Propionibacterium shermanii 1.3 S transcarboxylase domain (PSTCD), to enable metabolically biotinylation of the virus. We demonstrate here that the new adenovirus no longer shows CAR-dependent cell uptake and transduction. When metabolically biotinylated and avidin-coated, it forms a nano-complex that can be retargeted to distinct cells using biotinylated antibodies. This vector may prove useful in the path towards achieving targeted gene delivery.Gene delivery vectors based on adenovirus, particularly human adenovirus serotype 5 (hAd5) have great potential for the treatment of variety of diseases. However, the tropism of hAd5 needs to be modified to achieve tissue- or cell- specific therapies for the successful application of this vector system to clinic. Here, we modified hAd5 tropism by replacing the fiber knob which contains the coxsackievirus B and adenovirus receptor (CAR)-binding sites with a biotin acceptor peptide, a truncated form of  However, the ubiquitous expression of adenoviral primary receptor CAR in many tissues and the predominant liver tropism of the vector after systemic administration limit the application of hAd5 vector to clinical use . Therefoin vivo -8. The b[in vivo -6. Singl in vivo . The hAdIn the study reported here, we have ablated the native tropism of hAd5 by removing the fiber knob and part of the central shaft. We have added to the short fiber a truncated form of PSTCD as a biotin acceptor protein to allow the virus to be metabolically biotinylated. We demonstrated here that the N-terminal tail and 9 shaft repeats fused with PSTCD protein can be successfully incorporated into the adenovirus particles to form the required trimer and to be biotinylated. The resulting metabolically biotinylated adenovirus can be redirected to specific cells depending on the biotinylated antibody used.The hAd5 fiber proteins exist as homotrimers which contains an N-terminal tail, a central shaft comprising 21 repeating sequences of 15 amino acids, and a C-terminal globular knob domain . WithoutThe presence of the modified fiber and the successful biotinylation of the fiber were confirmed using immunoblotting analysis. As shown in Figure We then began to characterize our new viral vectors functionally. The interaction of the Ad5 fiber knob with cell surface CAR mainly determines the tropism of the virus in cell culture. Therefore, both the Ad.9R-GFP and Ad.9RPSTCD-GFP vectors should have decreased transduction efficiency to a cell line that has high expression level of CAR because the complete knob domain is deleted. We transduced MA104/APP cells  with indTo test the possibility that the Ad.9RPSTCD-GFP vector could be retargeted through biotin-avidin-biotin interaction in cells, we first pre-incubated the Ad9RPSTCD-GFP vector with neutravidin to generate the Ad.9RPSTCD-GFP(avidin) vector. This neutravidin-coated vector was purified by cesium chloride (CsCl) density gradient centrifugation. We then chemically biotinylated the surface proteins of MA104/APP cells and incubated them with the Ad.9RPSTCD-GFP(avidin) vector at different MOI before assessing transduction by counting GFP-positive cells. As shown in Figure Next, we tested whether the Ad.9RPSTCD-GFP(avidin) vector could be efficiently retargeted to cells by using biotinylated antibodies against a specific cell surface protein. We chemically biotinylated monoclonal antibody J310 which recognizes rat aminopeptidase P (APP) , and monAs a last test, we wanted to form preconjugated biotinylated adenovirus-avidin-biotinylated antibody nano-complexes in order to induce possible specific targeting. It is difficult to control the number and location of biotin residues when antibodies are chemically biotinylated. Without a defined biotin site, chemically biotinylated antibodies can be attached to avidin in any orientation, often interfering with their ability to bind antigens. In contrast, metabolically biotinylated Fab constructs can be genetically engineered to precisely control the number and location of biotin residues on the antibody. Our lab has developed metabolically biotinylated Fab fragments against rat APP (833cb Fab) which has one biotin residues located at the C-terminal side of the C1 domain of the heavy chain . We incuin vitro. This vector may become useful for in vivo retargting and screening with future modifications that reduce RES uptake. For instance, we plan to further modify this vector by introducing the point mutation in the hexon protein to ablate the interaction of hexon with coagulation factor FX according the methods described in reference [in vivo with specific biotinylated antibodies. The methods and new vectors described and characterized here may form a foundation for future modifications and studies attempting to further improve tissue- and cell- specific retargeting of adenoviral vectors.Construction of targeted adenoviral vector through modification of fiber protein can be challenging because the trimerization of modified fiber is essential for proper virus assembly, and the retargeting ligands need to be functional in the trimerized fiber structure. In this report, we demonstrated that the 9RPSTCD modified fiber can be successfully assembled onto hAd5 virus and can be biotinylated simultaneously with viral production. Because of the deletion of CAR-interaction, the Ad.9RPSTCD-GFP(avidin) complex can be directed to transduce only selected cells by binding to other cell surface receptors through biotinylated targeting ligands. This 9RPSTCD virus may now be suitable for high throughput screening of targeting ligands eference , and thehAd5: human adenovirus serotype 5; RES: reticuloendothelial system; CAR: coxsackievirus B and adenovirus receptor; PSTCD: Propionibacterium shermanii 1.3 S transcarboxylase domain; MOI: multiplicity of infection; CsCl: cesium chloride; APP: aminopeptidase P; RAEC: rat aortic endothelial cells.The authors declare that they have no competing interests.YZL conducted the major experiments related to this project and wrote the manuscript, PV provided the 833cb Fab, JES led the study and wrote the manuscript. All authors read and approved the final manuscript.


This new filter was tested in BMI experiments in which rhesus monkeys used their cortical activity, recorded through chronically implanted multielectrode arrays, to directly control computer cursors. The 10th order unscented Kalman filter outperformed the standard Kalman filter and the Wiener filter in both off-line reconstruction of movement trajectories and real-time, closed-loop BMI operation.Brain machine interfaces (BMIs) are devices that convert neural signals into commands to directly control artificial actuators, such as limb prostheses. Previous real-time methods applied to decoding behavioral commands from the activity of populations of neurons have generally relied upon linear models of neural tuning and were limited in the way they used the abundant statistical information contained in the movement profiles of motor tasks. Here, we propose an n-th order unscented Kalman filter which implements two key features: (1) use of a non-linear (quadratic) model of neural tuning which describes neural activity significantly better than commonly-used linear tuning models, and (2) augmentation of the movement state variables with a history of  Research on brain-machine interfaces (BMI) – devices that directly link the brain to artificial actuators neural tuning models). Our experiments demonstrate the increased accuracy of our quadratic model versus the previously-used linear model. Second, our filter takes advantage of the patterns of movements performed during the execution of tasks. For example, a prosthetic used to aid in feeding has to perform a stereotypical pattern of movements: the prosthetic actuator moves back and forth between the user's mouth and the food items placed on a tray. Our approach uses this stereotypic pattern to improve BMI output accuracy. Third, our filter allows the relationships between neural activity and arm movement at multiple time offsets to be used simultaneously.We propose a new computational approach for BMIs, the n-th order unscented Kalman filter (UKF), to improve the extraction of motor commands from brain activity. Our experiments showed that this new approach offers more accuracy compared to methods which use linear models while remaining computationally light enough for implemention in real-time. This filter offers three improvements upon previous designs of BMI decoding algorithms. First, our filter allows the use of non-linear models of neuronal modulations to movements  trained to perform two reaching tasks. In off-line comparisons, our method demonstrated significantly better accuracy compared to the Kalman filter, the Wiener filter, and the population vector method Our algorithm was tested both off-line and in real-time, closed-loop experiments in which cortical recordings were obtained from macaque monkeys  to perform reaching tasks that incorporated stereotypic patterns of movements. The monkeys manipulated a hand-held joystick to acquire visual targets with a computer cursor . In the e center . In the us curve .Both monkeys were implanted with multielectrode arrays in multiple cortical areas. Monkey C was implanted in M1, PMd, posterior parietal cortex (PP) and supplementary motor area (SMA) in the right hemisphere. Monkey G was implanted bilaterally in primary motor cortex (M1), primary somatosensory cortex (S1) and dorsal premotor cortex (PMd). Extracellular discharges of 94 to 240 (average 142) cortical neurons were recorded while each monkey performed the behavioral tasks.We applied the n-th order unscented Kalman filter to the data collected in 16 daily sessions: 6 sessions from Monkey C and 10 sessions from Monkey G. Data used from each session ranged from 9 to 25 minutes. After evaluating filter accuracy off-line, we conducted six on-line experiments, three with each monkey, while the monkeys controlled the BMI using the unscented Kalman filter and comparison methods in closed-loop operation. We treated the neurons recorded from different cortical areas as one ensemble; differences between individual cortical areas were not considered here.Our n-th order unscented Kalman filter (UKF) combined two extensions to the standard Kalman filter movement model, predicted the hidden state at the current time step given the state at the previous n time steps. The observation model or neural tuning model predicted the expected neuronal rates from the estimated desired movement via a non-linear function. We incorporated multiple taps of the state in the neural tuning model to relate neural activity with hand kinematics at multiple time offsets simultaneously. We used a nonlinear quadratic model of tuning to express neuronal rates as a function of hand position and velocity.Like the standard Kalman filter, the n-th order unscented Kalman filter inferred the hidden state (the position and velocity of the desired movement) from the observations . The state transition model or We analyzed the predictive accuracy of the quadratic tuning model used in our n-th order unscented Kalman filter. Firing rates of single neurons were predicted from hand position and velocity using the quadratic (with n = 1 and n = 10 taps) and the linear neural tuning models after the models were fit with linear regression using the Moore-Penrose pseudoinverse. A 10-fold cross-validation procedure was used to test predictive accuracy from 16 recording sessions with an average of 142 neurons recorded per session, and we report results using signal-to-noise ratios  and correlation coefficients (CC). The n = 1 tap quadratic model  was more predictive  than the linear model . 1753 out of 2273 units (approximately 77%) were better predicted using the quadratic model. The n = 10 tap quadratic model  was more predictive (P<0.001) than the n = 1 tap quadratic model (about 900 or approximately 40% of units were better predicted).The superior performance of the quadratic tuning model is illustrated in the contour plots of For velocity tuning , while dWe compared the ability of our method to reconstruct hand movements from neural recordings with several commonly used, real-time methods by performing 10-fold cross-validation on 16 previously recorded sessions. Parameters for the algorithms were fitted by ridge regression, a regularized form of linear regression, using recorded neural and behavioral (joystick position and velocity) data. The first cross-validation fold of each session was used to optimize ridge regression parameters and omitted from the results. The mean off-line reconstruction accuracy of the 10th order unscented Kalman filter (UKF), the 1st order unscented Kalman filter, the standard Kalman filter, the 10 tap Wiener filter fitted with ridge regression (RR), the 10 tap Wiener filter fitted with ordinary least squares (OLS), and the population vector method used by Taylor et al. are shown in In terms of position estimates, the 10th order UKF with our quadratic tuning model was consistently more accurate than the other algorithms. The two-sided, paired sign test with 288 observations  and significance level The 1st order UKF produced position estimates with significantly higher SNR than the standard Kalman filter  and 10 tap Wiener filter fitted with ridge regression . In terms of CC, no comparison was significantly different at the Performance was measured by comparing the position of the target  and the BMI-controlled cursor. past taps and future taps, in units of 100 ms, described the time offsets prior to and after the instant of neural activity between which tuning was modelled, respectively . For each setting of the number of neurons, 10 subsets of neurons were randomly selected and each algorithm was evaluated on these subsets using 10 fold cross-validation. The first fold was reserved for finding optimal ridge regression parameters, and the mean accuracy on the nine remaining folds are plotted in In terms of computational load, the MATLAB implementation of the 10th order UKF on an Intel Pentium 4 class computer used 0.012±0.005 seconds per iteration (mean±standard deviation), or around 80 Hz on average. The 30th order UKF (15 future and 15 past taps) used 0.0360±0.0001 seconds per iteration, or around 28 Hz on average. Our on-line implementation in C++ using Automatically Tuned Linear Algebra Software (ATLAS) easily executed faster than 10 Hz, our binning frequency.In this study, we achieved an improvement over previous closed-loop linear BMI decoding by implementing a more accurate decoding algorithm, the n-th order unscented Kalman filter (UKF). This filter modeled arm movement profiles better because it used the history of past movement, and it described neuronal modulations to movements better by using a quadratic model of neuronal tuning which included tuning at multiple time offsets. The filter performed well both in off-line reconstruction of previously recorded data and on-line, closed-loop BMI operation.Much work has been done investigating algorithmic methods for decoding continuous control signals from extracellular neural recordings for neuroprosthetics  Another class of decoding methods work directly from individual neuron spikes instead of instantaneous firing rate estimates. In this approach, spike trains are modeled as discrete events or point processes and decoding can operate at millisecond time scales. The point process analog of the Kalman filter, using a Gaussian representation for uncertainty in state estimates and an inhomogenous Poisson model of spiking, was derived by Eden et al.  and called the To improve decoding of simple reaching movements, tuning to the goal coordinates of reach trajectories has been used to augment tuning to movement. Kemere et al. (2004) included both movement tuning and target position tuning in a maximum-likelihood filter spikernelOther techniques have been investigated for decoding of continuous hand movements. Isaacs et al. (2000) used principle components analysis and the nearest-neighbor algorithm While there is a large variety of algorithms available for decoding desired movement from neural signals, only our approach and the KARMA algorithm of Shpigelman et al. In this study, we explored whether a quadratic model of neural tuning can improve BMI decoding accuracy. Our analysis showed that our quadratic model of neural tuning was significantly more predictive of neuron firing rate than a linear model. We then implemented an unscented Kalman filter which used this quadratic model to infer desired hand movements. The increased spike count prediction accuracy (0.02 dB) and off-line reconstruction accuracy of the (1st order) UKF versus the standard Kalman filter (0.39 dB) and 10 tap Wiener filter (0.25 dB) demonstrates the benefits of our quadratic model. By using the unscented transform, we were able to implement a non-linear filter without resorting to computationally expensive particle filtering techniques.Our decoding method was further enhanced by incorporating a short history of hand kinematics into the hand movement model. We implemented an n-th order UKF which used the hand movement in the n previous time steps to predict hand movements in the next time step. Adding a short history to the state space had the additional benefit of modeling neural tuning across multiple time offsets simultaneously. When using We explored the optimal history length, or number of taps, for the UKF. Our results suggest that the number of taps for best performance depends on the behavioral task. For the pursuit task, accuracy increased with the number of future taps and plateaued at n = 15 or slightly later, and accuracy increased slowly with the number of past taps. For the center-out task, a small number of future taps resulted in the highest accuracy, while the number of past taps had small effects. The improvement of the 10th order UKF versus the 1st order UKF was greater for the pursuit task (mean 1.23 dB) than for the center-out task (mean 0.48 dB).Based on these results, we conjecture that the richer movement model of the 10th order UKF was able to capture the hand movement patterns produced during the performance of the pursuit task better than those generated during execution of the center-out task. This is likely because hand movements for the center-out task are autocorrelated over shorter time spans than hand movements for the pursuit task. Hand movements during center-out reaches were brief and unrelated between reaches, while during the pursuit task the hand moved relatively smoothly. The n taps of our movement model can be viewed as extra smoothing, hence the pursuit task, with smoother movement trajectories, benefits more than the center-out task. Our analysis showed that the movement model of the 10th order UKF made large contributions to the accuracy of the filter (6.25 vs 3.88 dB in pursuit task), yet this movement model was unable to provide accurate estimates by itself, without the aid of the neural recordings  algorithm proposed by Shpigelman et al. Our n-th order unscented Kalman filter is particularly suited for use in cortically driven prosthetic devices because of its relatively high accuracy and unique features. Our algorithm takes advantage of a non-linear model of neural tuning in a computationally inexpensive implementation that is well suited for mobile, low-power prosthetic systems. Furthermore, our algorithm takes advantage of patterns of movement, abundantly found in typical tasks such as feeding, that a prosthetic may be engaged to do. Since this new approach is Bayesian, it allows the computation of the certitude of decoded movements. Thus, decoded movements with low probaility can be suppressed, and undesired movements caused by decoding errors or unexpected neural activity can be detected and prevented. The separation of the neural tuning and movement models also allows training data to be used more efficiently, making the prosthetic easier to calibrate.The unscented Kalman filter can be applied to learn neural tuning model parameters or adapt to time-varying neural tuning and time-varying patterns of movement through a technique called dual Kalman filtering for joint parameter and hidden state estimation The UKF can also compensate for degradation of neural recordings as this can be described as changes in the neural tuning model. Furthermore, models of movement can be improved over time to best predict movements produced during execution of particular tasks. These models can also be learned over time to handle novel tasks. Our future work will pursue these approaches toward the development of user-friendly, computationally efficient, and accurate algorithms for BMIs.Macaca mulatta) performing reaching tasks by moving a computer cursor using a hand-held joystick and by controlling the cursor directly through their cortical activity decoded by a BMI  and were approved by the Duke University Animal Care and Use Committee. Cortical recordings were collected from 2 rhesus monkeys , left SMA (9 sessions), left M1 (9 sessions), and right PP (1 session). Data from Monkey G were recorded from left PMd (13 sessions), left M1 (13 sessions), left S1 (8 sessions), and right PMd (7 sessions). Extracellular neural signals were amplified, digitized, and high-pass filtered using Multichannel Acquisition Processors . Neuronal action potentials were discriminated by thresholding and sorted on-line through waveform templates set by the experimenter using Plexon spike-sorting software or using templates produced by custom-built spike sorting software During the experimental sessions, each monkey sat in a primate chair. Their heads were unrestrained, and the recording system was connected to the implants using light flexible wires. A two degree of freedom (left-right and forward-backwards) analog joystick was mounted vertically at the monkey's waist level. The joystick was 30 cm in length and had a maximum deflection of 12 cm. The monkeys were trained to manipulate the joystick with their hands. Monkey C performed with the left hand, and Monkey G performed with the right hand. An electrical resistance-based touch sensor on the joystick handle measured whether the monkey was holding the joystick. An LCD projector projected visual images on a screen mounted 1.5 m in front of the monkeys . Using tEach behavioral task required placing the computer cursor over the target using the joystick. The monkeys performed two tasks: (1) center-out and (2) pursuit. The center-out task  used staThe pursuit task  used a mwhere For all algorithms, spike counts were calculated in 100 ms nonoverlapping bins to estimate the instantaneous firing rate. Joystick position was recorded at 1 KHz and down-sampled to 10 Hz to match the binning rate. Velocity was calculated from position by two-point digital differentiation. Position and velocity data were centered at their means. Spike counts were centered at their means for the Kalman-based filters. Data recorded while the monkey did not hold the joystick were disregarded. Off-line analysis was conducted using MATLAB . Real-time filters were implemented in a custom built BMI system running on a workstation with an Intel Xeon 2.2Ghz processor.Our n-th order unscented Kalman filter (UKF) can be described as a modification of the Kalman filter movement model, a linear function t given the state at the previous n time steps:In the hidden Markov model for BMI decoding using the n-th order unscented Kalman filter , the hidmovement model noise, which describes the uncertainty arising from approximations made in the model and intrinsic randomness in the movement process. This movement model is an autoregressive process of order n (AR n), as compared to the AR 1 movement models of the Kalman filters previously used for BMI decoding  spike counts:Finally, the state covariance was updated: Equations 6 through 17 implement one iteration of the algorithm. A side-by-side comparison of the equations for the Kalman filter and the n-th order unscented Kalman filter are shown in In off-line reconstructions, the initial values of We fitted the parameter matrices T matrix T is the number of data points (i.e. the time length of the training data). We then constructed a T matrix i of n columns of n columns of To fit where where An alternative method for setting the movement model is to use the equations describing motion, e.g. position is the integral of veloctiy over time. However, this method does not capture the patterns in the movements generated by the BMI user as well as movement models fit from kinematic data. In practice, our fits to The movement model noise covariance matrix where N by T matrix T matrix i of k future taps, k steps. This was done by removing the last k columns of k columns of To fit where N by N neural tuning model noise covariance matrix The N by where The n-th order unscented Kalman filter and several comparison methods were evaluated off-line using data collected in experiments in which monkeys moved a computer cursor using the joystick. The n-th order UKF used folds) and the testing procedure was repeated on each held-out portion in turn. Both the movement and neural tuning models were fit for each cross-validation fold. In this study we did not address the question of how to design a general movement model, instead we leave this for future work.For off-line reconstructions, cross-validation was conducted. In this procedure, a portion of the data for each session was held-out for testing and the rest was used to fit parameters. Performance of the algorithms was evaluated on the held-out portion to avoid fitting models and making predictions on the same data. The data for each session were divided into 10 equal-sized portions .The Kalman filter used for comparison had the same state variables as the 1For the population-vector method, the neuronal weights were fit via ordinary least squares without regularization. The original formulation of the population vector method predicted velocity and did not predict position directly. To make position predictions, we substituted the Cartesian position coordinates for the velocity components. We implemented the Taylor et al. To quantify filter performance, we compared algorithm estimated trajectories to joystick trajectories (in off-line reconstructions) and to target trajectories (in closed-loop BMI). We computed two metrics: the signal-to-noise ratio (SNR) and the correlation coefficient (CC). SNR was calculated as:not translation and scale invariant, unlike the CC. This is an advantage because translation and scale invariance imply that the CC may leave undetected certain unwanted filtering results. For example, a predicted hand trajectory that is incorrect by a large but constant displacement has the same CC as a trajectory without the erroneous displacement, since only deviations from the mean are analyzed by the CC. As indicated by its name, CC is a measure of correlation, but we are interested in measuring accuracy. Furthermore, as the CC saturates at 1, its scale is compressed as it approaches 1, making it more difficult to grasp intuitively and making similar increments at lower values of CC and higher values of CC incomparable. Short of benchmark datasets, we believe the SNR measure best facilitates direct comparison between algorithms developed by different authors.where To aggregate results for each session, mean SNR and CC among the cross-validation folds and between the x- and y-axis predictions were computed. Standard error of the mean was calculated for each session with 18 observations (9 folds×2 axes). To test for significant effects, we treated each cross-validation fold and each axis as a condition for paired, two-sided sign tests. We used an Materials S1(0.26 MB DOC)Click here for additional data file.


Previous studies have shown that the enzyme γ-glutamyl transpeptidase (GGT) is essential for the nephrotoxicity of cisplatin. This study was designed to determine whether GGT activity is necessary for the therapeutic effect of the drug. The relationship between GGT expression and clinical response to platinum-based chemotherapy was examined in 41 human germ cell tumours. Sections of formalin-fixed, paraffin-embedded tumours were immunohistochemically stained with an antibody directed against human GGT. There was no expression of GGT in any of the 17 seminomas or four dysgerminomas; whereas, 12/12 ovarian yolk sac tumours and 4/4 embryonal carcinomas of the testis were GGT-positive. In stage I tumours fewer tumour cells expressed GGT than in later stage tumours. In four germ cell tumours of mixed histology, the seminomatous and dysgerminoma areas were GGT-negative while the areas of the tumour with yolk sac or embryonal histology contained GGT-positive tumour cells. The patients with seminomas or dysgerminomas who were treated with cisplatin-based chemotherapy, all had a complete response despite the absence of GGT expression in these tumours. Fifteen of the 16 patients with yolk sac or embryonal carcinomas received cisplatin-based chemotherapy following surgery. Twelve had a complete response, while three failed to respond to platinum-based therapy. There was no correlation between the level of GGT-expression and response to therapy in this group. Three of the four patients with tumours of mixed histology were treated with cisplatin-based therapy, and had a complete response. Therefore, expression of GGT is not necessary for the therapeutic effect of cisplatin in germ cell tumours. The results from this study suggest that systemic inhibition of GGT would inhibit the nephrotoxic side-effect of cisplatin without interfering with its activity towards germ cell tumours. © 1999 Cancer Research Campaign


The recent development within high-throughput technologies for expression profiling has allowed for parallel analysis of transcriptomes and proteomes in biological systems such as comparative analysis of transcript and protein levels of tissue regulated genes. Until now, such studies of have only included microarray or short length sequence tags for transcript profiling. Furthermore, most comparisons of transcript and protein levels have been based on absolute expression values from within the same tissue and not relative expression values based on tissue ratios.Presented here is a novel study of two porcine tissues based on integrative analysis of data from expression profiling of identical samples using cDNA microarray, 454-sequencing and iTRAQ-based proteomics. Sequence homology identified 2.541 unique transcripts that are detectable by both microarray hybridizations and 454-sequencing of 1.2 million cDNA tags. Both transcript-based technologies showed high reproducibility between sample replicates of the same tissue, but the correlation across these two technologies was modest. Thousands of genes being differentially expressed were identified with microarray. Out of the 306 differentially expressed genes, identified by 454-sequencing, 198 (65%) were also found by microarray. The relationship between the regulation of transcript and protein levels was analyzed by integrating iTRAQ-based proteomics data. Protein expression ratios were determined for 354 genes, of which 148 could be mapped to both microarray and 454-sequencing data. A comparison of the expression ratios from the three technologies revealed that differences in transcript and protein levels across heart and muscle tissues are positively correlated.We show that the reproducibility within cDNA microarray and 454-sequencing is high, but that the agreement across these two technologies is modest. We demonstrate that the regulation of transcript and protein levels across identical tissue samples is positively correlated when the tissue expression ratios are used for comparison. The results presented are of interest in systems biology research in terms of integration and analysis of high-throughput expression data from mammalian tissues. High-throughput quantitative profiling of transcripts and proteins is a widely used approach for studying biological processes. As a consequence, technologies develop rapidly in order to improve quality, increase the throughput and reduce the cost of expression profiling. Currently, transcript profiling technologies include DNA microarray , Serial As new high-throughput technologies emerge and develop, more comparative expression studies across technologies and across transcriptomes and proteomes have been reported. At the transcript level, these studies have been dominated by comparisons of SAGE data with either Affymetrix short oligonucleotide microarrays -22 or cDHere we report a comparative study of three high-throughput technologies for multi-sample expression profiling using tissue samples from pig heart and skeletal muscle. We compare transcript profiles from 454-sequencing and cDNA microarray based on relative expression within tissues and expression ratios across tissues. Furthermore, we analyze the relationship between transcript and protein regulation between the two tissues by direct comparison of expression ratios from cDNA microarray, 454-sequencing and iTRAQ-based proteomics.Identical tissue samples were used in expression profiling with 454-sequencing, cDNA microarray and iTRAQ-based proteomics, three for heart and three for skeletal muscle. An overview of the experimental setup is shown in figure To determine the level of reproducibility of 454-sequencing and cDNA microarray for measuring gene expression in heart and skeletal muscle and to evaluate the level of correlation in expression across the two technologies, Pearson's correlations between RA for pairs of tissue samples for the 2.541 shared genes were computed. For each of the six samples and two technologies, a vector of RA expression values was prepared. The resulting 12 RA expression vectors were then used to compute pair-wise tissue sample correlations, of which the average values within and across the two tissues and technologies are plotted in figure The agreement between 454-sequencing and cDNA microarray in identifying differential gene expression was evaluated by comparing lists of differentially expressed genes between heart and skeletal muscle. A t-test was carried out to compute the fold changes and P-values corrected for multiple testing was set to 0.05 as a cutoff for significant regulation. Identification of differentially expressed genes using 454-sequencing produces the lowest number of significantly regulated genes, 306 out of 2.954 tested genes (10%). Out of the tested 12.563 genes, microarray produces a list of 5.888 genes that are significantly regulated, a much higher percentage (47%) compared to that of 454-sequencing. Of the 306 significantly regulated genes identified using 454-sequencing, 198 genes (65%) are overlapping with the 5.888 genes identified using cDNA microarray. Plotting the log2 of the expression ratios between heart and skeletal muscle for these 198 genes shows that the direction of the regulation is identical for a majority of the genes  in comparison to the corresponding correlation observed for 454-sequencing (0.23), which suggests a larger tissue difference. We speculate that the main reason for the difference between these two transcript-based technologies is the differences in the dynamic range of the expression profiles. Thus, the distribution of the RA values from 454-sequencing is considerably wider than that observed for microarray, indicating that sensitivity for detecting expression difference across tissue samples is highest for 454-sequencing. Whether the larger number of genes detected to be different across skeletal muscle and heart by 454-sequencing is reflecting real biological differences between these two tissues is uncertain. The correlation values across 454-sequencing and microarray at 0.25 and 0.51 for heart and muscle respectively was relatively low, but considerably higher than the correlation value at 0.13 across the two transcript-based technologies and across the two tissues. This means that even though the technologies are quite different they still measure the respective expression profiles within heart and muscle to be most similar.The number of differentially expressed genes identified in relation to the number of tested genes was much higher for cDNA microarray  than for 454-sequencing . Because it is not the same set of genes being tested, it will of course not lead to the same list of identified differentially expressed genes. However, microarray tends to predict more genes that are differentially expressed. Some might be false positives, even though P-values were corrected for multiple testing. If expression ratios across any given two tissues are close to one, they are vulnerable to random shifts between up-regulated, not-regulated and down-regulated genes. This has previously been suggested as a reason for discrepancy between cDNA- and oligonucleotide-based microarray platforms in expression ratios . CardiacTo investigate the relationship between the regulation of transcript and protein levels across tissues, the expression ratios across heart and skeletal muscle from microarray, 454-sequencing and iTRAQ-based proteomics were compared. Transcript and protein ratios across heart and skeletal muscle were shown to be positively correlated for 148 genes detectable by all three technologies. The correlation appear to be slightly more positive for the comparison with 454-sequencing (0.53) than for the comparison with cDNA microarray (0.49). The slope is 0.6 for cDNA microarray and 1.4 for 454-sequencing when ratios are compared against iTRAQ-based ratios. The positive correlation between transcript and protein ratios suggests that for most genes, a relative tissue difference across a given tissue pair in transcript level leads to a similar difference in the protein level. A list of all 148 genes with expression ratios for the three technologies and indications of concordance was generated. Genes, where the transcript-protein relationship deviates from this linearity might be interesting to study further, since they could be examples of differential regulation of either translation or mRNA- or protein-turnover among the tissues. Alternatively, translation of mRNAs from genes that generate high levels of transcripts might be limited by the availability of RNA-binding factor or the capacity of the translational machinery. Deviations may also result from gene annotation errors ,22 or alIn this study we have analyzed the reproducibility of expression data within and between microarray and 454-sequencing technologies. Furthermore, we have integrated expression data from both transcriptomic and proteomic profiling to analyze the gene regulation across two porcine tissues by comparing tissue expression ratios of transcripts and proteins. Both transcript-based technologies displayed a high degree of reproducibility within technology, but the reproducibility across these two technologies was modest. The majority of the differentially regulated genes identified by 454-sequencing was also found by the cDNA microarray platform. Most interesting was the comparison of data from both transcript-based technologies with relative expression values from iTRAQ-based proteomics. Integrative analysis revealed that the regulation of transcript and protein levels across the two tissues is positively correlated for most genes using tissue expression ratios for comparison. Some genes without transcript-protein concordance were identified, which may arise from annotation errors or differential regulation of translation, turnover or alternative splicing. The results presented here should be of high importance for integration and analysis of high-throughput expression data, in particular for studies of the regulation of transcript and protein abundances in mammalian tissues.Longissimus dorsi; LDO) were prepared by pooling equal amounts of tissue sampled from five healthy Hampshire gilts at age four to six months. Each pool was divided into six sub-samples, three for each tissue named HEA1, HEA2, HEA3, LDO1, LDO2 and LDO3. The exact same six tissue sub-samples were used for expression profiling with cDNA microarray, 454-sequencing and iTRAQ-based proteomics. A reference sample for the cDNA microarray experiment and iTRAQ-based proteomics was constructed by combining equal amounts of tissue from the six sub-samples.Tissue samples of heart (HEA) and skeletal muscle  [, the log2-transformed median intensity ratios of Alexa-647 to Alexa-555 were normalized within-slide using the printtip-loess method [ and given the accession ID GSE10122.Total-RNA extractions were carried out from each sample using the RNeasy Maxi Kit from Qiagen. Alexa Flour-labelled cDNA was synthesized from 20 μg of total-RNA using SuperScript Plus Direct cDNA Labeling System from Invitrogen. The reference sample was labelled with Alexa 555 and the individual tissue samples were labelled with Alexa 647. Each of the six labeled tissue samples was co-hybridized with the labelled reference sample to three 27 k pig cDNA microarray slides representing approximately 20 k genes. Microarray cDNA platform development was based on a large EST sequence resource established as part of the Sino-Danish Pig Genome Sequencing Project ,41. Deta)  using th)  from Bios method . The rawFrom the same total-RNA extraction batches used for the microarray experiment, poly A+ RNA was purified from ~200 μg total-RNA using the Oligotex mRNA Mini Kit from Qiagen. The poly A+ RNA isolates were used as templates in synthesis of high quality double-stranded cDNA with oligo(dT)12–18-priming of the first strand cDNA (SuperScriptTM Doubled-Stranded cDNA Synthesis Kit from Invitrogen). The final purification of the synthesized cDNA was performed on MinElute PCR Purification column from Qiagen, and an average cDNA fragment size of 300–400 bp was obtained by applying 45 psi (= 3 bar) of nitrogen for 50–60 seconds on 2.7–4.7 μg starting material using a nebulizer. Subsequently, the distribution of fragments was profiled on a BioAnalyzer DNA 1000 LabChip (Agilent Technologies). As part of the library preparation, the ends of the sheared cDNA fragments were polished by treatment with T4 DNA Polymerase and T4 Polynucleotide Kinase prior to ligation of sequencing adaptors as previously described. The fragments were immobilized on streptavidin beads, following nick repair with Bst DNA Polymerase, and the sstDNA libraries were eluted by alkali denaturation. Finally, the qualities and quantities of the libraries were assayed on Bioanalyzer RNA 6000 Pico LabChip (Agilent Technologies). A titration run, based on DNA/bead ratios of 4 and 16 copies per bead (cpb) in the emulsion-based clonal amplification, revealed the optimal experimental set-up for the large sequencing run using enriched beads. Each of the six libraries was sequenced on a full 70 × 75 PicoTiterPlate (PTP). In total 1.253.361 sequences were generated and submitted to the Short Read Archive (SRA) of NCBI and assigned the accession ID SRA000267.Eight samples (including two reference samples) were processed in parallel. Total protein fraction was purified from 200 mg tissues as previously described . The supThe microarray cDNA gene expression data and the 454-sequencing data was linked together by mapping all microarray cDNA sequences and 454-based sequences to NCBI's pig UniGene database release 28 and then linking the data by their shared UniGene IDs. The sequences were mapped to UniGene IDs by BlastN and collecting only IDs for the first hit with score at or above 100. For correct microarray cDNA mappings it was required for each UniGene Id that the corresponding UniGene sequence was able to identify the exact same microarray cDNA sequence as the first hit when it was compared back to a database of all microarray cDNA sequences. In total, 12.563 out of 26.877 microarray cDNA sequences were mapped to 12.563 UniGene IDs and 647.093 out of 1.253.361 454-sequences were mapped to 18.624 UniGene IDs. In total, 2.541 genes were identified with expression profiles from both cDNA microarray and 454-sequencing by using the overlapping UniGene IDs to merge the two data sets together. For iTRAQ-based proteomics, a database of predicted pig proteins from trEST based on translations of the latest release (30) of UniGene sequences was downloaded and searched [The expression profiles from 454-sequencing was achieved by counting the number of transcripts per gene, in this case represented by the number of BlastN query 454-sequences for each target UniGene ID in each of the six tissue samples. The minimum number of sequence counts in a tissue sample was set to one resulting in a total number of 2.954 UniGene IDs for which gene expression could be detected by 454-sequencing in all six samples. To calculate within-tissue relative expression profiles for each of the six tissue samples, taken into account the variation in total raw sequence counts, Relative Abundance (RA) values were computed for each gene (UniGene ID) in each tissue sample. The RA value for a given gene in a given tissue sample was computed as the 454-sequence count for that gene divided by the total 454-sequence count for the 2.541 genes in the whole tissue sample. To be able to make direct comparison of 454-based RA values with microarray-based expression profiles for the six tissue samples, corresponding microarray RA values were also computed using the same approach. The microarray RA value for a given gene was computed as previously proposed ,37 based2+B2). These distances were used to evaluate the concordance of genes across transcript and protein ratios.For comparison of relative expression profiles across 454-sequencing and cDNA microarray we computed tissue sample correlations for examining the reproducibility within and across technologies. Twelve RA vectors, six RA vectors from 454-sequencing and the six RA vectors from cDNA microarray, corresponding to the six tissue samples were paired in 12 × 12 = 144 possible combinations and the Pearson's correlation coefficient between the vectors in each combination was computed. The correlation values were grouped into nine bins corresponding to the three tissue comparisons  and the three technology comparisons . Average correlations were computed for these nine bins and used to evaluate the reproducibility within and across 454-sequencing and cDNA microarray. For comparing across-tissue mRNA expression ratios from the two transcript-based technologies against iTRAQ-based protein ratios we first prepared log2-transformed values of the expression ratios between heart and skeletal muscle for all three technologies. These values were organized in three vectors and pair-wise Pearson's correlation coefficients were calculated for the two comparisons 454-sequencing versus iTRAQ-based proteomics and cDNA microarray versus iTRAQ-based proteomics. In the comparison of expression ratios across transcripts and proteins a straight line was fitted for the data points described by the standard equation Ax+By+C = 0, where -A/B is the slope and C is the intersection between the line and the y-axis. Distances for all data points  to the fitted line were calculated based on the standard formula d = |Am+Bn+C|/√ and heart (white) generated using microarray and 454-sequencing. RA values were log2 transformed and binned for optimal visualization.Click here for fileList of 148 overlapping genes between microarray, 454-sequencing and iTRAQ-based proteomics with indication of concordance between transcript and protein expression ratios. Table file with details for 148 genes overlapping between microarray, 454-sequencing and iTRAQ-based proteomics. Table contains number, UniGene ID, name, log2 ratios for the three technologies and indications of concordance between transcript and protein expression ratios. Concordance was based on the distance between a point and the fitted straight line being less than one time the standard deviation of all distances. The table is sorted so that the genes that are not in concordance according to both 454-sequencing and microarray are listed first.Click here for file


Podospora anserina, three genes controlling this allorecognition process het-d, het-e and het-r are paralogs belonging to the same hnwd gene family. HNWD proteins are STAND proteins  that display a WD-repeat domain controlling recognition specificity. Based on genomic sequence analysis of different P. anserina isolates, it was established that repeat regions of all members of the gene family are extremely polymorphic and undergoing concerted evolution arguing for frequent recombination within and between family members.Genes involved in non-self recognition and host defence are typically capable of rapid diversification and exploit specialized genetic mechanism to that end. Fungi display a non-self recognition phenomenon termed heterokaryon incompatibility that operates when cells of unlike genotype fuse and leads to the cell death of the fusion cell. In the fungus het-R (HNWD2) and het-E (hnwd5) genes with altered recognition specificities. The vast majority of the mutants present rearrangements in the repeat arrays with deletions, duplications and other modifications as well as creation of novel repeat unit variants.Herein, we directly analyzed the genetic instability and diversification of this allorecognition gene family. We have constituted a collection of 143 spontaneous mutants of the We investigate the extreme genetic instability of these genes and provide a direct illustration of the diversification strategy of this eukaryotic allorecognition gene family. All living organisms have developed processes to discriminate self from non-self. Non-self recognition is required both in conspecific allorecognition  or recognition of pathogenic non-self. In eukaryotes non-self recognition relies on the production of polymorphic proteins able to react to a range of molecules. Typically, genes involved in non-self recognition are among the most polymorphic loci in eukaryotic genomes, a classical example being the MHC/HLA loci in mammals . Diversihet loci are co-expressed in the cytoplasm of the heterokaryon and leads to a cell death reaction [het genes remain unclear. VI is thought to restrict transmission of deleterious cytoplasmic elements such as viruses or abnormal mitochondria [In filamentous fungi conspecific non-self recognition happens in the heterokaryotic cell produced after the somatic fusion by anastomosis of cells of unlike genotypes -5. The vreaction . The biochondria ,8, and tchondria . It has chondria ,11. In tchondria ,13.het loci have been characterized in only two species, Podospora anserina and Neurospora crassa. In P. anserina the het-d and het-e loci are involved in non-allelic incompatibility interactions with het-c (unrelated to N. crassa het-c) [het-r locus is involved in non-allelic incompatibility with het-v [P. anserina genome [het-d, het-e and het-r belong to the hnwd gene family that includes 2 additional members (hnwd1 and hnwd3). They are part of the larger nwd family that comprises five additional members [hnwd genes encode for proteins including a N-terminal HET domain effector of cell death [hnwd genes encode for proteins of the STAND class thought to act as molecular switches with a closed inactive form and an open active form adopted upon binding to an activating molecule [nwd genes display the NACHT and WD domains but lack the HET domain.So far a het-c)  while thth het-v ,16  concfeatures . The comresponse ,30, rapiIt is well established that tandemly repeated sequences are unstable and undergo repeat number expansions and contractions. This repeat instability is at the origin of numerous cancers and neurological and developmental disorders in humans but in a number of cases this plasticity also appears advantageous as it can favour rapid genome evolution and adaptation ,32. RepeP. anserina to directly analyze rearrangements in intragenic tandem repeats in this eukaryotic non-self recognition gene family. We selected a collection of 143 loss of function specificity variants in two members of the hnwd family spontaneously generated under vegetative growth conditions  can be associated in a single haploid nucleus by appropriate genetic crosses, leading to the production of so-called self incompatible (SI) strains , or in extragenic suppressor which we did not observe. For each collection, we identified the mutated het gene by confronting each mutant against both parents used to set up the original cross. Incompatibility is characterized by the formation of an abnormal contact zone between incompatible strains called the barrage [het genes. All the mutants of the RV collection were affected at the het-r locus and expressed an inactive het-r allele. As well, all the mutants of the CE collection were affected at the het-e locus and expressed an inactive het-e allele. The combined 143 mutants from both collections are affected in the het genes that belong to the hnwd gene family suggesting a particular genetic instability at the hnwd loci.Mutations allowing for suppression of incompatibility could occur  barrage . For eachnwd genes. The presence of highly conserved tandemly repeated WD40 sequences both in the het-E and het-R genes led us to investigate the status of the repeat domains. The number of WD40 repeat units present in the WD repeat domains of the mutated genes (het-r or het-e) was estimated from the size of the PCR amplicon generated using locus specific primers , the number of excepted deletions per interval if they occurred at random along the sequence. E depends on the length of the interval, the length of the WD repeat domain and the number of mutants in the subclass .One of the striking features of the mutant allele sequences is that in the vast majority of the alleles, one finds new WD repeat units. We found 16 repeat units that differ from a parental unit by a single mutation and 22 repeat units that differ from the parental units by more than a single point mutation figure . We obsed genome . A high nogaster  or Daphnia obtus . A phylonwd loci apparently occur in the wild [From these observations, we conclude that although intergenic WD40 repeat unit exchanges between the wild , in our r10 and r11 of the RV collection were deleted of 2 or 4 WD40 repeat units respectively. The deletion event resulted from fusions between repeats 5 and 7 and between repeats 5 and 9 of the wild-type allele in mutants r10 and r11 respectively. This fusion event generates new WD40 repeat units sequences. The same was true for the two dWD mutants of the CE collection (e8 and e9). Again we found that in both cases a single fragment was deleted and resulted in new chimeric WD40 repeat sequences  in each repeat unit are under positive selection. Based on homology modelling, the corresponding amino acids all group at surface loops at the top of the β-propeller structure at the interaction surface . The amies table . Nine ofFive of the snWD mutants from the RV collection display a WD repeat domain identical to the wild type repeat domain. We then sequenced the remaining of these mutant genes after PCR amplification with primers C and D  even if one only considers the 54 different repeat units identified in the sequenced genome as the building blocks for a 7 repeat WD-domain repertoire. This likely represents an underestimation since additional repeat unit variants were found in a different isolate and novel repeat units can be generated through repeat instability. In addition, the β-propeller structures might be made of more than 7 repeats as exemplified by the 11 repeat WD-domains of wild-type het-e and het-v[In biotechnological applications, it has been demonstrated  targets ,41, the ognition ,51. Somehin loci ,53. Evolbination  preferenng genes . Similarng genes . It has and het-v.NBS-LRR genes and the sea lamprey lymphocyte receptors, the repeat proteins are involved in pathogen-detection and the extraordinary binding versatility of these proteins is related to the diversity of the pathogen associated patterns that are recognized. Now how should the evolvability of the hnwd genes be understand? The possibility to generate such a vast variety of binding specificities does not make obvious sense in the context of vegetative incompatibility especially considering that in N. crassa, incompatibility alleles are very stable, in finite number  and evolutionarily very ancient. The evolvability of the hnwd genes along with other features of the VI reaction recently lead us to suggest that the hnwd genes might be components of a fungal innate immune system with the function in response to pathogenic non-self [In the two examples cited above, the plant non-self . In thishnwd genes based non-allelic incompatibility in P. anserina leads to various degrees of pre and post-zygotic isolation [hnwd genes as well as the hybrid necrosis phenomenon caused by NBS-LRR genes in A. thaliana [The causes and the consequences of genetic incompatibilities currently raise a keen interest among evolutionary biologist because of their role in speciation processes ,57-59. Fsolation . The prethaliana ,13 illusP. anserina have been reviewed recently [P. anserina isolate called s harbours the het-r, het-V, het-c2 and het-e4 alleles at the het-r, het-v, het-c and het-e loci and is nearly isogenic to all other strains used in this work [s strain and a strain bearing the het-R and het-V1 alleles at the het-r and het-v loci will produce SI het-R het-V F1 progeny. As incompatibility between het-R and het-V alleles is thermosensitive, these SI strains can grow as wild type at 32°C, while transfer to 26°C triggers the incompatibility reaction [s strain and a strain bearing a single incompatible allele at the het-e locus (het-Ea) produce a het-C het-E SI strain. Incompatibility in these SI strains is partially suppressed by the addition of 6 g/l of dihydrostreptomycin in the culture medium [het-R het-V or het-C2 het-E SI strains in permissive conditions (32°C or dihydrostreptomycin containing medium) for 24 h until the mycelia reached 1 cm in diameter, before transfer to non permissive conditions (26°C or dihydrostreptomycin free medium). After 5 days of incubation we observed spontaneous mutant sectors arising. We collected a single mutant sector per starting colony to avoid analysing several times the same mutant. Vegetative incompatibility was assessed by barrage testing and sexual compatibility was assessed by spermatization as previously described [Conditions and media used for recently . The refhis work . Becausereaction . A crosse medium . We seleescribed .BglII (Biolabs).Routine genomic DNA extractions were performed as described by , or usinGene and loci names are italicized while protein names are not. Active alleles are indicated with capitals while inactive alleles and loci are indicated with lower cases.DC and MP carried out the experimental work. CC, SJS and MP participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.Details of the primers used.Click here for fileMapping of the deletions occurring in the dWD class of mutants of the RV collection.Click here for filehnwd gene family members.Schematic representation of the parental WD repeat domains of the Click here for fileP. anserina nwd gene family, along with the new complex WD40 repeat unit sequences from the RV and CE collections.Neighbor joining phylogenetic tree constructed with the nucleic sequences corresponding to WD40 repeat units from all members of the Click here for filehet-R allele (A and B) of from the CE collection with the original het-E allele (C and D) were compared.Protein sequences of WD repeat domains of randomly selected deletion mutants from the RV collection compared with WD repeat domain of the original Click here for fileEvidences for occurrence of gene conversion.Click here for fileEvidences for occurrence of unequal crossing overs.Click here for filehnwd gene family analysed here.Combinations of amino acids found at position 7, 9 25 and 27 of the WD40 repeat units in the Click here for filehet-R mutant alleles affected in the HET or NACHT domain.Nucleic and peptidic sequences of Click here for fileList of the accession numbers for the sequences used in this study.Click here for file


Retrograde peri-implantitis constitutes an important cause for implant failure. Retrograde peri-implantitis may sometimes prove difficult to identify and hence institution of early treatment may not be possible. This paper presents a report of four cases of (the implant placed developing to) retrograde peri-implantitis. Three of these implants were successfully restored to their fully functional state while one was lost due to extensive damage. The paper highlights the importance of recognizing the etiopathogenic mechanisms, preoperative assessment, and a strong postoperative maintenance protocol to avoid retrograde peri-implant inflammation. During the past decade, the use of osseointegrated implants has become an increasingly important treatment modality for the replacement of missing teeth in fully and partially edentulous patients The success of osseointegrated dental implants has revolutionized dentistry. With mor2The widespread use of these implants has in recent years, produced different types of complications. Retrograde peri-implantitis, a lesion occurring at the periapical area of an osseointegrated implant, has recently been described as a pos58The usual plaque related or occlusion related peri-implant failure is relatively easily identified when compared to the retrograde peri-implant lesions. With the ever increasing esthetic demands of patients, early loading of implants, especially in the anterior segment has become a necessity. As a number of teeth in the anterior segment are lost due to trauma and other non-periodontal causes, the clinician must be aware of the potential risk of developing retrograde peri-implantitis due to periapical pathology in the existing socket/adjacent teeth. This paper presents four cases of retrograde peri-implantits (each with different etiopathogenic mechanism), three of which were successfully managed and subsequently restored to function. The paper then discusses the possible etiopathogenic mechanism and suggests guidelines for the early identification and management of peri-implantitis.$. Adequate primary stability was obtained at the time of placement. When reviewed after six weeks, the site showed signs of abscess formation in the alveolar mucosa in relation to implant #31. There was no evidence of probing depth around the implant, but radiographs revealed peri-implant bone loss at the middle third region. The implant was found to be stable with no mobility. Open flap debridement was done and the implant surface decontaminated with universal implant deplaquer$$. The implant was subsequently followed up for a period of one year with regular three-month clinical and radiological reviews.A 42-year-old female patient presented with a history of failed endodontics followed by extraction and socket preservation in relation to #31 six months prior to reporting to the implant clinic. The adjacent teeth #32 and #41 showed endodontic restorations. After clinical and radiological evaluation it was decided to place a 3.0 × 12 mm single piece implantThe normal peri-implant sulcus depth and absence of other inflammatory signs in the peri-implant mucosa suggests that anterograde peri-implantits was not the cause for bone loss observed in this case. Even though endodontic therapy was performed in the adjacent teeth, placement of implant might have triggered latent periapical pathology12 from t13The process of implant placement could result in activation of this latent response either due to overheating or contamination or a combination of both. This activation could have resulted in the rapid bone loss in a short period of time. The importance of periodic clinical and radiographic examination of implants that are placed adjacent to endodontically treated teeth has to be emphasized and a shorter recall program has to be instituted to identify and manage retrograde peri-implant bone loss in its early stages.$ was placed with adequate primary stability. In two weeks’ time patient presented with signs of peri-implant abscess formation and mobility of the implant in relation to tooth #21. Radiograph revealed peri-implant bone loss at the apical third as well as the adjacent tooth #22. Open flap debridement was done with universal implant deplaquer$$ and the osseous defect was filled with calcium phosphate and Hydrase#. Tooth #22 was endodontically treated. The implant was subsequently followed up for a period of one year with regular three-month clinical and radiological reviews.An 18-year-old male presented with a history of trauma two weeks prior to presentation resulting in avulsion of tooth #21. After clinical and radiological evaluation a 3.0 × 15 mm single piece implantIn this case, the implant was placed in an avulsed site adjacent to tooth #22 that exhibited signs of trauma (Ellis class II fracture). The tooth was tested for vitality using the EPT (electric pulp tester), which revealed the presence of a vital pulp. Subsequently, the implant placement was undertaken without endodontic therapy in tooth #22. This case illustrates the limitations of using the EPT  when testing the vitality of the tooth. The undetected pericapical pathology flare following implant placement resulting in the retrograde peri-implant lesion is identified in this case. The fractured tooth adjacent to an edentulous site must be critically evaluated for evidence of dormant periapical lesions and the et al,[et al,[Management of the peri-implantitis was performed as stated by Tözüm M et al, and Peñal,[et al, by treat$ was placed in relation to #31 and #41 with adequate primary stability. At one-month review abscess formation was noticed around implant #31 with mobility. Implant #41 was intact with no signs of peri-implantits. Radiographic examination revealed peri-implant bone loss at the apical third as well as the adjacent tooth #32. A flap was raised and thorough debridement was done with universal implant deplaquer$$ followed by placement of bioceramic bone graft## and collagen membrane* along with endodontic restoration of tooth #32 on the same day. The implant was subsequently followed up for a period of one year with regular three-month clinical and radiological reviews.A 38-year-old male presented with a history of treated chronic periodontitis and missing teeth #31 and #41. Clinical and radiological examination revealed inadequate ridge width for implant placement; hence block graft augmentation was performed with a ramus block graft at the site. After 6 months, two 3.0 × 15 mm single piece implantExistence of active periodontal inflammation in the adjacent teeth is one possible etiopathogenic mechanism responsi$ was placed in relation to #11 with good primary stability. In two weeks’ time patient presented with signs of peri-implant mucositis and mobility. The implant was removed the subsequent week.A 25-year-old male patient presented with a history of failed endodontic therapy in relation to tooth #11 and subsequent extraction 6 months prior to presentation to implant clinic. Clinical and radiologic examination revealed adequate hard and soft tissue dimensions for implant placement. A 3.0 × 15 mm single piece implantThe existence of a periapical pathology necessitated extraction of the tooth after failure of endodontic therapy. Although there was no radiographic evidence of any pathology in the periapical region at the site of implant placement, the previous periapical pathology17 had obRetrograde peri-implantits constitutes an important source of implant failure. Careful preoperative evaluation of the site, adjacent teeth, and postoperative assessment of the implant placed could reduce the chances of development of retrograde peri-implantitis. Once diagnosed, the lesion has to be treated aggressively rather than by observation and conservative management.$ BioHorizons Maximus® Single Piece Implant$$ Universal Implant Deplaquer (Straumann)# Regen Biocement and Hydrase (Steiner Laboratories)## Grabio Glascera* Healiguide membrane


Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms.etc. were found tethered to the serine protease domains. Some domain combinations  were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes.A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes, suggesting distinct roles for serine proteases in prokaryotes. Many domain combinations were found unique to specific prokaryotic species, suggesting functional specialisation in various cellular and physiological processes. Proteolysis also contributes to the timely inactivation of proteins and is a major biological regulatory mechanism in living systems GDSA [S/T]) ) ,64. TheyA [S/T]) . In addiA [S/T]) .Rubrobacter xylanophilus) ; NP_623361.1 ; NP_953479.1  etc suggesting that some of the LonB-like proteins in bacteria were disseminated to different species through multiple lateral gene transfer events  and NP_578196.1 (Pyrococcus furiosus). Similarly, a Thermococcus kodakaraensis KOD1 Lon protease-like protein YP_184581.1 co-clusters with NP_126400.1 (Pyrococcus abyssi) and NP_579167.1 (Pyrococcus furiosus)  co-clusters with two archaeal Lon protease-like proteins NP_616787.1 (Methanosarcina acetovirans) and NP_635142.1 (Methanosarcina mazei), suggesting the possibility of the lateral transfer of Lon protease genes between bacteria and archaea  were identified in the present analysis. They display a higher representation in Euryarchaeota than trypsins, DD-peptidases and Clp proteases (Tables  SPHs wer) Figure . YP_6446ccus sp.  suggestiSerine protease-like domains often exist as a part of multi-domain polypeptides. In several enzyme families, changes in the domain alliances are known to modulate the enzyme function, usually by altering the substrate specificity or enzyme efficiency. The co-existing domains may also play a key role in the substrate specificity of these proteins, either by facilitating protein-protein interactions or their specific involvement in pathways ,68. SuchOf the 966 serine protease-like proteins identified in the present study, 311 32%) were found to carry co-existing domains. However, the distribution of the multi-domain proteins is not uniform across the five families, which display unequal preferences to enter into domain alliances. While, trypsins and subtilisins have a significant proportion of the multi-domain representatives, DD-peptidases and Clp proteases are overwhelmingly single-domain polypeptides. Most Lon protease representatives are multi-domain proteins  were multi-domain polypeptides, where 15 different domain architectures were observed. The impact of some unique domain combinations observed in prokaryotic trypsin-like proteins and their likely influence on the biological functions of these proteins are discussed.Hahella chejuensis  or two (49 of 112) PDZ domains tethered to the C-terminus of the trypsin-like domain  domains N-terminal to the protease domain, FHA domain which is found in diverse proteins associated with metabolic processes such as DNA repair, signalling, transport etc [Streptomyces avermitilis was associated with a CBM_5_12 module C-terminal to the protease domain; these are presumed to have a carbohydrate-binding function. YP_273108.1 in Pseudomonas syringae was associated with TerD module N-terminal to the protease domain; this domain, found in tellurite resistance proteins, is required for growth in toxic medium. This is not functionally characterised to our knowledge. NP_604177.1 in Fusobacterium nucleatum carries an Endonuclease_NS domain, which encodes an endonuclease that acts on double and single-stranded nucleic acids [Some trypsin homologues were found associated with regions most similar to modules likely to function in signalling and metabolism Table . Trypsinport etc . NP_8221Streptomyces avermitilis were associated with Alpha lytic protease prodomain (Pro_Al_prot), usually associated with Alpha-lytic endopeptidases – a subset of trypsins involved in lysing and degrading soil organisms (Table Trypsin homologues (NP_822175.1 and NP_827729.1) from Pelodictyon luteolum was found associated with six Sel1 repeats that were originally identified in a negative regulator of Notch signalling pathway in Caenorahbditis elegans [Modules of indeterminate function were also found associated with some trypsin-like proteins. For example YP_374752.1 from  elegans . HoweverSubtilisins were found to be the most versatile of the five serine protease families. 85 out of 227 37%) proteins with subtilisin-like domains were identified as multi-domain polypeptides and a total of 38 different domain combinations were discerned, many of which are specific to bacteria or unique to certain prokaryotic species (Table 7% proteiIdiomarina loihiensis) or two  PPC domains C-terminal to the protease domain were identified in different prokaryotic lineages. While the single PPC-domain containing subtilisin-like proteins were identified only in archaea and Gram-negative bacteria , subtilisin-like proteins with two PPC domains were identified in both Gram-positive and Gram-negative bacteria as well as archaea (Tables Bacillus subtilis) were identified that carry PA domain inserts in the subtilisin protease domain. PA domain is suggested to form a lid-like structure that covers the active site in the protease and is believed to be involved in protein interactions or mediate substrate recognition by proteases [Natromonas pharaonis) associated with PKD domain were also identified. PKD domains are predicted to be involved in protein-protein and protein-carbohydrate interactions [Several subtilisins identified in the present study reveal their association with domains that contain regions facilitating protein-protein interactions Table . Subtiliroteases . Subtiliractions  (Table 3Pseudomonas fluorescens) were identifiedthat carry an Autotransporter beta-domain, C-terminal to the subtilisin domain . NP_689039.1 in Sterptococcus agalacticae additionally carries a closely related motif called YSIRK type signal peptide [Halobacterium. Cleaved_Adhesin domain found in hemagglutinins and peptidases that in Porphyromonas form components of extracellular virulence complex RgpA-Kgp [Symbiobacterium thermophilum. Big_2 domain possibly associated with cell adhesion in bacteria was found encoded by subtilisin-like protein NP_969490.1 in the predatory bacterium Bdellovibrio bacteriovorus where it is likely to be associated with the hydrolytic machinery that facilitates the bacterium's predatory lifecycle [Thermoanaerobacter tengcongenesis, which also carries a pair of SLH domains believed to anchor the peptidoglycans [Pseudomonas putida; NP_927988.1 in Photorhabdus luminescens); CARDB-cell adhesion related bacterial domain (NP_954260.1 in Geobacter sulfurreducens) [Salinibacter ruber)  carries a NosD module C-terminal to the subtilisin domain; NosD is a periplasmic protein believed to insert copper into exported reductase apoenzyme [Lactobacillus johnsonii, encodes a multi-domain protein with five FIVAR modules, a putative sugar binding domain mostly found in cell-wall associated proteins [Bdellovibrio bacteriovorus was found to carry a CUB domain, an extracellular module associated with diverse functions in development and signalling in eukaryotes, however, its role in prokaryotes is not clear [Many subtilisin-like proteins were identified with the regions most similar to the modules likely to function in signalling and metabolism flanking the protease domain Table . NP_6169poenzyme . NP_9658ot clear  . NP_967370.1 gene product in Bdellovibrio bacteriovorus codes for a Proprotein convertase P-domain C-terminal to the subtilisin domain. It is associated with the kex2/subtilisin endopeptidases in eukaryotes, gammaproteobacteria and few others and is believed to be necessary for the folding and maintenance of the subtilisin domain and regulating its calcium pH specificity [Thermoplasma acidophilum (archaea) encodes for a thermopsin module, N-terminal to the subtilisin domain, similar to those found in the thermostable acid proteases in archaebacteria [Some subtilisin-like proteins were found associated with the domains that likely modulate the function of the adjacent subtlisin protease domain Table . A subticificity . NP_3942bacteria   in NP_951948.1 in Geobacter sulfurreducens; BNR repeats in NP_824495.1 in Streptomyces avermitilis; PilZ domain in NP_969350.1 in Bdellovibrio bacteriovorus; Tryptophan rich (W_rich_C) domain in YP_382882.1 in Synechococcus [Several modules of unknown or indeterminate function were also found associated with subtilisin-like proteins in prokaryotes Table . These itilisins  seen in Hahella chejuensis was found to encode a region most similar to ABC transporters that function in translocation of diverse compounds across biological membranes [Streptomyces avermitilis carries three each of Condensation (associated with enzymes that synthesise peptide antibiotics [Bacteroides thetaiotaomicron) and YP_444518.1  were found associated with the Glyco_hydro_3 module found in the O-Glycosyl hydrolases that hydrolyse the glycosidic bond between two or more carbohydrates. YP_444518.1 also carries a Glyco_hydro_3_C module, often found in association with Glyco_hydro_3 and is involved in catalysis and binding β-glucan [DD-peptidase-like proteins were found to be extremely rigid in terms of domain combinations. Only five of 254 proteins carrying DD-peptidase-like domains were identified as multi-domain polypeptides, in sharp contrast to other serine protease families analysed here, except Clp proteases Table . These eembranes . Anotheribiotics ), AMP-biβ-glucan  (Table 3Aeropyrum pernix), NP_126341.1 (Pyrococcus abyssi) and NP_579262.1 (Pyrococcus furiosus) were identified in hyperthermophilic archaea and are associated with NfeD-like module C-terminal to the protease domain (Table ATPases Associated with diverse cellular Activities) modules, which are one of the most diverse and promiscuous modules known to associate with diverse domains and function in a wide range of physiological processes [Clp proteases show an overwhelming preference for existence as single domain polypeptides. Only three of 121 Clp protease homologues identified in the current study were found to carry additional domains  and the Sigma54_activat-AAA-S16 in gene products such as YP_183677.1 (Thermococcus kodakaraensis) (Table Methanococcus jannaschii). An archaeal Lon protease-like protein NP_578196.1 (Pyrococcus furiosus) carries a Magnesium chelatase and a Sigma54_activat domain N-terminal to the predicted Lon protease domain  N-terminal to Lon protease domain, which may facilitate interactions with specific substrates  gene products were found to retain co-existing domains and display a conserved domain architecture  suggests their probable functions as virulence factors and in antibiotic resistance. Interestingly, while trypsins are also well represented in the eukaryotes, subtilisins (with the exception of plants) and DD-peptidases are less abundant in higher organisms suggesting that such enzymes were likely lost during the evolution as an adaptation to the cellular (and the extracellular) environment in the eukaryotes. Phylogenetic analysis suggests putative lateral transfer of serine protease genes between different bacterial and archaeal species and also between some bacteria and archaea. It is likely that some serine protease-like proteins may have been disseminated in the different prokaryotic species through probable horizontal gene transfer events. The lateral transfer of the serine protease genes in bacteria may possibly confer an evolutionary advantage on the recipient [Genome-wide studies reveal a large number of serine proteases belonging to the trypsin, subtilisin, DD-peptidase, Clp protease and Lon protease families in prokaryotes. However, there is only a limited knowledge available about their probable biological functions. Trypsins, subtilisins and the DD-peptidases have a higher number of representatives than the Clp protease and the Lon protease families in the genomes considered for the present analysis. The differences in the representations of the five serine protease families probably arose due to the selection of specific classes of serine proteases during evolution as an adaptation to different cellular and extracellular environments. For instance, the high abundance of the trypsins and the subtilisins in ecipient .etc were found tethered to the serine protease domain. Addition of new domains would permit these enzymes to acquire new functions and specificities contributing to the functional diversities of these gene families. However, a lack of significant repertoire of accessory domains does not necessarily indicate lack of functional diversity. Enzyme families may adopt alternative mechanisms to expand their functional repertoire, such as associating with limited but functionally diverse modules and other proteins or effecting changes in key amino acid residues. For instance, the Clp proteases form extensive complexes with the functionally diverse AAA+ modules that would enable them to modulate various physiological processes. The presence of multiple copies of the same accessory domain  in many serine protease-like proteins is another likely approach to expand their functional repertoire. Some domain combinations  were found to be widespread and conserved in prokaryotes suggesting a critical roles. Unique domain combinations of some prokaryotic serine protease-like proteins suggest their involvement in species-specific functions. Several domain architectures identified in prokaryotic serine proteases in the present analysis are very different from those reported in eukaryotic serine proteases. This highlights the distinct biological roles for the prokaryotic serine proteases compared to those in the eukaryotes. Some of these prokaryotic serine protease-like proteins with atypical domain combinations are attractive targets for experimental characterisation. Some pathogen peptidases identified in the present analysis with no identifiable homologues (unique domain architectures) in their hosts may be promising drug targets [Streptococcus pneumoniae, a human pathogen, are postulated to function in pathogenesis based on the domains associated with the serine protease domain. Some serine protease-like proteins such as NP_967057.1 (CUB domain) in Bdellovibrio bacteriovorus and YP_374752.1 in Pelodictyon luteolum (Sel1 repeats) are associated with domains similar to eukaryotic signalling modules with no known functions in prokaryotes. A systematic deletion of the one or more co-existing domains in the gene products with atypical domain combinations and the resulting phenotypes may help understand their roles in pathogenesis and other prokaryotic physiological processes and the role of the co-existing domains in modulating the functions of these serine proteases. Similarly, a phylogenetic cluster of the trypsin-like proteins (such as NP_302493.1 in Mycobacterium leprae) that contain a Colicin_V domain known to function in pathogenesis, tethered C-terminal to the protease domain, suggests an acquisition of the unique patterns in the interface region of the trypsin domain in these gene products. Identification of the conserved domain-domain interface regions and mutagenesis may help understand the function of these gene products and the role of the interactions between the adjacent domains.In the absence of the experimental characterisation for the most of the proteins sequences, an approximation of their biological functions is often inferred based on their sequence similarities to the proteins of known function. Studies have shown that the overall biological functions and the interactions of the multi-domain proteins are conserved by the retention of the domain composition and sequential arrangement . Therefo targets . For exaThe systematic analysis of the five serine protease families in the representative prokaryotic genomes is expected to enable a better understanding of the previously uncharacterised serine proteases encoded in the various genomes. The numbers of the serine protease-like proteins is likely to increase with the increasing amounts of the prokaryotic genomic data and the present analysis should help provide paradigms that would be useful in extending such analyses to a broader repertoire of the prokaryotes. The diversity of the functional domains co-existing with the protease domain in the serine protease-like proteins has provided clues to their biological functions, much of which are yet to be characterised experimentally. Experimental characterisation of some of these gene products as proposed here may help uncover the specific functional roles for the serine proteases in various cellular and physiological processes and help understand their influence on growth and development in the prokaryotic species.ATP: Adenosine triphosphate; NCBI: National Center for Biotechnology InformationLT and RS conceived of the study and participated in its design and coordination. LT carried out the computational sequence analysis. LT authored the first draft of this manuscript and RS provided comments and revisions to the final version of this text. Both authors read and approved the final manuscript.An inventory of the serine protease-like proteins belonging to the five chosen families identified in the various prokaryotic genomes and their inferred domain architectures. An inventory of serine proteases belonging to five chosen families identified in various prokaryotic genomes using the multi-fold approach (see Methods for details) and the details of co-existing domains (adjacent to the serine protease domain) identified for each putative serine protease.Click here for fileA list of the various prokaryotic genomes considered for the present study. The species abbreviations employed in the illustrations of the phylogenetic trees accompany the species name in parentheses. A list of the genomes in which serine protease-like proteins were identified in the current study. The genomes have been categorized accoding to their taxonomic lineages and the abbreviated species IDs (in parenthesis) that have been suffixed to the protein identifiers in the phylogenetic trees accompany the species name.Click here for file