Traditionally, treatment of dysphagia and dysphonia has followed a specificity approach whereby treatment plans have focused on each dysfunction individually. Recently however, a therapeutic cross-system effect has been proposed between these two dysfunctions. At least one study has demonstrated swallowing improvement in subjects who completed a dysphonia treatment program. However, we are unaware of any evidence demonstrating the converse effect. In this paper, we present a case-report of a 74 year old male who demonstrated improvement in selected vocal parameters after completion of a dysphagia therapy program.Dysphagia therapy resulted in improved laryngeal function in this subject. Results implicate improved vocal fold tension with increased glottal closure. Further investigation into the potential for this cross-system effect is warranted. Swallowing and phonation represent different functions of the aerodigestive tract that share a common subsystem. Consequently, an injury or disease process that impairs either of these functions has the potential to impair the other. Traditionally, therapy programs adhered to a treatment specificity principle addressing these dysfunctions as separate concerns. However, recent evidence suggests that a cross-system interaction may exist between them , implyinExploring potential cross-system treatment effects between dysphagia and dysphonia is appropriate among patients with head and neck cancer, as both disorders may occur concomitantly in 44% to 47% of this patient population ,4. This A 74-year old, retired, white male with treatment refractory dysphagia following chemo-radiation therapy for treatment of a T3N1M0 base of tongue squamous cell carcinoma, enrolled in an experimental dysphagia treatment research program at a university-affiliated speech and hearing clinic. Nine months prior to initiating this dysphagia therapy program he completed 40 doses of radiation therapy (7200 cGray to the primary tumor site and 5040 cGray to a secondary neck lymphnode) in conjunction with cisplatin chemotherapy. Following chemoradiation he completed two separate courses of dysphagia therapy. At time of enrollment in this dysphagia treatment program, he received nearly all nutrition and hydration via percutaneous gastrostomy tube. Daily oral intake was limited to approximately two ounces of pureed foods. Vocally, he complained of fatigue, pitch breaks, difficulty being understood on the telephone, and reduced singing ability.® NMES unit . The VitalStim device uses two pairs of electrodes placed vertically along the midline of the anterior neck to supply the electro-stimulation.He completed 15 days of an experimental swallowing exercise program supported with transcutaneous neuromuscular electrical stimulation (NMES) . No significant effects were observed for lowest attainable pitch  = 0.949, p = .513). Mean scores for those voice parameters demonstrating significant change within or across treatment sessions are presented graphically in Figures Endoscopic, perceptual, and instrumental vocal results supported the existence of a cross-system interaction between dysphagia rehabilitation and improved laryngeal function. Baseline endoscopic examination revealed significant supraglottic compression and a glottal gap during phonation, while post therapy examination revealed decreased supraglottic compression and glottal closure during phonation. Perceptually, the subject reported that his voice was louder, that he was able to sing in church again, and that others reported being able to better understand him on the telephone. Instrumentally, significant between session main effects were observed in both MPT  and the end of treatment measurements (session 15) were of primary focus, post hoc analysis was completed for MPT, highest attainable pitch, and habitual pitch. Analysis revealed that mean MPT increased significantly between session 6 and session 11 (p = .003) with this increase maintained through session 15 and the three follow-up sessions; mean highest attainable pitch increased significantly between treatment sessions 6 and 11 (p = .004), and demonstrated a trend toward significance between sessions 6 and 15 (p = .017) with the increase maintained through the one-week and one-month follow-up sessions, but not at six months post treatment. Furthermore, the increase in habitual pitch noted in session 11 was significant when compared to both baseline session 6 (p < .001) and last treatment session 15 (p < .001), but no significant change was noted between sessions 6 and 15 (p = 1.00).This patient demonstrated improvement in all swallowing measures immediately after completion of the therapy program Table . Total oThis case-report documented improvement in MPT and highest attainable pitch in one individual who completed a dysphagia therapy program. Although phonation and swallowing represent two different laryngeal functions, the improvements seen in this case report support recent evidence that treating deficits in one function may result in cross-system effects on the function not being actively rehabilitated .One interpretation from this case is that this dysphagia therapy program improved laryngeal muscle functioning. Since the dysphagia therapy included swallowing exercise paired with concurrent transcutaneous electrical stimulation over the larynx, either, or both of these treatment components may have resulted in improved laryngeal function. Swallowing exercise incorporated the "effortful swallow" technique which has been shown to prolong elevation of the larynx pre-swallow , and incTiming of the observed vocal changes supports the impact of the treatment program on laryngeal functioning. Voice changes were initially noticed at the end of the first week of dysphagia therapy. Next, significant increase in all voice parameters was noted at session 11, a time point corresponding with an increase in therapy program difficulty; e.g., increase in laryngeal exercise. Last, deterioration in vocal function noted at six months corresponded clinically with a decline in swallowing function, possibly reflecting a decrease in laryngeal maintenance exercise associated with a reduction of swallowing activity. When the timing of improvement and decline in voice characteristics is considered in conjunction with the improvement and decline in swallowing function, one can speculate that the combined effect of intensive exercise and NMES contributed to an underlying increase in laryngeal muscle function.This case-report raises important clinical questions regarding treatment effects which must be tested in future controlled studies. Future studies of potential cross-system effects should include replication in larger samples, across different diagnoses, with appropriate control conditions. Appropriate controls would include assessment of respiratory capacity before/following treatment, separation of exercise vs. NMES effects, and comparison to traditional voice therapy techniques.To our knowledge, this report is the first to document improvements in voice function during dysphagia therapy, supporting emerging evidence for a potential cross-system rehabilitative effect between dysphagia and dysphonia. The pattern of change observed in this case suggests a positive impact of intensive dysphagia therapy combined with transcutaneous NMES on laryngeal muscle function. Observations made in this case report suggest a new and interesting program of clinical research.NMES: Neuromuscular electrical stimulation. MPT: Maximum phonation time. MASA: Mann Assessment of Swallowing Ability . FOIS: F® device and all electrodes used to deliver the electrical stimulation were supplied by the Chattanooga Group . At the time of this study, Dr. Crary was the recipient of an educational grant from Chattanooga Group to support general research efforts in this area.The VitalStimLL treated the subject, analyzed the data under GC-M's supervision, and wrote the draft of the manuscript. GC-M and MC developed the treatment protocol, conceptualized the study, supervised the data analysis, and edited the manuscript. Additionally, MC completed baseline and follow-up dysphagia evaluations and supervised LL (a PhD student) in all aspects of the case study. All authors read and approved the final manuscript.All procedures used in this experimental dysphagia treatment protocol were approved by the local Institutional Review Board. The subject signed an informed consent prior to initiating the dysphagia therapy protocol.


The effects of different sensitization and allergen provocation regimens on the development of allergen-induced bronchial hyperreactivity (BHR) to histamine were investigated in conscious, unrestrained guinea-pigs. Similar early and late phase asthmatic reactions, BHR for inhaled histamine after the early (6 h) as well as after the late reaction (24 h), and airway inflammation were observed after a single allergen provocation in animals sensitized to produce mainly IgG or IgE antibodies, respectively. Repeating the allergen provocation in the IgE-sensitized animals after 7 days, using identical provocation conditions, resulted in a similar development of BHR to histamine inhalation. Repetition of the allergen provocation during 4 subsequent days resulted in a decreased development of BHR after each provocation, despite a significant increase in the allergen provocation dose necessary to obtain similar airway obstruction. The number of inflammatory cells in the bronchoalveolar lavage was not significantly changed after repeated provocation, when compared with a single allergen provocation. Finally, we investigated allergen-induced bronchial hyperreactivity by repetition of the sensitization procedure at day 7 and 14 (booster), followed by repeated allergen provocation twice a week for 5 weeks. Surprisingly, no BHR to histamine could be observed after either provocation, while the number of inflammatory cells in the bronchoalveolar lavage fluid after 5 weeks was enhanced compared with controls. These data indicate that both IgE and IgG sensitized guinea-pigs may develop bronchial hyperreactivity after a single allergen provocation. Repeated allergen exposure of IgE sensitized animals causes a gradual fading of the induced hyperreactivity despite the on-going presence of inflammatory cells in the airways, indicating a mechanism of reduced cellular activation.


We present an ambulatory cognitive state classification system to assess the subject's mental load based on EEG measurements. The ambulatory cognitive state estimator is utilized in the context of a real-time augmented cognition (AugCog) system that aims to enhance the cognitive performance of a human user through computer-mediated assistance based on assessments of cognitive states using physiological signals including, but not limited to, EEG. This paper focuses particularly on the offline channel selection and feature projection phases of the design and aims to present mutual-information-based techniques that use a simple sample estimator for this quantity. Analyses conducted on data collected from 3 subjects performing 2 tasks (n-back/Larson) at 2 difficulty levels (low/high) demonstrate that the proposed mutual-information-based dimensionality reduction scheme can achieve up to 94% cognitive load estimation accuracy. Following the successful demonstration of a P300oddball detector, many brAccurate assessment of cognitive load from ambulatoryelectroencephalogram (EEG) could lead to a wide variety of applications for braininterface systems . Of specThefocus of this paper is on feature and channel selection for real-time cognitivestate classification based on EEG in order to address items (1) to (4) inthis list. Note that nonstationarity could also be partially addressed to theextent that training session provided sufficiently rich data to representvarious sources of nonstationarity.The use of EEG, as the basis of assessment inbrain-computer interface (BCI) and AugCog systems, is predicated oncharacteristics such as good temporal resolution, non-invasiveness, low cost,and portability .Howeverinthis paper, our focus will be on dimensionality reduction , because criteriasuch as accuracy, real-time performance, and wireless networking require allrely on a set of compact features. Furthermore, choosing the most informativeand stable feature subset can also partly solve the subject-to-subjecttransfer, session to session transfer, and nonstationarity problem. The othermodules of the classification system were designed following well-establishedtechniques. For example, we employed a standard adaptive filtering techniquefor the removal of eye artifacts. We used FFT based power spectrum density(PSD) estimation procedures to estimate the power at various frequency bandsbroadly accepted to be associated with cognitive activity—theseestimates served as the primary features for classification. Additionally, weused Gaussian mixtures model (GMM), K nearest neighbor (KNN), and Parzen windowdensity estimate (Parzen) methods for classification. The PSD featuresconstitute a high-dimensional vector that contains information pertinent to theclassification of cognitive states, as well as irrelevant components and noise.Direct classification using such input features is undesirable since theunwanted components have an adverse effect on the overall classificationperformance and the generalization ability of the system. Consequently, apractical technique for extracting the relevant information from these featuresis necessary.From a machine learning point-of-view, an EEGcharacterization system (such as a BCI) requires a robust pattern recognitionsystem to assess the cognitive states or the intent of the operator. A typicalclassification system contains five parts: preprocessing, feature extraction,dimensionality reduction, classification, and postprocessing. Although anyimprovement in one of these parts can boost the performance of the system, We present the following: (1) a nonparametric sample estimatorfor mutual information that combines fast linear ICA solutions withsample-spacing entropy estimators to achieve computational simplicity; (2) EEGchannel selection and linear feature projection techniques based on mutualinformation to achieve dimensionality reduction for computational andgeneralization benefits.A mobile wireless sensor suite was assembled using avariety of off-the-shelf components. EEG was collected from 32 channels using aBioSemi Active Two system . VerticaAll channels reference the right mastoid. EEG isrecorded at 256 Hz sampling frequency while the subject is performing tasks withvarious cognitive loads. EEG signals are preprocessed to remove eye blinksusing an adaptive linear filter based on the Widrow-Hoff training rule. InformaParzen classifier is anonparametric approach to estimate the posterior probability of a featurevector . Please check. belonging to a given class, using Gaussian kernels inthis case. The estimate is a mixture-of-Gaussians with smooth contributionsfrom all samples and this represents a compromise between discrete votes fromnearest neighbors and the small number of Gaussian components of the parametricmodel. The details of the classifiers are discussed in the appendix. We nowdescribe the EEG channel selection and feature projection procedures in moredetail, as this is the main focus of this paper.In the design phase, the PSD features are used to rankand select EEG channels to reduce dimensionality. For this purpose, we assumethat training patterns are representative of the spectral patterns one wouldexpect in the performance environment. The final feature vector, with a muchlower dimensionality than the original input, is then fed to a committee ofthree classifiers. Since the distribution of the feature vectors is unknown, weused both parametric and nonparametric classifiers in the committee: GMM, KNN,and Parzen. The classification component signal flow is illustrated in feature extraction to mean dimensionalityreduction via linear or nonlinear projections. In our terminology, featureextraction is the process of determining candidate features from rawmeasurements .Feature extraction is the process of discovering astatistical pattern that can differentiate various classes that lead todistinct observations. In contrast, dimensionality reduction is a process offinding optimal feature vectors with reduced dimensionality from a large poolof candidates to keep the useful information and eliminate irrelevantinformation. This reduces the computational load and increases the robustnessof the classification system. Both feature extraction and dimensionalityreduction are important steps in classifying EEG signals. Note that someresearchers use the term The PSD features of EEG signals constitute ahigh-dimensional vector (5 frequency bands for 32 EEG channels yield 160features) that contains information pertinent to the classification ofcognitive states, as well as irrelevant components and noise. Directclassification using these raw input features yields poor generalizationperformance. We therefore propose a mutual information based technique topreserve channels and feature subspaces with maximal generalizable. We,therefore, propose a mutual information based learning technique for finitesize training sets to preserve channels and feature subspaces that maximize thegeneralization of discriminative power. Dimensionality reduction can beachieved by feature transformations. The transformation generates either a newfeature space, which is called feature projection; or generates a subset of theoriginal feature space, which is called feature selection. Feature selection isa special case of linear projections where the projection matrix is sparse withonly one unit per row. Linear transformations are widely used due to theirsimplicity and robustness. Therefore, they are often preferred tocomputationally complex and more fragile nonlinear counterparts, especiallywith small training sets.wrapper approach, is computationallyvery complex ; thus, is in feasible for large number of features. In contrast, afilter-based approach, which selects features by optimizing a given criterion,is independent of the classifier and is more flexible, but might not yieldclassifier-tuned optimal results. Since we use a committee of classifiers, thefilter approach is found more suitable.Optimal feature selection coupled with a specificclassifier topology, namely the Principal component analysis (PCA) is a widely useddimensionality reduction technique , 31;howquality of a low-dimensional feature vector f (either inselection or projection) is the mutual information (MI) between f and the classlabel c as definedbypc is the classprior, HS and IS denoteShannon's definitions of entropy and mutual information , Rf tr (Rf), E[ffT]E[ffT], RfRf. Given these matrices, the ICA solution can be easilydetermined using efficient generalized eigendecomposition algorithms.There are several efficient algorithms for solving thelinear ICA problem based on a variety of assumptions including maximization ofnon-Gaussianity, minimization of mutual information, nonstationarity of the sources,and so forth –48. The y such that(yi with the classlabel c can be computedusing (HS(yi) using allsamples regardless of class labels, and the conditional entropy of each classusing only the samples from the corresponding class.Once the ICA transform is determined and employed toobtain uch that holds ≤ ⋯ ≤ y(N). The m -spacingentropy estimator is given in terms of the sorted samples by . IS can be estimated using the method described in In real-time brain interface applications such as theambulatory cognitive load estimation problem considered in this work, thereduction in the number of input features is further motivated by the limiteddata acquisition and processing capabilities of the hardware. While collectingmeasurements from all EEG channels and then projecting their combined featurevector to a lower-dimensional linear or nonlinear manifold would be desirable,the hardware limitations and the prohibitive cost of collecting and processingeach additional EEG channel signal beyond the capacity of the hardware imposesus to focus on identifying the salient EEG channels that contain the mostuseful information for accurate estimation of the cognitive state in the designphase. Each channel yields several (five in our case) features and our goal isto find a quasi-optimal subset of EEG channels such that the MI betweenfeatures obtained from the selected channels and class labels is maximized forthe given number of channels (our hardware can handle up to 7channels):In order to determine an effective subset of theavailable features or channels (which encompass multiple features), we rank thechannels using a forward incremental strategy. We first select the channelwhose features have maximum mutual information with class labels and assign itrank 1. Rank 2 is assigned to the channel that has maximum MI when used inconjunction with the previously selected rank-1 channel. The procedure thenranks iteratively all features or channels taking into account the joint mutualinformation with previously ranked channels.d channel is theoptimum choice given the previous d -1 channels.While the top d channels do notnecessarily have to be the best d -subset,determining the latter requires a combinatorial search, and is infeasible forvery large dimensional situations (such as with 32 EEG channels or 160features). Using the incremental ranking strategy, the computational complexityis (n+1)n/2  instead of the (2n− 1) of exhaustivesearch. The search procedure could be modified easily to include a channelsubtraction phase where a previously ranked channel is removed to the unrankedset if it does not contribute to the joint information of the current rankedsubset. Another advantage of this method is that, using MI for ranking resultsin classifier-independent EEG channel ranking, thus it is computationallyefficient compared to wrapper techniques (it uses a simple MI estimator anddoes not require repeated classifier training).The procedure results in an ordering of EEG channelssuch that the rank- IS = IS for y = WTf. Furthermore, since  for i = 1,…,n, n is thedimension of the transformed features y. The projection matrix then consists of the d columns of theICA matrix W thatcorresponds to the top d components of y. This projection scheme is illustrated in Even after channel selection, further dimensionalityreduction might be desirable to improve classifier generalization performance.This can also be achieved using the maximum MI framework because an invertibletransformation does not change the mutual information. In particular, thelinear invertible ICA mapping guarantees that e, since  holds foy = WTf is the ICAtransformation.The approximations in Section 2 introduce anestimation bias to each MI evaluation step. From the derivation, we can seethat the bias, defined as the expected difference between the estimation andthe true MI, isIn this section, we present analyses carried out ondata collected from three subjects performing two tasks in multiple sessions(used for training and testing). Note that in many BCI experiments, reports areprovided in terms of leave-one-out performance on the complete data set due toscarcity. However, in our experience, this overestimates actual generalizationperformance (due to nonstationarity being nulled by the leave-one-outprocedure).S1 – S3 executed twomental tasks called Larson and n-back [attention, encoding, rehearsal, retrieval, and match. Thecomplexity of this task was manipulated by varying the interstimulus interval(low and high). In the n -back task,subjects are required to match the letter in either spatial location or verbalidentity in the previous trials. The easy task only requires comparing thecurrent stimuli with the first one, involving the combination of mentalactivities include attention and match. The difficult task requires comparingthe current stimuli with stimuli presented two trials previously, and involvesa complex combination of mental activities that includes attention, encoding,rehearsal, retrieval, and match. All three subjects performed both tasks at thetwo designated difficulty levels. Each case consists of about 3000 data samplesin a 150-dimensional feature space (30 EEG channels × 5 frequency bands)with two classes: low and high workloads. We applied the EEG channel-rankingalgorithm to the data to study the subject and task dependency of the selectedchannels. Prior work suggested that the optimal EEG channels may vary fordifferent mental tasks and different subjects.In this experiment, we demonstrate the performance ofthe channel selection procedure outlined to examine the effectiveness of theselection procedure outlined in Section 3.2. Based on hardware limitations forreal-time processing of EEG, the goal of identifying up to 7 channels out ofthe 30 available ones (we omitted 2 extremely noisy channels in this dataset)is set. Three subjects  n-back , 51, 52.Localn (n is the numberof the selected EEG channels). To examine the ability to select optimalchannels for all tasks and all subjects, we also used data from all subjectsand tasks to get another ranking called Globaln. An instance of Local 10  and Global 10  EEG channels are shownin Phy 7. Notethat the individual best channels vary for each subject and task combination asexpected. Nevertheless, the global ranking strongly coincides with theseindividual rankings as observed from We first applied the approach on individualsubject-task combinations, and obtained specialized EEG channel rankings,designated as To validate the proposed method, we employed acommittee of 3 classifiers: GMM, KNN, and Parzen, with majority vote anddecision fusion on the selected EEG channels. For jackknife evaluation ofperformance, the data for each case is partitioned to five sets and each set issaved for testing using the other four for training. The confusion matrices areestimated and the correct classification rates are calculated. Theclassification accuracies averaged over the five test sets are shown in To provide a wrapper-benchmark for the proposed ICA-MIchannel selection method, we also apply error-based ranking to the ICAprojections on the same EEG datasets. The error based ranking method uses thesame forward search strategy described in the algorithm of slow walking, navigating and counting, communicating with radio, and studyingwritten information while standing . Tasks are assigned class labels from 1to 4, corresponding to the assigned task. After preprocessing and featureextraction, approximately 6000 data samples were obtained, each with35-dimensional feature vectors (7 EEG channels with 5 frequency bands each) anda desired class label. In this experiment, the channels corresponded to sitesCZ, P3, P4, PZ, O2, P04, F7. These were selected based on a saliency analysisof EEG collected from various subjects performing cognitive test battery tasks[In this section, we demonstrate how an optimalICA-feature . Please check. subspace selected according to the mutualinformation criterion performs in reducing feature dimensionality withoutadversely affecting classification performance. Data was collected from onesubject as four predetermined ambulatory tasks were executed: ry tasks. A randoijth entry ofconfusion matrix P shows P(decide class i| true class is j)). Although inthis particular experiment keeping all 35 features yielded the bestperformance, the classification results illustrated here shows that thisfeature selection method is able to capture the low-dimensional relevantcomponents in the original feature space. This suggests that the additionalfeatures may introduce irrelevant and confusing information that might impairthe classification accuracy. In conclusion, mutual information based featureprojections are expected to eliminate unnecessary dimensions from the featurevector if not improve performance.The classification results for different featureranking methods are shown in The classification result for ICA-error ranking expectedlyexhibits better performance than that of ICA-MI,however, it takes much longer time.We described a framework based on mutual informationmaximization to solve the EEG feature/channel selection anddimensionality reduction problems in order to perform cognitive stateclassification. The initial real-time and offline experiments suggest that thedeveloped practical and fast algorithm that combines ICA transformations andsample-spacing entropy estimators can classify a small set of discretecognitive states with a reasonable accuracy when combined with 3 parametric andnonparametric classifiersThe experiments demonstrated that the important EEGsites are consistent with prior physiological knowledge —frontal sitesassociated with working memory tasks are rated high . Some clbetter features.As a comparison, we also implemented the wrapperapproach for feature/channel selection: use classification error as thecriterion. As expected, the wrapper approach exhibited better performance thanfilter approach because it is optimal to specific classifiers; however, it ismuch more slower, which makes it infeasible in practice with dense array EEGsystems that are becoming increasingly popular in BCI research.


Polymerase chain reaction (PCR) is very useful in many areas of molecular biology research. It is commonly observed that PCR success is critically dependent on design of an effective primer pair. Current tools for primer design do not adequately address the problem of PCR failure due to mis-priming on target-related sequences and structural variations in the genome.We have developed an integrated graphical web-based application for primer design, called RExPrimer, which was written in Python language. The software uses Primer3 as the primer designing core algorithm. Locally stored sequence information and genomic variant information were hosted on MySQLv5.0 and were incorporated into RExPrimer.RExPrimer provides many functionalities for improved PCR primer design. Several databases, namely annotated human SNP databases, insertion/deletion (indel) polymorphisms database, pseudogene database, and structural genomic variation databases were integrated into RExPrimer, enabling an effective without-leaving-the-website validation of the resulting primers. By incorporating these databases, the primers reported by RExPrimer avoid mis-priming to related sequences  as well as possible PCR failure because of structural polymorphisms ). To prevent mismatching caused by unexpected SNPs in the designed primers, in particular the 3' end (SNP-in-Primer), several SNP databases covering the broad range of population-specific SNP information are utilized to report SNPs present in the primer sequences. Population-specific SNP information also helps customize primer design for a specific population. Furthermore, RExPrimer offers a graphical user-friendly interface through the use of scalable vector graphic image that intuitively presents resulting primers along with the corresponding gene structure. In this study, we demonstrated the program effectiveness in successfully generating primers for strong homologous sequences.http://www4a.biotec.or.th/rexprimer.The improvements for primer design incorporated into RExPrimer were demonstrated to be effective in designing primers for challenging PCR experiments. Integration of SNP and structural variation databases allows for robust primer design for a variety of PCR applications, irrespective of the sequence complexity in the region of interest. This software is freely available at  Polymerase Chain Reaction (PCR) is a common laboratory technique in biological and medical sciences, with a wide range of applications such as DNA cloning, DNA resequencing for single nucleotide polymorphism (SNP) discovery and quantification of gene expression. In the design of any PCR experiment, the first step of designing oligonucleotide primer pairs is crucial for the success of the experiment. Selection of inappropriate primers can result in no amplification (PCR failure) or amplification of non-targeted regions (mis-priming). Therefore, the primer pair is tailored to be specific to the desired target sequence.The design of target-specific primers for PCR experiments typically requires consideration of different types of genomic information besides the target DNA sequence, such as repetitive DNA elements, intron/exon boundaries, and SNPs, which must be retrieved from various databases. All information is then combined to construct a sequence template for a primer design program. To confirm their specificity, designed primers are usually aligned against the corresponding genome sequence using tools like BLAST , BLAT 22, and PrTo resolve this situation, a number of automated primer designing tools have been developed based on Primer3 , ELXR 55, ExPrimWhile these programs provide some solutions related to the aforementioned primer design process, they are not always able to effectively design primers for two main problems, namely 1) no amplification due to severe mismatching, or lack of target and 2) mis-priming (non-specific binding besides the target sequence). These two problems lead to increased PCR failure rate . The firde facto standard [To address these issues, we present a graphical web-based tool, named RExPrimer, which allows users to automatically design PCR primer pairs for amplifying human genomic sequence without leaving the website. RExPrimer uses Primer3 as the design core, since this open source software has been continuously adopted by research communities as the standard -9. The nRExPrimer offers three modules of primer design: 1) for resequencing genomic DNA , 2) for SNP genotyping (gene based and region based), and 3) for oligonucleotide checking. The following types of identifiers are supported as input: HUGO gene name, NCBI Gene ID, and chromosomal locations. Since there are also other interesting non-gene regions, such as regulatory regions and intergenic regions, our program also supports arbitrary genomic regions as input based on chromosome location. For the SNP genotyping module, the program allows one additional input format as a SNP ID (rs-number) or set of SNP IDs. Because this application supports batch design for SNP genotyping primers, it can enhance productivity of high-throughput SNP sequencing projects.After the input is received, the local human genome database module is interrogated and the corresponding target sequence information, including the genomic sequence, annotations  and polymorphisms is subsequently identified and retrieved using query language. When a gene name is used as an input, more than one associated sequence, such as splicing variants may be found in the database. In this case, users have an option to choose the desired isoform. Then, the program supplies the option of selecting the gene regions to be amplified: whole gene , region of interest .CYP2D6, which has highly similar pseudogenes is illustrated.Non-unique PCR primers are one of the main factors that lead to PCR failure. Multiple regions of homologous, or near-homologous sequence in the genome can compete with the target sequence for primer hybridization leading to PCR artifacts. The human genome contains many pseudogenes, which can have near-perfect homology to their functional counterparts. To avoid designing non-specific primers for genes with pseudogene counterparts, the input sequence's pre-defined pseudogene sequences are retrieved from the pseudogene database module. Multiple sequence alignment of these pseudogene sequences and target gene including alignment scores by the MUMmer program  are dispAfter all of the target DNA sequence information is retrieved from the local database module, it is passed as input to Primer3 for the automatic generation of primer pairs. The RExPrimer user interface allows the users to specify parameters for Primer3 primer selection, such as product size, melting temperature (Tm), GC content. If desired, each designed primer could be screened against a repeat database to reduce nonspecific priming by choosing this available option. The amplicon size is user-defined according to the constraints of the experiment, e.g. accurate sequencing limits amplicons to a few hundred base pairs. If the input sequence is larger than a user-specified product size limit, the program automatically subdivides the template sequence into smaller segments with user-defined segment overlap size in order to ensure that the overlapping sequences can give high quality sequencing data. Primers are then designed separately for each overlapping fragment.Sequence variation in the 3' end, particularly the last three bases can severely disrupt primer hybridization and thus decrease PCR efficiency to the point of PCR amplification failure . Hence, Results reported from RExPrimer include two parts: SVG graphical display and HTML primer information summary. The graphical representation displays resulting primer pairs with their respective locations in the target gene in the same window Figure . If presWhen strong primer-destabilizing effects such as SNP-in-Primer are reported, users can immediately redesign primers without repeating the same procedure again. Optionally, the users can directly specify the approximate range of primer or target region, e.g., to avoid regions of high SNP density. The redesign of primer pairs can then be forced to a confined user-specified region.CYP2D6), which is thought as one of the most important enzymes for the metabolism of many clinically used drugs. CYP2D6 is highly polymorphic, which can be expressed as variation in CYP2D6-related drug metabolism among individuals. Besides a growing number of SNPs, numerous CNVs have been reported worldwide for this locus. Therefore, extensive population-wide study of genetic variations at CYP2D6 has medical importance. There are several problems that could interfere with the accuracy of genotyping, including:To validate whether RExPrimer is effective in designing primers in challenging PCR applications, RExPrimer was used to design primers for the Cytochrome P450 2D6 locus • CNVs from unequal crossing-over• SNPs reported to influence the enzyme activityCYP2D6 pseudogenes, SNPs, and CNVs. Mis-priming to pseudogenes can lead to artifactual PCR. SNPs and CNVs can lead to mismatching and non-primer binding to the desired region. The number of potential primers that are specific to CYP2D6 are thus limited.Existing primer design tools could have failed to consider the effect from CYP2D6 locus are shown in Table CYP2D6 before the primer designing step  and dystrophin (DMD) genes were used as internal controls . These results demonstrate that the primers designed by RExPrimer are suitable for CNV genotyping.In this study, primers were cautiously designed by considering every possible problem mentioned above using RExPrimer. The primers designed by RExPrimer for the p Figure . In ordeIn this study, we have developed a comprehensive tool for PCR primer design which covers a broad range of functionalities including resequencing target genes and SNP genotyping. The primer design pipeline is composed of three key components: 1) pseudogene detection, 2) primer design core, and 3) SNP-in-Primer/genomic variation notification. Additional file RExPrimer utilizes the publicly available sequence information including the human genome and annotation database, SNP databases, and the pseudogene database. By incorporating these databases and the Primer3 program, reliable and accurate primer designs can be achieved. The current pseudogene database module comprises of approximately sixteen thousand pseudogenes  while thCYP2D6 case study provided in the result section.Most primer design tools verify the uniqueness of the PCR target sequence by using UCSC In-Silico PCR  after thRExPrimer appends several key features before and after the primer design process using Primer3, enabling the selection of unique primer sequences and reliably amplifiable targets, which other currently available software cannot match. However, with the caveat that the primers designed are unique and the targets are amplifiable (no CNV or SNP-in-Primer), no extra claims for the actual performance of the primers beyond what is predicted by Primer3 are made. Hence, we have not attempted to determine the success rate of RExPrimer for designing primers, since the cost of performing multiple PCR experiments is not justified. Finally, RExPrimer has a major strength on its graphical web interface, especially the gene structure visualization, which makes RExPrimer intuitive and user friendly.Currently, RExPrimer can offer the oligonucleotide primer design for human genomic sequence in which SNP and pseudogene data are largely available. In the future, the program and locally built databases will be expanded to support primer generation for different organisms once their pseudogene and polymorphism data are readily available. To enhance the quality of primer design, the local SNP databases will be automatically updated to make use of the latest information, including as wide a range of population diversity as available.CYP2D6. We expect that RExPrimer is able to fill the gap and accomplish current needs for automated primer design procedure.RExPrimer is a one-stop tool for PCR primer design, which can support high-throughput resequencing and mutation screening research. The incorporation of large SNP and genomic variation databases make it possible to efficiently detect sequence variation in the designed primers that might cause PCR failure. The notification system of target pseudogenes before the primer design step helps users to select the appropriate target regions, which can significantly shorten the design process. RExPrimer is shown in this study to be indeed effective for designing primers for RExPrimer is a web-based application that was written in Python language. The web interface was created by Python Webware framework . This ap1. Users provide input parameters required to design primer pairs such as gene name, SNP ID, genomic location.2. The input parameters are used to query the targeted sequence from the local human genome sequence database, which was retrieved from NCBI and stored locally on our server.3. The target sequence is checked using MUMmer  for pseu4. RExPrimer prepares the input file for Primer3 from the primer conditions and target sequence. If the target sequence is greater than the target size parameter set in Primer3, the system will construct a set of overlapping fragments that covers the entire target sequence.5. Primer3 processes the input files prepared in step 4 and generates the primer pair results in Primer3 format.6. The primer pair results are then crosschecked against different locally constructed SNP and genomic variation databases covering a wide range of human SNP variation, CNV, and indel polymorphisms.7. Primer pairs are visualized on the locus-specific region using scalable vector graphics (SVG). The software also provides primer information in HTML format : Bioinformatics, available online at http://www.biomedcentral.com/1471-2105/10?issue=S15.Other papers from the meeting have been published as part of The feature comparison of existing primer designing software. The table displays the features provided in currently available primer designing software compared with those present in RExPrimer program. The cells with bold italic letters indicate unique features offered by RExPrimer.Click here for file


Campylobacter jejuni infection represents the most frequent antecedent infection triggering the onset of the neuropathic disorders Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). Although sialylated ganglioside-mimicking lipo-oligosaccharide (LOS) structures are the strongest neuropathogenic determinants in C. jejuni, they do not appear to be the only requirement for a neuropathic outcome since strains capable of their production have been isolated from patients with uncomplicated cases of enteritis. Consequently, other pathogen and/or host-related factors contribute to the onset of neurological complications. We have used comparative genomic hybridization to perform a detailed genomic comparison of strains isolated from GBS/MFS and enteritis-only patients. Our dataset, in which the gene conservation profile for 1712 genes was assayed in 102 strains, including 56 neuropathogenic isolates, represents the largest systematic search for C. jejuni factors associated with GBS/MFS to date and has allowed us to analyze the genetic background of neuropathogenic C. jejuni strains with an unprecedented level of resolution.The majority of GBS/MFS strains can be assigned to one of six major lineages, suggesting that several genetic backgrounds can result in a neuropathogenic phenotype. A statistical analysis of gene conservation rates revealed that although genes involved in the sialylation of LOS structures were significantly associated with neuropathogenic strains, still many enteritis-control strains both bear these genes and share remarkable levels of genomic similarity with their neuropathogenic counterparts. Two capsule biosynthesis genes (Cj1421c and Cj1428c) showed higher conservation rates among neuropathogenic strains compared to enteritis-control strains. Any potential involvement of these genes in neuropathogenesis must be assessed. A single gene (HS:3 Cj1135) had a higher conservation rate among enteritis-control strains. This gene encodes a glucosyltransferase that is found in some of the LOS classes that do not express ganglioside mimics.Our findings corroborate that neuropathogenic factors may be transferred between unrelated strains of different genetic background. Our results would also suggest that the failure of some strains isolated from uncomplicated cases of enteritis to elicit a neuropathic clinical outcome may be due to subtle genetic differences that silence their neuropathogenic potential and/or due to host-related factors.The microarray data has been deposited in NCBI's Gene Expression Omnibus under accession number GSE3579. Campylobacter jejuni infection is a leading cause of acute bacterial gastroenteritis worldwide  from the replicates for each gene represented on the microarray. The filtered data exported from BASE contains the average log ratio data for 1712 reporters. Log Ratio data was visualized and analyzed in TIGR's MultiExperiment Viewer (MEV version 3.0) [et al. [Microarrays were scanned using a Chipreader laser scanner  according to the manufacturer's recommendations. Spot quantification, visual inspection of potential outliers, and flagging of anomalous spots was performed using the program ArrayPro Analyzer . The microarray data exported from ArrayPro was imported into the BioArray Software Environment (BASE version 1.2)  and is aion 3.0) . Cluster [et al. , as impl [et al. .For statistical analysis of differential Log Ratio averages between groups of isolates we used the T-test implemented in TMEV, using a modified Bonferroni-corrected significance threshold of P < 0.05. For statistical analysis of differential gene conservation rates between groups of strains, gene conservation profiles were obtained from Log Ratio data by categorizing genes into "present", "divergent", and "absent" according to thresholds that were empirically determined previously ,37 To deGBS, Guillain-Barré syndrome;MFS, Miller Fisher syndrome; LOS, Lipo-oligosaccharide; CGH, comparative-genomic hybridization.The author(s) declares that there are no competing interests.ENT designed M-CGH experiments, carried out downstream data analysis, and drafted the manuscript. RRA performed hybridizations, performed preliminary data analysis and assisted with downstream data analysis. MK characterized the Japanese strains. PCRG characterized the Dutch and Curaçao strains. JHEN, MG, and AvB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. HPE, and NY participated in the conception and supervised the design of the study. All authors submitted comments on drafts and read and approved the final manuscript.Statistical assessment of genes displaying differential conservation rates among enteritic and neuropathogenic isolates. The table represents the statistical assessment of the conservation rates among the genes that have been observed to be differentially conserved enteritic versus neuropathogenic isolates.Click here for file


Medicaid recipients are disproportionately affected by tobacco-related disease because their smoking prevalence is approximately 53% greater than that of the overall US adult population. This study estimates state-level smoking-attributable Medicaid expenditures.We used state-level and national data and a 4-part econometric model to estimate the fraction of each state's Medicaid expenditures attributable to smoking. These fractions were multiplied by state-level Medicaid expenditure estimates obtained from the Centers for Medicare and Medicaid Services to estimate smoking-attributable expenditures.The smoking-attributable fraction for all states was 11.0% . Medicaid smoking-attributable expenditures ranged from $40 million (Wyoming) to $3.3 billion (New York) in 2004 and totaled $22 billion nationwide.Cigarette smoking accounts for a sizeable share of annual state Medicaid expenditures. To reduce smoking prevalence among recipients and the growth rate in smoking-attributable Medicaid expenditures, state health departments and state health plans such as Medicaid are encouraged to provide free or low-cost access to smoking cessation counseling and medication. Medicaid is a means-tested entitlement program that provides health care coverage to approximately 58 million low-income Americans, many of whom would otherwise be uninsured ,2. The MAs a percentage of state budgets, Medicaid expenditures increased from 8% in 1985 to 21.5% in 2006, surpassing elementary and secondary education as the largest single budget item  and the Behavioral Risk Factor Surveillance System (BRFSS) to update previous estimates of Medicaid smoking-attributable medical expenditures at the state level . These ewww.meps.ahrq.gov/mepsweb/.We used the 2001 and 2002 MEPS to develop a model that predicts smoking-attributable medical expenditures for the Medicaid population. MEPS is a nationally representative survey of the civilian, noninstitutionalized population that quantifies each participant's total annual medical spending, including expenditures from public- and private-sector health insurers and out-of-pocket payments. The data also include information about each participant's source of health insurance  and sociodemographic characteristics . Information about MEPS is available at The MEPS sampling frame is drawn from participants in the National Health Interview Survey (NHIS). NHIS is a nationally representative survey that collects data on selected health topics. Although MEPS does not capture information on smoking, self-reported smoking variables are available for a subset of adult NHIS participants (the Adult Sample File) and can be merged with MEPS data. We used responses to the question "Have you smoked at least 100 cigarettes in your entire life?" to differentiate between ever smokers and nonsmokers. We excluded from the analysis sample respondents with missing data on smoking variables  and those who did not receive Medicaid coverage. Our final MEPS-NHIS population included 1,588 adults with weighting variables that allowed us to generate nationally representative estimates of the adult, civilian, noninstitutionalized Medicaid population .Before constructing our national model, we used the Medical Care component of the Consumer Price Index to inflate all MEPS annual medical spending data to 2004 dollars.www.cdc.gov/BRFSS/. Although BRFSS does not collect medical expenditure data, it includes information about each participant's smoking status, insurance status (before 2001), and sociodemographic characteristics . Because these variables match those from MEPS-NHIS, we were able to construct an expenditure prediction model with MEPS-NHIS data and use the results to generate expenditure estimates for smokers and nonsmokers on the basis of state-representative population characteristics of BRFSS participants.The BRFSS is a state-based telephone survey of the adult (aged ≥18), noninstitutionalized population that tracks health risks in the United States. The most recent BRFSS surveys do not allow for stratifying participants by type of health insurance. This information was, however, available before 2001. Therefore, we used 1998-2000 BRFSS data to predict state-level medical expenditures for the Medicaid population. Information about BRFSS is available at As we did with our MEPS-NHIS restrictions, we excluded those with missing smoking data (≈1%) and those who did not receive Medicaid coverage. Our final BRFSS population included 16,201 adults with weighting variables that allowed us to generate state-representative estimates of the adult, noninstitutionalized Medicaid population .Estimating state-specific smoking-attributable medical expenditures for the Medicaid population involved 3 steps. First, we used MEPS-NHIS data to create a model that predicts annual medical expenditures for Medicaid recipients as a function of smoking status, body weight, and sociodemographic characteristics. Second, we used state-representative BRFSS data and results from our MEPS-NHIS national model to estimate the fraction of medical expenditures for Medicaid recipients that was attributable to smoking for each state. Third, we multiplied these fractions by previously published estimates of state-specific Medicaid expenditures to compute smoking-attributable Medicaid expenditures for each state. These steps are described in detail below.EXP = Pr(C × IPEXP + [1 − C]NIPEXP), where EXP represents predicted annual expenditures; Pr represents the predicted probability of positive medical expenditures during the year and is estimated with a logistic regression model; C represents the conditional probability of positive inpatient expenditures, given positive expenditures, and is estimated with a logistic regression model; IPEXP represents ordinary least squares (OLS)-predicted medical expenditures, given positive inpatient expenditures during the year; and NIPEXP represents OLS-predicted medical expenditures, given positive expenditures but no inpatient expenditures.We used a 4-part regression model to predict annual medical expenditures for each MEPS-NHIS Medicaid recipient. The 4-part regression approach was pioneered by authors of the RAND Health Insurance Experiment to control for several unique characteristics of the medical expenditures distribution and is now commonly applied to medical expenditures data ,13. The All OLS regression models are estimated on the logged expenditure variable to adjust for the skewness in annual expenditures . Logged expenditures are converted back to expenditures by using the homoscedastic smearing factor .Including dummy variables that indicate smoking status  in each regression model allowed us to quantify the effect of smoking on annual medical expenditures. In addition to smoking status, all regressions controlled for other variables assumed to influence annual medical expenditures, including self-reported body weight, sex, race/ethnicity, age, region of residence, education, and marital status. Regression models were estimated by using SUDAAN version 8  to control for the complex survey design used in MEPS-NHIS. We used the coefficient estimates from the MEPS-NHIS models to predict annual medical expenditures for each BRFSS Medicaid recipient. To do this, we multiplied each person's characteristics (the independent variables) by his respective coefficients generated from the 4 MEPS-NHIS regression models and combined the results with the equation above. Using the BRFSS weighting variables and each person's predicted medical expenditures, we computed total predicted medical expenditures for each state's Medicaid population.We estimated smoking-attributable medical expenditures as the difference between predicted expenditures for ever smokers and predicted expenditures for nonsmokers, leaving all other variables unchanged. This method allowed us to isolate the effect of smoking while maintaining any other population characteristics that may contribute to higher annual medical expenditures among smokers.For the Medicaid population in each state, the percentage of aggregate medical expenditures attributable to smoking was calculated by dividing aggregate predicted expenditures attributable to smoking by total predicted expenditures for adult Medicaid recipients in each state. Because BRFSS is limited to adults, our results should be interpreted as the fraction of adult medical expenditures that are attributable to smoking among adults in each state.For a variety of reasons, including the lack of data on institutionalized populations, MEPS national spending estimates (and state-level spending estimates based on MEPS) underestimate actual US health care spending . TherefoState-specific estimates of smoking prevalence among Medicaid recipients vary considerably across states and range from 35% (Mississippi) to 80% (New Hampshire) . NationaSmoking-attributable medical expenditures in the adult Medicaid population total $22 billion. State-level smoking-attributable medical expenditures among adult Medicaid recipients range from $40 million (Wyoming) to $3.3 billion (New York) .The 2000 Public Health Service (PHS) clinical practice guideline for treating tobacco dependence recommends individual, group, and telephone counseling, as well as 5 medications . TreatinThe growth rate in Medicaid expenditures has led the National Governors Association to propose a bipartisan plan to reform the program. A key element of this plan is to make Medicaid more effective and efficient by developing policies that will "maintain or even [improve] health outcomes while potentially saving money for both the states and the federal government" . One wayThe MEPS-NHIS national model that was used to calculate our state-level estimates is an improvement on a previous study that used data from the 1987 National Medical Expenditure Survey (NMES) to estimate smoking-attributable Medicaid expenditures . ResultsDespite these strengths, our study has several limitations. First, both the MEPS-NHIS and BRFSS are limited to noninstitutionalized populations, but we apply the resulting smoking-attributable fractions to expenditure estimates that include both institutionalized and noninstitutionalized populations. If these fractions are different for the institutionalized population, our expenditure estimates would be biased. Second, data limitations precluded us from quantifying smoking-attributable medical expenditures for smokers younger than 18 years and nonsmokers exposed to secondhand smoke. The effects of secondhand smoke on children's health are considerable . SecondhAn estimated 443,000 Americans die prematurely each year as a result of smoking or exposure to secondhand smoke . Medicai


We report a case of an unexpected coexisting anterior myocardial infarction detected by delayed enhancement MRI in a 41-year-old man following a presentation with a first episode of chest pain during inferior acute myocardial infarction. This second necrotic area was not initially suspected because there were no ECG changes in the anterior leads and the left descending coronary artery did not present any significant stenoses on emergency coronary angiography. Unrecognised myocardial infarction may carry important prognostic implications. CMR is currently the best imaging technique to detect unexpected infarcts. Myocardial infarcts are routinely detected by single photon emission computed tomography (SPECT) myocardial perfusion or CMR imaging. Rioufol et al. showed with intravascular coronary ultrasound (IVUS) that although only one lesion is clinically responsible during an acute coronary syndrome (ACS), ACS seems to be associated with pan-coronary destabilisation . CMR is A 41-year-old man was referred to our department with an inferior acute myocardial infarction. He was smoker with hyperlipidaemia (low-density lipoprotein cholesterol was 169 mg/dL). The time symptom onset to admission to the intensive care unit was ten hours. His 12-lead electrocardiogram revealed a normal sinus rhythm at 60 beats/min with persisting ST elevation and Q waves in the inferior leads. Emergency coronary angiography revealed an acute thrombotic occlusion of the second segment of the right coronary artery, a severe stenosis of a non-dominant and small circumflex coronary artery and diffuse plaques in all segments of the left anterior descending coronary artery (The patient remained well and asymptomatic during follow-up. CMR and myocardial scintigraphy were also performed in this patient 3 months later. CMR again showed persistent areas of delayed enhancement in the inferior wall transmurally and subendocardial enhancement in the anterior wall at the mid-ventricular level. Myocardial scintigraphy suggested myocardial ischaemia in the inferior, lateral and basal walls in favour of a restenosis, but this study did not detect any myocardial necrosis in the anterior wall (In a patient with acute inferior myocardial infarction, CMR was able to detect an unexpected coexisting area of necrosis in a different vascular territory. CMR findings in animals confirm that the subendocardial infarcts detected by CMR correspond to histologically defined myocardial infarcts . In our To conclude, the incidence of unrecognised coexistent myocardial infarcts may be underestimated and not infrequent. Of particular interest, this case highlights the possibility to detect these coexisting subendocardial infarcts using CMR. We believe that CMR has advantages, at least in patients with discrepancies between therapeutic success and missing clinical improvement.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.The authors declare they have no competing interests.EG, PC, HCG, MM Data acquisition, or analysis and interpretation, EG, ME, PC, MM Critical revision for important intellectual content, EG, ME Manuscript drafting, EG, HCG, ME, PC, MM Final approval of the manuscript.


In plants and animals innate immunity is the first line of defence against attack by microbial pathogens. Specific molecular features of bacteria and fungi are recognised by pattern recognition receptors that have extracellular domains containing leucine rich repeats. Recognition of microbes by these receptors induces defence responses that protect hosts against potential microbial attack.A survey of genome sequences from 101 species, representing a broad cross-section of the eukaryotic phylogenetic tree, reveals an absence of leucine rich repeat-domain containing receptors in the fungal kingdom. Uniquely, however, fungi possess adenylate cyclases that contain distinct leucine rich repeat-domains, which have been demonstrated to act as an alternative means of perceiving the presence of bacteria by at least one fungal species. Interestingly, the morphologically similar osmotrophic oomycetes, which are taxonomically distant members of the stramenopiles, possess pattern recognition receptors with similar domain structures to those found in plants.The absence of pattern recognition receptors suggests that fungi may possess novel classes of pattern-recognition receptor, such as the modified adenylate cyclase, or instead rely on secretion of anti-microbial secondary metabolites for protection from microbial attack. The absence of pattern recognition receptors in fungi, coupled with their abundance in oomycetes, suggests this may be a unique characteristic of the fungal kingdom rather than a consequence of the osmotrophic growth form. Drosophila melanogaster. At least 11 families of toll-like receptors (TLR) have been discovered in mammals and the MAMP recognised by each TLR has been well characterised Arabidopsis thaliana which recognises bacterial flagellin, though there are examples of plant pattern-recognition receptors that have no characterised motifs in their intracellular domain Arabidopsis thaliana number over 400 proteins Plants and animals regularly encounter large numbers of potentially harmful micro-organisms, yet the vast majority of these bacteria, fungi, or protozoa are unable to cause disease. The first line of defence for both of these groups of eukaryotes is known as innate immunity Cryphonectria parasitica is infected by a hypovirus, that alters its behaviour and pathogenicity Lysobacter enzymogenes can, for instance, act as a biological control agent for fungal diseases of plants Basal immunity is thought to represent the most basic method for an organism to protect itself against potential microbial pathogens, although both animals and plants also possess more specific immune responses to protect themselves against pathogens that have evolved the ability to circumvent this first line of defence Given the widespread nature of pattern recognition receptor proteins, we decided to test whether basal defence from infection by microbial pathogens occurs via leucine-rich repeat containing pattern recognition receptors across all eukaryotic kingdoms, or alternatively, whether osmotrophic organisms such as fungi and oomycetes have independently evolved alternative strategies for contending with microbial attack.In this study, we utilised a wide cross-section of available eukaryotic genomic sequence data to investigate the conservation of LRR-receptors among eukaryotic organisms and, in particular, to test for the occurrence of such receptors in a large selection of taxonomically diverse osmotrophic species for which completed genome sequences are available. In this way we have been able to investigate the potential innate immunity mechanisms that occur in the kingdom Fungi and in the distantly related oomycetes.Paramecium tetraurelia to just 5 in Cryptosporidium parvum. The numbers of LRR-containing proteins encoded by the genomes of fungi are comparatively low, ranging from six in the budding yeast Saccharomyces cerevisiae and Mycosphaerella fijiensis to 37 in Postia placenta  had less than ten of these genes within their genomes. Differences between taxonomic groups are even more striking when the number of LRR-receptors encoded by the genome of each species was compared  have one gene encoding an LRR-domain containing adenylate cyclase, but none of them are predicted to have a transmembrane helix. Alignment of fungal adenylate cyclase protein sequences showed that the sequence from M. fijiensis had an additional 560 amino acids at the N-terminus compared to closely related sequences, which is likely to be due to an error in the gene model. This region contains the predicted transmembrane helix. CNAG_04363 from Cryptococcus neoformans encodes an LRR-domain containing protein which is predicted to have one transmembrane helix, although there are no other closely related fungal sequences. The protein encoded by this gene is predicted to have an LRR N-terminal domain in the middle of the protein. If we assume the position of this domain shows the true N-terminus of the protein, then this gene model also has an incorrect extension at the 5′-end which contains the predicted transmembrane domain. Removing these two genes from the list of fungal LRR-receptors leaves us with just five fungal examples of these genes from four species . None of these sequences are predicted to have any other functional domains. There are examples of secreted LRR domain-containing proteins involved in innate immunity, for example, plant polygalacturonase inhibiting proteins We set out to investigate the occurrence of leucine-rich repeat (LRR) domain containing receptors throughout the available eukaryotic genomic data sets. The 101 species selected for this study , represeplacenta . In factcompared . LRR-recng genes  and thesMonosiga brevicollis and all species of oomycetes. There are also examples of LRR-protein kinase receptors in some metazoan species. The number of LRR-protein kinase receptors in plants range from 70 in Selaginella moellendorffii to 301 in Populus trichocarpa, consistent with their importance as receptors in a number of physiological roles including defence against pathogens and development Chlorella NC64A. Interestingly, we identified Toll-like receptors in most lineages of metazoa, including Ecdysozoa, Lophotrochozoa and Deuterostomia, but not in the choanoflagellate Monosiga brevicollis, the placozoan Trichoplax adhaerens or the cnidarian Nematostella vectensis. To confirm the lack of LRR-kinase receptors in fungi, we identified all fungal genes encoding proteins containing both LRR and protein kinase domains . It is difficult to provide phylogenetic evidence showing the evolutionary relationship between LRR-domains, due to problems in sequence alignment because of the repetitive nature and high diversity of the sequences Phytophthora ramorum and chlorophyte species. None of these closely related sequences contain any other functional domains or transmembrane regions.As mentioned previously, the number of LRR-domain encoding genes within fungal genomes is less than other eukaryotes sampled. Furthermore, these genes can be related, based on sequence similarity, to genes of known function. These include adenylate cyclase, protein phosphatase 1 regulatory subunit, septation initiation network scaffold protein and glucose-repressible alcohol dehydrogenase transcriptional effector. The structure of adenylate cyclases in fungal species is particularly interesting. Fungi are unique amongst the species studied, in that their genomes contain adenylate cyclase-encoding genes that contain LRR-domains Saccharomyces cerevisiae, the adenylate cyclase directly interacts with the alpha subunit of a heterotrimeric G protein and RAS proteins through the G-alpha binding and Ras association domains, respectively Magnaporthe oryzae, the protein phosphatase domain of the adenylate cyclase MAC1 has been shown to interact with both a MAP kinase and serine/threonine kinase Candida albicans, the adenylate cyclase Cyr1p is involved in detecting the presence of bacteria using the LRR motif The typical domain structure of a fungal adenylate cyclase is shown in Podospora anserina could also be involved in innate immunity Podospora anserina, Cochliobolus heterostrophus, Botrytis cinerea, Sclerotinia sclerotiorum), although proteins containing NACHT and WD-repeat domains but not HET domains were found in another 13 species. So if STAND domain proteins were involved in innate immunity, this type of immunity is also restricted to only certain species of filamentous ascomycete.In fungi, cell fusion between genetically unlike individuals triggers cell death, which is known as an incompatibility reaction. It has been proposed that five genes that control this process in the fungus Phytophthora sojae) also has a protein kinase domain, whereas in the other stramenopile species none of the LRR-receptors have attached functional domains. There also seems to be little sequence similarity, based on BLASTP Phytophthora ramorum that had incorrect gene models where an in-frame intron had been introduced that removed the region containing the transmembrane helix. Hyaloperonospora parasitica but also a number of paralogous duplications within the Phytophthora lineage. The transmembrane domains in the oomycete LRR-receptor kinases are between the LRR and protein kinase domains, suggesting the latter are likely to be extracellular, as is the case with the plant LRR-receptor kinases Analysis of sets of predicted proteins showed that all species of stramenopiles that were analysed encode LRR-receptors in their genomes . There iThe innate immunity system in plants and animals involves recognition of microbial specific molecules (MAMPS) by pattern recognition receptors containing extracellular LRR-domains Candida albicansC. albicans adenylate cyclase can detect the breakdown products of bacterial peptidoglycan C. albicans may have the capacity to detect and respond to bacteria, which may be potential pathogens or competitors. In our study we have shown that possession of these unusual LRR-containing adenylate cyclases is uniquely found in fungi and is widespread among diverse fungal species, suggesting that it may constitute a conserved attribute of these signalling proteins within fungi. Adenylate cyclase is responsible for catalysing the synthesis of cyclic AMP from ATP. In fungi, cAMP-dependent protein kinase A signalling pathways play roles in control of morphogenesis and the response to starvation C. albicans, but the possession of such domains in adenylate cyclase genes from such a wide group of fungal species suggests that the biological significance of the LRR domains should be explored further.Our comparative genomics study has shown that classical LRR receptors are largely absent from the kingdom Fungi, being completely absent from 45 of the 49 genome sequences that we sampled. The restricted distribution of LRR-receptor encoding genes in four genome sequences was, furthermore, largely confined to chytrids and zygomycota, with normally only a single example per genome. This is strikingly different from the situation in both plants and animals where dramatic gene family expansion has occurred and this suggests that widespread use of LRR receptors is not a feature of fungi. How then do fungi perceive and respond to microbial pathogens? One clue to how this might occur comes from the recent functional study of the role of LRR domains in the adenylate cyclase protein of The large-scale absence of LRR receptors in fungi is, however, also consistent with fungi possessing alternative mechanisms to detect potential microbial pathogens and defend themselves from invasion. Filamentous fungi are well known to produce a wide range of secondary metabolites, some of which have anti-microbial or antibiotic properties, such as the well-known β-lactam antibiotics like penicillin Interestingly, our analysis also showed that all oomycete species in this study possess LRR-receptors with intracellular protein kinase domains that appear similar to those found in plants. This striking contrast with the absence of pattern recognition receptors in fungi, suggests that the osmotrophic growth habit, shared by these distinct organisms For each species used in this study, sets of predicted proteins were downloaded from the sources listed in ftp://ftp.sanger.ac.uk/pub/databases/Pfam). This library contains 10,340 protein models constructed from manually curated multiple alignments and covers 73% of proteins in UniProt http://hmmer.janelia.org/). Default thresholds were used, which were hand-curated for every family and designed to minimise false positives The Pfam-A library from release 23.0 of the Pfam database was downloaded from the Pfam website (http://www.cbs.dtu.dk/services/TMHMM/). TMHMM uses a membrane topology prediction algorithm based on a hidden Markov model The sequences of predicted proteins for all 101 genomes were analysed for the number and position of transmembrane helices using a local version of TMHMM , producing 329 clusters and 3,562 singletons.The protein sequence containing the leucine-rich repeat domain was extracted for each protein. These 8,001 sequences were clustered into groups of similar sequences using OrthoMCL To rule out the possibility that any sequences of interest came from contaminating bacterial or plant DNA, rather than the species for which a given genome had been sequenced, the DNA sequence of all fungal and oomycete LRR-containing genes was compared to the genome sequences of 1,337 species of bacteria and three species of higher plant using BLASTN Figure S1Phylogenetic tree constructed using sequences that show the greatest homology to a set of LRR-protein kinases found specifically in a few species of filamentous ascomycetes. Bootstrap support scores of 50 or above shown for each branch. Taxa are labelled with protein IDs  or GI numbers for other sequences from NCBI database. Fungal sequences are indicated by a blue bar. Note: CHG08425 from Chaetomium globosom has an LRR domain, but lacks a protein kinase domain.(2.64 MB TIF)Click here for additional data file.Figure S2Phylogenetic tree constructed using adenylate/guanylate cyclase domains from representative species in each of the taxonomic groups used in this study. Bootstrap support scores of 50 or above shown for each branch. Protein IDs (from database sources listed in (4.46 MB TIF)Click here for additional data file.Figure S3Phylogenetic tree constructed using LRR-domain sequences from ORTHOMCL1 cluster (see text for details). Bootstrap support scores of 50 or above shown for each branch. Protein IDs (from database sources listed in (0.86 MB TIF)Click here for additional data file.Table S1Species used in this study(0.02 MB XLS)Click here for additional data file.Table S2The number of LRR-domain containing proteins identified in each species(0.02 MB XLS)Click here for additional data file.Table S3The number of LRR-receptors identified in each species(0.02 MB XLS)Click here for additional data file.Table S4LRR domain containing receptors in fungi(0.02 MB XLS)Click here for additional data file.Table S5LRR domain proteins containing a signal peptide in fungi(0.02 MB XLS)Click here for additional data file.Table S6The number of Toll-like receptors identified in each species(0.02 MB XLS)Click here for additional data file.Table S7The number of LRR-receptor kinases identified in each species(0.02 MB XLS)Click here for additional data file.Table S8LRR kinase proteins in fungi(0.02 MB XLS)Click here for additional data file.Table S9Protein domain structure of fungal LRR-domain containing adenylate cyclases(0.04 MB XLS)Click here for additional data file.Table S10Protein domain structure of fungal NACHT domain containing proteins(0.05 MB XLS)Click here for additional data file.


Transitional cell carcinoma affecting the upper urinary tract, though uncommon, constitutes a serious urologic disease. Radical nephroureterectomy remains the treatment of choice but has undergone numerous modifications over the years. Although the standard technique has not been defined, the laparoscopic approach has gained in popularity in the last two decades. The most appropriate oncological management of the distal ureteral and bladder cuff has been a subject of much debate. The aim of the nephroureterectomy procedure is to remove the entire ipsilateral upper tract in continuity while avoiding extravesical transfer of tumor-containing urine during bladder surgery. A myriad of technical modifications have been described. In this article, we review the literature and present an overview of the options for dealing with the lower ureter during radical nephroureterectomy. Primary urothelial carcinoma of the upper urinary tract accounts for about 5% of all renal and urothelial malignancies. Though relatively uncommon, the incidence of upper tract transitional cell carcinoma (TCC) appears to be slowly increasing –3. WhileLe Dentu and Albarran performed the first complete open nephroureterectomy (ONU) for upper tract TCC in 1898 , but it The procedure may be undertaken open or laparoscopically. The “standard” laparoscopic nephroureterectomy (LNU) technique has not been defined and continues to evolve. Variationsinclude utilization of a pure laparoscopic technique versus a hand-assisted technique;transperitoneal versus retroperitoneal; and a myriad of approaches to deal withthe lower ureter . Indeed,en bloc, “closedsystem” specimen following controlled occlusion of the ureteral orifice.Continuity of the specimen, though desirable, may be conceded to aid ease ofextraction as long as the distal ureter is ligated and divided at tumor-freelocation. The key issue is to avoid extravesical urine contact and adherence tothese principles will reduce risk of spillage or seeding of tumor cells.The risk of tumor recurrence within the residual ureteralstump/periureteral meatal region in cases of incomplete upper tract removal isoften cited as between 30 and 64% –12. TotaThe following techniques, along with some minor variations, have been advocated in order to accomplish distal ureteral removal:“pluck” technique,intussusception technique,pure laparoscopy,open resection.This involves the transurethral disarticulation of the intramural ureter along with the ureteralorifice (UO) using a standard resectoscope loop or Collin's knife, usuallyprior to performing the laparoscopic nephrectomy. The UO is resected deep intoextravesical fat allowing subsequent “plucking” of the entire ureter fromabove. Though the original intention was to decrease morbidity by avoiding asecond lower abdominal incision during open surgery, this is a less compellingargument with laparoscopic surgery as specimen extraction mandates a largerincision.Following UO resection, the patient is put in the flank position for LNU. The kidney is mobilized along with the ureter down to the level of the pelvic brim. Gentle traction on theureter should result in the removal of the entire length of ureter down to thelevel of the detached distal ureter. An indwelling Foley catheter is left inthe bladder for at least 7 days. The major concern related to this technique isthe risk of tumor cell spillage into the retroperitoneum with subsequentseeding and local recurrence.A number of authors have published small retrospective series employing the pluck technique and report no local disease recurrences –15. Palon = 30) or the pluck technique (n = 20) [Nevertheless, the propensity for urological malignancies, including TCC, to seed is wellrecognized . Not sur(n = 20) . The traFurthermore, utilizing the pluck technique is likely to permit continued urine extravasation from the cancer-bearing ureter , 25.Add is then passed through thecystoscope to ligate and occlude the ureteral orifice. The bladder cuff is thencompletely circumscribed down to perivesical fat and subsequently removed enbloc following kidney mobilization. The Endoloop also acts as a markerensuring complete specimen removal. A similar procedure has been describedusing a transurethrally placed 5-mm laparoscopic hem-o-lok clip on the ureteralstump, as an alternative to the Endoloop, to ensure a closed system [Various modifications on the pluck theme have beendescribed in an attempt to minimize tumor spillage. Tan et al. advocatedcompletion of the laparoscopic nephrectomy first with the clipping of theureter to prevent distal migration, followed by transurethral Collin's knifemobilization of the distal ureter and bladder cuff . Recentd system . Boththd system , 27.unroofing technique has been describedand popularized by the Washington Universitygroup [The ureteral tygroup . This catygroup . Employmtygroup . LNU toHALNU offers distinct advantages when consideringhow best to manage the distal ureter. Firstly, the requirement of a longerincision to facilitate a hand port will allow improved access to the bladderand distal ureter, offering the surgeon the option of either an extravesicalapproach, open transvesical cystotomy, or detaching the ureter using atransurethral technique . In addIn summary, therefore, pluck techniques arecontraindicated in the presence of lower ureteral tumor and widespread urinarytract carcinoma in situ. Coexistent bladder TCC should preclude the situation,where the bladder is left “open” with potential exposure of the perivesicaltissues to malignant-cell laden urine. Patients with previous pelvicirradiation and active inflammatory conditions of the bladder are probably notideal candidates for endoscopic procedures. Blind pulling of the ureter isdiscouraged to minimize ureteral tearing and the possibility of residualtissue. While retrospective studies have not confirmed the superiority of theopen over the pluck method, the oncologically safe practice of maintaining a “closed” system is preferred and retroperitoneal exposure to potentially tumor cell-laden urine for any durationof time, in our opinion, is best avoided.This technique, first described by McDonald in 1953, has undergone various subsequent modificationsaround a central theme of ureteral ligation and removal either by stripping orintussusception . PrincipThough its long-term safety during LNU has not beeninvestigated, even after 5 years' followup, Clayman et al. reported no pelvictumor recurrences in 14 patients undergoing ureteral stripping during ONU . This isSince the ureter is transected, it is contraindicated for ureteral tumors and primarily confined to low-grade renal pelvic tumors. Additionally, any cause for pelvic fibrosis,such as previous surgery or irradiation and retroperitoneal fibrosis, mayfurther increase the risk of retention of ureteral remnant. Bladder mucosa isexposed to ureteral mucosa with the potential for seeding. Its main drawback isits failure to guarantee adequate excision of the intramural ureter and bladdercuff, potentially resulting in tumor recurrence, and is thus unlikely to gainuniversal acceptance following LNU.A completely laparoscopic approach offers distinct advantages in termsof blood loss, postoperative pain, recovery times, and equivalent short- andintermediate-term oncologic efficacy. The kidney and ureter are mobilized inthe standard fashion and the distal ureter may be secured by one of 2 main techniques:laparoscopic extravesical stapling ofthe distal ureter , 39;transvesical laparoscopic detachment and ligation (Clevelandapproach) , 41.This method is usually combined with a ureteral unroofing procedure. Theureter is clipped early and dissected caudally until it diverges to merge withthe detrusor muscle fibers at the ureterovesical junction (UVJ). Gentletraction on the ureter will tent up the wall of the bladder at the UVJ enablingplacement of a 12-mm laparoscopic GIA tissue stapler  or a large hem-o-lok clip. If desired, an ontable bladder fill with saline/indigo carminesolution may be performed to exclude extravasation and/or a delayed cystogramis obtained before catheter removal. A more recent trend has been to performstapling of the bladder cuff as the initial step, followed by a transurethralresection of the ipsilateral UO till the staple line is reached .Laparoscopic stapling has manifest advantages. It may help reduceoperative time and facilitates a minimally invasive procedure while maintaininga “closed” urinary tract, thus preventing tumor spillage. There are, however,numerous concerns related to staple usage. Deployment of the stapler may prove awkward in the restricted pelvic space. An error in judgment may result in either part of the intramural ureterbeing left behind or inadvertent injury to the contralateral UO. In addition,the stapled margin cannot be assessed histologically. The Nagoya group reported stone formation at the staple line in 3 (5.7%) of 53 patients at an average of 20 monthspostoperatively . Using an = 12) with the traditional transvesical open excision(n = 12) at nearly 4 years followup [Romero et al. compared long-term safety and oncologic efficacy of extravesicallaparoscopic stapling  . TheblaThis novel technique of securing the distal ureter and bladder cuff usingtransvesically placed laparoscopic ports was described by Gill et al. and isalmost exclusively employed by the Cleveland clinic group , 41. In The authors claim that the transvesical technique adheres to general oncologicalprinciples of complete and controlled en bloc specimen extraction. The ureteralcatheter and Endoloop occlude the ureter, thereby reducing urine leakage. Anindwelling ureteral catheter may aid identification and mobilization of theureter during the laparoscopic procedure. Complete retrieval is confirmed byvisualization of the Endoloop. However, this may be a difficult technique tomaster for most urologists and operating time is usually lengthened by 60–90 minutes , 47.Othn = 12) or transvesical laparoscopic detachment of the distal ureter(n = 36) [LNU with the transvesical technique has been evaluatedagainst ONU in a retrospective series . Gill e(n = 36) . FollowiRecently, Cheng et al. described a similar technique utilizing a pneumovesicum to securethe UO and bladder cuff . FollowiThe open technique (either2-incision or extended single incision) forms the standard against which alltechniques are measured. Typically performed after nephrectomy, it can beperformed through a lower midline, modified Pfannenstiel, or Gibson incision.The lower ureter is clipped, dissected free, and removed in continuity with thebladder cuff. The bladder cuff may be secured extravesically (using a rightangle clamp) or via an anterior cystotomy. The en bloc specimen is deliveredthrough the same incision.In the review of 252 patients performed by Hall et al., 194 patients had undergone ONU with openbladder cuff excision for TCC and showed excellent long-term local control . KlingleA recent multicenter retrospective Belgian study analyzed 100 patients following LNU for TCC . Of thesThe open technique is not without its pitfalls. The “blind” extravesical clamping may compromise the contralateral UO and does not inevitably guarantee adequate bladder cuffretrieval . An anteGiven the uncommon nature of upper tract TCC, there is a scarcity of prospective randomized studies withlong-term followup comparing both modalities of nephroureterectomy. The “goldstandard” open surgery offers excellent access but at the expense of increasedpatient morbidity. Since being first described in 1991 by Clayman et al.,increasing surgical experience and equipment quality has seen LNU emerge as aviable option with the express intention of minimizing surgical morbiditywithout compromising oncology outcomes.Rassweiler et al. performed a meta-analysis comparing ONU and LNU, which included 1365 patientsfrom 85 studies . LNU wasn = 67) [n = 49) [n = 60) [n = 46) [n = 224) [Further, recent smaller retrospective comparisons by McNeill et al. (n = 67) , Tsujiha[n = 49) ,Taweemo[n = 60) , Rouprêt[n = 46) , and Mann = 224)  havedemn = 224) .Port-site seeding following LNU remains a concern but is fortunately rare if appropriate surgicaltechniques are adopted and have been mainly confined to individual case reports, 61.Hand-assisted laparoscopic nephroureterectomy referred to as (HALNU) is often seen as a compromise between the open and laparoscopic technique. Arguments in its favor include a shorter learningcurve, facilitates tactile feedback and the eventual requirement of a longerincision following LNU for specimen extraction. In a prospective, but nonrandomizedstudy with 27 subjects, patients undergoing an HALNU could expect a quickerdischarge from hospital, faster recovery, and fewer complications with anequivalent intermediate-term oncologic outcome compared to the open approach .On the Clearly, in the absence of prospective, randomized studies comparing ONU, LNU, and HALNU, it isinjudicious to draw robust conclusions regarding the superiority of onetechnique over the other. Nevertheless, though radical ONU still represents thegold standard for upper tract TCC, laparoscopic nephroureterectomy appears tooffer the advantages of minimally invasive surgery without deteriorating theoncological outcome in most cases.TCC of the upper urinarytract, though rare, constitutes a serious urological disease. Even though it iscurable in its early stages, there has been little improvement in disease-specificsurvival in high-risk patients over the last three decades. Radicalnephroureterectomy, with en bloc removal of the entire ureteral length and cuffof bladder, remains the procedure of choice, and the technique has undergonenumerous modifications in recent years. The integration of laparoscopy intourological practice has seen LNU emerge as a viable option for the managementof upper tract TCC.Given the relative rarityof the disease and a lack of robust multicenter effort, it is unlikely that theissue of LNU versus ONU for upper tract TCC will be resolved in a prospectiverandomized manner. Nevertheless, a multitude of small prospective studies withintermediate followup have clearly demonstrated the benefits of minimallyinvasive surgery associated with LNU, along with comparable oncological efficacy in the hands ofappropriately trained and experienced laparoscopic urologists. Long-term datafrom these studies would be beneficial.The issue of the distalureteral remains unresolved, and number and complexity of available techniqueswill undoubtedly continue to evolve. The existing data does not confirm theoverwhelming superiority of one technique over the other. Each method has itsdistinct advantages and disadvantages and it is essential that the responsiblesurgeon adopts a meticulous, safe, reproducible, and oncologically soundtechnique.