.To accommodate skewed distributions of SAQ scores (approximately 70% of patients had no angina symptoms at one year), and to improve scale interpretability, the SAQ angina frequency, physical limitation and HRQoL scores were dichotomized into the presence or absence of angina, presence or absence of cardiac-related physical limitation and the presence or absence of HRQoL deficits, respectively. The physical limitation section of the SAQ asks patients to indicate if they were limited due to reasons other than chest pain or angina. For analytic purposes, patients whose limitations were due to non-cardiac causes were given a score of 100 on the SAQ sub-scale  health status was evaluated using the Short Form-12 (SF-12) health status survey. The SF-12 is reliable and valid, consisting of 12 items that reflect overall physical and mental well-being (i.e. does not contain disease-specific questions). Scoring for the PCS and Mental Component Summary (MCS) scores of the SF-12 followed the methods described by Ware et al. The PCSThe primary independent variable of interest for this study was diabetes mellitus. Patients were considered to have diabetes if it was recorded in the medical record or if they were on diabetes medication at the time of ACS admission. A sensitivity analysis including the 44 patients newly diagnosed with diabetes during their ACS hospitalization, did not significantly alter the results. Additional variables available for risk adjustment included the demographic, cardiac factors, noncardiac factors, and treatment variables listed in Tables 2 tests for categorical variables. The unadjusted associations between diabetes and primary cardiac-specific health status outcomes were evaluated by comparing the proportion of patients with any angina, any degree of physical limitation or any HRQoL deficits between those with and without diabetes using χ2 tests. Secondary univariate analyses compared mean SAQ scores for each subscale and mean PCS and MCS scores one year following ACS between participants with and without diabetes using t-tests.Statistical analyses were performed using SAS version 9.1 . Baseline characteristics of patients with and without diabetes were compared using t-tests for continuous variables and χMultivariable logistic regression was used to evaluate the risk-adjusted association between diabetes and one-year post-ACS health status. In addition, analyses using multiple linear regression with SAQ scores as continuous outcomes were performed to ensure that categorization did not obscure any effects and yielded results similar to the primary analyses. Finally, multivariable linear regression was used to evaluate the association between diabetes and overall mental health status as measured by MCS scores from the SF-12.Primary multivariable models were constructed using forward selection on the candidate pool of independent variables listed in Tables Missing follow-up health status assessments can potentially produce a selection bias from survey non-responders that influences the results of health status studies . SeveralOf the 1,199 patients in the cohort, 326 patients (27%) had diabetes. Baseline characteristics of patients with and without diabetes are listed in Table ACS characteristics and treatments are presented in Table At the time of the index hospitalization, patients with diabetes had lower mean SAQ scores for angina frequency  and physical limitation , but no significant difference in HRQoL scores . One year following ACS, patients with diabetes had significantly lower SAQ angina frequency , physical limitation  and HRQoL  scores compared to patients without diabetes.In unadjusted analysis, patients with diabetes, when compared to those without diabetes, were significantly more likely to have angina of any degree , cardiac-related physical limitation , and HRQoL deficits , cardiac-related physical limitation  and HRQoL deficits  at one year compared to patients without diabetes.In multivariable models, additional predictors of having angina were age , history of MI , hypercholesterolemia , history of alcohol or substance abuse , chronic lung disease , and history of peripheral vascular disease . Additional predictors of physical limitation were female gender , Caucasian race , history of MI , history of alcohol or substance abuse , discharge prescription of an angiotensin converting enzyme inhibitor or an angiotensin receptor blocker  and chronic lung disease . The additional predictors of HRQoL deficits were age , ejection fraction (OR 1.12 (1.01–1.27 per 10% decrease), acute thrombolytic therapy , and chronic lung disease .In multivariable linear regression analyses, diabetes remained significantly associated with worse overall physical function as measured by the SF-12. PCS scores were, on average, three points lower for those with diabetes compared to those without diabetes . Diabetes was not independently associated with differences in MCS scores .The goal of this study was to examine the association between diabetes and patients' health status outcomes one year after an ACS. We found that patients with ACS and coexisting diabetes were more likely to present with unstable angina, and were less likely to undergo coronary angiography during ACS hospitalization. At one year following ACS, patients with diabetes were significantly more likely to have angina, cardiac-related physical limitation and HRQoL deficits compared to patients without diabetes. These associations remained significant after adjustment for a wide array of demographic, disease severity, comorbid condition, and treatment variables. Diabetes was also negatively associated with overall physical health status, but was not significantly associated with overall mental health status.It may be surprising that patients with diabetes were more likely to have angina one-year post ACS. Previous studies have shown that patients with diabetes have a higher rate of silent myocardial ischemia -17, althThe results of this study support the potential for more aggressive identification and treatment of angina in patients with diabetes following ACS in order to improve outcomes. Angina decreases patients' quality of life, is associated with worse patient satisfaction, and increases the risk for subsequent ACS and death,3,20-22.or invasive approach can be taken with similar morbidity and mortality outcomes [We also found that patients with diabetes and unstable angina were less likely to receive coronary angiography, raising the possibility that greater use of an early invasive strategy in diabetic patients could improve outcomes. The discrepancy in angiography rates did not appear to be wholly or directly attributable to clinician concerns over impaired renal function given that 94% of diabetic patients had admission creatinine levels <2.0 μg/ml. Current American Heart Association/American College of Cardiology guidelines for ACS state that in the absence of high-risk indicators such as new ST-segment depression, symptoms of heart failure or high-risk findings on non-invasive testing, an early conservative outcomes . Given tThe results of this study expand the previous literature on diabetes and health status in cardiac populations. Prior studies have suggested that diabetes is associated with greater overall physical limitation and worse quality of life following CABG surgery or PCI ,32. PrevPotential limitations to this study should be considered. First, missing follow-up health status assessments can bias quality of life studies . HoweverIn conclusion, we found that diabetes was associated with more angina, greater cardiac-specific physical limitation, and worse HRQoL one year after an ACS. Future studies should evaluate interventions to reduce angina burden and improve the health status of patients with diabetes following ACS. Potential strategies include an early invasive approach for unstable angina in patients with diabetes and more aggressive surveillance and therapy for angina in the post-ACS period. It is hoped that such interventions will optimize the patient-centered outcomes of symptom burden, physical function, and quality of life for the growing population of patients with diabetes and CAD.1) Dr. Frederick Masoudi serves on the speaker's bureau of AstraZeneca, the speaker's bureau of Takeda NA and the advisory board of Takeda NA. Dr. John Spertus serves as a consultant for CV Therapeutics; and 3) Dr. John Rumsfeld served as a consultant for CV Therapeutics and Pfizer.PP participated in the design of the study, performed statistical analyses and drafted the manuscript. JS participated in the study design and coordination and helped draft the manuscript. DM, FM, RH and JR participated in the design of the study helped draft the manuscript. KR performed statistical analyses and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Information needs and resource preferences of office-based general pediatricians have not been well characterized.Data collected from a sample of twenty office-based urban/suburban general pediatricians consisted of: (a) a demographic survey about participants' practice and computer use, (b) semi-structured interviews on their use of different types of information resources and (c) semi-structured interviews on perceptions of information needs and resource preferences in response to clinical vignettes representing cases in Genetics and Infectious Diseases. Content analysis of interviews provided participants' perceived use of resources and their perceived questions and preferred resources in response to vignettes.Participants' average time in practice was 15.4 years (2–28 years). All had in-office online access.Participants identified specialist/generalist colleagues, general/specialty pediatric texts, drug formularies, federal government/professional organization Websites and medical portals (when available) as preferred information sources. They did not identify decision-making texts, evidence-based reviews, journal abstracts, medical librarians or consumer health information for routine office use.patient-specific  and general medical  information. They identified specialists and specialty textbooks, history and physical examination, colleagues and general pediatric textbooks, and federal and professional organizational Websites as information sources. Participants with access to portals identified them as information resources in lieu of texts.In response to clinical vignettes in Genetics and Infectious Diseases, participants identified Question Types about For Genetics vignettes, participants identified questions about prenatal history, disease etiology and treatment guidelines. For Genetics vignettes, they identified patient history, specialists, general pediatric texts, Web search engines and colleagues as information sources. For Infectious Diseases (ID) vignettes, participants identified questions about patients' clinical status at presentation and questions about disease classification, diagnosis/therapy/referral guidelines and sources of patient education. For ID vignettes, they identified history, laboratory results, colleagues, specialists and personal experience as information sources.Content analysis of office-based general pediatricians' responses to clinical vignettes provided a qualitative description of their perceptions of information needs and preferences for information resource for cases in Genetics and Infectious Diseases. This approach may provide complementary information for discovering practitioner's information needs and resource preferences in different contexts. Information behaviors of pediatricians have been examined in different clinical environments: in general patient care at an academic medical center ,2, in nePublished studies on pediatricians' information use have focused on hospital-based practitioners and house staff, although most pediatricians practice in office settings, away from medical libraries, librarians and hospital-based specialists . Office-In contrast to these constraints has been the increasing adoption of information and communication technologies (ICT) into practice . In addiDomain competencies for US pediatricians have traditionally been defined by formal training curricula and board certification , but infThis pilot study explores information needs and resource preferences of (a) a characterized group of practitioners  within (b) a characterized clinical workflow (office-based practice) for (c) problems in characterized domains (genetics and infectious diseases) using short clinical vignettes. Vignettes  have beeIn this study, we explore several questions:• Can vignettes be used to elicit information needs and resource preferences from a group of practitioners with common attributes, clinical environments and patient problems?• What are information needs and resource preferences of office-based general pediatricians, and are there commonalities with pediatricians in other settings?We conducted semi-structured interviews of twenty office-based general pediatricians to elicit their: (a) demographic and computer use data, (b) verbalized uses of specified information resources and (c) "think aloud" perceptions of questions and identification of information resource preferences in response to short vignettes representing clinical cases in genetics and infectious diseases. This study was approved by the appropriate institutional review board.Participants were interviewed individually by telephone by one of the authors (GRK).A sample of twenty general pediatricians was recruited from urban/suburban office-based practices responding to general calls for subjects through two professional pediatric group newsletters (Winter-Spring 2004), through hospital pediatric department and joint community practice affiliations and through direct telephone/e-mail solicitation. Participants were enrolled by telephone.Sixteen short vignettes See  represenThe vignettes were randomized into non-repeating sequential groups of four vignettes and presented to participants by telephone in a standardized fashion. Participants' verbal responses were recorded and transcribed immediately post-interview for content analysis. Interviews lasted approximately 30 minutes each. One participant was replaced after it was discovered post-interview that the audio recorder had malfunctioned.For each of the twenty participants, after a standard introduction to the study and consent, data was collected in a continuous three-part fashion:1. In response to each of four sequential, randomized vignettes, the participant was asked to state perceptions of the questions presented by the vignette and how s/he would approach the problems in a "think-aloud" fashion , indicat2. In response to a list of medical and pediatric information resources, the participant was asked to state the types of questions, problems or information needs for which s/he would use each resource type.3. Information about demographics  was elicited.Demographic information was tabulated for descriptive analysis [see Results: Demographics].Indicated use of specific information resource types and their relative frequencies of indicated use  were tabulated for descriptive analysis [see Transcribed responses to vignettes were coded consecutively according to the constant comparison method using open coding  to identCodes representing questions perceived from vignettes were sorted into nine categories forming two broad categories of Question Types about patient-specific and general medical information see . QuestioValidation of the results was by member-checking. A summary of the results [see Results] was presented to all participants with a 90% return. All returns concurred with the summary.Twenty pediatricians  in urban/suburban pediatric practice participated. Average time in practice was 15.4 yrs ). Twelve participants classified their practices as private practice, 4 as managed care and 4 as free clinic/federally qualified health center. All 20 classified their clinical work as general pediatrics, with 7 adding adolescent medicine, 2 adding adult medicine and 1 adding developmental pediatrics. Thirteen worked in private offices, 4 in multiple specialty settings, 2 in multi-site clinics and 1 in an expanded urgent care (with limited well child care).All participants shared office computers with Internet access in clinical areas. All had billing functions performed by separate computer systems. Twelve had access to a private networked computer, 10 used handheld computers, 6 had wireless access, 4 had mobile computers and 4 used electronic prescribing. Three used an EMR, 4 had access to an EMR that provided read-only capability. Three used computers in consultation rooms. Two had computers for other projects (research and patient education). Twelve had the capability to communicate with their patients by e-mail, but only 2 did so more than very rarely.The most frequently identified resources were specialists, generalist colleagues and general pediatric texts, followed by drug formularies and specialty textbooks. Professional organization and federal health Websites (in particular the American Academy of Pediatrics and the Centers for Disease Control and Prevention) were identified as sources of guidelines and other information. When commercial medical portals were available , they were frequently identified as a consulted information resource.Resources rarely or not identified as sources of information in the office included: algorithmic/flow-chart decision-making texts, evidence-based medical reviews, medical journal abstracts, medical librarians or consumer health information. Several participants identified evidence-based medicine resources for specific problems or for self-education.Pediatric handbooks (such as the Harriet Lane Handbook ) were idGeneralist colleagues were identified for confirmation of findings and for discussion of diagnostic and management decisions. Specialists were identified as information resources in anticipation of referral. Contact with generalist colleagues was usually face-to-face (in the office) while contact with specialists was via telephone and with a person or institution with which the participant had prior interaction or contact through an established referral network.The CDC Travel Health Website  was the Collectively, patient-specific Question Types regarding details about history, findings , diagnosis, and treatment plans were the most frequently perceived, followed by Question Types regarding current patient state , social support and patient/family understanding of a condition. Participants also perceived pragmatic questions about legal and insurance issues regarding minors in non-custodial care.Patient-specific questions about maternal, prenatal and birth history were perceived more frequently in response to Genetics vignettes and questions about patients' appearance/condition were perceived more frequently in Infectious Disease vignettes.Collectively, general medical Question Types regarding etiology, and treatment/diagnosis guidelines for a condition were the most frequently perceived.General medical questions about disease etiology occurred more frequently in response to Genetics vignettes and questions about diagnosis/referral criteria and disease classification were perceived more frequently in Infectious Disease vignettes.Most general medical Question Types posed by participants had correspondences to Generic Medical Questions in the taxonomy developed by Ely et al , however• "What are the classifications of a disease?" (to decide on an approach to management):• "...what type of sickle cell [the patient] has, if he has SS [a type of sickle cell disease] or one of the more favorable ones..."• "...what type of school failure, is it in one area, speech?"• "What is available treatment?" (to locate new and uncommon resources):• "...what treatments are available, what's experimental? If it exists, where is it provided? Would [the patient] qualify for participation in a study?"• "What is the latest therapy available for patients with a disease X?"Collectively, specialists were the most frequently identified Information Resource Type. They were also the most frequently identified last resource when an answer could not otherwise be found.The most frequently identified patient-specific resources were patient history and examination, followed records from and communication with previous providers and institutions , which were similar for both Genetics and Infectious Diseases vignettes.After specialists, the most frequently identified general medical resources were specialty and general pediatric texts and colleagues , although participants with access to medical portals indicated them as resources in lieu of texts. Participants also identified Web search engines, guidelines from professional organizations (American Academy of Pediatrics) and local institutions .For Genetics vignettes, genetic specialists, dysmorphology texts/atlases and genetic databases (Online Mendelian Inheritance in Man (OMIM) ) were moThe information process of physicians in patient care has been described as iterations of inquiries/interventions  and responses to updates of a patient's clinical status and/or a practitioner's information state . ParallePractitioners anticipate information needs by organizing and maintaining "personal information collections, defined as subsets of... information...that individuals [build] conceptually and physically over time" , includiCharacterization of such collections and their use by practitioners may be helpful on several fronts. First, it can help plan placement of resources to promote their effective use. It has recently been suggested that the way to promote evidence-based medical practice is to promote information management . Second,In response to vignettes, patient-specific questions focused on patient history, examination and management. General medical questions focused on etiology, diagnostic/therapeutic management and guidelines. For Genetics vignettes, participants perceived questions about disease etiology more frequently than for Infectious Disease vignettes, for which they perceived questions about diagnostic and referral guidelines, disease classification and sources of patient education materials.In comparison to an analysis of patient care questions asked at an academic medical center (AMC), the office-based (OB) participants perceived a higher relative frequency of questions about diagnosis and no questions about drugs. Reasons to explain this may include: 1) the OB group used hypothetical patients whereas the AMC-derived group used actual patients, 2) the OB group used only outpatient settings  whereas more than half (58.8%) of the AMC-derived questions were generated from inpatient settings (where patients' admitting diagnoses are known), 3) the OB group used hypothetical encounters (where details of therapy may not be specified), whereas the AMC-derived group used actual patient cases and 4) the vignettes elicited questions about hypothetical cases in genetics and infectious diseases, whereas the AMC-derived questions came from patients from a number of different domains.As noted in the AMC study of pediatricians' questions, the participants' questions fell within a few Generic Questions from the taxonomy derived from ethnographic observation of family practitioners. Interestingly, participants perceived two Question Types that did not have specific matches within the taxonomy: questions about disease classifications and about sources of the best and latest care for a disease. One suggestion that has been made is that pediatricians may have different information needs from family practitioners .perspectives , the inventory (of objects/relationships) relevant to that perspective  and the granularity (of knowledge) needed  to express concepts and information needs easily and clearly [To express information needs of a group of practitioners, a question taxonomy should: a) match their  clearly . The GenWithin the context of Genetics and Infectious Diseases (as represented in these vignettes), participants identified a relatively small, but common set of defined resource types . They named a few specific, trusted online resources, although some were comfortable with "open" Web searching. They identified trusted and established sources of specialty care , reachable by phone.In comparison to AMC pediatricians, office-based general pediatricians have similar resource preferences, but also identified community resources, including the patient's family and social environment . Participants identified specific Websites  and medical portals when available (through academic affiliations) as trusted sources. In contrast to AMC-defined roles of "faculty" and "resident," participants identified in-office colleagues as informal consultants and specialists (by phone) as formal consultants. Specialists were frequently identified as "last" resource if an answer could not otherwise be found.The described use of vignettes can generate a description of information resource use, but it does not provide information on what users actually do. It also does not provide information on barriers to pursuing answers to questions. It is not intended to supplant other methods of discovering information needs , but as a way of exploring information needs and resource preferences in a flexible and low-cost fashion. It may be useful in "fine-tuning" exploration of information needs and resource preferences:• Vignettes about rare or new conditions may be used to identify practitioners' unrecognized information needs and/or to guide the development and deployment of new information resources to fulfill needs• Vignettes about new morbidities may be used to identify the unrecognized practitioner information needs and/or to guide the development of practitioner education and awareness programsIn addition to the qualitative frequency analyses described in this paper, exploratory factor analysis of the results may be used to discover categories of resources that may help determine their selection by practitioners in different circumstances (using a Hierarchy of Abstraction Model ).Using content analysis of semi-structured responses to short clinical vignettes in the domains of genetics and infectious diseases, we have explored information needs and resource preferences of office-based general pediatricians. This approach yielded descriptive information that demonstrates commonalities and distinctions with other studies on the information needs of general pediatricians.The author(s) declare that they have no competing interests.GRK participated in the design of the study and obtaining IRB approval, developed the vignettes and questionnaire, organized agreement on attribute assignments, recruited participants, interviewed the participants, coded transcripts , and drafted the manuscript. ELB participated in the design of the study and obtaining IRB approval, tested the vignettes and reviewed the questionnaire and reviewed the manuscript. HPL participated in the design of the study and obtaining IRB approval, helped in the development of vignettes and questionnaire, participated in agreement on attribute assignments, participated in coding of transcripts and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Vignettes used in semi-structured interviews Sixteen vignettes listed by domain (Genetics (8), Infectious Diseases(8))Click here for fileInformation resource survey Relative frequency of resource use identified by participants. Cited uses of specific resourcesClick here for fileQuestion types perceived in vignettes Patient-specific and general medical question types perceived by participants in response to vignettes with closest match of general medical question types to Generic Question taxonomy [taxonomy Click here for fileInformation resource preferences identified in vignettes Patient-specific and general medical information resources identified by participants in response to vignettesClick here for fileFrequency of perceived question types in vignettes Patient-specific and general medical question types perceived by participants in response to vignettes according to frequency of reportClick here for fileFrequency of identified information resource preferences in vignettes Information resources identified by participants in response to vignettes according to frequency of report Click here for file


An analysis of the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome sequences, identified 310 selenoprotein genes that clustered into 25 families. This included 101 new selenoprotein genes that belonged to 15 families, doubling the number of prokaryotic selenoprotein families. Selenocysteine (Sec) is a rare amino acid which occurs in proteins in major domains of life. It is encoded by TGA, which also serves as the signal for termination of translation, precluding identification of selenoprotein genes by available annotation tools. Information on full sets of selenoproteins (selenoproteomes) is essential for understanding the biology of selenium. Herein, we characterized the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome project.We identified 310 selenoprotein genes that clustered into 25 families, including 101 new selenoprotein genes that belonged to 15 families. Most of these proteins were predicted redox proteins containing catalytic selenocysteines. Several bacterial selenoproteins previously thought to be restricted to eukaryotes were detected by analyzing eukaryotic and bacterial SECIS elements, suggesting that eukaryotic and bacterial selenoprotein sets partially overlapped. The Sargasso Sea microbial selenoproteome was rich in selenoproteins and its composition was different from that observed in the combined set of completely sequenced genomes, suggesting that these genomes do not accurately represent the microbial selenoproteome. Most detected selenoproteins occurred sporadically compared to the widespread presence of their cysteine homologs, suggesting that many selenoproteins recently evolved from cysteine-containing homologs.This study yielded the largest selenoprotein dataset to date, doubled the number of prokaryotic selenoprotein families and provided insights into forces that drive selenocysteine evolution. Selenium is a biological trace element with significant health benefits . This micis-acting stem-loop structure residing within selenoprotein mRNAs [trans-acting factors dedicated to Sec incorporation [The mechanism of selenoprotein synthesis in prokaryotes was elucidated extensively by Böck and colleagues ,9. Transin mRNAs ,10, and poration . In eukaporation . Bacteriporation ,14.Drosophila genomes and in several EST databases. However, due to lack of bacterial consensus SECIS models, prediction of bacterial selenoproteins in genomic sequences is difficult. Instead, these proteins can be identified through searches for Sec/Cys pairs in homologous sequences [As UGA has the dual function of inserting Sec and terminating translation, and only the latter function is recognized by available annotation programs, selenoprotein genes are almost universally misannotated in sequence databases . To addrequences .We report here the use of a modified search strategy to characterize the selenoproteome of the largest prokaryotic sequencing project, the 1.045 billion nucleotide whole genome shotgun sequence of the Sargasso Sea microbial populations . This daThe Sargasso Sea genomic database contains the largest collection of microbial sequences derived from a single study . No geneThis procedure identified 209 selenoprotein genes, which belonged to ten known selenoprotein families and 101 selenoprotein genes, which belonged to 15 new selenoprotein families (each represented by at least two sequences)  and glycine reductase selenoprotein B (grdB), were represented by single sequences. Interestingly, although all known selenoproteins present in the dataset were identified, only nine of the ten families had Cys homologs in the NR database. One selenoprotein, grdA, did not have known Cys homologs . NeverthSeveral selenoprotein families had a particularly high representation in the Sargasso Sea dataset. The most abundant family was SelW-like, which contained 48 genes. Although the function of this protein is unclear, a conserved CXXU motif (Cys separated from Sec by two other residues) suggests a redox function. In addition, this protein was previously found to interact with glutathione, a major redox thiol compound in cells ,25. A peAmong 15 new selenoprotein families, 13 contained at least two individual TGA-containing ORFs Table  highlighAll new selenoproteins contained stable stem-loop structures downstream of Sec-encoding TGA codons that resembled bacterial SECIS elements. Representative predicted SECIS elements found in several new selenoprotein families are shown in Figure Among 25 known and new bacterial selenoprotein families identified in the Sargasso Sea dataset, three families, SelW-like, GPx and deiodinase, were previously thought to be of eukaryotic origin. However, multiple sequence alignments Figure  stronglyDeiodinase is known to activate or inactivate thyroid hormones via the reaction of reductive deiodination . This prSelW and GPx homologs were recently detected in some bacteria, but the number of these sequences was small and their origin not clear . DetectiS-sulfoxide reductase (MsrA). Except for GPx, these families were represented by single selenoprotein genes. No bacterial SECIS elements were found in these genes. In SelM and SelT sequences, typical eukaryotic SECIS elements were present in 3' UTRs as detected by SECISearch [In addition, we identified five eukaryotic selenoproteins: SelM, SelT, SelU, GPx and methionine-CISearch , whereasPrevious analyses of selenoprotein sets in the three domains of life revealed that bacterial and archaeal selenoproteomes significantly overlap, whereas eukaryotes had a different set of selenoproteins ,20. The An exhaustive search of Sargasso Sea selenoproteins against 260 completely sequenced prokaryotic genomes revealed that these selenoproteins were present in a limited number of genomes, which contrasted with the widespread occurrence of their Cys-containing homologs  were different, the majority of detected selenoproteins showed scattered occurrence. Indeed, the Sec-containing forms of proteins were rare compared to homologous Cys-containing forms, which were widespread. It appears that that most detected selenoproteins evolved recently from Cys-containing homologs in organisms, which already had the system for Sec insertion. It can be predicted that as searches of additional prokaryotic sequence datasets identify new selenoprotein genes, many of these will be present in only a small number of species. At present, Sec evolution is not fully understood, but it is clear that Sec/Cys interchanges are possible in both directions depending on the need for particular redox properties and on the restriction imposed by the dependence of species on the trace element selenium.Further analysis of both Sargasso Sea and completely sequenced prokaryotic genomes revealed that essentially all selenoproteins with known function were redox proteins, which used Sec either to coordinate redox-active metals or for thiol/disulfide-like redox catalysis. Among 25 selenoprotein families detected in the Sargasso Sea, 14  were homologs of known thiol-dependent redox proteins Table , and mosWhole-genome shotgun sequencing projects have been applied extensively to determine genomic sequences of a variety of organisms, and recently this approach was used to sequence the microbial community of the Sargasso Sea. Many of the Sargasso Sea organisms represent phyletic groups previously not known or poorly characterized, including organisms that could not be isolated from the microbial community or be cultured . Identifin silico approaches that identify full sets of selenoproteins in organisms provided powerful new tools for determining identities of selenoproteins as well as their expression characteristics and functions [Previous functional information on selenoproteins has been derived largely from wet-lab experiments. More recently, several unctions -20,38. Munctions ,22.In the present study, we used a similar procedure, but supplemented it with additional filters to improve performance. All known prokaryotic selenoprotein families present in the Sargasso Sea genomic dataset were identified by this approach (209 genes that clustered into ten prokaryotic selenoprotein families). In addition, 101 sequences that belonged to 15 new selenoprotein families were identified. Thus, our study has approximately doubled the list of known prokaryotic selenoprotein families and generated the largest selenoprotein dataset to date.On the basis of the presence of SECIS elements specific to major domains of life, we could determine the origin of detected selenoproteins . All ten known and 15 new prokaryotic selenoprotein families had predicted bacterial SECIS elements. Interestingly, both selenoprotein forms and Cys-containing homologs of thyroid hormone deiodinase, a protein previously thought to be restricted to the animal kingdom and present exclusively in the selenoprotein form, were identified in prokaryotes. The detected deiodinase-like proteins were prokaryotic as they contained bacterial SECIS elements.Detection of prokaryotic deiodinase-like proteins and several other bacterial selenoproteins thought to be restricted to eukaryotes suggests a revision of the view that eukaryotic and prokaryotic selenoproteomes do not overlap. Although this idea was consistent with the previous selenoprotein analyses, at least four selenoprotein families are now known that occur in both prokaryotes and eukaryotes: SelW, GPx, selenophosphate synthetase and deiodinase. We also detected homologs of five additional eukaryotic selenoproteins, but the absence of bacterial SECIS elements, presence of eukaryotic SECIS elements or introns, and homology to eukaryotic proteins argued that these selenoproteins were eukaryotic in origin.Surprisingly, sets of selenoproteins in the Sargasso Sea database and in the combined set of 260 completely sequenced prokaryotic genomes were quite different in regard to both identities and number of selenoprotein genes. The Sargasso Sea dataset was rich in selenoprotein genes, most of which were homologs of known thiol-dependent redox enzymes. In contrast, the proportion of selenoprotein genes in completely sequenced prokaryotic genomes was approximately three times lower, and the majority of detected genes used Sec for metal coordination. Thus, even with the availability of 260 genomes, the roles of selenium in nature are just beginning to be understood. For example, our current analysis of the Sargasso Sea dataset implicated selenium in arsenate reduction, molybdopterin biosynthesis, sulfurtransferase function and other processes, which were not known to be dependent on this trace element.We also observed common features in the two sets of selenoproteins. For example, most of the detected selenoproteins had a large number of Cys homologs. The scattered occurrence of selenoproteins in both datasets suggests a highly dynamic nature of Sec evolution. As long as the system for Sec insertion is maintained, Sec may appear when required by the changing environment and disappear when this requirement recedes. Thus, the analysis of selenoproteomes and the compensatory sets of Cys-containing proteins provides a unique model system to examine evolutionary forces to a changing environment.The whole-genome shotgun sequence database of the Sargasso Sea was obtained from the National Center for Biotechnology Information (NCBI) ftp server with the project accession number AACY00000000 . Unlike The NR protein database was downloaded from the NCBI ftp server . This daTo enable selenoprotein searches automatically, we developed a set of programs as discussed below. A UNIX/LINUX platform was used. The entire search process was performed on a Prairiefire 128-node, 256-processor Beowulf cluster supercomputer at the Research Computing Facility of the University of Nebraska - Lincoln.Each Cys-containing sequence in the NR protein database was searched against the Sargasso Sea database of nucleotide sequences for possible TGA-containing hits using TBLASTN. E-value cutoff was set to 10.0. TBLASTN output for each protein sequence was parsed and Cys/TAA or Cys/TAG pairs were filtered out. Only local alignments, in which Cys in a query sequence was aligned with TGA in the nucleotide sequence from the target Sargasso Sea database, were further analyzed. As Sec is typically located in enzyme active sites, additional filters were added. Specifically, local alignments were discarded if they contained more than two stop codons , two stop codons of which one was not TGA, or two TGA codons with one aligned to a non-Cys residue. A total of 38,446 local redundant alignments  were identified which corresponded to 19,410 proteins in the NR protein database.For each TGA-containing sequence in the local alignment set, regions upstream and downstream of the TGA were analyzed to identify minimal ORFs with the assumption that in-frame TGA coding for Sec must be inside predicted ORFs. If stop codons were encountered closer to TGA codons than candidate start codons (ATG or GTG), such TGA-containing sequences were discarded. Conservation of TGA-flanking regions in all 6 reading frames was also analyzed with BLASTX and screened against a database of conserved domains using RPS-BLAST. These criteria were also used to filter out false positive hits. Finally, redundant sequences were removed. These filters reduced the set to 2,131 unique TGA-containing candidate ORFs.To cluster protein sequences into different protein families or groups, the pairwise alignment tool in the BLAST program package, BL2SEQ, was used. 1,045 clusters were obtained with 1 to 63 sequences in each cluster.Considering that Cys/TGA pairs in most false-positive hits were not expected to be conserved, whereas conservation was expected for true-positive Cys/Sec pairs, all clusters were automatically searched against NCBI NR and microbial databases using BLASTX and TBLASTX. Each predicted ORF containing an in-frame TGA was considered further only if at least two corresponding Cys-containing homologs were detected and the proportion of TGA/Cys pairs in the set of homologs was greater than 50%. This procedure resulted in 331 clusters containing 1,072 ORFs.All 331 clusters were analyzed for the presence of potential bacterial SECIS elements immediately downstream of the TGA codons using mfold  or RNAfoA simple classifier was developed to divide clusters that contained bacterial SECIS-like structures into three groups: known selenoproteins, new selenoproteins and selenoprotein candidates. Except for known selenoproteins, clusters containing at least two different sequences with conserved in-frame TGA codons were considered as new selenoproteins. Clusters containing only one sequence were considered selenoprotein candidates because of the possibility of a sequencing error causing an in-frame TGA. Finally, clusters that could be aligned such that their TGAs also aligned, were joined into larger clusters.The complete set of predicted selenoprotein sequences with annotations  is available as a text file Click here for file