Radiological and histological evaluations are affected by subjective interpretation. This study determined the level of inter- and intraobserver variation among radiologists for detection of abnormal parenchymal lung changes on high resolution computed tomography (HRCT).HRCT images of 65 patients known to have systemic lupus erythematosus  were retrospectively reviewed by four nonthoracic radiologists . Each radiologist read the scans twice, with an interval between readings of at least 6 months. The interobserver variation among the first and second readings of the four radiologists and the intraobserver variation of each radiologist's two readings were assessed by the kappa statistic.There was good agreement between the first and second readings of each radiologist. There was moderate agreement between the two readings of one MR radiologist (kappa=0.482); the other three radiologists had kappa values that were good to excellent . There was a clinically acceptable level of interobserver variability between all radiologists. The agreement was fair to moderate between the MR radiologist and the other observers (kappa range: 0.362-0.519) and moderate to good between the other three radiologists (0.508-0.730).The interpretation of imaging findings of abnormal parenchymal lung changes on HRCT is reproducible and the agreement between general radiologists is clinically acceptable. There is reduced agreement when the radiologist is not involved on a regular basis with thoracic imaging. Difficult or indeterminate cases may benefit from review by a chest radiologist. High-resolution computed tomography (HRCT) of the lungs is being increasingly used in the diagnosis and treatment of diffuse parenchymal lung disease (DPLD). Until recently, open lung biopsy was the most reliable guide to the likely outcome, with histological appearance suggesting inflammation indicating a relatively good prognosis and fibrotic change implying a poor outcome.4The emergence of HRCT as a versatile diagnostic test is due to its diagnostic accuracy. Radiological and histological evaluations are affected by subjective interpretation and this observer variation can affect the reproducibility of a diagnostic test. With the increasing popularity and accessibility of HRCT, the majority of patients with interstitial lung disease are currently managed on the basis of HRCT observations, without histological evaluation. Thus, knowledge of observer variation in the interpretation of HRCT is needed.The HRCT images of 65 patients known to have systemic lupus erythematosus (SLE) and treated at the chest clinic in a general hospital were retrospectively reviewed by four radiologists. The four radiologists included two magnetic resonance radiologists (HK and OS) and two general radiologists (RA and PS). These participating radiologists, all working at a teaching hospital, had completed their general radiological training 8-16 years earlier at different institutions. The two MR radiologists had received special training in MRI for 1-2 years and had been mainly involved in MRI reporting for the past 4-6 years. All four radiologists were aware that the patients in the study had SLE, but no additional clinical information related to the respiratory system was made available to them when reviewing the HRCT images. Each radiologist read the scans twice, with an interval of at least 6 months between the two readings.The radiologists determined whether the following radiological lung features of SLE were present: diffuse thickening, nodular thickening, alveolar honeycombing, perivascular nodularity, peribronchial nodularity, calcification, bronchiectasis, scarring, a ground-glass appearance, emphysematous bullae, prominent vasculature, pleural calcification, diffuse pleural thickening, nodular thickening with small and large nodules. After assessment, the results were recorded as normal lung parenchyma or abnormal lung parenchyma . The findings were described based on the location of the abnormalities , and lower lobe) and also according to the central or peripheral (outer third of the lung) nature of the abnormalities. The interobserver variation between the first and second readings of the four radiologists and the intraobserver variation between each radiologist's two readings were identified and the results assessed by the kappa statistic.The images were obtained on a CT scanner , with 1-mm collimation at full inspiration. Scans were obtained at 10-mm intervals from the apices to the bases, with the patient in the supine position. The images were reconstructed with a high-spatial-frequency algorithm and photographed at window settings appropriate for viewing the lung parenchyma. All the images were evaluated on hard copy.Interobserver and intraobserver variability in grading appearances on HRCT were quantified Figures  and 2 us7P value of all four radiologists was.001 which indicates there is significant agreement between the observations of all the observers.The first and second readings (intraobserver variability) for each radiologist were compared . There wAgreement between radiologists (interobserver variability) was also compared for the two readings . There wObserver variability has been a problem ever since imaging began to contribute to the diagnosis of diffuse parenchymal lung disease, beginning as far back as the 1940s.8We used the kappa coefficient of agreement to evaluate observer variability as this accounts for chance agreement. The clinical significance of a kappa value depends upon its context and the values cannot be always compared between studies as it is dependent on disease prevalence.A few of the early studies performed to evaluate the diagnostic accuracy of HRCT also incorporated an evaluation of observer variation. However, these studies included many unusual diagnoses12The role, if any, of the better quality images produced by the newer and more versatile CT machines in the reduction of inter- and intraobserver variability has not been specifically studied, although this might be of some significance.Significant variability between experienced histopathologists has been documented in the semiquantitative grading of interstitial fibrosis, intra-alveolar inflammation, and interstitial inflammation in open-lung biopsy samples.5Although we did not classify our patients based on the referral pattern from peripheral clinics, there would no doubt be a lower degree of agreement in tertiary referral cases. This highlights the fact that in difficult cases of DPLD it will not be sufficient to rely on either imaging or pathology in isolation. It is important in these situations to integrate the clinical information with HRCT and pathology findings (if available) before initiating treatment or formulating a final diagnosis.In conclusion, our study quantifies the level of inter- and intraobserver variability in the detection of abnormal parenchymal lung changes of SLE. The interpretation of imaging findings is reproducible and comparable. The agreement between general radiologists is clinically acceptable. There is some variability in interpretation and reduced agreement between one MR radiologist (who was involved in thoracic CT imaging only infrequently) and the others. Difficult or indeterminate cases may benefit from review by a dedicated thoracic radiologist and, in some situations, a lung biopsy.


Paraneoplastic cerebellar degeneration is part of a rare spectrum of neurological syndromes whereby gynaecological, lung or breast cancers present primarily with neurological manifestations. The presence of onconeural antibodies and PET scanning help in the challenging diagnosis of these conditions but despite the treatment of the primary cancer, the prognosis for the neurological symptoms is poor. Paraneoplastic syndromes are non metastatic neurological manifestations associated with onconeural antibodies that arise in less than 1% of patients with an underlying malignancy. These syndromes are well defined in cases of small cell carcinoma of the lung and less well known but described in cases of breast and gynaecological cancers . We presThe patient was a 40-year-old female who developed dysarthria and ataxia in late 2006. These symptoms lasted for two months after which she made a complete recovery. Unfortunately she relapsed in March 2007, presenting with impaired coordination, speech difficulties and ataxia and was soon after confined to a wheelchair. Breast examination was normal, and neurological examination confirmed the presence of ataxia, dysarthria and impaired coordination.MRI scans of the spine and brain were normal and blood tests were positive for anti-Yo antibodies. A subsequent whole body PET CT although showed no clear primary lesion, indicated an avid node in the left axilla. Bilateral mammograms were normal and ultrasound of both breasts was normal except for the pathological node in the left axilla. Fine needle aspiration of the left axillary lymph node showed metastatic carcinoma, although extensive immunocytochemical studies could not confirm or exclude breast or lung carcinoma. MRI scan of the breast showed a single 6 mm nodular lesion and a targeted ultrasound of the left breast showed several hypoechoic nodules. Core biopsies of two of the visualised nodules showed high-grade ductal carcinoma in situ and poorly differentiated invasive carcinoma Figure .The patient underwent a left skin-sparing mastectomy combined with implant-based immediate reconstruction plus axillary node clearance. Histological analysis showed a 4 cm area of grade 3 invasive ductal carcinoma and DCIS associated with extensive immunological response that had broken the tumour into small islands Figure . There wThe patient made a good postoperative recovery and was subsequently referred for adjuvant chemotherapy and herceptin treatment and post-mastectomy radiation. Her neurological condition remained stable during treatment.Paraneoplastic cerebellar degeneration is classified as one of the paraneoplastic syndromes. These are rare, non-metastatic complications in patients with cancer, commonly gynaecological, breast or small cell lung in origin.The pathogenesis of these syndromes is not entirely understood, but evidence suggests that certain autoantibodies expressed against tumour cells may interact with cells in the nervous system -3. The aAnti-Yo antibodies are anti-Purkinje cell autoantibodies that act against the antigens common to the tumour and Purkinje cells in the cerebellum and are produced as an immune response to some tumours . Not allCases have been reported where cerebellar degeneration has preceded the tumour by as long as 5 years after expression of the anit-Yo antibody ,7.Rojas et al evaluated the long-term outcome of PCD and anti Yo antibodies in 2000. Of a total of 34 patients with PCD and anti-Yo antibodies, tumour progression was the cause of death in 52% of cases, whilst in 29% of patients it was the neurological condition. The failure to cure the cancer in 52% of patients was due to the fact that by the time the diagnosis had been made, most tumours had already metastasised to regional lymph nodes  or distant organs.In particular, all patients with breast cancer had axillary lymph node metastases. This resembles early mediastinal lymph node metastases in patients with small cell carcinoma of the lung and anti-Hu associated syndromes. This suggests that the invasion of the regional nodes causes the tumour to invoke an immune response, which leads to the neurological syndromes. Of note, it was found in the same study, that although anti-Yo antibody associated syndromes favour the cerebellum, the disease is much more aggressive with most patients becoming bed bound within 3 months of the diagnosis . The hisAttempts to treat the neurological symptoms on the whole, using chemotherapy, immunosuppression or immunoglobulins have not been reported to achieve any significant improvement . In addiThe authors declare that they have no competing interests.KM and CC were involved with the clinical management of the patient. KM and AN were involved in the design of the study and the preparation of the manuscript. VAT carried out the histological analysis. AN, ZS and NM performed the literature search. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


We describe a 42-year-old age woman who accidentally swallowed her lower denture, which was composed of eleven teeth. The daily descent of the denture was followed by plain abdominal radiography and physical examination. The image was localized at the left upper quadrant on admission day, but it stopped on its way at the right lower quadrant on day two and three. Since the patient's complaints increased we planned surgical removal of the denture. In this report, we had discussed the diagnosis, follow up and treatment options of swallowed partial denture with current literature review. Foreign body aspiration is a common pediatric problem that commonly affects children of all ages and has been a recognized cause of accidental deaths. In adults aspiration of teeth and dental restorations is a recognized, yet an infrequent happening in the literature. Main reasons of aspiration are maxillofacial trauma, dental treatment procedures or ethanol intoxication and dementia -3.The majority of foreign bodies entering the oropharynx will pass into the alimentary canal and pass without incidence, though there is a danger of perforation of the gut which can have very serious consequences including death.If the material used in the construction of denture is methylmethacrylate, which is radiolucent, early diagnosis of an impacted or ingested denture in many cases is complicated. Herein, we present surgical treatment of accidentally swallowed partial denture composing of eleven units in a 42-year-old woman.A 42-year-old Turkish woman with a history of swallowing of lower partial denture while eating her meal 4 hours ago presented to emergency department with complaints of abdominal pain and nausea. On physical examination her vital signs were stable and there was just abdominal tenderness.Complete blood count and blood chemistry were normal. Plane abdominal radiograph of the patient on left upper quadrant, revealed foreign body in accordance with denture Figure . On follBecause of the persistence of denture at the same region and increase in her complaints we decided to perform surgical exploration. The edema and distention of intestines and hyperemia at the distal ileum were observed. The denture was palpated at entrance of ileocecal valve Figure . The denAspirated or swallowed partial dentures can present a diagnostic challenge. Accidental swallowing is much more common in pediatric group and rare in adults especially reported at with learning and mental health disorders ,5.The most likely presenting symptom after swallowing of a denture is dysphasia, with other complaints related to how far the denture has progressed and time since swallowing. Thus further reports may also be anticipated of sore throat, choking sensation, retrosternal pain, sweating and a raised temperature and coughing up blood. Early diagnosis and treatment will avoid the edematous reaction and mucosal infection and necrosis that heighten the risk of rigid oesophagoscopy .Reported late complications of the undiagnosed swallowed denture include extraluminal migration from the esophagus causing either a diverticulum or perforation (once a perforation has occurred, further severe squeal may be anticipated, e.g. tracheo-oesophageal fistula, the need to resect 18 cm of ileum, enterocolonic fistula and sigmoid colon perforation -11. PolyWe have followed physical examination and radiological findings of this patient for three days. Because of persistence of foreign body image at the same localization and increase of patient's symptoms we have performed surgical exploration to extract the partial denture via enterotomy.In conclusion, medical personnel, especially those called upon to manage emergencies, should likewise be aware of the multiple hazards. They need to know that certain people will be unable to perceive or report the disappearance of a denture; therefore they should be alert to the possibility of swallowing or inhalation. The follow up of patients must be under the supervision of the same clinician team.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.YT collected and interpreted the patient data and performed the surgery with SE and EKO. SE drafted the manuscript. MS and KK helped in collecting of references, figures and drafting of manuscript. MAA critically revised the manuscript. All authors read and approved the final manuscript.


MouseCyc is a database of curated metabolic pathways for the laboratory mouse. . MouseCyc has been integrated with genetic and genomic data for the laboratory mouse available from the Mouse Genome Informatics database and with pathway data from other organisms, including human.Linking biochemical genetic data to the reference genome for the laboratory mouse is important for comparative physiology and for developing mouse models of human biology and disease. We describe here a new database of curated metabolic pathways for the laboratory mouse called MouseCyc  The availability of the nearly complete genome sequence for the laboratory mouse provides a powerful platform for predicting genes and other genome features and for exploring the biological significance of genome organization . HoweverBiochemical interactions and transformations among organic molecules are arguably the foundation and core distinguishing feature of all organic life. Most of these transformations are understood as sequential interactions among molecules. Thus, biochemical pathways, rather than individual reactions and molecules, are often the most useful 'units' of investigation for biomedical experimentalists by providing conceptual reduction of biological system complexity. Biochemical pathways in mammalian systems historically have been characterized and defined with little or no genetic information, making the present day task of connecting metabolism and genomics a challenging enterprise.The Kyoto Encyclopedia of Genes and Genomes (KEGG) was one of the first projects that addressed the integration of small molecule biochemical reaction networks with genes, and it includes graphical representations of these reactions ,6. KEGG Another resource that seeks to integrate pathway and genomic data is Reactome ,9. ReactSaccharomyces cerevisiae , green hlamyCyc ), thale  (AraCyc ), rice, (RiceCyc ), plants(RiceCyc ), human,HumanCyc ) and, veattleCyc ), as welattleCyc . All datHere, we describe the implementation and curation of the MouseCyc database  using thThe initial implementation of the MouseCyc pathway genome database using the PathoLogic prediction software with Pathway Tools resulted in the prediction of 304 pathways, 1,832 enzymatic reactions, and 5 transport reactions. Following the automated build of MouseCyc, the predicted reactions and pathways were evaluated and refined manually. The initial manual curation effort focused on identifying pathways and reactions, predicted by PathoLogic, that were not relevant to mammalian biochemistry . The manual curation process resulted in the elimination of 135 non-mammalian pathways (45% of the pathways predicted for mouse by PathoLogic) from the database. The high percentage of predicted pathways in MouseCyc that required manual re-assignment was not surprising given that, for historic reasons, the MetaCyc reference database ,12 used ADC) has been reported to encode the enzyme in the first step of this pathway [Adc) of the human enzyme, however, lacks amine decarboxylating activity and, instead, appears to function as an ornithine decarboxylase antizyme inhibitor (oazin) in the superpathway of ornithine degradation [RGD1564776 has not been biochemically characterized yet. The example of arginine degradation illustrates two important points relative to the MouseCyc project. First, the orthology of enzymes does not always translate to functional equivalency. Second, ongoing investigation into the details of biochemistry necessitates regular manual curation and refinement for effective and error-proof 'translation' of advances in biochemistry to genomics.One of the obstacles that complicates unambiguously linking enzymes to genes is that protein products of orthologous genes do not necessarily have common biochemical functions . Moreove pathway . The mouradation . A more radation ; howeverradation , the autBecause of the limited amount of data on vertebrate organisms within the reference database that PathoLogic relies on for its predictions of metabolic potential , a number of important pathways were missing from the initial build of MouseCyc. Examples of curated biochemical pathways for the mouse that have been also submitted for inclusion in the MetaCyc reference database include biosynthesis of androgens, biosynthesis of corticosteroids, biosynthesis of estrogens, biosynthesis of prostaglandins, biosynthesis of serotonin and melatonin, ceramide biosynthesis, cyclic AMP biosynthesis, cyclic GMP biosynthesis, Leloir pathway, sphingomyelin metabolism, sphingosine and sphingosine-1-phosphate metabolism, and L-ascorbate biosynthesis VI . Thus, one of the major ongoing manual curation processes for MouseCyc is the creation of records for biochemical pathways that are specific to mammalian systems or the laboratory mouse that were not predicted by PathoLogic.Adh3 (Adh5 in current nomenclature), is a 'true' liver ethanol dehydrogenase, while the neofunctionalization of other family members during vertebrate evolution resulted in the changes to substrate specificity, expression pattern and enzymatic properties [The manual review of PathoLogic-predicted pathways for MouseCyc revealed numerous individual enzymatic reactions that cannot currently be associated with mouse-specific pathways. These reactions were not removed from MouseCyc; instead, they have been retained for possible incorporation into MouseCyc pathways at a later date. The rationale for retaining 'orphan' enzymatic reactions in the database is two-fold. First, there are a number of reactions that have been identified enzymatically in mammalian systems  for which no corresponding mammalian gene has yet been reported. Second, the majority of the 'extraneous' pathways contained one or more reactions for which a mouse enzyme has been either identified or predicted. They could be structural units of not yet curated pathways. One of the primary ongoing curation tasks for MouseCyc involves discerning valid enzymes for reactions within pathways from those erroneously assigned by PathoLogic. The main sources of errors in PathoLogic predictions are the protein sequence similarity-based inference of gene/protein function used in genome annotations. This curation process includes a review of published biochemical literature and protein sequence-based analysis of gene families. A notable example is the alcohol dehydrogenase gene family (EC 1.1.1.1), in which an 'ancestral' enzyme, operties . In thisoperties .GULO) gene [Asmt) gene [Uox (EC 1.7.3.3) and Urah (EC 3.5.2.17) leads to formation of allantoin, a much more soluble and less toxic compound [One of the primary benefits of using Pathway Tools for building PGDBs is that the software supports comparative metabolomics by allowing users to display the same pathway from different PGDBs simultaneously. In addition to side-by-side evaluation of individual pathways Figure , MouseCyLO) gene . Melatonmt) gene . Purine compound .GALT) are associated with classic galactosemia [Galt gene exhibit high levels of galactose-1-phosphate and galactose but are otherwise phenotypically normal [One of the main goals for the MouseCyc database initiative was to integrate the pathway-centered view of the mouse genome with the extensive biological knowledge about mouse genes and human disease phenotypes represented in the MGI databases . The intctosemia , a severy normal . In Mousy normal  and mousThe MouseCyc OmicsViewer  is the sIn addition to serving as a mouse-specific reference database of biochemical pathways, MouseCyc can also be used for generating hypotheses about biological processes using genomic data. To test the value of the OmicsViewer for hypothesis generation, we utilized the previously published data set of genes expressed in the mouse oocytes  to exploDocumenting the similarities and differences of biochemistry and metabolism between mice and humans is particularly important for investigators seeking to use the laboratory mouse in animal studies related to drug therapies, toxicology, and human disease. In our curation of MouseCyc to date we have documented, and formally represented, differences in metabolic potential among mammals that are due to the absence of critical enzymes or to functional divergence of putative orthologs. Connecting mouse genes and pathways to human diseases in MouseCyc highlights differences in biochemistry that cannot yet be clearly associated with specific genes and proteins. For example, the Leloir pathway Figure  is the mA primary value-added aspect of the MouseCyc project relative to other pathway databases lies in the extent to which pathways in MouseCyc have been integrated with the comprehensive functional and phenotypic knowledge of mouse genes and the associations of mouse genes with human disease phenotypes that are available through the MGI resources. In addition to the reciprocal hypertext links between genes and pathways that are available in MGI and MouseCyc, researchers can rapidly visualize the literature-curated functional and phenotypic annotations of genes and gene products available from MGI in the context of all biochemical pathways known for mouse. As illustrated by the mouse oocyte transcriptome study described in this manuscript Figure , supportThe initial implementation of the MouseCyc database required substantial manual refinement to make the presentation of pathway knowledge more representative of mammalian biology. The degree of manual refinement required was due, in part, to the fact that most vigorous biochemical genetics research has been performed using microorganisms such as bacteria and yeast. As a result, the MetaCyc reference database that was used for pathway prediction is somewhat biased toward biology of unicellular microorganisms. The ongoing incorporation of curated data from MouseCyc into MetaCyc, as well as expansion of curatorial efforts for other projects using mammalian systems, specifically HumanCyc  and CattAn important future direction for the MouseCyc resource will be to represent explicitly the cell and tissue-type specificity of particular pathways and their reactions. In the current implementation of the database, all genes encoding enzymes with the same function are assigned to the same biochemical reaction, making it impossible to discern the network of enzymes executing a particular pathway in one tissue versus another. For example, ethanol metabolism Figure  depends The Pathway Tools development kit software (version 10.0) was downloaded from Stanford Research Institute and installed on each of two Sun Fire X4100 servers  running SUSE Linux. One of the servers is devoted to development and curation activities; the second server is the dedicated host for the public instance of the MouseCyc database  and HumaThe Pathway Tools software system has four main components . The PatA catalog of mouse genes and annotations was downloaded from the MGI FTP site (6 November 2007). The gene annotations included gene name and symbol, EC numbers, Gene Ontology annotations, genome coordinates (for NCBI build 36) and accession identifiers for EntrezGene, UniProt, and MGI. RNA genes and pseudogenes were not included in the annotation file.A total of 47 files were created as input to the PathoLogic algorithm following the format specifications outlined in the Pathway Tools installation guide. Annotation files were created for 19 mouse autosomes, 2 sex chromosomes, the mitochondrial genome, and for genes with unknown chromosome location. For each annotation file, a separate chromosome sequence file was created in FASTA format. Finally, a file (the genetic elements file) to guide the instantiation of the chromosomes and their annotations was also created.Following the automated build of MouseCyc, the data-editing tools built into the Pathway Tools software system were used for manual refinement and annotation of pathways and reactions.Pre-compiled OmicsViewer files for phenotype annotations of mouse genes from the MGI database are available via FTP . These fUpdates to the Pathway Tools software are implemented as they become available. MouseCyc currently runs on Pathway Tools version 13.0.The MouseCyc database is updated bi-monthly with new and revised manually curated pathways. Updates to mouse genome annotations  are propagated to MouseCyc using the PathoLogic incremental update utilities. With each genome annotation update, potential new pathways and reactions are generated automatically and reviewed manually. Information on the current content and history of updates to MouseCyc can be found by following the 'History of updates to this database' link on the MouseCyc home page.EC: Enzyme Commission ; KEGG: Kyoto Encyclopedia of Genes and Genomes; MGI: Mouse Genome Informatics; PGDB: pathway genome database; TIP: Transport Inference Parser.CJB conceptualized the study, EP and CJB performed the initial PathoLogic build of the MouseCyc database, AVE conducts the ongoing curation of MouseCyc, MED provides ongoing synchronization of MouseCyc with MGI, MPG provides ongoing software and hardware updates and support for MouseCyc and underlying Pathway Tools platform, and AVE, MED and CJB wrote the manuscript.The following additional data are included with this article: a table listing biochemical pathways created by MouseCyc group (Additional data file Biochemical pathways created by MouseCyc group.Click here for file


The epidermal growth factor receptor (EGFR) signaling pathway plays a key role in regulation of cellular growth and development. While highly studied, it is still not fully understood how the signal is orchestrated. One of the reasons for the complexity of this pathway is the extensive network of inter-connected components involved in the signaling. In the aim of identifying critical mechanisms controlling signal transduction we have performed extensive analysis of an executable model of the EGFR pathway using the stochastic pi-calculus as a modeling language.Our analysis, done through simulation of various perturbations, suggests that the EGFR pathway contains regions of functional redundancy in the upstream parts; in the event of low EGF stimulus or partial system failure, this redundancy helps to maintain functional robustness. Downstream parts, like the parts controlling Ras and ERK, have fewer redundancies, and more than 50% inhibition of specific reactions in those parts greatly attenuates signal response. In addition, we suggest an abstract model that captures the main control mechanisms in the pathway. Simulation of this abstract model suggests that without redundancies in the upstream modules, signal transduction through the entire pathway could be attenuated. In terms of specific control mechanisms, we have identified positive feedback loops whose role is to prolong the active state of key components , and negative feedback loops that help promote signal adaptation and stabilization.The insights gained from simulating this executable model facilitate the formulation of specific hypotheses regarding the control mechanisms of the EGFR signaling, and further substantiate the benefit to construct abstract executable models of large complex biological networks. Important cellular processes, such as differentiation and division, rely on signal transduction cascades that occur throughout the cell. The signal transduction cascade, initiated by the binding of epidermal growth factor (EGF) to the epidermal growth factor receptor (EGFR), is essential for normal cell growth and development . ConversThe EGFR signaling cascade is initiated by the binding of EGF to the transmembrane EGFR at its extracellular domain. The binding induces dimerization and autophosphorylation of its intracellular domains. The dimers form docking sites for the recruitment of cytoplasmic enzymes and adapter proteins, starting a complex signaling cascade . The subin silico experimentation, and provide guidance for future experimentation. In addition, this modelling approach may be amenable to detailed analysis of the stochastic behavior of a network using model checking .Previous studies of the EGFR signaling pathway have combined mathematical modeling and quantitative experimentation. These studies revealed insights into the functionalities of the pathway ,17 and fThe ability of the EGFR signaling cascade, as with many other receptor tyrosine kinase signaling systems, to maintain a reproducible output despite input variations can be attributed to the module diversity and redundancy that allows for compensatory functioning in case of component failure . It is tHere we present a modularized model of the EGFR signaling cascade within a single cell in the pi-calculus framework. Our model is adapted from previous mathematical models describing the same pathway ,21. ModuThe original kinetic model of the EGF-induced signaling pathway ,21 was uet. al. [signaling module. Signaling modules are important for modularization of the EGFR signaling pathway as well as more generally for reuse and integration of modules in larger system models. Perturbations to the model were done by knocking-out reactions feeding in and out of the components highlighted in Figure The original model by Schoeberl et. al.  is modulet. al. ,6,20,23.m on the same channel x. (3) an output action !x(n) describes sending a message to another molecule whose receiving an input n on same channel x. The specification do π1.P1 or ... or πN.PN, where πi stands for one of the three previous actions, represents the ability of a molecule to react in N different ways. The parallel composition P|Q represents the existence of molecules at the same time. By defining X(m) = P, we can define a molecule of type X parameterized by m. Each type of molecule will have its definition recorded in a constant environment E. For a more complete introduction to stochastic pi-calculus, we refer the reader to [Similar to the procedure of modeling concurrent computational systems, biological systems can be described in the stochastic pi-calculus language. The behaviour of a molecule, such as a protein or a signaling complex, can be described as a process P, and actions describe possible interactions between molecules. Possible actions π include: (1) a delay action delay@r describes a change in the internal structure of a molecule with a delay whose rate is r. (2) an input action ?x(m) describes receiving a message from another molecule whose sending an output eader to . EssentiOur model was executed using the Stochastic Pi Machine (SPiM) simulator version 1.13. The stochastic pi-calculus code for executing the unperturbed model in SPiM was modified to run simulations on a perturbed EGFR signaling pathway. Each simulation of the EGFR model was carried over 3,600 time units, which is the equivalent of 60 minutes real time, and 100,000 sampling events took place over this time range. Observables generated by SPiM from the state variables were converted into absolute numbers of molecules for each species (each complex of different compounds was considered a unique species). Simulation of each perturbed model was carried over ten replicates and the number of molecules of ERK-PP was averaged over each time unit. The molecular profiles were plotted using the R-statistical platform, and a LOWESS method was applied to smooth the graphs.In these simulations, we identify active signal response as the molecular profile of components in the signaling pathway when 50 ng/ml of EGF is added as a stimulus, and the inactive signal response as the molecular profile when no EGF is stimulating the system.Our model of the EGFR signaling pathway is an updated version of previous models ,21. It iAnalysis of these initial simulations reveals interesting features that hint to the results of later deeper analysis of the model. The activation of the EGF-receptors, in the dimerized and phosphorylated form, clearly depends on the concentration of EGF, responding with greater amplitude and earlier peaks. Although EGF concentration heavily affects membrane receptor activation, there is less concentration sensitivity as we trace down the signaling cascade. In particular, the amplitude and duration of the signal response in ERK-PP is hardly affected, even at low levels of stimulus. As suggested from the sharper and much shorter duration in the signal response of Ras-GTP and Raf-P, it is likely that the degree of activation in upstream components have little effect on the downstream signaling components. Following activation of Raf and Ras, there is considerable signal amplification occurring in the pathways leading to MEK and ERK activation. Even though the amplitude of the signal response does not seem to be significantly affected by the amount of EGF stimulation, the sustainability of the signal response in most components, including ERK-PP, seems to be attributed to the high EGF concentrations.We partition the model into functional modules based on the common biological concept of the importance of substances in the pathway. This partition is highlighted in Figure An important characteristic of the EGFR signaling cascade is its ability to maintain output reproducibility, despite input variation or system perturbation. Design features, like network redundancy, may attribute to this functional robustness. Our first analysis consists of systematic inhibition of each reaction in the model separately. We include in Figure In the middle of the pathway, the Ras modules seem to maintain both fragile and robust network elements. Robustness of the two modules may be attributed to their extensive feedback mechanisms, and fragility may be due to the criticality of Ras-GTP for persistent signal transduction. Inhibition of v31 does not affect ERK from attaining maximal activation, but does affect the sustainability of its activated state Figure . The obsOur next analysis is by simulation of the pathway under inhibition of multiple reactions Figure . Reactioone of the substances in orange or red does not affect activity of the output. In the case that a substance has only orange connections then one is required for activation but not both. The red connections denote inhibition and all others activation. Therefore, this representation of the EGFR signaling pathway provides a summary of the key mechanisms controlling signal transduction between components, and also suggests an intuitive way of understanding how the activation or inactivation of each component can affect the whole system. This representation captures the relations between substances but is not intended to be perceived as purely Boolean. We stress that deactivation of a red/orange input may still affect the amplitude or duration of activity of the signal, however, it remains active.As a result of our simulations under extensive perturbation of the computational model, we can generate dependence relationships between key components in the EGFR signaling pathway. We exhaustively examined all conditions across the EGFR signaling pathway where components achieve active and inactive states. Activity/inactivity values for the molecular activities were compiled for each key component in order to map dependency of activity between these components. The resulting dependencies are summarized in Figure From the connectivity diagram in Figure The connectivity diagram is justified by pi-calculus (SPiM) simulations. We have also created a smaller stochastic pi-calculus model that includes only these key molecular components and their connectivity as presented in Figure This work presents a computational model of the EGFR signaling pathway. The model, written in the stochastic pi-calculus language, represents our current understanding of EGFR signaling dynamics and the contribution of important signaling components to this pathway. Execution of the model allowed us to simulate and observe signal transduction through the pathway under various conditions. Some simulations match the experimental observations and increase our confidence in the model and its consistency with previous models ,27,21. OThe insights gained from simulation and analysis of the kinetic behaviour of the EGFR signaling pathway helps us understand not only the terminal signal response to EGF stimulus, but also the response at various levels of the signaling cascade. The analysis suggests that there is high sensitivity in upstream components to variations in transient EGF stimulus, but this sensitivity does not translate to differential signal response in downstream components. Specifically, sensitivity analysis reveals greater sensitivity in the EGF binding reaction and the Raf dephosphorylation reaction . In addiPartitioning of the EGFR signaling network into modules is not a trivial task, but it leads to a simpler model of the signaling cascade which offers clarity and computational tractability . IdentifBy comparing the results of the simulations in terms of signaling modules, we further verify differences between modules based on network characteristics, like redundancy and robustness to perturbations. Some of these characteristics, such as the dependence of ERK-PP signal response on the activation of EGF receptors in the EGF module, may be an indication of why contact and tyrosine kinase inhibitors continue to be an elective means of preventing EGFR signaling . Our knoin silico experimentations on computational models. More importantly, computational models have been widely used in the fight against numerous detrimental diseases by helping to determine possible targets for drug therapies. Interestingly, through our tests to determine the effectiveness of targeting multiple inhibition sites as a means of combination therapy, we discovered a higher level of system sensitivity to targets in the downstream modules  as opposed to targets in the upstream modules (EGF and Grb2). Specifically, we did not detect any evidence of significant attenuation of EGFR signaling when phosphorylation reactions in the upstream modules are targeted for partial inhibition, contradicting the results of analysis done on other mathematical models [The ability to perturb a biological system without any cost, except for those that are computational, truly attests to the important practicality of conducting l models . Since hl models , it woulAs a means of compiling our observations of the various states that the components in the EGFR signaling pathway achieved under different conditions, and as a way of demonstrating model abstraction with the aid of models in the pi-calculus language, we produced a simplified connectivity diagram Figure . The diaIn order to perform our analysis we have chosen to use the SPiM simulator, which is based on the pi-calculus formalism. There is a wide range of existing tools based on process calculi, such as BetaWorkbench , Bio-PepThrough simulation of the EGFR signaling model under different perturbation conditions, we can discover regions of network robustness and fragility. The molecular profiles of the important signaling components presents evidence for the increasing insensitivity to low EGF stimulus as we move down the signaling pathway. In contrast, greater sensitivity is exhibited by the signaling system when downstream signaling modules are perturbed. There seems to be significant redundancy that is contributing to network robustness in Grb2 and Ras signaling modules. Further study of partial inhibition at the important reactions demonstrates that high signal attenuation is achieved when multiple sites in downstream signaling modules are targeted. By analyzing the behaviour of interface components as the signal passes through the modules, we are able to produce a mapping of signaling modules to simplified operations, and thereby, generating a connectivity diagram for the EGFR signaling pathway. From the diagram, we can see important feedback mechanisms, some of them crucial to the maintenance of activated response in ERK and could explain the irreversible maturation of tumour cells. As is shown, the results from these simulations of the EGFR signaling pathway can be used to help formulate hypotheses, regarding the dynamics of EGFR signaling, to be tested in the 'wet lab'. Furthermore, the experimental results can be used to eliminate spurious assumptions in the model and hence further verify it.DW, NP, and JF conceived and designed the computational simulations. AP and LC participated in crucial discussions. AP provided technical advice with designing the SPiM experiments and running the SPiM simulations. LC implemented an earlier version of the EGFR signaling pathway model, which served as a basis for the current modular version of the model that was implemented by AP. DW edited the model, performed simulations and analyzed the data. DW, NP, LC, and JF wrote the manuscript. AP contributed to the additional files. All authors read and approved the final manuscript.Stochastic pi-calculus model of the biochemical interactions involved in the EGFR signalling pathways. The model is based on the reaction map of Figure http://research.microsoft.com/en-us/um/people/aphillip/egfr09/ and the SPiM simulator is available at http://research.microsoft.com/spim/Click here for fileBehaviour of ERK-PP in response to complete inhibition of key reactions in the EGF module.Click here for fileBehaviour of ERK-PP in response to complete inhibition of key reactions in the Grb2 module.Click here for fileBehaviour of ERK-PP in response to complete inhibition of key reactions in the Ras Shc-independent module.Click here for fileBehaviour of ERK-PP in response to complete inhibition of key reactions in the Ras Shc-dependent module.Click here for fileBehaviour of ERK-PP in response to complete inhibition of key reactions in the Raf module.Click here for fileBehaviour of ERK-PP in response to complete inhibition of key reactions in the MEK module.Click here for fileBehaviour of ERK-PP in response to complete inhibition of key reactions in the ERK module.Click here for fileBoolean table for inferring logical inputs for (EGF-EGFRi)2*.Click here for fileBoolean table for inferring logical inputs for (EGF-EGFR*)2-GAP.Click here for fileBoolean table for inferring logical inputs for (EGF-EGFR*)2-GAP-Shc*.Click here for fileBoolean table for inferring logical inputs for (EGF-EGFR*)2-GAP-Grb2-Sos.Click here for fileBoolean table for inferring logical inputs for (EGF-EGFR*)2-GAP-Shc*-Grb2-Sos.Click here for fileBoolean table for inferring logical inputs for Ras-GTP.Click here for fileBoolean table for inferring logical inputs for Raf*.Click here for fileBoolean table for inferring logical inputs for MEK-PP.Click here for fileMolecular profiles of interface components as a result of perturbations on two different input signals. See highlighted rows in Additional Files Click here for fileMolecular profile of ERK-PP in response to signal from the EGF-EGFR complex as simulated by the abstract pi-calculus model.Click here for fileMolecular profile of ERK-PP in response to signal from the EGF-EGFR complex as simulated by the abstract pi-calculus model, but without abstraction of the Grb module.Click here for file


We report the first four North American cases of Candida dubliniensis fungemia, including the first isolation of this organism from the bloodstream of an HIV-infected person. All isolates were susceptible in vitro to commonly used antifungal drugs. This report demonstrates that C. dubliniensis can cause bloodstream infection; however, the incidence of disease is not known.


The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best.While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex—all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea.This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota. DNA replication is one of the defining processes of modern life. The spread of DNA replication likely represents a major evolutionary transition in early life. Duplication of DNA content allows organisms to pass genetic information onto future generations. Mutations during the duplication process enable populations to evolve and adapt. The centrality of DNA replication to such important life processes makes the evolution of the DNA replication machinery all the more significant for understanding the evolution of life.Chromosome replication in Archaea and eukaryotes requires three ancillary complexes—the proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance complex (MCM) de novo DNA synthesis. Therefore, once the single stranded DNA template is generated by the replicative helicase, an RNA primer is initially synthesized by a DNA primase to create a primer/template junction. The primer/template junction is recognized by the clamp loader, which loads the clamp onto this DNA structure. The clamp then recruits the DNA polymerase to the single stranded DNA to perform the actual template guided process of DNA replication. The function of PCNA is to encircle the DNA and affix, or clamp, the polymerase to the template. In a role analogous to the bacterial beta clamp, PCNA enhances the speed and efficiency of DNA polymerase by enabling the polymerase to synthesize the complementary strand continuously without frequent dissociation.We refer the interested reader to Refs. Saccharomyces cerevisiae genome appears to be the result of the duplication of a smaller ancestral genome In all cases where distinct sequence types are observed within a complex, the proteins are sufficiently similar to imply a common ancestry. For over 40 years it has been observed that gene duplication followed by divergence is an important source of new or modified protein functions The extensive role and implications of gene duplication in the evolution for increasing complexity speak to a larger puzzle. The question of emergence of complexity Complexes consisting only of repeated identical subunits are simpler than complexes consisting entirely of homologous, but not identical, subunits. As such, the number of distinct sequence types in each complex serves as a proxy for the overall level of complexity. We trace the emergence of the distinct sequence types in order to put together a picture of how such complexity arose. For instance, where did the distinct subunits come from? Were more specialized subunits invented once and subsequently horizontally gene transferred (HGT) or did complexity increase independently in different lineages? Did simpler complexes with less specialized subunits beget the more specialized subunits in the complexes consisting of distinct subunits, or vice-versa?With these questions in mind, we examine the phylogeny of the PCNA, RFCS, and MCM subunits. The phylogenetic data is then compared in detail with the known biochemistry of each subunit, in particular, a subunits interaction partners within each complex.PCNA was so named after it was found to be highly abundant in proliferating cells Nitrosopumilis and Cenarcheaum in Korarchaeum and Nanoarchaeum sequences are grouped together within those of the Crenarchaeota. Given the general agreement between the PCNA phylogeny and the organismal taxonomy, HGT does not appear to have occurred.The maximum likelihood phylogeny of the PCNA subunits is shown in Drosphila, Arabidopsis, and Thermococcus  and four small (RFCS). The RFC complex opens between the the RFCL  in orderthe RFCL .Cenarcheaum deep in the branching of archaeal sequences, again consistent with proposals that it be a member of a distinct phylum. The Korarchaea and Nanoarchaea sequences cluster with those of the Euryarchaea. The rooting between the eukaryotes and Archaea follows the canonical pattern, dividing the crenarchaeotes and the Euryarchaea at the base of the archaeal clade.The maximum likelihood phylogeny of the RFCS subunits is shown in The phylogeny of the RFCS subunits shows that a RFC with four distinct RFCS sequence types seems to have been present in a common eukaryotic ancestor. This can be seen from the four eukaryotic RFCS clades—one for each RFCS position. On the other hand, the archaeal RFC consists of one or two distinct RFCS subunits In Euryarchaeota, the specialization of RFCS into RFCS1 and RFCS2 appears to have occurred before the split between Methanomicrobia and Halobacteria. Following the RFCS1-RFCS2 divergence, there appear to be two independent losses of RFCS2 in the Methanomicrobia, indicated by stars in Note that the long branch of RFCS2 corresponds to a change of function. Unlike RFCS and RFCS1, RFCS2 is unable to further extend the small subunit chain since it contains only one RFCS-RFCS binding site The results for RFCS are consistent with a simpler ancestral RFC complex containing RFCL and four identical RFCS subunits. In the Archaea, we see subsequent multiple independent duplications and divergences of the distinct subunit types in both crenarchaeotes and Euryarchaea. In eukaryotes, we do not see any intermediate forms with fewer than four distinct RFCS types.MCM complex plays a role in replication licensing Nitrosopumilus and Cenarcheaum is consistent with a distinct phylum level group, the proposed Thaumarchaeota The phylogeny of the MCM subunits is shown in The phylogeny of the MCM subunits shows that MCM with six distinct sequence types seems to have been present in a common eukaryotic ancestor, a result previously noted by Liu et al. Methanococcus aeolicus appears to have lost MCM III while Methanococcus maripaludis C6 has five MCM V sequences.The largest number of MCM genes can be found in the Methanococci. The Methanococci subunits in Pyrococcus furiosus requires the presence of accessory protein GINS for unwinding DNA activity Methanococcus maripaludis S2 result in the formation of a heterohexameric complex M. maripaludis has a very robust genetic system, we anticipate that subsequent studies will reveal the need for multiple MCM homologs in this archaeon, instead of the usual single homolog in most archaea.There are multiple eukaryotic MCM complexes. At least two different complexes are known to play a role in unwinding dsDNA These results are consistent with an ancestral homohexameric MCM complex. In the Archaea, we see subsequent multiple independent duplications and divergences of the distinct subunit types in the Euryarchaea. The crenarchaeotes, on the other hand, retain the simpler ancestral configuration. In eukaryotes, we do not see any intermediate forms with fewer then six distinct sequence types implying a common eukaryotic ancestor containing six distinct MCM subunits.M. maripaludis, we anticipate that subsequent studies will reveal the need for multiple MCM homologs in this archaeon, instead of the usual single homolog in most archaea. Similarly specialized roles have yet to be identified in the archaeal analogs of these proteins, but hints of additional function exist. Crenarchaeota exhibit differences in the PCNA interacting protein (PIP) box of proteins such as FEN1 and DNA polymerase B1-differences that are not found in the exclusively homotrimeric PCNA-containing eukaryotes, Euryarchaeota, Cenarchaeum, and NitrosopumilusThe different numbers of distinct but homologous subunits utilized in the formation of these three complexes in different taxa represent different levels of refinement in the structure and interactions of the complexes. Complexes made up of identical subunits retain the least possibilities for refinement and specialization, while complexes made up entirely of distinct subunits hold the most possibilities for refinement and specialized interactions of each subunit. For example, the eukaryotic RFCS subunits have been shown to play a role in cell cycle regulation, serving as sensors for important processes such as cell cycle arrest and DNA damage repair increasing. These refinements sometimes occur independently in multiple archaeal lineages with no evidence for HGT of distinct subunit types between different species. The agreement among our phylogenies and the concurance with other results supports the conclusions of Brochier et al. Nitrosopumilus and Cenarcheaum data are consistent with the proposal for an additional archaeal phylum, the Thaumarchaeota The archaeal branch always begins with complexes formed from exactly one PCNA, RFCS, or MCM distinct subunit type. Thereafter, the archaeal subunits duplicate and diverge, resulting in complexes with a greater level of refinement. In other words, the number of distinct subunits is always On the other hand, eukaryotes exhibit no changes in the number of distinct subunits. Instead, the level of refinement remains that of an ancestral Eukaryote from which the modern eukaryotes derive. In two of the cases, RFC and MCM, the ancestral eukaryotic complexes contained the maximum number of possible distinct subunits. In the other case, PCNA, the ancestral eukaryotic complex was made from three identical copies of a single distinct subunit. The same level of refinement has been retained in all modern eukaryotes surveyed in the literature When the number of distinct subunits increases, the duplication is followed by an initially faster evolution. This can be seen from the longer branch lengths that lead into some subunit clades, for example, the long branches of RFCS2 in Similar patterns of early complexity increase (subunit differentiation) in the common ancestral line of eukaryotes, followed by relatively stable conservation of the composition throughout subsequent speciation has been previously observed in other complexes including the Finally, it is interesting to consider the role of DNA processivity within the larger scheme of evolution in early life. Processivity was likely a requirement for the replication of large chromosomes on competitive timescales. One consequence of increased processivity in DNA replication would be the ability to retain additional copies of genes that could then potentially specialize and form more refined complexes. Ironically, the initial evolution of these three complexes may have provided themselves with the means necessary for their own subsequent refinements.Sequences were collected from the NCBI database and identified using BLAST ./raxmlHPC-PTHREADS -T 8 -f a -x 57843 -p 83755 -N 10000 -m PROTMIXDAYHOFF -s alignment_file.phyhttp://www.atgx-montpellier.fr/phyml/) the RAxML-generated trees with their corresponding multiple alignments as the initial input The trees presented in the main article were condensed in ARB Figure S1Uncondensed PCNA phylogeny.(0.03 MB TIF)Click here for additional data file.Figure S2Uncondensed RFCS phylogeny, rooted by RFCL.(0.03 MB TIF)Click here for additional data file.Figure S3Uncondensed MCM phylogeny.(0.04 MB TIF)Click here for additional data file.Figure S4Genome context for the Methanomicrobiales, Methanosarcinales, Methanosaeta thermophila, and uncultured archaeon RC-I. The key shows the genes that are conserved across contexts. Uncolored genes denote that there was no homolog among these seven contexts.(0.27 MB TIF)Click here for additional data file.Table S1List of sequences used in this study.(0.10 MB PDF)Click here for additional data file.


The low incidence of tetanus in developed countries has resulted in a decreased vigilance of this disease. This raises concern, as the prodromal stadium of a generalized tetanus infection may lack the characteristic paroxysmal muscle spasms. Tetanus can rapidly progress into life-threatening muscle spasms accompanied by respiratory insufficiency and/or autonomic dysfunction. This emphasizes the need for early diagnosis and treatment.A 4-year-old Caucasian boy presented with a one-week history of general malaise, mild fever, indolence and anorexia. He subsequently developed dysphagia, sialorrhoea, difficulties opening the mouth and eventually dehydration. Due to parental concerns about the boy's refusal of fluids, a pediatrician was consulted. At that time of presentation he showed signs of trismus and muscle rigidity. Together with the lack of immunization and a toe nail infection, this lead to the suspicion of a generalized tetanus infection. After sedation, endotracheal intubation and ventilation, passive immunization and initiation of antimicrobial treatment, he was immediately transferred to a pediatric intensive care unit (PICU) for further treatment. The frequency and severity of paroxysmal muscle spasms increased progressively during his PICU stay, despite high doses of sedatives. Not before two weeks after admittance, extubation and careful weaning off sedatives was achieved.Tetanus infection remains a rare but potentially lethal disease in developed countries. As the full scope of classical symptoms may be absent at first presentation, tetanus should always be considered in non-immunized patients with an acute onset of dysphagia and trismus. Clostridium tetani, of which its spores are ubiquitously distributed in our environment. This causes the inevitable increased risk of tetanus infection after wound contamination. Although tetanus has become rare in developed countries due to the successful implementation of primary immunization series, infants that are not immunized on religious or philosophical grounds are still at risk   and opisthotonus [In our patient, dysphagia, sore throat and difficulty opening the mouth (with the suspicion of a peritonsillar abscess) were the main complaints in the prodromal stadium of severe generalized tetanus. These and other complaints thotonus .In literature, only two adult case reports demonstrate the diagnostic challenge with oropharyngeal symptoms in generalized tetanus in developed countries ,6. In boAnother complicating matter is the distinction with localized tetanus, in which the distribution of tetanus toxin and muscle spasms is limited to specific body areas. Although these cases are usually associated with good outcome, rare cases with involvement of the cranial nerves  have a high risk of progressing to generalized tetanus with a high mortality . In tetaC. tetani bacilli can be achieved by either penicillin or metronidazole based regimens. A randomized clinical trial that compared these antibiotics for the treatment of generalized tetanus showed no differences with regard to in-hospital mortality or autonomic dysfunction [Appropriate treatment of generalized tetanus consists of neutralization of free circulating tetanus toxin, surgical debridement, eradication of the bacterial load and advanced supportive care. Human tetanus-specific immunoglobulin (TIG) is available for neutralization of tetanospasmin , althougfunction . In factfunction . Autonomfunction . Multidrfunction .The diagnosis of generalized tetanus in children remains a diagnostic challenge in developed countries, as the classical symptoms may be absent at presentation. Early recognition and immediate initiation of advanced critical care are necessary to prevent rapid clinical deterioration. Therefore, the differential diagnosis of non-immunized children with an acute onset of dysphagia and trismus should always include generalized tetanus.Written informed consent was obtained from the parents of the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.PRJ wrote the manuscript and was involved in the clinical care. TAHG and NJGJ provided clinical care to the patient and revised the manuscript. CHS was involved in the design and preparation of the manuscript. All authors read and approved the final manuscript.


Arteriovenous malformations of the lower urinary tract are uncommon lesions, usually presenting as scrotal masses. A case of recurrent acute scrotal pain mimicking testicular torsion that was attributed to the presence of an arteriovenous malformation of the spermatic cord is described. To our knowledge this is the first reported case of an arteriovenous malformation of the spermatic cord presenting with acute scrotal pain. Arteriovenous malformations (AVMs) occur mainly in the central nervous system, although they have been described in other organs as well. AVMs rarely involve the testis or the scrotal components, presenting mainly in the form of para-testicular or intra-testicular masses. This case reveals an uncommon clinical presentation of a non-palpable AVM of the spermatic cord. To our knowledge we present the first reported case of an arteriovenous malformation of the spermatic cord being the cause of recurrent acute scrotal pain.An otherwise healthy 22-year-old man presented with acute pain in his right hemiscrotum. He recalled having similar episodes of self-limited scrotal pain since his adolescence. The pain had been attributed by attending physicians to episodes of intermittent testicular torsion. He denied any history of trauma, urinary tract infection, sexually transmitted diseases, or voiding symptoms.Clinically both testes were intrascrotal, there was no swelling or signs of inflammation and the cremasteric reflex was intact. The right testis was, however, very tender in palpation. Both epidydimis were also normal and non-tender in palpation. Routine laboratory test and urinalysis were normal, and urine culture was sterile.Grey scale scrotal ultrasonography and Color Doppler Ultrasound (CDU) were performed. Sonography revealed no evidence of testicular tumor, varicocele or other pathology of the testis or epidydimis. Figure The patient underwent a diagnostic right scrotal exploration due to his persistent scrotal pain. Scrotal exploration revealed a macroscopically normal testicle, epididymis and spermatic cord. There was no evidence suggestive of intermittent testicular torsion, e.x "bell clapper" deformity of the testis, or torsion of testicular appendages.Four months later the patient presented with the same intractable right scrotal pain. Clinical examination and ultrasound investigation were insignificant. Orchiectomy was suggested this time and was performed through an inguinal incision after obtaining patient's consent. The surgical specimen consisted of a 16 ml testicle with its spermatic cord structures. The testis and spermatic cord were macroscopically unremarkable. Detailed pathology examination of the testicle and vas deferens revealed insignificant pathological changes, except for a small, smooth vascular mass (0.5 cm maximum diameter) in the lower part of the spermatic cord with the characteristics of an arteriovenous malformation Figure . The patArteriovenous malformations (AVMs) represent defects of the circulatory system that are generally believed to arise during embryonic or fetal development or soon after birth. The characteristic of AVMs is that arteries and veins are tangled and not connected by capillaries. The lack of capillaries allows blood traveling through these abnormal vessels to flow rapidly and under high pressure, thus preventing arterial blood from reaching the tissues leading to various degrees of ischemia and resulting pain. Histologically, the irregular vascular spaces are lined by nonproliferating and quiescent endothelial cells and are separated by fibrous stroma . The abnAVMs involving the lower urinary tract are uncommon as opposed to AVMs located in the Central Nervous System (CNS). Even more rare is the case of an AVM of the urinary tract presenting with pain. There is a report of ejaculatory pain caused by an AVM located between the prostate and seminal vesicles , while tArteriovenous malformations of the spermatic cord and testis are benign lesions consisting of complex tangles of enlarged dilated arteries and veins without intervening capillaries. In the cases published so far AVMs of the scrotal compartments present as either painless paratesticular masses ,5-7 or aScrotal AVMs appearing as masses can be detected by pelvic arteriography and managed by subsequent superselective embolization, however success is not always guaranted , necessiHowever in our case the small size of the lesion would make it impossible to detected by means of arteriography and to embolize successfully. The decision for orchiectomy was justified by the patient's intractable pain, suggesting some degree of testicular torsion considering his age, and the absence of findings on imaging studies.The case presented highlights a rare and unique cause of recurrent acute scrotal pain, attributed to the presence of an arteriovenous malformation of the spermatic cord. Therefore there is reason to believe that AVMs of the scrotum should be considered in the aetiology of otherwise inexplicable, recurrent scrotal pain. In that case consideration should be given to a trial of superselective angio-embolization of the lesion before one resorts to orchiectomy.The author(s) declare that they have no competing interests.PS was the treating urologist of the patient and performed together with AB both surgical procedures. PS has also drafted the manuscript. IR was the anaesthesiologist in charge in both operations performed. HA was the pathologist performing the stains and making the final pathology report for the case.All authors have have read and approved the final manuscript.


Patients in SLE2 (46 patients) and RA2 (31 patients) were not vaccinated. Throughout the following year, we studied parameters of disease activity and the occurrence of viral respiratory and bacterial infections in our patients. The vaccine was well tolerated in all cases. Vaccinated patients had significantly fewer occurrences of infections. Every viral and bacterial infection resulted in the worsening of the main disease. We believe that influenza vaccine is indicated for SLE and RA patients in stable condition. However, this decision must be made on a patient-by-patient basis. We plan to continue our study with the goal of formulating a better protocole for the clinical practice.The role of influenza vaccination in patients suffering from autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), has long been a subject of discussion. The risk of exacerbation of the main disease following vaccination is of particular concern, and needs to be carefully evaluated against the risk of disease flares as a result of infections. Our study included 69 SLE patients and 54 RA patients, all in stable condition. We split the groups into two subgroups each: patients in SLE


Currently, notalgia paresthetica (NP) is a poorly-understood condition diagnosed on the basis of pruritus, pain, or both, in the area medial to the scapula and lateral to the thoracic spine. It has been proposed that NP is caused by degenerative changes to the T2-T6 vertebrae, genetic disposition, or nerve entrapment of the posterior rami of spinal nerves arising at T2-T6. Despite considerable research, the etiology of NP remains unclear, and a multitude of different treatment modalities have correspondingly met with varying degrees of success. Here we demonstrate that NP can be caused by long thoracic nerve injury leading to serratus anterior dysfunction, and that electrical muscle stimulation (EMS) of the serratus anterior can successfully and conservatively treat NP. In four cases of NP with known injury to the long thoracic nerve we performed transcutaneous EMS to the serratus anterior in an area far lateral to the site of pain and pruritus, resulting in significant and rapid pain relief. These findings are the first to identify long thoracic nerve injury as a cause for notalgia paresthetica and electrical muscle stimulation of the serratus anterior as a possible treatment, and we discuss the implications of these findings on better diagnosing and treating notalgia paresthetica. Notalgia paresthetica (NP) is a poorly-understood condition presenting with pruritus, pain, and paresthesias in an area medial to the scapula and lateral to the thoracic spine. In addition, patients commonly report hyperpigmentation of the skin and other skin abnormalities. It was first described by Astwazaturow in 1934, but both etiology and prevalence of NP are unclear . PreviouThe long thoracic nerve arises from branches of cervical nerve roots C5-C7 and innervates the serratus anterior muscle. Injury to the long thoracic nerve or its cervical roots leads to dysfunction of the serratus anterior, with consequent scapular winging or loss of normal scapular protraction. We describe four cases of NP with known injury to the long thoracic nerve or the cervical roots giving rise to the long thoracic nerve, where transcutaneous electrical muscle stimulation (EMS) to the serratus anterior in an area far lateral to the area of pain and pruritus Figure  resultedAll patients reported pain with or without pruritus between the thoracic spine and medial scapula which grew progressively worse throughout the day. Furthermore, all reported exacerbation with activities requiring forward flexion of the arms such as driving, typing, washing dishes, or putting objects on a shelf. Patients did not report skin abnormalities, and no hyperpigmentation was observed. When temperature sensation in the area of maximal pain was tested with ice and compared to the contralateral region, all patients reported reduced sensation of cold without frank numbness. All had injury to the long thoracic nerve or the cervical roots at C5-7 giving rise to the long thoracic nerve, despite the absence of clinically-apparent scapular winging. There was no evidence of hyperpigmentation or other dermatologic symptoms. Prior to treatment with EMS all patients had seen multiple physicians and physical therapists, and tried multiple medications without relief. EMS of the serratus anterior muscle was conducted at 70 Hz with a pulse width of 300 μs to induce tetany of the serratus anterior. Stimulation was prescribed to be 30 seconds on and 30 seconds off for 15 minutes twice a day. All patients reported relief of pain beginning within days upon starting EMS, recurrence of pain if they discontinued stimulation for any prolonged period, and maintenance of analgesic effects with only intermittent stimulation necessary.The patients described in this case series provided informed consent for the manuscript and/or accompanying images to be published.A 39-year-old male presented with pain and pruritus medial to the left scapula several months after sustaining an ipsilateral broken clavicle and multiple rib fractures in a bicycle accident. He reported a marked increase in pain with activity utilizing his arms. Unlike his other pain complaints which improved following the accident, over time his left mid-scapular pain became more persistent and refractory. At 60 mg of duloxetine his medial scapular pain was replaced by a sense of itching, and then at 120 mg disappeared completely, although he ultimately could not tolerate duloxetine due to "personality changes". Electrodiagnostic studies revealed cervical root dysfunction at C5, C6, and C7 suspected to be a traction injury from landing on his shoulder after going over the handlebars of his bicycle. The patient began electrical stimulation of the serratus anterior muscle in fall 2007. He reported using the stimulation approximately 15 minutes once a day. He discontinued using it after attaining significant relief only to find that within several weeks the pain reappeared. Upon resuming intermittent stimulation his pain relief returned. By follow-up email in April 2009, he characterized his ongoing relief as "a highly significant improvement in the quality of my life.... Since I started to use [the stimulator] again I have been largely pain free". Treatment with muscle stimulation on an intermittent basis continues after 20 months follow-up.A 52-year-old male presented with chronic right shoulder and mid-scapular pain and pruritus of 20 years duration, status post ipsilateral thoracotomy and multiple shoulder surgeries, the most recent of which was 2 years previous. The thoracotomy scar ran perpendicularly across the expected path of the long thoracic nerve. He had failed therapy with amytriptyline, valium, skelaxin, fentanyl, dilaudid, methadone, gabapentin, and tizanidine. He reported no intercostal pain, and his notalgia paresthetica scapular pain was located several inches medial to the posterior margin of the thoracotomy scar. He began electrical stimulation of the serratus anterior muscle in July 2008. In a follow up by email in April 2009, he reported pain relief as an "8 on a scale of 10" and continues daily use of the stimulator. Follow-up after 9 months demonstrated ongoing use of the stimulator and continuing relief.A 40-year-old male physician presented with itching, paresthesias, and when symptoms were severe, pain medial to the right scapula of 20 years duration, status post a thoracotomy performed while a child. The ipsilateral thoracotomy scar ran across the expected course of the long thoracic nerve. He reported no intercostal pain, and his NP symptoms were located several inches medial to the posterior margin of the thoracotomy scar. Past treatments included topiramate and over-the-counter analgesics. He reported 70% relief of symptoms within the first two weeks of using EMS to the serratus anterior muscle and continues daily use as of September 2009. He continues to use the stimulator intermittently after 9 months of follow-up.A 34-year-old female presented with pain and pruritus medial to the right scapula, status post cervical fusions of C4-5 and C5-6 in 2006 due to degenerative joint disease. She also presented with neural foraminal stenosis in C6-7. Electrodiagnostic studies were consistent with dysfunction of C5, C6, and C7. Nonetheless, she denied radicular symptoms into the arms. Past treatments included gabapentin, desipramine, and multiple opioids. Stimulation of the serratus anterior muscle began in fall 2007. As of April 2009 she uses the stimulation for 15 minutes a day but is only intermittently compliant. When she discontinues stimulation the pain gets steadily worse prompting her to reinitiate stimulation, and continue until improvement of symptoms once again leads to discontinuation. Follow-up at 20 months following initiation of serratus stimulation revealed the patient to be using the stimulator intermittently with continued benefit.The effectiveness of EMS, applied directly to the serratus anterior, in providing significant pain relief supports our conclusion that notalgia paresthetica can be caused by long thoracic nerve injury and consequent serratus anterior dysfunction. These findings are the first to identify the association of long thoracic nerve and serratus anterior dysfunction as a possible cause for NP, with EMS as a possible treatment. Serratus dysfunction following long thoracic nerve injury and the resulting loss of scapular protraction is well understood to lead to scapular winging. Although scapular winging was not clinically apparent in these cases, subtle shoulder girdle asymmetry was appreciated following knowledge of the diagnosis. We hypothesize that even in the absence of obvious winging, loss of protraction may result in subtle retraction of the scapula. This in turn may generate either traction or compression of the cutaneous medial branches of the thoracic dorsal primary rami of spinal nerves as they course to the skin through muscles attached to the non-protracted scapula such as the rhomboid and trapezius. Traction on these nerves would be expected to refer pain to the area between the scapula and the spine, the region of symptoms in NP.This hypothesis would explain 1) the finding of long thoracic nerve and serratus anterior dysfunction in patients presenting with pain medial to the scapula; 2) the finding that the pain was exacerbated by activities with the arm flexed in front of the body, which loads the serratus; 3) the loss of temperature sensation in the area of pain medial to the scapular edge; 4) analgesia medial to the scapula in response to muscle stimulation of the much more lateral serratus; and 5) previous reports of the efficacy of anti-neuropathic pain medications such as gabapentin  and oxcaWe suspect that EMS contracts the denervated serratus which is over-stretched under the load of the arm in the chronic absence of physiologic contraction. The intermittent contraction associated with the direct electrical muscle stimulation results in a durable but ultimately reversible shortening of these overstretched muscle fibers leading to a serratus muscle of shorter length, more closely approximating its normal resting length. This shorter serratus muscle, while still unable to contract physiologically, may nonetheless hold the scapula in a more anatomically correct position compared to the completely stretched, unstimulated serratus seen following denervation. Further work is needed to confirm or refute these speculations.Our results are consistent with a case of NP and comorbid scapular winging reported in 2004, in which NP naturally resolved several months before scapular winging [Although our reports focus on long thoracic nerve and consequent serratus anterior dysfunction as an etiology for the clinical syndrome of NP, it is possible that serratus dysfunction is sufficient but not necessary. We have seen pain of similar presentation among those with high thoracic disk disease, and in two patients with severe scoliosis in whom the scoliosis appeared to result in impingement of the dorsal primary rami between two adjacent transverse processes. Our experiences, along with radiographic findings by others , also suAdditionally, while serratus anterior dysfunction may lead to improper scapular stabilization, an imbalance of other stabilizers such as the trapezius or other posterior scapular stabilizers may provide the same symptoms. The authors suggest a defect in scapular stabilization is just one of a variety of causes of irritation to the medial cutaneous branches of the dorsal primary rami, yielding pain and pruritus in the region between scapula and spine. This hypothesis on the pathophysiology of NP would predict that the injury to other motor nerves stabilizing the scapula  or the stabilizing muscles they innervate might lead to a similar clinical syndrome.Our findings highlight the need to establish normal scapulothoracic stabilization in patients presenting with NP. In cases of NP with a well-defined etiology of injury to the serratus anterior, the long thoracic nerve, or the cervical roots it arises from, EMS of the serratus anterior muscle may be a promising treatment modality for notalgia paresthetica.While pain and pruritus were both present in our patients, we note the lack of hyperpigmentation or other visible skin abnormalities which commonly accompany descriptions of notalgia paresthetica. It is unclear whether these skin abnormalities cause or are a result of the pain and pruritus in the symptomatic area. Immunohistochemical findings in NP have been inconclusive, with discordant conclusions on distributions of nerves in the symptomatic area -11. FurtIt is possible that analgesia from electrical muscle stimulation is a result of transcutaneous electrical nerve stimulation (TENS). A study investigating TENS for relief of NP symptoms where TENS was applied to the symptomatic area medial to the scapula found roughly 30% improvement in pruritus over two weeks , highligWe describe a novel and well-defined etiology of long thoracic nerve injury with consequent serratus anterior dysfunction in notalgia paresthetica, and EMS of the serratus anterior as a long-acting and effective treatment conferring the advantage of a once-daily treatment regimen. Future work might expand on this case series by: 1) conducting randomized blinded trials of serratus stimulation for NP; 2) conducting case-control studies among patients with NP to evaluate the association of NP and long thoracic nerve injury; 3) performing quantitative sensory testing (QST) to document the presence of nerve injury in the area of pain among patients with NP and potential QST improvements after treatment; and 4) exploring if loss of scapular stabilization by other means, e.g. injury to the dorsal scapular nerve and spinal accessory nerve, leads to a similar clinical syndrome with respect to NP. We also note that future studies of notalgia paresthetica would benefit immensely from a multidisciplinary approach integrating the expertise of dermatologists, neurologists, orthopedic surgeons, and pain management specialists.Notalgia paresthetica is a poorly-understood condition in which patients present with pain with or without pruritus and paresthesias in an area lateral to the spine and medial to the scapula. To date, there have been various proposed etiologies of NP and a multitude of different treatment approaches which have met with varying degrees of success. We describe a novel and well-defined etiology of long thoracic nerve injury with consequent serratus anterior dysfunction in notalgia paresthetica, and EMS of the serratus anterior as a long-acting and effective treatment conferring the advantage of a once-daily treatment regimen. The authors hypothesize that abnormal scapulothoracic stabilization creates traction or compression of the cutaneous medial branches of the thoracic dorsal primary rami. This then gives rise to the symptoms of this syndrome. EMS of the serratus anterior muscle is a possible treatment for NP that deserves further study.The authors declare that they have no competing interests.AG conducted all electrodiagnostic studies and treated patients clinically. IRC treated patients clinically. MB and SM helped with manuscript preparation, literature review, and background context for the manuscript. SM additionally provided material resources. CKW led the literature review and prepared the manuscript. All authors critically read and approved the final manuscript.


A spectrum effect was defined as differences in the sensitivity or specificity of a diagnostic test according to the patient's characteristics or disease features. A spectrum effect can lead to a spectrum bias when subgroup variations in sensitivity or specificity also affect the likelihood ratios and thus post-test probabilities. We propose and illustrate a methodological framework to distinguish spectrum effects from spectrum biases.Data were collected for 1781 women having had a cervical smear test and colposcopy followed by biopsy if abnormalities were detected (the reference standard). Logistic models were constructed to evaluate both the sensitivity and specificity, and the likelihood ratios, of the test and to identify factors independently affecting the test's characteristics.For both tests, human papillomavirus test, study setting and age affected sensitivity or specificity of the smear test (spectrum effect), but only human papillomavirus test and study setting modified the likelihood ratios (spectrum bias) for clinical reading, whereas only human papillomavirus test and age modified the likelihood ratios (spectrum bias) for "optimized" interpretation.Fitting sensitivity, specificity and likelihood ratios simultaneously allows the identification of covariates that independently affect diagnostic or screening test results and distinguishes spectrum effect from spectrum bias. We recommend this approach for the development of new tests, and for reporting test accuracy for different patient populations. It As the literature became increasingly confused, the recent paper by Goehring et al.  represenGoehring et al.  only proHere, we propose a methodological framework, derived from the approaches described (both applied together for the first time), to distinguish spectrum effects from spectrum biases. Our purpose is to isolate factors independently affecting the diagnostic accuracy of a test. This approach is illustrated by an application to the Papanicolaou smear test for detection of cervical cancer.We undertook a secondary analysis of the study by the French Society of Clinical Cytology to compare the efficiency of the conventional Papanicolaou smear, ThinPrep liquid-based cytology and the Hybrid-Capture II human papillomavirus test (HPV test) -26. The Sensitivity, specificity and likelihood ratios were used as indicators of test accuracy. Stratified analyses of these indicators were performed for the following variables: HPV test, study setting , age (< or ≥ 35 years), current smoking, European origin, educational level (higher education or less), menopausal status and contraception status . Confidence intervals for sensitivity and specificity were produced with the Wilson score method without continuity correction . ConfideThe logistic model for sensitivity and specificity proposed by Coughlin et al.  was usedThe approach proposed by Janssens et al.  was usedAll multivariable regression models initially included covariates modifying the test accuracy indicators with a p-value of 0.20 or less in logistic regression univariable analyses and first-order interactions between these covariates and the disease status (according to the diagnostic test or the reference standard). Final models were obtained by a grouped backward stepwise selection procedure. At each step, the variable with the least significant main effect was removed from the model if its interaction terms were associated with a p-value greater than 0.05. Even if not significant, all first-order interactions (for variables with a significant main effect) were conserved in the final model to obtain less biased estimations of group-specific likelihood ratios, as recommended by Janssens et al. .All analyses were performed using SAS software version 8 .Among the 1781 women included, 355 scored positive with the conventional Papanicolaou smear test (20%). Table Table Additional files We propose a methodological framework for identifying factors independently responsible for spectrum effects (i.e. which affect the sensitivity and specificity only) and for spectrum biases (i.e. which affect the likelihood ratios and post-test probabilities). This framework consists of double modelling, of sensitivity/specificity and positive/negative likelihood ratios respectively and therefore extends the stratified analysis of spectrum effects and biases proposed by Goehring et al. , taking Many authors report differences in diagnostic or screening test accuracy between subgroups, but few have used a multivariable modelling approach to identify factors responsible for differences in the performance of tests and confounding factors. A review of current practice, including investigations of so-called spectrum bias Table , shows tOur framework nevertheless presents some difficulties, mainly due to having to use non-trivial regression modelling. In particular, the simultaneous fitting of prior and posterior odds of the disease could be considered complex, as could the use of bootstrapping methods to construct confidence intervals for coefficients. Another difficulty is the management of interaction terms and the risk of colinearity between covariates included in the models. We chose to include only first order interactions between covariates and the disease status (according to the diagnostic test or the reference standard) because these interactions were the only ones relevant in the context of the differences in performance of a diagnostic or screening test. Usual recommendations concerning the practical implementation of regression analysis methods remain helpful in this context ,42. In pIn conclusion, we have shown the value of complementary and simultaneous modelling of sensitivity, specificity and likelihood ratios in logistic regression models: this approach can identify covariates that independently affect the accuracy of a diagnostic or screening test and can distinguish spectrum bias from spectrum effects. This approach appears preferable to subgroup analyses, which are classically recommended ,16 but fThe author(s) declare that they have no competing interests.JC conceived the study and its design, obtained funding source, provided study material, and administrative, technical, or logistic support. CE performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Logistic regression models for sensitivity, specificity and likelihood ratios of tests. The table presents Coughlin et al.'s model for sensitivity and specificity and Janssens et al.'s model for likelihood ratios .Click here for fileClinical reading: final multivariable regression model for sensitivity and specificity. The table presents the coefficients of the final regression model  and the method to calculate sensitivity and specificity from these coefficients.Click here for fileClinical reading: final multivariable regression model for the likelihood ratios. The table presents the coefficients of the final regression models  and the method to calculate likelihood ratios from these coefficients.Click here for file