IPF is typically progressive and leads to significant disability. The median survival is 2 to 5 years from the time of diagnosis. Medical therapy is ineffective in the treatment of IPF. New molecular therapeutic targets have been identified and several clinical trials are investigating the efficacy of novel medication. Meanwhile, pulmonary transplantation remains a viable option for patients with IPF. It is expected that, during the next decade, considerable progress will be made toward the understanding and treatment of this devastating illness.Idiopathic pulmonary fibrosis (IPF) is a non-neoplastic pulmonary disease that is characterized by the formation of scar tissue within the lungs in the absence of any known provocation. IPF is a rare disease which affects approximately 5 million persons worldwide. The prevalence is estimated to be slightly greater in men  than in women . The mean age at presentation is 66 years. IPF initially manifests with symptoms of exercise-induced breathless and dry coughing. Auscultation of the lungs reveals early inspiratory crackles, predominantly located in the lower posterior lung zones upon physical exam. Clubbing is found in approximately 50% of IPF patients. Cor pulmonale develops in association with end-stage disease. In that case, classic signs of right heart failure may be present. Etiology remains incompletely understood. Some environmental factors may be associated with IPF . IPF is recognized on high-resolution computed tomography by peripheral, subpleural lower lobe reticular opacities in association with subpleural honeycomb changes. IPF is associated with a pathological lesion known as usual interstitial pneumonia (UIP). The UIP pattern consists of normal lung alternating with patches of dense fibrosis, taking the form of collagen sheets. The diagnosis of IPF requires correlation of the clinical setting with radiographic images and a lung biopsy. In the absence of lung biopsy, the diagnosis of IPF can be made by defined clinical criteria that were published in guidelines endorsed by several professional societies. Differential diagnosis includes other idiopathic interstitial pneumonia, connective tissue diseases ,  Idiopathic pulmonary fibrosis (IPF).Synonym: cryptogenic fibrosing alveolitis (CFA) was the preferred term in Europe until terminology was simplified by consensus conference .Idiopathic pulmonary fibrosis (IPF) is a chronic disease that manifests over several years and is characterized by scar tissue within the lungs, in the absence of known provocation. Exercise-induced breathlessness and chronic dry cough are the prominent symptoms.IPF belongs to a family of lung disorders known as the interstitial lung diseases (ILD) or, more accurately, the diffuse parenchymal lung diseases (DPLD). Within this broad category of diffuse lung diseases, IPF belongs to the subgroup known as idiopathic interstitial pneumonia (IIP). By definition, the etiology of IIP is unknown. There are seven distinct IIPs, differentiated by specific clinical features and pathological patterns . IPF is The incidence and prevalence of IPF are difficult to determine because uniform diagnostic criteria have only recently been defined . HistoriThe best available data suggests an incidence of approximately 10.7 per 100,000 persons for men; and 7.4 per 100,000 persons for women. The prevalence of IPF is slightly greater at 20.2 men per 100,000 and 13.2 women per 100,000 . Data frCigarette smoking is strongly associated with IPF. One study reported a correlation between smoking history (20–40 pack-years) and risk for IPF, with an odds ratio of 2.3  for smokers .IPF affects men more than women. In addition, the incidence of IPF increases with age. IPF most commonly appears between the fifth and seventh decades of life, with two-thirds of all cases arising in patients over 60 years of age . The meahTERT and hTR in a cohort of patients with familial IPF [Familial cohorts of IPF are described in dozens of reports, though sporadic cases constitute the majority of disease. Clinical features of familial IPF are indistinguishable from those of the sporadic form, excepting for an earlier age of onset . Familialial IPF . These tIPF patients experience breathlessness upon exertion. They are often bothered by a dry cough which interferes with daily activities. The onset of symptoms is slow, but symptoms become progressively worse over time. The initial presentation of breathlessness is commonly attributed to aging, cardiac disease, or emphysema which results in typical delays of diagnosis. Retrospective analysis of IPF patients suggests that symptoms precede diagnosis by a period of 6 months to 2 years . SymptomVelcro.Auscultation of the lungs reveals early inspiratory crackles, predominantly located in the lower posterior lung zones upon physical exam. These rales have a fine acoustic character reminiscent of the sound made by Clubbing is found in approximately 50% of patients with IPF. There are no other physical manifestations, unless cor pulmonale has developed in association with end-stage disease. In that case, classic signs of right heart failure may be present.The examination of patients with IPF should attempt to identify those signs suggesting an alternative diagnosis such as systemic sclerosis or polymyositis that can be associated with secondary pulmonary fibrosis. To that end, the examiner should look for sclerodactily, scleroderma, proximal muscle weakness and telangiectasias. The history should exclude Raynaud's phenomenon.There are no specific laboratory abnormalities in IPF. However, mild elevation of the erythrocyte sedimentation rate, a low-positive titer of anti-nuclear antibody (ANA) and/or low-positive rheumatoid factor can be seen and are thought to represent a general state of inflammation. In advanced disease, blood counts may reveal polycythemia.A high titer of autoantibodies suggests an alternative diagnosis such as connective tissue disease. In order to exclude secondary causes of pulmonary fibrosis, a broad panel of autoantibodies should be ordered during the initial evaluation.1). The ratio of FEV1/FVC remains normal (or increased) in IPF, consistent with restrictive physiology. Lung volume measurements confirm restrictive physiology, usually manifest in reduction of total lung capacity (TLC). Restrictive physiology is the consequence of reduced pulmonary compliance. Changes in compliance can be attributed to the accumulation of parenchymal scar tissue and the subsequent distortion of normal lung architecture.Routine spirometry reveals decreased measures of forced vital capacity (FVC) and forced expiratory volume in one second  mismatching, as opposed to anatomic shunting or reduced diffusion capacity .The natural history of IPF is incompletely known. IPF usually assumes a course of relentless physiologic deterioration. However, some patients remain stable for extended periods and individual outcomes can be highly variable . Still, New insight into the natural history of IPF has been gleaned from secondary analysis of the placebo groups assembled for recent multi-center clinical trials -23. It aJapanese physicians were the first to describe acute, unexpected deterioration in patients with IPF. This phenomenon has been called the "acute exacerbation" or, more euphemistically, the "terminal complication" of IPF ,25. AcutPulmonary hypertension has been reported to occur in 32 to 84% of patients with IPF. The exact prevalence remains unclear because triggers for the evaluation of pulmonary pressures and the best method to detect pulmonary hypertension in IPF remain unsettled ,34. HoweSeveral groups have described a syndrome in which IPF coexists with pulmonary emphysema -39. This1)CPI = 91 - (0.65 percent predicted DLCO) - (0.53 percent predicted FVC) +  is a similar process which has recently been demonstrated as an important pathway mediating fibroproliferation in certain renal diseases . PulmonaAn overlooked feature of pulmonary fibrosis is the presence of increased angiogenic activity, reminiscent of tumorigenesis. This has been well-established in both animal and human studies ,62. An iThe chest roentograph is abnormal in most patients with IPF Figure . NeverthRoentographic images of IPF can demonstrate reticular markings (net-like curvilinear opacities). These markings are distributed in a bilateral, asymmetric pattern with predilection toward the lower lobes. A particular pattern of course reticular lines juxtaposed between areas of focal round translucency is known as honeycombing. Roentographic honeycombing emerges late in the course of disease and portends poor prognosis .Use of standard roentographs lacks diagnostic accuracy. A correct diagnosis of IPF (true positive) is made by a roentograph in less than 50% of cases. Furthermore, the radiographic interpretation of interstitial patterns has the characteristic of poor interobserver agreement. Studies examining this particular test characteristic have reported meager 70% concordance amongst radiologists ,66.The development of HRCT revolutionized the diagnostic evaluation of interstitial lung disease. HRCT utilizes x-ray technology, along with computerized algorithms, to construct images of virtual thin axial slices through the chest. These high-fidelity images allow a detailed examination of the pulmonary parenchyma. Subsequently, interobserver agreement and overall diagnostic accuracy has been improved with this technology. HRCT permits identification of alternate patterns of diseases. The primary role of HRCT during the diagnostic evaluation of interstitial lung diseases has become the discrimination of "typical" radiographic IPF from that of other ILD.The "typical" appearance of IPF on HRCT consists of patchy, predominantly peripheral, predominantly subpleural and necessarily bibasilar reticular opacification Figure . Ground Certain characteristics need be absent from HRCT in order to label the images as consistent with the "confident" IPF pattern. The HRCT cannot feature predominant ground glass opacities, nodular infiltrates or significant lymphadenopathy. A predominance of upper lobe infiltrates is also inconsistent with "confident" IPF. These findings may suggest alternative diagnoses. For example, ground glass implies heart failure, non-specific interstitial pneumonia, cryptogenic organizing pneumonia, desquamative interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease or hypersensitivity pneumonitis. A pattern of fine nodules might suggest hypersensitivity pneumonitis, granulomatous infection or lymphangitic spread of malignancy. Upper lobe disease is found in pulmonary Langerhans' cell histiocytosis, hypersensitivity pneumonitis, several pneumoconioses, sarcoidosis, ankylosing spondylitis, rheumatoid nodules and eosinophilic pneumonia syndromes. Significant hilar lymphadenopathy is associated with sarcoidosis, infection and malignancy.Several studies examined the accuracy of HRCT utilizing histopathology as the "gold standard" ,69. StudHowever, testing for the "confident" HRCT pattern is not a sensitive tool for case finding of IPF. The full spectrum of the "confident" HRCT pattern can only be found in 4-out-of-5 cases of biopsy-proven IPF. In other biopsy-proven IPF cases, a less specific reticular pattern is seen on HRCT which has been called "possible" IPF Figure . The radThe role of bronchoalveolar lavage (BAL) in the diagnosis of IPF remains limited. While the cell count of BAL fluid from patients with IPF has an expected differential distribution (increased numbers of neutrophils and/or eosinophils), the diagnosis of IPF can not made solely on the basis of BAL fluid analysis. Though much effort has been invested in evaluating the clinical utility of BAL, the results of many studies are contradictory ,71. StilA surgical lung biopsy is close to the "gold standard" for diagnosis and is recommended to confirm all suspected cases of IPF. Biopsy from two sites is recommended based on data indicating a substantial risk of sampling era unless specific effort is made to reflect the gamut of gross disease .In cases presenting with a "confident" HRCT pattern, biopsy can be avoided because the results of biopsy can be predicted ,69. A siThe decision to perform surgical lung biopsy must be carefully considered. Advanced lung disease, poor functional status and older age are relative contraindications to surgery. The absolute risk associated with biopsy is controversial and all of the evidence related to this issue is derived from retrospective data, thus colored by inherent selection bias. While some studies have noted a high short-term mortality, other studies have demonstrated that surgical lung biopsy can be performed safely ,75. VATSThe gross pathology of IPF may be normal, but often a distinctive nodular appearance of the pleural surface is found. This has been likened to cirrhosis of the liver. The histopathological lesion associated with IPF is known as usual interstitial pneumonia (UIP). This lesion is defined by a distinctly variegated pattern. UIP features normal lung architecture alternating with patchy areas of histologically apparent pulmonary parenchymal fibrosis Figures  and 5. Fe.g.: bleomycin, amiodarone and nitrofurantoin toxicity). Distinguishing IPF from other disorders that contain UIP requires correlation with the clinical history.It is important to note that the UIP pattern is found in several diseases and is not limited to IPF. UIP can be associated with connective tissue disease, asbestosis, hypersensitivity pneumonitis, the Hermansky-Pudlak syndrome and drug toxicities .The actual "gold standard" diagnosis of IPF consists of clinical-radiological-pathological correlation and was defined by consensus conference in the year 2000 and adopted by the American Thoracic and European Respiratory Societies (ATS/ERS) in a statement of guidelines published in the same year . Accordidefinite diagnosis of IPF requires all of the following: [The llowing: • Surgical lung biopsy revealing a histologic pattern consistent with UIPe.g.: connective tissue disease, environmental exposure, etc.)• Exclusion of other known causes of interstitial lung disease • HRCT demonstrating a pattern of "confident" or "possible" IPF.probable diagnosis of IPF in cases in which a surgical biopsy is not possible. These clinical criteria were endorsed by the ATS/ERS consensus statement on IPF [In the absence of a surgical biopsy, the diagnosis of IPF remains uncertain. Yet, a set of reproducible clinical criteria were developed to define the t on IPF . By consMajor criteria • Exclusion of other known causes for interstitial lung disease 1/FVC ratio)and/or impaired gas exchange (increased A-a gradient or decreased diffusion capacity)• Abnormal pulmonary function testing that includes evidence of restriction • Transbronchial lung biopsy or bronchoalveolar lavage (BAL) does not support an alternative diagnosisMinor criteria • Age > 50 yr• Insidious onset of otherwise unexplained dyspnea on exertion• Duration of illness ≥ 3 months• Bibasilar, inspiratory crackles These criteria have never been subjected to a prospective analysis and, over time, diagnostic algorithms have continued to evolve. As HRCT technology has improved and the utility of this modality has been consistently demonstrated in clinical trials, HRCT has become a more important tool in diagnostic algorithms. Meanwhile, transbronchial biopsy and BAL have fallen from favor, mostly due to low diagnostic yield. A new ATS/ERS-sponsored consensus statement should address these issues and publication of such is expected in 2008.The clinical-radiological-pathological "gold standard" diagnosis of IPF is flawed due to several issues, such as: 1) lack of standardized tests to exclude known causes of interstitial lung disease; 2) poor interobserver agreement regarding the interpretation of radiographic images; and, 3) poor interobserver agreement as regards the recognition of histological patterns. Interobserver agreement amongst radiologists reading HRCT has been reported to be no better than 80% . AgreemeSince the mid-1990s, attention has focused on the division of idiopathic pulmonary fibrosis into histologically-defined subgroups. This practice stems from the description of NSIP in 1994 . NSIP pai.e.: UIP vs. NSIP); in fact, pathological pattern conferred no independent prognostic value. The last bit of evidence comes from a cohort of families affected by familial pulmonary fibrosis where both NSIP and UIP were often found within a single family [Three lines of evidence suggest that NSIP and UIP are different ends of a spectrum resulting from the same disease. The first piece of evidence is found in the examination of patients undergoing multiple surgical lung biopsies. One such study found that 26% of patients with IPF have NSIP pathology in one lobe while simultaneously displaying UIP in a sample from another lobe . The sece family .The differential diagnosis of IPF includes other idiopathic interstitial pneumonias. HRCT is useful for excluding disease with predominantly ground glass opacity or nodular patterns. Non-specific interstitial pneumonia (NSIP) will always remain in the differential and, in some cases, can only be excluded by biopsy.Connective tissue diseases such as systemic sclerosis, polymyositis or rheumatoid arthritis can mimic IPF, both clinically and radiographically. The great majority of patients with systemic sclerosis will present with HRCT scan features more closely resembling NSIP . Yet, reforme fruste autoimmune disorders which can be difficult to recognize. These entities comprise autoimmune-mediated lung disease without a constellation of signs and symptoms to fulfill diagnostic criteria for defined rheumatologic illness. The presence of one or more symptoms, such as Raynaud's phenomenon, proximal muscle weakness or sicca features, coupled with laboratory features of systemic inflammation (antinuclear and other specific autoantibodies) define the syndrome of undifferentiated connective tissue disease that can accompany pulmonary fibrosis and resemble IPF [There are also mble IPF .e.g.: bleomycin, nitrofurantoin, amiodarone, carmustine and methotrexate) and several congenital disorders  are also in the differential.Chronic hypersensitivity pneumonitis and other environmental  exposures can also be difficult to differentiate. The clinical history can serve to discriminate this condition but is oftentimes equivocal. Nonetheless, a history of exposure to asbestos, grain dust and mold should be sought during the initial evaluation of IPF. Radiation pneumonitis, end-stage sarcoidosis, certain drug toxicities  in predicting mortality [Three separate groups of investigators observed a relationship between physiologic measures and survival amongst well-defined cohorts of IPF patients -5. Of paOther investigators have examined IPF patients and identified interval decline of forced vital capacity as a characteristic that is predictive of survival. A 10% decline of forced vital capacity, at either six or twelve months, had poor prognostic implications and was more accurate than predictions based upon baseline physiologic parameters alone .The ability of HRCT to predict the outcome of IPF was also demonstrated. When biopsy-proven IPF patients were followed for three years by HRCT, it was found that radiographic honeycombing predicted the worst survival . In addiAnother interesting study examined the relationship between the "confident" IPF pattern and survival. A cohort of patients with biopsy-proven IPF were analyzed by HRCT and divided into groups based upon radiographic/pathologic concordance. UIP pathology was shown to confer a worse prognosis when seen in combination with the "confident" IPF pattern. It was found that the an "indeterminate" pattern of HRCT conferred better prognosis despite the presence of UIP on biopsy .In the past, unremitting inflammation was thought to cause progressive pulmonary fibrosis. Therefore treatment regimens were designed to suppress the immune system with the goal of halting subsequent fibroproliferation. However, large randomized and placebo-controlled trials were never performed to assess the efficacy of this strategy. The only evidence in support of immunosuppressive therapy for IPF is a handful of small studies. Nonetheless, the ATS/ERS consensus statement recommends the use of corticosteroids combined with a cytotoxic agent for carefully selected IPF patients . The conThe best evidence in support of the prednisone/azathioprine regimen comes from a prospective, randomized, double-blind study of only 27 IPF patients . This stA single randomized study of 43 IPF patients compared prednisolone alone to prednisolone/cyclophosphamide . This stTwo recent studies re-evaluated the utility of prednisone/cyclophosphamide in the treatment of IPF but both studies failed to support its use ,91. The Another potential therapy for IPF is N-acetylcysteine (NAC) which is a molecular precursor to the naturally occurring antioxidant glutathione. Glutathione is known to be depleted in the lungs of patients with IPF . TheoretRecently, a panel of experts were asked to rate the evidence for the various treatment options for IPF . The panSeveral clinical trials are presently assessing the utility of novel agents in the treatment of IPF. One promising drug is pirfenidone which has already been tested in phase I and phase II studies in the United States and Japan. A study examining the use of pirfenidone enrolled 105 IPF patients to receive either the study drug or placebo . The priγ (IFN-γ). In the first multicenter study, 330 patients with IPF were randomized to receive either IFN-γ or placebo [γ as measured by the primary endpoint. However, analysis of secondary endpoints revealed a trend toward improved survival in the group receiving IFN-γ. This trend did not reach statistical significance (p = 0.08), but the study was not powered to detect an effect on survival. Therefore, a second trial was designed to specifically evaluate survival, with a plan to enroll over 800 IPF patients. Unfortunately, this second trial was recently discontinued after a planned interim analysis determined a lack of benefit from IFN-γ relative to placebo (unpublished data).A word of caution: experience has shown that, although a drug may appear promising in small phase II trials, large trials with additional power to determine efficacy may, in fact, reveal that a drug is ineffective. This was recently demonstrated by trials investigating the medication interferon- placebo . PatientA survival benefit has been demonstrated for lung transplantation in IPF patients . HoweverThe timing of pulmonary transplantation poses additional challenges. Until recently, early referral was advocated for all patients with IPF because of long pre-transplant waiting times exceeding the median survival time of patients with IPF. However, new allocation scores in the U.S., devised to alleviate transplant waiting list mortality, have dramatically reduced waiting times for patients with IPF thus removing the impetus for early referral . Now theThe decision to perform lung transplantation in a patient with IPF requires careful consideration of the risks and benefits of such an undertaking. Advanced age precludes many patients with IPF from serious consideration of lung transplantation. Lung transplantation should be reserved for those with adequate social support and limited comorbidities, in order to face the rigors of post-transplantation medical management. Lung transplantation, on the whole, is best performed at specialized centers that employ experienced surgeons and physicians who are familiar with post-transplantation management.Regardless of the primary therapy, patients with IPF need to be treated with supportive measures as clinically indicated by their condition. For example, exercise-induced hypoxemia warrants a prescription for supplemental oxygen. While supplemental oxygen has been shown to improve exercise performance in chronic obstructive pulmonary disease (COPD), it has not been rigorously evaluated for the treatment of IPF. Nevertheless, a study that examined quality of life (QOL) in patients with IPF found no difference in QOL between patients receiving supplemental oxygen compared with those not receiving oxygen . This isPatients with IPF should be encouraged to enroll in a program for pulmonary rehabilitation. Pulmonary rehabilitation has not been rigorously examined in IPF, though quadriceps strength has been correlated with exercise capacity amongst patients with IPF . This imIPF remains a disease for which the etiology is unknown. The pathogenesis is only poorly understood and the natural history of the disease is just beginning to reveal itself through observation of placebo groups from several large multi-center clinical trials. There is no definitive approach to the treatment of IPF because evidence for effective medical therapy is still lacking. Future directions for research should include programs that encourage the search for new molecular targets for therapy; and research to identify genetic susceptibility factors ,102. Sev


Analysis of intron gain and loss in fungal genomes provides support for an intron-rich fungus-animal ancestor. Eukaryotic protein-coding genes are interrupted by spliceosomal introns, which are removed from transcripts before protein translation. Many facets of spliceosomal intron evolution, including age, mechanisms of origins, the role of natural selection, and the causes of the vast differences in intron number between eukaryotic species, remain debated. Genome sequencing and comparative analysis has made possible whole genome analysis of intron evolution to address these questions.We analyzed intron positions in 1,161 sets of orthologous genes across 25 eukaryotic species. We find strong support for an intron-rich fungus-animal ancestor, with more than four introns per kilobase, comparable to the highest known modern intron densities. Indeed, the fungus-animal ancestor is estimated to have had more introns than any of the extant fungi in this study. Thus, subsequent fungal evolution has been characterized by widespread and recurrent intron loss occurring in all fungal clades. These results reconcile three previously proposed methods for estimation of ancestral intron number, which previously gave very different estimates of ancestral intron number for eight eukaryotic species, as well as a fourth more recent method. We do not find a clear inverse correspondence between rates of intron loss and gain, contrary to the predictions of selection-based proposals for interspecific differences in intron number.Our results underscore the high intron density of eukaryotic ancestors and the widespread importance of intron loss through eukaryotic evolution. Homo sapiens with 8.1 introns per gene [Caenorhabditis elegans with 4.7 [Drosophila melanogaster with 3.4 [Arabidopsis thaliana with 4.4 [Unlike bacteria, the protein-coding genes of eukaryotes are typically interrupted by spliceosomal introns, which are removed from gene transcripts before translation into proteins. Eukaryotic species vary dramatically in their number of introns, ranging from a few introns per genome to several introns per gene. The reasons for these vast differences, as well as the explanation for the particular pattern of intron number across species, remain obscure. The first genomes with characterized intron densities suggested the possibility of a close association between intron number and organismal complexity. The initial animal and land plant species studied had high intron densities, for instance, with 4.7 , Drosophwith 4.4 . Howeverwith 4.4 ,7, with Schizosaccharomyces pombe (0.9 per gene) [Saccharomyces cerevisiae (0.05 per gene) [Neurospora crassa and Aspergillus nidulans have much higher intron densities (2-3 per gene) [Cryptococcus species [The case of fungi is particularly striking. The first fungal genomes characterized, the yeasts er gene)  and Sacc pombe 0. per geneer gene) ,11, and er gene) ,13. Gene species  or more  species . ConservS. pombe and S. cerevisiae, a pattern that is due to some combination of loss in fungi [S. pombe, S. cerevisiae, and a variety of other species [Many intron positions are shared between eukaryotic kingdoms. In particular, many intron positions are shared between plants and animals but not the intron-sparse fungi in fungi -19, and in fungi ,17. Sepain fungi ,17,19, o species ,20. ThisWe used comparative genomic analysis of the gene structures of 1,161 sets of orthologs among 21 fungal species and four outgroups. We found that studied fungal species share many intron positions with distantly related species; both the fungal ancestor and fungus-animal ancestor (Opisthokont) were very intron rich, with intron densities matching or exceeding the highest known average densities in modern species of fungi and approaching the highest known across eukaryotes. Fungal evolution has been dominated by intron loss and we identify independent nearly complete intron loss along three distinct fungal lineages in addition to overall patterns of intron loss.S. cerevisiae with 7 total introns) to 6.7 introns per kb , although the trend is not significant when the hemiascomycete fungi are excluded . This finding of much longer introns in the very intron-poor hemiascomycetes is intriguing, particularly in light of other peculiarities of evolution in very intron poor lineages [To study fungal intron evolution, we identified 1,161 orthologs among 21 fungal species and 4 outgroups . We constructed a phylogenetic tree using Dollo parsimony ,23 from together . As suchWe next studied intron loss and gain in fungi in CORs of 1,161 genes. Four previously proposed methods showed very similar pictures, with large numbers of introns present in ancestral genomes and widespread subsequent intron number reduction along various fungal lineages , hemiascomycetes (0.01-0.07 introns per kb) species have reduced their intron number by at least 94%, S. pombe has reduced its intron number by 81% (0.52 introns per kb), and even relatively intron-rich euascomycete species (0.81-1.16 introns per kb) have undergone a 60% reduction in intron number. Interestingly, following dramatic intron number reduction in the euascomycete ancestor, intron number has remained relatively unchanged within the clade  as most euascomycetes; euascomycete species' 50-100% more introns than S. pombe are thus primarily due not to greater retention of ancestral introns but to recent gain. Likewise, Cryptococcus neoformans retains fewer shared plant-animal introns than does Rhizopus oryzae, yet has 70% more introns, apparently due to more intron gain.On the other hand, our results also attest to ongoing intron gain. Most species have experienced hundreds of intron gains in CORs  since the fungal ancestor, and nearly every studied species is estimated to have gained more than one intron per kb since the intron ancestor. Differences in intron gain are sometimes the central determinant of modern differences in intron number. For instance, et al. [Intron evolution within hemiascomycetes provides insights into the evolution of nearly intronless lineages. The extensive loss of introns in hemiascomycetes corresponds to the position in the fungal phylogeny with a significant shift in intron structure. Intron structure in hemiascomycetes requires a six base sequence at the 5' splice site and a seven base pair site at the branching point . The othet al.  suggesteet al.  is that S. cerevisiae introns encode functional elements such as small nucleolar RNAs (snRNAs) [S. cerevisiae), indicating their conservation since divergence from the fungal ancestor. However, only 8 of 76 snRNAs are found in the 275 nuclear introns in S. cerevisiae [cis-regulatory containing elements or encoding factors necessary for post-transcriptional regulation, but they may also persist by chance due to low rates of loss.Why have all of the introns then not been lost in hemiascomycete species? Some of the (snRNAs)  or promo(snRNAs) . snRNAs revisiae . Intronsrevisiae , perhapsrevisiae . Some ofYarrowia lipolytica hemiascomycete species examined is shared with any species more distant than euascomycetes. However, six of the seven are broadly shared within the hemiascomycete lineage, suggesting either that the remaining introns are very hard to lose or that loss rates have greatly diminished within the lineage. By contrast, 14 of 23 introns present in Y. lipolytica but no other hemiascomycete are shared with a non-euascomycete, and 10 are shared with plants and/or animals; thus, widely shared introns have been preferentially lost among hemiascomycetes after the divergence with the Y. lipolytica ancestor.On the other hand, our results show that hemiascomycete intron positions are not in general widely shared. Only one of the seven intron positions in non-Eukaryotic species vary in their numbers of introns by orders of magnitude. These differences have traditionally been attributed to alleged differences in the intensity of selection against introns across eukaryotes ,32. AddiThese results provide an excellent opportunity to compare different previously proposed methods for reconstruction of intron evolution. There are five previously proposed methods. Dollo parsimony assumes a minimal number of changes but that once an intron is lost at a position, it is never regained . Roy andPreviously, application of four methods  to intron positions in conserved regions of 684 sets of orthologs showed very different pictures of early eukaryotic evolution. Roy and Gilbert estimated the animal-fungus and plant-animal ancestors had some three-fifths as many introns as vertebrates (among the most intron-dense known modern species) , while RWe used all five methods to reconstruct intron evolution for the current data set. In contrast to the previous discordance, all methods now provide similar estimates for the numbers of introns in the animal-fungus ancestor. Dollo parsimony tended to be very different from the rest of the estimates for deep nodes in the tree. The Carmel and NYK methods show the most striking agreement, with less than 2% difference across all nodes except for the Opisthokont ancestor (3.3% difference). The NYK and Csűrös methods also show striking agreement, giving estimates within 2% of each other for 13 out of 18 (non-hemiascomycetes) nodes, and to within 10% for 17 out of 18. The RG method agreed with the other three methods to within 15% for all nodes except six and was not more than 30% higher than either of the other methods for any node other than the Ascomycete node. Notably, the three nodes on which RG was comparatively highest for the current data set are deep nodes near very long branches in this tree. Thus, further taxonomic sampling would likely bring even these nodes into better agreement (see below). Numbers of intron losses and gains in CORs along each branch were also estimated using all four methods. Though absolute numbers of estimated intron losses and gains along each branch varied more considerably between methods, there was a striking agreement in the relative incidence of intron loss and gain, with Csűrös (2.03 losses per gain), evolutionary reconstruction by expectation-maximization  and NYK (2.12) nearly identical and RG only 21% higher (2.66). Notably, overall estimated numbers of gains were very similar, with only 19 more gains by RG than NYK. Results for all methods are given in Additional data files 4 and 5.Strikingly, all four methods now estimate that the fungus-animal ancestor had at least 70% as many introns as vertebrates, 15% more than estimated by Roy and Gilbert and more than twice that previously estimated by Csűrös and NYK. Thus, it appears that the previous difference in estimated intron density in the animal-fungal ancestor was not due to overestimation by the RG method, but to a 2.5-fold underestimation by the other methods. Indeed, even the estimates of Roy and Gilbert appear to have been conservative .Why should this be? Following the original authors , we suggWe performed simulations of intron evolution that included variations in intron loss rate across sites, and reconstructed intron loss/gain evolution on each set using four of the five methods . We considered a four-taxa case in which taxa A and B are sisters, and taxa C and D are sisters , and in which there were 1,000 introns in CORs in the common ancestor and allowed loss rates to vary between intron positions Figure . In thesThere are four clear observations, each of which held over all sets of parameters. First, all methods underestimated ancestral intron density. Second, for each data set RG was closest to the real value, followed by EREM, then by Csűrös, then by Dollo parsimony. Third, the Csűrös and EREM methods consistently estimated significant numbers of parallel insertions even though none were included in the simulations - that is, both methods overestimated parallel insertions. Fourth, these trends typically increased with overall branch length. An exception to this was the lack of clear dependency of EREM on branch length.S. pombe and S. cerevisiae, both of which have lost the vast majority of their ancestral introns . Under such long branch conditions, the RG method somewhat underestimated ancestral intron density, while the other methods considerably underestimated intron density and overestimated parallel insertion. In the new data set, the inclusion of fungal species that retain many more of their ancestral introns shortened the fungal branch, leading to a convergence of the four methods on better estimates .Together, these observations suggest the following explanation for the discrepancy between previous and current estimates. In the previous data sets , the funIndeed, the difference between NYK's estimation of the incidence of parallel gain between the present and previous data sets is striking. According to the NYK method of calculating parallel intron insertions, our data set showed very little evidence for parallel intron gain. Their method estimated 93.08 total parallel gains; thus, only 2.2 % of 4,228 shared introns were due to parallel gain. This is much less than the previous estimate that 18.5% of shared positions in the Rogozin data set were due to parallel gains. This is despite the fact that the overall number of estimated intron gains, as well as the overall number of estimated gains per kb, was higher in our data set than in the Rogozin data set. Thus, it seems that parallel gains were previously overestimated, and given the near identity of results from Csűrös method to NYK's, the same is very likely true of Csűrös' method.This decrease in the estimated incidence of parallel gain is all the more striking given the increased number of taxa across data sets, which presumably brings with it an increased number of real gains and real parallel gains, although the implications are not entirely clear given that the species present in the current data set are not a superset of the species in the previous set. Our simulations suggest here that there will be countervailing effects of greater taxonomic sampling, with a decrease in the overestimation of parallel gains due to long-branch effects coinciding with an increase in the overall number of true parallel gains. The decrease in estimated incidence of parallel gain seen here implies that currently the former effect dominates; however, with better and better sampling the latter effect may come to dominate in future data sets. More thorough simulation studies will be necessary to more completely understand this issue.What of other ancestral nodes of key biological interest for which the different methods gave very different estimates? The three methods' previous estimates based on the Rogozin data set also differed significantly for the fungi-animal-plant ancestor and the bilateran ancestor. In the previous data set, both ancestors were flanked by at least one very long branch, suggesting that all methods might have underestimated intron densities. The finding of intron-rich protostomes and apicomplexans would make resolution of this issue possible in the near future. This argument suggests that intron density was very high even in very early eukaryote ancestors.These results resolve a debate over the intron density of the fungal-animal ancestor. All proposed methodologies now agree that this ancestor was very intron rich, and that all modern fungi have experienced more intron loss than gain since divergence. These results underscore that intron evolution in eukaryotic evolution often defies common assumptions of organismal and gene structure complexity and requires new models of intron loss and gain evolution.ab initio and evidence based gene predictions and combined into a single composite gene call with the tool GLEAN [ab initio gene prediction programs SNAP [Aspergillus fumigatus, Aspergillus terreus, Coprinus cinereus, Podospora anserina and Rhizopus oryzae were not available so automated annotations were generated so that these species could be included. We generated a new annotation of the v1 P. chrysosporium genome [Annotated genomes of many of the fungi analyzed were obtained from GenBank or directly from sequencing centers and are listed in Additional data file 3. For unannotated genomes, gene predictions were generated using a combination of ol GLEAN . The ab ams SNAP , AUGUSTUams SNAP , and Genams SNAP  were firams SNAP  and Exonams SNAP . At the m genome  as we fom genome . Frozen m genome  and Web m genome  interfacm genome .A. thaliana annotations (Feb 2005) [Fugu rubripes  [Mus musculus (Ensembl 30.33f) [H. sapiens annotations (Ensembl 30.35c) [-5. The output was processed with a custom Perl script to generate, for each pair of species, pairwise orthologs via best-mutual-FASTP hits. The pairwise orthologs were combined via single-linkage clustering for all sets of species into multi-way orthologs only if they formed clusters that contained exactly one protein member from each species.The predicted proteins from the 21 fungal genome annotations , were combined with the eb 2005)  availablembly 2) , Mus mus 30.33f) , and H.  30.35c)  searched 30.35c)  with an The protein sequences for these orthologs were then aligned using the multiple sequence alignment program MUSCLE . The proThe alignments were evaluated for these intron positions in order to build a matrix of all intron positions. Similar to methodology in previous work , each obStagonospora nodorum is basal to the euascomycetes for consistency with more exhaustive phylogenetic methods using larger sampling of taxa [A random sampling of 30 of the protein alignments were used to generate a species tree by concatenating the aligned sequences and removing all gap columns from the alignment. The tree was computed and bootstrapped with MrBayes . The fun of taxa . Other t of taxa .Dollo parsimony was computed with dollop from the PHYLIP package  using deThe resulting matrix of classified intron positions was evaluated using the RG method computed along the species tree to compute intron densities, numbers of intron gains and losses, and the fraction of introns present at different internal nodes in the tree. The NYK method was also used to construct intron loss and gain rates and densities in ancestral nodes after modification of the authors' C code. The modified RG Perl code and the NYK C code is available in Additional data file 6. The Csűrös method, implemented in intronRates.jar program ,61, was We simulated a four-taxa case in which taxa A and B are sisters, and taxa C and D are sisters, and in which there were 1,000 introns in CORs in the common ancestor . Different introns were assigned different loss rates as given by a standard gamma distribution, with varying gamma-values. The internal branch was set to length zero . External branch lengths were set to be of equal length, with a length chosen for each gamma value such that, on average, a given fraction (70% or 30%) of all introns present at the ancestral node were retained in each descendent taxon. We generated data sets for gamma values from 2.0 (most variation in intron loss rate) to 10.0 (least) in increments of 0.5. No insertion, parallel or otherwise, was assumed. For each set of parameters we generated expected numbers of introns with each phylogenetic distribution, and used these values, rounded to the nearest integer, as inputs for all three methods.COR, conserved orthologous region; EREM, evolutionary reconstruction by expectation-maximization; NYK, Nguyen, Yoshihama, and Kenmochi method of intron reconstruction; RG, Roy-Gilbert method of intron reconstruction.JES conceived of the project, collected the genome data and annotations, annotated genomes, wrote and ran necessary software, performed analyses, created the figures and tables, and wrote the paper. FSD contributed to the content and writing of the paper, provided access to computational resources, and supervised JES. SWR provided intron reconstruction methods, conceived and performed the simulations, analyzed the data, created figures and tables, and wrote the paper.et al. [et al. [et al. [et al. [The following additional data are available with the online version of this paper. Additional data file et al. , Csűrös et al. , Roy andet al. ,20, EREM [et al. , and Dol [et al. , Csűrös  [et al. , Roy and [et al. ,20, EREM [et al. , and DolS. nodorum constrained based on previously published phylogenies. The tree on the right is the strict consensus tree of 116 MP trees built using Dollo parsimony and the matrix of presence or absence of intron positions. Nodes that are not present in all 116 trees are collapsed. Species groups are colored so that Euascomycota are in dark green, Hemiascomycota in red, archiascomycete S. pombe in yellow, Basidiomycota excluding U. maydis in blue, U. maydis in purple, zygomycete R. oryzae in orange, vertebrates in pink, and green plant A. thaliana in light green.The left tree was built with a MrBayes using 30 orthologous proteins with the position of Click here for fileThe phylogenetic tree used in the simulation data analysis.Click here for fileGenomes and annotations used for this analysis with the source and version of the annotation indicated, with references for previously published annotations.Click here for fileNot all methods reconstruct a value for the Crown ancestor as this requires an outgroup and additional assumptions.Click here for fileNot all methods estimate gain and loss from Crown ancestor as this requires an outgroup and additional assumptions.Click here for fileSummary statistics for intron length, intron frequency, total length per genome, total intron count, total length of coding sequence, and genome size.Click here for fileThe file also contains the customized software for running NYK and the RG intron calculations.Click here for fileIntrons are represented by numbers in the alignment indicating the phase of the intron  as defined by the position in the codon the intron falls within. Coding sequence alignments and unaligned sequences are available from the authors.Click here for file


Nyctotherus ovalis is a single-celled eukaryote that has hydrogen-producing mitochondria and lives in the hindgut of cockroaches. Like all members of the ciliate taxon, it has two types of nuclei, a micronucleus and a macronucleus. N. ovalis generates its macronuclear chromosomes by forming polytene chromosomes that subsequently develop into macronuclear chromosomes by DNA elimination and rearrangement.N. ovalis. We determined the telomeres, subtelomeric regions, UTRs, coding regions and introns by sequencing a large set of macronuclear DNA sequences  and cDNAs  and comparing them with each other. The telomeres consist of repeats CCC(AAAACCCC)n, similar to those in spirotrichous ciliates such as Euplotes, Sterkiella (Oxytricha) and Stylonychia. Per sequenced chromosome we found evidence for either a single protein-coding gene, a single tRNA, or the complete ribosomal RNAs cluster. Hence the chromosomes appear to encode single transcripts. In the short subtelomeric regions we identified a few overrepresented motifs that could be involved in gene regulation, but there is no consensus polyadenylation site. The introns are short (21–29 nucleotides), and a significant fraction (1/3) of the tiny introns is conserved in the distantly related ciliate Paramecium tetraurelia. As has been observed in P. tetraurelia, the N. ovalis introns tend to contain in-frame stop codons or have a length that is not dividable by three. This pattern causes premature termination of mRNA translation in the event of intron retention, and potentially degradation of unspliced mRNAs by the nonsense-mediated mRNA decay pathway.We examined the structure of these gene-sized macronuclear chromosomes in The combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides. Paramecium  -dATP by PCR with M13-forward and -reverse primers.The α2-tubulin clone  and the Nyctotherus was separated on a 0.7% (w/v) native agarose gel and blotted onto a Hybond N+ membrane (Amersham) by downward alkaline southern blotting. The filter was pre-hybridized for 1 h at 65°C in hybridization solution ). After pre-hybridization, the hybridization solution was refreshed and the 32P-labeled probe was added. The filter was hybridized at 65°C for 16–19 h. The hybridized filter was washed twice in 2× SSC, 0.1% SDS (w/v) and twice in 0.5% SSC, 0.1% SDS (w/v) at 65°C. Depending on the signal on the filter an extra washing step with 0.2% SSC, 0.1% SDS at 65°C was included. Autoradiograms were exposed for 0.5–4 h at -70°C with intensifying screens.Genomic DNA from Nyctotherus ovalis was prepared by dissolving living Nyctotherus ovalis cells, purified by electromigration CCCC)/and eventually refined their removal by hand.From the Princeton-set (see above), we could determine that macronuclear telomeres of N. ovalis sequences contain only one gene, we compared the 2,841 gDNA from the nr-set against UniProt release 9.1 database, with a SWX search and an e-value threshold of 1e-05. For all N. ovalis gDNAs, we retrieved the boundaries of the "best hit" to a known protein. Subsequently we examined whether, in the hit list there were any hits that did not overlap with the first hit. We never observed cases of such non-overlapping hits, arguing for the presence of only a single gene per macronuclear chromosome.To determine whether or not the Sequences were compared against UniProt database . Sequences with a match on the reverse strand were reversed and in the case of the presence of one or the two telomeres, the upstream and/or downstream regions were extracted. 1,459 gDNAs could be oriented this way representing 937 different clusters/non-redundant sequences.As we discovered that the coding strand had a higher purine/pyrimidine ratio than the reverse strand, we used this ratio to orient the sequences that could not be oriented with UniProt hit. Sequences having a purine/pyrimidine ratio < 1 were reversed.The 300 first nucleotides after the telomeres 5' and the 300 nt before the telomeres 3' from the sequences of the UniProt-set were extracted. We measured the Shannon entropy  introns were known, using SWX (Smith-Waterman X) on a Decypher machine. Subsequently GeneWise was run for the best hits of Nyctotherus sequences with the Paramecium proteins in the "-splice flat-intron tied" mode to identify likely introns in the Nyctotherus sequences. From the GeneWise results we extracted the locations of the Nyctotherus introns in the Paramecium proteins. The latter were then compared with the experimental locations of the Paramecium introns to establish which ones were conserved. Introns were considered conserved when the Nyctotherus introns mapped to the same location in the aligned Paramecium proteins as the Paramecium introns, allowing for a maximum shift of 2 amino acids. Supporting the conservation of the 48 thus determined introns was the observation that they had a conserved phase in all cases except one. Experimentally-determined intron locations in Paramecium were kindly provided by Laurent Duret and Jean-François Goût (University de Lyon).1,914 cDNAs were compared to 2,841 gDNAs of the nr-set using GeneDetective with the default values except "filter off" on Decypher and Exonerate , with thGenewise . First, N. ovalis against the position of the introns in their closest homologs  in another ciliate T. thermophila and a member of the Apicomplexa P. falciparum. Ciliates and Apicomplexa are both Alveolata. The genome of T. thermophila was recently published [Tetrahymena protein and genomic sequences were obtained from the Tetrahymena Genome Database on . We worked with Assembly2 – Nov_2003_scaffolds and 27,769 cDNAs from 08-30-2006. By comparing the N. ovalis sequences to known proteins , we identified homologs of the spliceosome complex.We compared the positions of the introns of ublished . TetrahyWe used MEME , an expecis-acting regulatory DNA elements and trans-acting factors that covers the whole range from yeast to human, to search these motifs in our set.We also used TRANSFAC , a databWe detected tRNAs using tRNAscan-SE  with theWe determined the stop codon usage by examining those cDNAs that aligned with 1) at least 40% identity over 2) at least 60 amino acids that 3) did not have unknown nucleotides (N) in the alignment and 4) aligned until the N-terminus of a known protein. For the 328 cDNAs that fit those criteria we examined the next codon. TAA (141 cases) is preferentially used as stop codon, TAG (5 cases) also occurs in those locations, but less than e.g. GAC (40 cases) or GCC (59) cases.Symbiodinium was used as an outgroup to root the tree.rRNA sequences were extracted from Genbank; the sequence of the dinoflagellate Phylogenetic trees were constructed using an alignment provided by Muscle ,75. SequtRNA: transfer RiboNucleic Acid; TFBS: Transcription Factor Binding Site; nr: non-redundant; nt: nucleotide; SW: Smith-Waterman.GR did the main sequence analyses and drafted the manuscript. BD and ID determined the genetic code. RdG isolated and sequenced several complete genes and participated in drafting the manuscript. TvA, BB, GvdS, SM-vdS, isolated and sequenced several complete genes. AvH isolated and sequenced several complete genes and made southern blots. WJC sequenced 13 full length chromosomes, LFL supervised WJC and contributed to the manuscript as a whole and the genetic code discussion in particular. JH isolated the cells, initiated and coordinated parts of the study and participated in drafting the manuscript. MH supervised the sequence analysis, determined the intron conservation and finalized the manuscript.N. ovalis introns found by comparison between cDNAs and gDNAs. Comparison with the homologous sequences of ciliate T. thermophila and the apicomplexan P. falciparum.Click here for fileSelection of 3'UTRs. Eight 3'UTRs carefully selected by hand. No polyadenylation signal can be observed.Click here for file


Cryptococcus genomes suggests that recombination causes internal intron loss and that DNA repeat expansion can create new introns in a population.Comparison of five relatively closely related yeast  Cryptococcus.Genome comparisons across deep phylogenetic divergences have revealed that spliceosomal intron gain and loss are common evolutionary events. However, because of the deep divergences involved in these comparisons, little is understood about how these changes occur, particularly in the case of intron gain. To ascertain mechanisms of intron gain and loss, we compared five relatively closely related genomes from the yeast Cryptococcus. Some genes preferentially lose introns and a large proportion of intron losses occur in the middle of genes . Finally, we identify a gene that displays a differential number of introns in a repetitive DNA region.We observe a predominance of intron loss over gain and identify a relatively slow intron loss rate in Based the observed patterns of intron loss and gain, population resequencing and population genetic analysis, it appears that recombination causes the widely observed but poorly understood phenomenon of internal intron loss and that DNA repeat expansion can create new introns in a population. Cryptococcus species complex offers the ideal system to study the mechanisms of intron gain and loss.The evolutionary dynamics of spliceosomal introns has remained a subject of considerable debate since their discovery over 30 years ago . AlthougCryptococcus neoformans and Cryptococcus gattii speciated less than 100 million years ago, so comparing these two species should avoid the problems of deep divergence while allowing enough time for intron gain and loss to occur . If. If10]. karyotes  and a sukaryotes . Given tSeveral mechanisms could explain internal intron loss via a spliced mRNA intermediate. First, the points of homologous recombination between the cDNA and the gene may occur upstream of the 3' most intron, resulting in its persistence by being excluded from the double cross-over event. Given that the mechanism proposed for intron loss involving mRNA is the same as that proposed for the origin of processed pseudogenes, if internal recombination was common, one would expect to find processed pseudogenes that are internal segments of genes. However, to our knowledge, there are few examples of processed pseudogenes that are internal gene segments . Another4f-rnp intron loss polymorphism in Drosophila [Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, N. crassa, Plasmodium falciparum, S. pombe, and U. maydis) [Cryptococcus suggests a different mechanism contributes to internal intron loss. Because there is no known bias regarding where, along the length of a transcript, the 3' end can complementary pair, this hairpin-based loss mechanism should produce intron losses along the length of a gene with equal probability. The majority of losses, however, cluster toward the 3' end of the gene . cDNA is then synthesized from the region upstream of this base-pairing. This mechanism is suspected to cause the osophila  and is i maydis) . While se Figure . Additios Figure , all loss Figure . If, witC. neoformans serotype D [To determine if this mechanism is likely, we employed an allelic sojourn time density function derived from a diffusion approximation to estimate the frequency with which an initially single-copy, 3' poly-A primed intron loss allele recombines with a wild-type allele before being absorbed or fixed in the population . While we cannot be certain about historical recombination rates, we can make a reasonable estimation by employing the measured meiotic recombination rate determined for rotype D . Our anaCryptococcus species, we did identify a gene that has gained introns within C. neoformans. The D1 gene CNN00420 contains unique intron sequences relative to orthologs in A and D2 (no orthologs could be identified in serotype B). Resequencing strains from the serotype D population confirmed that these introns in the D1 allele are unique, implicating a sequence gain. Because each gained intron has exact identity to three other introns in the gene (and near exact identity to two others), it is likely that unequal crossing over, gene conversion or repeat expansion through replication strand slippage caused the gains. While we cannot discern which of these DNA duplicating mechanisms caused the intron gains in question, several studies have shown that replication strand slippage is a common and frequent means of varying intergenic repeat copy number. In Escherichia coli, RecA independent duplications and deletions between repeats of several hundred bases have been shown to occur at high frequency (10-5 to 10-4 per cell generation) [et al. [CNN00420 .While we found no intron gains between the two eration) . In an e [et al.  concludeTOUCH3 gene relative to its paralog in Arabidopsis. However, though there is debate on this point [Arabidopsis whole genome duplication on gene structure evolution is uncertain. Our analysis verifies that this mechanism can create introns in orthologs and contributes to gene structure divergence between species. Furthermore, this is the first time a DNA repeat expansion has been observed to create an intron polymorphism.To our knowledge, there is only one other example of an intron gain occurring through repeat duplication. Knowles and McLysaght  suggesteis point , paralogis point . AdditioIn his study of intron gain rates, Roy noted that certain ancient lineages underwent big bursts of intron gain while subsequent lineages experienced a precipitous decline in intron gain rates . This suC. neoformans serotype D strain JEC21 (D1) genome sequence and annotation was obtained from GenBank as deposited by TIGR . A second serotype D strain sequence, B3501 (D2), was obtained from the Stanford Genome Technology Center . The C. neoformans serotype A strain H99 (A) was sequenced by the Broad Institute . The Broad Institute also sequenced the C. gatii serotype B strain R265 (B1) . The second serotype B strain WM276 (B2) was obtained from the BC Genome sequencing center .The Whole genome alignments were created through a multi-step process using D1 as a reference. First, pairwise alignments between D1 and the other sequenced strains were created using PatternHunter . Blocks As described in , the annClusters of orthologs meeting these criteria were aligned separately from the whole genome alignment using the mLAGAN multiple sequence alignment algorithm and the phylogeny shown in Figure The unrooted phylogenetic topology among the five sequenced strains was determined by a neighbor-joining analysis  on a conGenome sequenced strains JEC21(D1) and B3051(D2), as well as additional serotype D individuals TP0603, WM629, JEC20 and B3502, were generously provided by W Meyer. The method of Bolano was used to extract genomic DNA from strains grown in liquid culture . Conservy represent the vector {x, N, s, h}, where x, 0 ≤ x ≤ 1, represents the frequency of an element in a population of N individuals, s is the intensity of selection acting on the allele and h is a heterozygous fitness factor. A newly created element thus appears in the population at a frequency of 1/2N. Under standard assumptions of the diffusion approximation, the amount of time, in N generations, an initially single copy element spends in the population prior to being absorbed (x = 0) or fixed (x = 1) is:We applied a sojourn time density function derived from a diffusion approximation to estimate the probability that a recombination event between a 3' generated intron loss allele (defined in Results) and a wild-type allele could result in an allele missing only internal introns  -44. Let where:s = 0), that the population is sufficiently large (2N = 10e5), and that the only value for any x < 1/2N is x = 0. Because s = 0, we need not estimate h, the dominance coefficient. Under these assumptions, the frequency spectrum of alleles is given approximately by:as in . Here, wOf interest is the frequency that an allele missing 3' introns recombines with a wild-type allele (no missing introns) to create a recombinant allele that is missing only internal introns. This can be estimated by defining the probability of recombination between dissimilar alleles as a function of allele frequency and integrating over the frequency spectrum as follows:r is the recombination rate and L is the average exon length. In Cryptococcus, these values are 7.58e-7 recombination events per base-pair per generation [Here, neration  and 253 Resampling analysis was conducted to determine intron loss rate 95% confidence intervals. A total population of 33,473 introns  was established, where 49 introns in the population corresponded to loss events and the remainder were intron conservation events. We sampled, with replacement, 10,000 introns from this population and then calculated the frequency and subsequent rate. Calculations were conducted 10,000 times and a normal curve approximation of the subsequent rate distribution was used to calculate the 95% confidence interval.Resampling analysis was also conducted for each gene demonstrating adjacent intron loss to determine the probability that the adjacent losses were caused by random, independent events. For each gene, a population equal to the number of introns in the gene was established, where each individual in the population has a value corresponding to an intron position within the gene. Samples equal to the number of observed losses in the gene were taken from this population, without replacement, and the longest consecutive run of adjacent introns was counted. After 10,000 runs, a histogram of the longest runs was created and used to evaluate the null hypothesis that the observed data were due to random, independent loss events.C. neoformans serotype A strain H99; B1, C. gattii serotype B strain R265; B2, C. gattii serotype B strain WM276; D1, C. neoformans serotype D strain JEC21; D2, C. neoformans serotype D strain B3501; dS, silent substitution rate; EST, expressed sequence tag.A, TJS wrote and ran necessary software, performed computational and wet lab analyses, created the figures and tables and wrote the paper. DEN generated syntenic alignments for ortholog detection, generated the species phylogeny and contributed to the content and writing of the paper. JEG conceived of the computational project and provided access to appropriate resources. JWT conceived of the wet lab experiment and provided necessary resources, contributed to the content and writing of the paper and supervised TJS.CNN00420 against itself. Additional data file CNN00420 orthologs from the serotype D population. Additional data file CNN00420 gained introns along the D1 lineage. Additional data file The following additional data are available. Additional data file Consecutive intron loss events.Click here for fileIntrons are binned according to the position (in base-pairs) of their occurrence in a coding sequence divided by the coding sequence length.Click here for fileIntron loss in regard to intron order within the gene is well distributed, not requiring the 3'-most intron to be lost.Click here for fileThe large number of off-center, parallel lines illustrates this gene's highly repetitive sequence.Click here for fileThe strain relationships are designated in Additional data file 6. Positions with a sequence of 'X' designate the boundaries of D1 annotated introns.Click here for fileCNN00420 ortholog for each strain is graphically represented in the cartoon next to each strain identifer. Horizontal bars correspond to sequence alignment positions: red lines indicate nucleotide sequence while white gaps indicate indels. Vertical blue bars correspond to annotated D1 introns. The phylogeny and the presence of unique sequence in the alignment corresponding to introns 3 and 5 in D1, its sibling JEC20, and parent B3502, suggest the gain of introns 3 and 5 in D1.The strain phylogeny is determined based on the history of strain synthesis as described in Results. The corresponding aligned Click here for fileExons (black lines) and introns (gold lines) are synthesized in the daughter strand during DNA replication. Repetitive sequences (red boxes) may create a looping out of the daughter strand and subsequent re-synthesis of the looped out region . After meiosis, the result is an allele with an extra repeat copy and, subsequently, an extra (acquired) intron.Click here for fileMathematica source code for the sojourn time density approximation.Click here for file


While the phylogenetic relationships of selenoprotein families and the evolution of selenocysteine usage are well documented, the evolutionary history of SECIS binding proteins has not been explored.The co-translational incorporation of selenocysteine into nascent polypeptides by recoding the UGA stop codon occurs in all domains of life. In eukaryotes, this event requires at least three specific factors: SECIS binding protein 2 (SBP2), a specific translation elongation factor (eEFSec), selenocysteinyl tRNA, and a in vitro. An in-depth phylogenetic analysis of the conserved L7Ae RNA binding domain suggests an ancestral relationship with ribosomal protein L30. In addition, we describe the emergence of a motif upstream of the SBP2 RNA binding domain that shares significant similarity with a motif within the pseudouridine synthase Cbf5.In this report we present a phylogeny of the eukaryotic SECIS binding protein family which includes SBP2 and a related protein we herein term SBP2L. Here we show that SBP2L is an SBP2 paralogue in vertebrates and is the only form of SECIS binding protein in invertebrate deuterostomes, suggesting a key role in Sec incorporation in these organisms, but an SBP2/SBP2L fusion protein is unable to support Sec incorporation Our analysis suggests that SECIS binding proteins arose once in evolution but diverged significantly in multiple lineages. In addition, likely due to a gene duplication event in the early vertebrate lineage, SBP2 and SBP2L are paralogous in vertebrates. Eukaryotic SECIS elements are stable stem-loop structures that are comprised of two helices separated by a kink-turn containing a conserved GA quartet (SECIS core) and an apical AAR motif that is present either as a terminal loop (Form 1) or a terminal bulge (Form 2)  UTP-labeled SECIS elements and the complexes resolved by non-denaturing gel electrophoresis. This amount of CT-SBP2 is within the linear range of this binding assay (data not shown). As expected, CT-SBP2 shifted wild-type but not mutant SECIS elements in which the AUGA motif in the SECIS core was deleted . Using Figure Having established the phylogenetic relationship of metazoan SBPs, we sought to determine whether we could elucidate the origins of SBP2. As mentioned above, SBPs contain an L7Ae RNA binding motif. This motif was identified by Koonin and colleagues  as a 32 de novo. Regardless of its origin, the conservation of the SID core motif in the eukaryotic taxa presented here (K517-R543) suggests that it arose once in eukaryotic evolution and that organisms such as D. discoideum and Plasmodium vivax with poorly conserved SID sequences likely diverged to adapt to as-yet unidentified unique requirements . Notably, the only region of significant similarity is that between RFQDR654-658 and the universally conserved Gly residue in the L7Ae RNA binding motif (G676). Figure Caban and colleagues recently performed structure/function studies on the SBP2 RNA binding domain including its L7Ae motif . In thisynthase  upstream of the  at NCBI  for thesD Figure . AdditioThis report represents the first phylogenetic analysis of SBPs across eukaryotic taxa. Since vertebrates possess two SBPs, we annotated determinants that serve as identifying motifs in SBP2L and SBP2. We showed that SBP2L is the likely progenitor of mammalian SBP2 and as the only form of SECIS binding protein in invertebrate deuterostomes, SBP2L is likely required for Sec incorporation in these organisms. However, the role of SBP2L in vertebrates will require further experiments to elucidate its function, and we expect forthcoming experimental evidence to support this hypothesis. Additionally the retention and loss of LSAD and PVFQ motifs in several taxa suggest that SBPs are subject to diverse evolutionary pressures. Conservation of the SID and RBD in unicellular eukaryotes and metazoans suggests that SBP2 arose once in eukaryotic evolution.A. aegypti, showed that its SID and RBD are coded by separate genes. Future use of A. aegypti cell culture could provide a unique system in which to study Sec incorporation. Based on the taxa sampled here, we propose that SECIS binding proteins arose once in eukaryotic evolution and diverged substantially as in the case of P. vivax, T. gondii, and D. discoideum. Additionally, the presence of SBP2L in some protostomes (C. sp. I and L. gigantia) but not in others (i.e. insects) suggests that SBP2L sequences occurred in the last common ancestor of protostomes and deuterostomes followed by divergence in different lineages. Lastly, a gene duplication event, possibly as the result of whole genome duplication, generated the paralogous pair of SBP2 and SBP2L in vertebrates.The origins of the SID and RBD, the essential domains for SBP2 function, may provide insight into the mechanism of Sec incorporation. The apparent phylogenetic relationship between the SBP L7Ae motif and RPL30 could provide insight into SBP2-ribosome interactions or reiterate a possible role for RPL30 in Sec incorporation as suggested by Chavatte . The oriab initio gene prediction using GenomeScan or the FGenesH webserver -Met as directed by the manufacturer. Protein quantitation was performed by PhosphorImager analysis as previously described -labeled wild-type and mutant GPX4 SECIS elements were transcribed as described by Caban [2007]. SECIS binding of in vitro translated proteins was assayed by incubating 8 fmol with 20 fmol of wild-type or mutant (ΔAUGA) SECIS probes in 1 × PBS supplemented with 250 μg/mL yeast tRNA (Sigma), 10 mM DTT, and 5 μg soybean trypsin inhibitor (Sigma). Final reaction volumes were 20 μL. EMSA reactions were incubated for 30 min at 37°, resolved on 4% non-denaturing polyacrylamide gels, and visualized by PhosphorImaging. Percent shifted probe was determined with ImageQuant software by subtracting background from the RRL only lane and normalizing to CT-SBP2.[aa: Amino acid; Sec: Selenocysteine; SECIS: Sec Insertion Sequence; SBP: SECIS binding protein; SBP2L: SBP2-like protein; SID: Sec incorporation domain; RBD: RNA binding domain; RP: Ribosomal protein; EMSA: Electrophoretic mobility shift assay; RRL: Rabbit reticulocyte lysate; UTR: Untranslated region; EST: Expressed Sequence Tag;JD performed all wet-lab experiments, sequence alignments, gene predictions and phylogenetic analysis, contributed to the writing of the manuscript and preparation of figures and PRC initiated the project, performed sequence alignments and phylogenetic analyses and contributed to the writing of the manuscript and preparation of figures. Both authors read and approved the final version.Multiple sequence alignment of all SBP2 and SBP2L sequences used for Figures Click here for fileA generalized workflow for identification of SBP2 or SBP2L in organisms for which these proteins were not annotated.Click here for fileList of SBP2/2L sequences used in this paper. Accession numbers are provided for sequences available at NCBI and predicted sequences are provided in full. Underlined residues indicate regions supported by ESTs. NCBI annotated sequences marked with an asterisk were refined based on EST and/or genomic DNA data . Note that some predicted sequences are likely to be partial due to lack of EST coverage or inability of gene prediction programs to correctly identify a start codon.Click here for fileAlignment of the core L7Ae motifs used to generate the phylogenetic tree in Figure Click here for file


Some patients benefit from topical corticosteroids.Sarcoidosis is a multisystemic disorder of unknown cause characterized by the formation of immune granulomas in involved organs. It is an ubiquitous disease with incidence  estimated at around 16.5/100,000 in men and 19/100,000 in women. The lung and the lymphatic system are predominantly affected but virtually every organ may be involved. Other severe manifestations result from cardiac, neurological, ocular, kidney or laryngeal localizations. In most cases, sarcoidosis is revealed by persistent dry cough, eye or skin manifestations, peripheral lymph nodes, fatigue, weight loss, fever or night sweats, and erythema nodosum. Abnormal metabolism of vitamin D3 within granulomatous lesions and hypercalcemia are possible. Chest radiography is abnormal in about 90% of cases and shows lymphadenopathy and/or pulmonary infiltrates (without or with fibrosis), defining sarcoidosis stages from I to IV. The etiology remains unknown but the prevailing hypothesis is that various unidentified, likely poorly degradable antigens of either infectious or environmental origin could trigger an exaggerated immune reaction in genetically susceptible hosts. Diagnosis relies on compatible clinical and radiographic manifestations, evidence of non-caseating granulomas obtained by biopsy through tracheobronchial endoscopy or at other sites, and exclusion of all other granulomatous diseases. The evolution and severity of sarcoidosis are highly variable. Mortality is estimated at between 0.5–5%. In most benign cases (spontaneous resolution within 24–36 months), no treatment is required but a regular follow-up until recovery is necessary. In more serious cases, a medical treatment has to be prescribed either initially or at some point during follow-up according to clinical manifestations and their evolution. Systemic corticosteroids are the mainstay of treatment of sarcoidosis. The minimal duration of treatment is 12 months. Some patients experience repeated relapses and may require long-term low-dose corticosteroid therapy during years. Other treatments (immunosuppressive drugs and aminoquinolins) may be useful in case of unsatisfactory response to corticosteroids, poor tolerance and as sparing agents when high doses of corticosteroids are needed for a long time. In some strictly selected cases refractory to standard therapy, specific antiTNF- SarcoidosisSarcoidosis is a multisystemic disorder of unknown cause that is characterized by the formation of immune granulomas in involved organs -4.Sarcoidosis affects primarily young and middle-aged adults. A prevailing hypothesis is that various unidentified antigens, either infectious or environnemental, could trigger an exaggerated immune reaction in genetically susceptible hosts . The cliα (TNF-α) at the sites of disease plays a pivotal role [α properties, either specifically or as part of broad effects [Although not always required, the mainstay of sarcoidosis treatment is systemic corticosteroids ,8. Cortital role . Interst effects .Sarcoidosis is an ubiquitary disease with an incidence varying according to age, sex, race and geographic origin ,3,4,9. Svia transplantation all suggest the role of transmissible agents either of infectious or of environnemental origin, although not identified yet [Time and space-related clusters, tracing case contacts and cases of possible transmission of sarcoidosis fied yet .α, IL-12 and IL-18 play a critical role in the formation of granulomatous lesions [Propiniobacterium acnes has also been incriminated as a causative agent but its role has not yet been ascertained. Finally, it has been shown that the incidence of sarcoidosis is influenced by genetic susceptibilities and particularly by polymorphisms involving immunity [Sarcoidosis is considered as the consequence of a chronic immunological response associating a genetic susceptibility and specific infectious or environmental factors -17. Sarc lesions . Patholo lesions . Howeverimmunity ,17.No cause of sarcoidosis has yet been comfirmed, despite numerous searches using most emerging diagnostic tools. Several reasons could explain the difficulty to determine an etiology in such a context . First, The presentation of sarcoidosis depends on epidemiological factors such as age, sex and race, the duration of the disease and the sites of involvement. Asymptomatic presentations, erythema nodosum and hypercalcemia are more frequent in Europeans, while symptomatic and multivisceral presentations are more frequent in Afro-Americans -4,9. OveDetailed occupational and environmental history is usually unremarkable and exposure to beryllium dusts and to drugs inducing granulomatosis must be excluded. As a general rule, no abnormality is audible at chest physical examination. By contrast, chest X-ray is abnormal at some point in 86–92% of cases and remains a key investigation for diagnosis ,20. RadiStage I, the most frequent presentation, is defined as isolated intrathoracic lymphadenopathy, stage II as lymphadenopathy accompanied by lung infiltration and stage III as lone parenchymal infiltration. Stage IV refers to overt lung fibrosis. Typical stage I and II are highly reliable for diagnosing sarcoidosis, while stage III and IV are far less accurate . This clPulmonary function tests typically demonstrate decreased volumes and CO diffusing capacity with functional alteration tending to be more frequent and marked from stage I to stage IV . Using tFiberoptic bronchoscopy yields granulomas by means of mucosal or transbronchial biopsy in 57–88% of cases ,24,25. LChest high resolution computed tomography (HRCT) has a better diagnosis accuracy than chest X-ray ,20. The Apart from intrathoracic lymph nodes and lung, the most frequent sites of sarcoidosis involvement include peripheral lymph nodes, eyes, skin and liver, each being noted in about 10–25% of cases in most series ,9. VirtuPeripheral lymphadenopathies are easily palpable and their frequency varies between series up to 70%. They are asymptomatic, firm, of various size and every site of lymph nodes may be affected. Peripheral lymphadenopathies are readily accessible to biopsy. They can be localized in the abdomen where they are recognizable by abdominal echography, computed tomography (CT) or magnetic resonance imaging (MRI) and they can be associated with liver or splenic nodules or enlargement.ocular sarcoidosis is comprised between 10 and 50% according to published studies. This wide range is due to recruitment bias in series reported by ophtalmologists and to epidemiological factors with an increased incidence in Japanese patients [The frequency of patients . Any parpatients . AnterioSkin manifestations of sarcoidosis are heterogeneous. Erythema nodosum is a non specific association of sarcoidosis realizing typically Löfgren's syndrome in the presence of bilateral hilar lymphadenopathy. The incidence of Löfgren's syndrome varies according to epidemiological factors (see "Epidemiology"). The frequency of specific skin manifestations of sarcoidosis ranges from 10 to 40% cases [etc. Skin lesions supply a plain and proper site for biopsy with the exception of erythema nodosum. Lesions of the face such as lupus pernio are very unpleasant and often linked with a longstanding evolution of sarcoidosis and osseous and sinonasal localizations. They are often difficult to control with treatments.0% cases . They ap0% cases . The cliwhile granulomas are found in up to 60–80% of liver biopsy specimens, abnormalities of biological tests, primarily cholestasis, are evidenced in only about 20% of cases. Clinical enlargement of the liver is far less frequent. Chronic intrahepatic cholestasis, hepatic dysfunction, cirrhosis and portal hypertension are all severe but rare complications of sarcoidosis.Localizations of sarcoidosis in heart, central nervous system, larynx or kidney are less frequent but potentially serious.Cardiac involvement of sarcoidosis appears to be much more frequent in Japanese population, particularly in females > 50 years, than in Europeans and Americans in which it concerns approximatively 5% of cases [18FDG PET can be helpful tools for diagnosis . Endomyocardial biopsy is theoretically the most confident mean to ascertain the diagnosis but in clinical practice it lacks sensitivity and it is an invasive procedure, which constitutes major limitations. Thus, the diagnosis of cardiac sarcoidosis usually relies on the conjunction of multiple arguments: (i) evidence of sarcoidosis, (ii) presence of cardiac abnormalities compatible with cardiac sarcoidosis and (iii) exclusion of any other cause of cardiac disease.of cases ,30. It cnervous system can be involved in sarcoidosis with a frequency around 10% [Any part of the ound 10% ,32: menikidneys is extremely rare, cited in 0.7% of cases [Clinically significant involvement of of cases ,33. HistParotid enlargement is seen in 5–10% of cases. Sarcoidosis of the upper respiratory tract occurrs in 0.7 and 6 % and may assume various features in relation to the involvement of sinonasal mucosa, pharynx and larynx. Laryngeal sarcoidosis is potentially serious by provoking airway obstruction. Sinonasal sarcoidosis is well-recognised to be a chronic and recalcitrant form of the disease, which is associated with lupus pernio in half cases [Articular involvement may be acute and transient or chronic and persistent [Osseous involvement is associated with characteristic abnormalities on radiography [Gastrointestinal tract is involved in less than 1.0%. The stomach is the most commonly involved part of gastronintestinal tract, sometimes diagnosed incidentally [Splenic enlargement is observed in 5–10%, it is usually minimal and asymptomatic and causes rarely decrease in the count of platelets, red and white cells [lf cases ,35. Typirsistent . Yet, whiography . Sarcoiddentally . Sarcoidte cells . In the te cells . HoweverHypercalcemia is present in around 11% and hypercalciuria in around 36% of cases . Serum aSarcoidosis does not influence pregnancy adversely. Whether or not the disease improves during pregnancy remains debated. On the other hand, it is clear that sarcoidosis may exacerbate again in the puerperium, which justifies a close surveillance within a period of 6 months after delivery. Pregancy should be avoided when treatments other than corticosteroids are necessary because of potential foetal toxicity or teratogenicity . It is aSarcoidosis is rare before the age of 15 and exceptional before 4. The disease of chidren resembles that of adults with respect to the distribution of organs involved. In old literature, sarcoidosis with an onset in children under the age of 4 has been reported to have an original presentation characterized by the combination of polyarthritis, uveitis and skin rash, and the rarity of intrathoracic involvement. It is likely that most of reported cases had actually Blau syndrome.Co-existence of sarcoidosis with diverse autoimmune conditions, other granulomatosis and proliferations in the same individual is classical, even though still controversial. These include all connective tissue diseases but principally scleroderma and Sjogren's syndrome, as well as ankylosing spondilitis, primary biliary cirrhosis and auto-immune disorders of the thyroid . InteresThe diagnosis of sarcoidosis relies on clinico-radiographical presentation and evidence of non caseating granulomas on biopsy specimens after appropriate exclusion of other granulomatous disorders ,24. DiagMost recommended sites to obtain a histological confirmation are mucosal or transbronchial biopsy through fiberoptic bronchoscopy ,24, skin67Ga) scintigraphy has long been used as a diagnostic tool in sarcoidosis [67Ga is seen in intrathoracic and various extrathoracic localizations of the disease. The current indications of 67Ga scintigraphy are as follows: (i) to substantiate the diagnosis in difficult cases, particularly in those with normal chest-X ray and a suspicion of sarcoidosis, when the "lambda plus panda" patterns is found, which is almost pathognomonic of the disease, (ii) to identify other clinically silent extra-pulmonary uptakes and provide potential sites for biopsies, (iii) and to assess activity in difficult cases such as stage IV disease in which whether or not to initiate potentially toxic therapy is a dilemma. Recently, an increasing literature on 18F-fluoro-2-deoxyglucose positron emission tomography (18FDG PET) in sarcoidosis has been published, but its role remains to be delineated [Gallium-67-citrate (coidosis  but is olineated . Moreoveα and β or intravesical BCG therapy or associated with common variable immunodeficiency are those conditions which can best mimic sarcoidosis. In infants under four years old, the Blau syndrome has to be considered before sarcoidosis, specially in the absence of thoracic involvement. Loco-regional granulomatous reactions satellite of breast or lung carcinoma and of lymphomas are rarely difficult to differenciate from sarcoidosis as well as other granulomatous disorders of unknown cause like Crohn's disease or Wegener granulomatosis.Other granulomatous diseases need to be ruled out before the diagnosis of sarcoidosis is ascertained ,24. In aSarcoidosis is generally a time-limited disease that lasts for 12–36 months in half cases, for less than 5 years in most remaining cases and occasionnally for multiple decades [The most valid prognostic factors remain the mode of onset, the disease extent and chest-X ray staging. An acute onset with Löfgren's syndrome or asymptomatic bilateral hilar lymphadenopathy usually confers a self-limiting course -4. On thThe most severe and frequent complication of sarcoidosis is the occurrence of pulmonary fibrosis. This is usually associated with chronic dyspnea and frank impairment of pulmonary function. Pulmonary fibrosis is the most frequent cause of respiratory failure and results in the majority of deaths related to sarcoidosis in western countries. Pulmonary fibrosis is the consequence of chronic unremitting granulomatous process in the lung. The concern about the development of pulmonary fibrosis is the reason for treating cases of sarcoidosis with persistant pulmonary infiltration.Airway obstruction can be due to several mechanisms in sarcoidosis: bronchial distortion secondary to pulmonary fibrosis, direct localization of granulomas in the airways, or more seldom extrinsic bronchial compression by hilar or mediastinal lymphadenopathy.i.e. increased pulmonary arterial pressure usually occurs in end-stage fibrotic cases. The severity of pulmonary hypertension is often out of proportion to pulmonary function impairement, which implies the role of alternative mechanisms including specific involvement of small lung vessels by sarcoidosis [Pulmonary hypertension, coidosis . PulmonaMycetoma formation is a recognized complication of advanced pulmonary fibrosis and it is believed to be associated with an increased risk of death because of severe bleeding or underlying respiratory insufficiency.etc.Some other severe localizations may also punctuate the evolution of sarcoidosis. In cardiac sarcoidosis, episodes of sustained ventricular tachycardia coupled with left ventricular dilatation are associated with an increased risk of death . CentralThe appropriate therapy for sarcoidosis has not yet been well defined for all patients ,7,9. BetCardiac, neurological, renal, ocular sarcoidosis not responding to topical therapy and malignant hypercalcemia always necessitate systemic therapy. Incapacitating general symptoms may lead to short-course corticosteroid therapy -4,7,8. Fα inhibitors have led to a dramatic improvement in small series or several case reports with refractory lupus pernio, uveitis or central nervous system localization. Recently, a double-blind study demonstrated a short-timed efficiency of infliximab in severe pulmonary localizations [Antimalarial drugs are a treatment of choice for mild isolated cutaneous lesions ,8. Aminoizations . Yet, etCurrently available agents for the prevention of glucocorticoid-induced osteoporosis may not be safe in sarcoidosis. Vitamin D should be avoided. Calcium supplementation may be given to patients without hypercalciuria under periodic survey of calcium metabolism. Bisphosphonates are also efficient in this setting.Symptomatic treatments and recommendations may be given in sarcoidosis: low calcium diet and avoidance of sunlight exposure, supplemental oxygen, cardiac drugs, implantable pacemakers or cardiovertible-defibrillator, hormonal substitution, anti-epileptic agents.Lung, liver and heart transplantation have been successfully performed in patients with end-stage refractory sarcoidosis . Medical