To improve image and diagnostic quality in dynamic CT myocardial perfusion imaging (MPI) by using motion compensation and a spatio-temporal filter.Dynamic CT MPI was performed using a 256-slice multidetector computed tomography scanner (MDCT). Data from two different patients–with and without myocardial perfusion defects–were evaluated to illustrate potential improvements for MPI . Three datasets for each patient were generated: (i) original data (ii) motion compensated data and (iii) motion compensated data with spatio-temporal filtering performed. In addition to the visual assessment of the tomographic slices, noise and contrast-to-noise-ratio (CNR) were measured for all data. Perfusion analysis was performed using time-density curves with regions-of-interest (ROI) placed in normal and hypoperfused myocardium. Precision in definition of normal and hypoperfused areas was determined in corresponding coloured perfusion maps.The use of motion compensation followed by spatio-temporal filtering resulted in better alignment of the cardiac volumes over time leading to a more consistent perfusion quantification and improved detection of the extend of perfusion defects. Additionally image noise was reduced by 78.5%, with CNR improvements by a factor of 4.7. The average effective radiation dose estimate was 7.1±1.1 mSv.The use of motion compensation and spatio-temporal smoothing will result in improved quantification of dynamic CT MPI using a latest generation CT scanner. Dynamic contrast enhanced CT scans enable collection of kinetic parameters . For instance, dynamic CT myocardial perfusion imaging (MPI) could help assess the hemodynamic significance of coronary artery stenosis on the associated myocardial tissue. In the last decade, coronary CT angiography (CCTA) has gained increased importance for non-invasive evaluation of the coronary arteries and the detection of coronary artery disease (CAD) The aim of this study was to assess the potential of MPI on a wide detector CT scanner in combination with motion correction and a spatio-temporal filter for improved quantification and reduced sampling resulting in radiation dose savings.In this study data from two different patients – with and without myocardial perfusion defects – were evaluated to illustrate potential improvements for MPI. Institutional review board approval  and written informed consent was obtained from all patients before enrollment in the study. Both patients with known CAD underwent magnetic resonance (MR) perfusion imaging and also invasive coronary angiography (ICA) within 4 weeks following perfusion imaging.Patient 1 is a 57-year-old male with known coronary atherosclerosis and significant (>90%) stenosis of the left circumflex artery (LCA) and the intermedial branch with prior unsuccessful interventional recanalization. The distal RCA is functionally occluded. MR imaging showed perfusion deficits in the inferior, inferolateral and lateral wall of the left ventricle with accompanying hypokinesia of the left ventricular (LV) wall and a reduced ejection fraction. Patient 2  has known CAD without interventional therapy in the past. No significant perfusion deficits were seen in MR perfusion imaging, and ICA showed coronary atherosclerosis but no significant coronary stenosis.Dynamic MPI was performed using a 256-slice multidetector CT scanner (MDCT) scanner with 80 mm z-axis-coverage  at 80 kVp, 250 mAs, 0.27 sec gantry rotation time and 250 mm field-of-view (FOV), with 360° reconstructions of 3 mm thick slices using the CA (smooth) kernel. Data was acquired during pharmacologically induced stress  in end-expiratory breath-hold. ECG-triggered axial scans with zero increment were performed at a specific cardiac location following an injection of 40 ml contrast medium  using a dual injector . Scans were triggered at alternate cardiac cycles at end-systole , resulting in 15 acquisitions over a period of 30 cycles. On this note, the option of scanning every alternate cardiac cycle was chosen to reduce the radiation exposure to the patient.The aim of motion compensation is to spatially align the entire set of acquisitions such that all scans are in the same reference frame. Here, a two-stage approach is chosen: in the first stage, the motion between any two adjacent scans (time frames) is estimated resulting in a displacement vector for every single voxel. The second stage concatenates the displacement vectors from all time frames in order to transform each scan towards the designated scan Motion estimation between two scans is also known as an “image registration” task. For clarity we define a set of A registration based only on a similarity measure may yield a deformed template image which perfectly matches the reference image as long as all gray values are present in both images. However, the problem is ill-posed and the underlying deformation does in general may not be applicable in a physical context. Therefore, an additional smoothness constraint (or regularizer) is considered which can be chosen to model the application specific physical properties. It should be noted that the regularization term prevents the resulting vector field from an unwanted physiologically implausible behavior . In addition, by regularization the registration algorithm is less affected by image noise, imaging artifacts or abrupt inflow of contrast agent. In this note we employ a regularizer based on the popular linear elastic potentialIts elastic properties are modeled by Lamé parameters By combining the similarity measure and the regularizing term, the problem can be formulated as finding a displacement field The computation of the Gâteaux derivative of (1) and (2) yields a necessary condition for For the discretization of (4) finite differences in conjunction with Neumann boundary conditions have been chosen. The resulting system of linear equations consists on one hand of a sparse, symmetric and highly structured matrix arising from the regularizer and, on the other hand, of a so-called force vector corresponding to the similarity measure . This filter configuration was chosen to ensure a satisfactory tradeoff between noise removal and preservation of the shape of the perfusion curves.2 were manually placed in the hypoperfused areas. As a control, equally sized ROIs were defined in remote myocardium of the lateral wall of the left ventricular chamber and the interventricular septum. Precision in definition of normal and hypoperfused areas was evaluated by three experienced radiologists (range 5–7 years) in corresponding coloured perfusion maps for peak enhancement (PE). Using the 17 segment model recommended by ACC/AHA/ASNC To emphasize possible improvements three datasets for each patient were generated: (i) original data, with no correction, (ii) motion compensated data, and (iii) motion compensated data with spatio-temporal filtering. All images were analyzed on a CT workstation  equipped with a prototype post-processing application . Image noise was calculated by the standard deviation of the mean CT attenuation values (HU) of the LV myocardium. Contrast-to-noise ratio (CNR) was calculated as mean HU within a specific ROI divided by image noise. Therefore, several ROIs of area 1 cm−1*cm−1) The effective dose of CT perfusion analysis was estimated by the product of the DLP (given by the scanner) and a conversion coefficient for the chest  to improve the quality of dynamic CT of the myocardium in a reduced radiation dose setting. The principal findings of our study have been that the use of motion correction and spatio-temporal filter results in: (a) an enhanced image quality, (b) better differentiation between normal and hypoperfused areas and (c) improved diagnostic quality of perfusion examinations. Additionally, we limited the radiation exposure by scanning every other cardiac cycle.For perfusion analysis, contrast enhancement for each voxel is assessed over a period of time. If the target organ (myocardium) shifts over time, standard perfusion evaluation tools cannot identify this discrepancy. As a result such tools are prone to including HUs of adjacent tissues that could appear because of spatial mis-alignment. Thus, the non-registered images suffer from artifacts caused by motion of the heart and motion of the whole chest e.g. due to breathing, and possible imperfect matching of the scan with the ECG interval. Those artifacts distort dynamic volume CT assessment of myocardial perfusion and may lead to misinterpretation of assessed values for perfusion measurements, for example in coloured perfusion maps.In this preliminary study, we did not observe any loss of diagnostic information with the use of motion compensation and the spatio-temporal filter. Instead, the use of these post-processing algorithms actually reduced the blurring of boundaries between normal and small and hypodense regions of the myocardium, thereby improving the quality of perfusion maps. For instance, using these algorithms, we were able to better detect the extent of hypoperfused territory (endocardial versus transmural, With regard to z-coverage, compared to the previous generations of MDCT scanners, the increased detector width of 256 or more slices offers also for perfusion imaging the possibility to image the whole left ventricular myocardium without table movement. So the complete target volume can be imaged within each scanning circle, and there is no need of conjunction of adjacent scan volumes for assessing a complete perfusion scan of the heart.This study was designed as a feasibility study for first evaluation of motion compensation and a spatio-temporal filter in CT MPI. Therefore, only two patients were included for clinical evaluation. This is a major limitation of our study, and these findings have to be proved in a clinical trial with a larger sample size.We demonstrated that dynamic CT MPI is a fast and accurate tool for detection of perfusion abnormalities. With the proposed post-processing steps the quality of MPI increases and the accuracy for diagnostics increases. So this technique for the first time offers the possibility to differentiate between subendocardial and transmural infarction in patients with myocardial ischemia in CT imaging. With this in mind a future step could be to further optimize radiation dose to the patient while using the proposed post-processing tools in combination the modern reconstruction algorithms, such as iterative reconstruction techniques.


Raf-1 kinase inhibitor protein (RKIP) has emerged as a significant metastatic suppressor in a variety of human cancers and is known to inhibit Ras/Raf/MEK/ERK signaling. By suppressing the activation of the NFkB/SNAIL circuit, RKIP can regulate the induction of epithelial-mesenchymal transition (EMT). The aim of this study was to evaluate RKIP expression and to determine its association with clinicopathological features, including EMT in form of tumor budding in pancreatic ductal adenocarcinoma (PDAC).114 well-characterized PDACs with clinico-pathological, follow-up and adjuvant therapy information. RKIP-expression was assessed separately in the main tumor body and in the tumor buds. Another 3 TMAs containing normal pancreatic tissue, precursor lesions  and matched lymph node metastases were stained in parallel. Cut-off values were calculated by receiver operating characteristic (ROC) curve analysis.Staining for RKIP was performed on a multipunch Tissue Microarray (TMA) of We found a significant progressive loss of RKIP expression between normal pancreatic ductal epithelia (average: 74%), precursor lesions , PDAC (average 20%) and lymph node metastases . RKIP expression was significantly lower in tumor buds (average: 6%) compared to the main tumor body . RKIP loss in the tumor body was marginally associated with advanced T-stage (p = 0.0599) as well as high-grade peritumoral (p = 0.0048) and intratumoral budding (p = 0.0373). RKIP loss in the buds showed a clear association with advanced T stage (p = 0.0089).The progressive loss of RKIP seems to play a major role in the neoplastic transformation of pancreas, correlates with aggressive features in PDAC and is associated with the presence of EMT in form of tumor budding. RAF kinase inhibitor protein  is considered a metastasis suppressor protein, acting as an endogenous inhibitor of the Raf–mitogen-activated protein (MAP) kinase (MEK)–extracellular signal-regulated kinase (ERK) pathway by inhibiting the phosphorylation and activation of MEK by Raf-1 . It has A reduced RKIP expression has been shown to be associated with tumor progression and unfavourable prognosis in a variety of human malignant tumors -9. In rePancreatic ductal adenocarcinoma (PDAC) is a common cause of cancer death and has a dismal prognosis . Most paTumor budding reflects a type of diffusely infiltrative growth frequently observed in gastrointestinal carcinomas and it is defined as the presence of detached, isolated single cells or small cell clusters (up to 5 cells) scattered in the stroma at the invasive tumor front -25 and hDue to our previous findings on colorectal cancer, we hypothesized that loss of RKIP may be associated with the frequent occurrence of tumor budding in pancreatic cancer and may play a role in pancreatic carcinogenesis. We therefore undertook the analysis of RKIP expression on a multi-punch TMA from a well-characterized cohort of 120 pancreatic ductal adenocarcinomas (PDACs), their precursor lesions (PanINs) and matched lymph node metastases. RKIP expression was correlated with clinicopathological data and especially with the presence of tumor budding. The REMARK guidelines were used as a basis for this biomarker study .120 non-consecutive PDAC patients surgically treated between 2000 and 2010 were randomly selected. Paraffin-embedded tissue blocks of primary tumors were retrieved from the Department of Pathology, Aretaieion University Hospital, University of Athens Medical School, Greece. All histomorphological data were reviewed from the corresponding hematoxylin and eosin (H&E) stained slides, while clinical data were obtained from chart reports. Clinicopathological information for all patients included age, gender, tumor diameter, number of positive lymph nodes and total number of lymph nodes harvested, TNM stage (7th Edition), perineural, as well as blood vessel and lymphatic invasion and resection margin status (R-status). Information on post-operative therapy was available for all patients. The use of this material was approved by the local ethics committees of the University of Athens and University of Bern.For each patient, the hematoxylin and eosin slides of the primary tumor from the corresponding whole tissue sections were evaluated and representative areas of the tissue were marked using a felt-tip pen for easy detection. To exclude bias because of possible tumor heterogeneity, each patient had 4 tumor punches (2 tumor center + 2 tumor front) taken from formalin-fixed, paraffin-embedded blocks using a tissue cylinder with a diameter of 0.6 mm that were subsequently transferred into 1 recipient paraffin block (3 × 2.5 cm) using a homemade semiautomated tissue arrayer. Tissues were obtained from the tumor center and the invasive tumor front so that each patient had at least 4 tumor punches included on this array . Three additional one-punch TMAs were constructed including normal pancreatic tissue (147 punches), precursor lesions  and matched lymph node metastases (94 punches).TMA blocks were cut at 4 μm and immunostained for pan-cytokeratin AE1/AE3 , that served to highlight areas of tumor budding and RKIP . Sections were de-waxed and re-hydrated in dH2O. RKIP staining was performed using a Bond Max Autostainer from Leica Microsystem  with antigen retrieval performed in citrate buffer at 100 for 20 min. Normal pancreatic tissue and nerve tissue served as an internal positive control. Negative control was obtained by omitting the primary antibody. Whole tissue sections of 5 randomly selected cases were stained in parallel as validation of the TMA results.Tumor budding was defined as detached single cells or clusters of < 5 cells. Cases were evaluated for tumor budding using a 10-in-10 approach . BrieflyImmunohistochemistry was evaluated by estimating visually the percentage of positive cells per tissue microarray punch in 5% intervals . In the case of multiple tumor punches per localization, the average protein expression was calculated across all punches from the same localization. The end result was that each patient had a final protein expression score for the main tumor body, the tumor buds, the matched lymph node metastases, the precursor lesions (PanINs) and the normal pancreatic tissue . Evaluation was performed blinded to clinical endpoints.In order to determine a valid cut-off score for RKIP expression (low/high), receiver operating characteristic (ROC) curve analysis was performed, using the endpoint of tumor budding. A threshold value of 10% was identified. Association of RKIP expression with categorical clinicopathological features was performed using the Chi-Square test with continuity correction and the Fisher’s Exact tests; for continuous variables such as age and tumor size, the non-parametric Wilcoxon’s Rank Sum test was used. For matched analyses, the Wilcoxon’s Signed Rank test for pairs and the Cochran-Mantel-Haenszel test for three of more groups were used. Logistic regression analysis was used to determine the odds ratio (OR) and 95%CI for loss of RKIP expression with certain clinicopathological features. Missing data were few and were assumed to be missing at random. No imputation for missing values was performed. Univariate survival time analysis was performed using the log-rank test and differences plotted using Kaplan-Meier curves. P-values <0.05 were considered statistically significant. Correction for multiple hypothesis testing was not carried out . AnalyseOne hundred and twenty patients with PDAC were included in this study. RKIP expression could be successfully evaluated in 114 and included 33 cases (29%) with low-expression and 81 cases (71.1%) with high-expression. Median overall survival (OS) for the cohort of 114 patients was 11 months (95%CI: 10–13), while the median disease-free interval (DFI) was 5.0 months (95%CI: 4–6). Patient characteristics are outlined in Table A panel of representative examples of RKIP expression can be seen in Figure RKIP expression was restricted to the cytoplasm. A significant and striking progressive loss of RKIP expression was found from the normal pancreatic tissue including normal ductal epithelia 73.8%) to PanIN (36.7%), then to carcinoma (20%) and finally to lymph node metastases (8.3%) (p < 0.0001). Interestingly, within tumor budding cells of the primary carcinomas, RKIP expression was nearly absent (average expression 6%) , precursor lesions , pancreatic cancer (average 20%) and matched lymph node metastases . This implies that progressive RKIP loss may play a key role in the pancreatic neoplastic transformation and that it is taking place even before invasion occurs. Similar findings were observed in a previous study concerning esophageal Barrett mucosa, where a down-regulation of RKIP was found in high-grade dysplasia compared with non-dysplastic lesions . Our finRKIP has been found to play a major role in the regulation of EMT by intervening in Raf-1/MEK/ERK and NF-kB mediated signaling -6. NF-kbFurther, we identified a variation of RKIP protein expression between the main tumor body and the tumor buds. RKIP expression was found to be significantly and consistently lower in the tumor buds (average 6%) compared to the main tumor body . Moreover, after using a matched pairs analysis a statistically significant loss of RKIP expression (p < 0.0001) was observed between the tumor and corresponding tumor buds. Our results suggest that this variation in the localisation of RKIP loss within the cancer tissue is relevant for the association with specific aggressive phenotypic features. RKIP loss in the main tumor body was marginally associated with higher T-stage (0.0599) as well as with the presence of peritumoral (0.0048) and intratumoral budding (p = 0.0373), while RKIP loss in the tumor buds exhibited a significant association with increased T stage (p = 0.0089). This finding underlines the predictive role of RKIP loss in the main tumor body concerning the presence of high-grade budding throughout the tumor. Additional RKIP loss in the tumor buds themselves seems to be involved in the propagation of the neoplastic process and to lead to increased aggressiveness of the neoplasm.RKIP, by inhibiting the Raf-MEK-ERK, NFκB, GRK and activating the GSK3β signaling pathways is implicated in the sensitization of cells to therapeutic drugs. When its expression is reduced or lost this could lead to significant resistance to cancer therapy . HoweverOur present results should be understood in the context of the study limitations. Although TMAs provide an efficient and cost-effective tool for testing a comprehensive panel of potential biomarkers on a large number of tumor specimens, the TMA technique could raise concerns related to the sampling of large, heterogeneous tumors. The effect of tumor heterogeneity was minimized by sampling at least two punches from the center and two from the invasive front and evaluating the average protein expression across the total number of samples. Our study may further be limited by the relatively small number of PDAC patients and the fact that all cases come from a single center. Nonetheless, our study benefits from complete clinicopathological data with information on adjuvant therapy and follow-up and the adherence to the REMARK guidelines which are essential for proposing prognostic biomarkers.In conclusion, our findings suggest that progressive loss of RKIP may play a key role in the neoplastic transformation of pancreas and correlates with aggressive features of PDAC. Moreover, RKIP loss seems to be strongly associated with EMT in pancreatic cancer, as reflected by the presence of high-grade tumor budding. Further characterization of the budding cells is needed in order to identify a “budding-promoting profile” and to underline the similarities between budding cells and EMT process in pancreatic cancer.The authors declare that they have no competing interests.EK has made substantial contributions to conception and design, to acquisition and interpretation of data and has drafted the manuscript. IZ performed the statistical analysis and has been involved in revising the manuscript critically for important intellectual content. BG has been involved in revising the manuscript critically for important intellectual content. AKP has made substantial contributions to acquisition of data and has been involved in revising the manuscript critically for important intellectual content, AL participated in the design and coordination of the study, has been involved in revising the manuscript critically for important intellectual content and has given the final approval of the version to be published. AP participated in the design and coordination of the study, has been involved in revising the manuscript critically for important intellectual content and has given the final approval of the version to be published. All authors read and approved the final manuscript.


Drosophila eye in which two primary pigment cells (PPCs) with a characteristic ‘kidney’ shape enwrap and eventually isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that in the developing Drosophila eye the ligand Delta was transcribed in cone cells and Notch was activated in the adjacent PPC precursors. In the absence of Notch, emerging PPCs failed to enwrap cone cells, and hibris (hbs) and sns, two genes coding for adhesion molecules of the Nephrin group that mediate preferential adhesion, were not transcribed in PPC precursors. Conversely, activation of Notch in single IOCs led to ectopic expression of hbs and sns. By contrast, in a single IOC that normally transcribes rst, a gene coding for an adhesion molecule of the Neph1 group that binds Hbs and Sns, activation of Notch led to a loss of rst transcription. In addition, in a Notch mutant where two emerging PPCs failed to enwrap cone cells, expression of hbs in PPC precursors restored the ability of these cells to surround cone cells. Further, expression of hbs or rst in a single rst- or hbs-expressing cell, respectively, led to removal of the counterpart from the membrane within the same cell through cis-interaction and forced expression of Rst in all hbs-expressing PPCs strongly disrupted the remodeling process. Finally, a loss of both hbs and sns in single PPC precursors led to constriction of the apical surface that compromised the ‘kidney’ shape of PPCs. Taken together, these results indicate that cone cells utilize Notch signaling to instruct neighboring PPC precursors to surround them and Notch controls the remodeling process by differentially regulating four adhesion genes.Sporadic evidence suggests Notch is involved in cell adhesion. However, the underlying mechanism is unknown. Here I have investigated an epithelial remodeling process in the  Drosophila eye in which two primary pigment cells (PPCs) with a characteristic ‘kidney’ shape enwrap and isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that cone cells utilize Notch signaling to communicate with neighboring PPC precursors. In response to Notch signaling, PPC precursors activate transcription of hbs and sns, two genes coding for adhesion molecules of the Nephrin group that bind Rst and Kirre, adhesion molecules of the Neph1 group. At the same time, PPC precursors inactivate transcription of rst and kirre genes. In addition, binding of Hbs or Rst to its counterpart from the same cell (cis-interaction) destabilizes the protein complex and promotes removal of the counterparts from the membrane, leading to complementary distribution of four adhesion molecules within two populations of cells. Thus, Notch controls epithelial remodeling by differentially regulating four adhesion genes.In developing tissues, one way to isolate a group of cells from the rest of the tissue is to induce a few neighboring cells to surround them. How centrally localized cells communicate with neighboring cells and how neighboring cells respond to signaling is not well understood. This work describes a mechanism underlying an epithelial remodeling process in the  Drosophila wing, Hh signaling regulates cell segregation between anterior and posterior compartments  and Sticks-and-Stones (Sns) from the Nephrin group; Roughest (Rst) and Kin of Irre (Kirre) from the Neph1 group Evidence accumulated to date supports the notion that cell adhesion plays a direct role in tissue remodeling. As first noted by J. Holtfreter and later formulated in “Differential Adhesion Hypothesis” (DAH) by M. Steinberg: sorting behaviors of cells are driven by interfacial free energy arising from differential adhesion among cells Drosophila eye in which two primary pigment cells (PPCs) enwrap and isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that Notch signaling controls transcription of two groups of adhesion genes in the Drosophila eye. Notch activates adhesion genes of the Nephrin group but suppresses those of the Neph1 group. Differential distribution of two groups of adhesion molecules is further facilitated by removal of one group of adhesion molecules by another group through cis-interactions, leading to complementary distribution of four adhesion molecules within two populations of cells. This work uncovers a link between cell signaling and tissue remodeling.This work describes a mechanism underlying an epithelial remodeling process in the Drosophila eye derives from an invaginated epithelium at the embryonic stage The fa-gN Dl reporter, the Notch activity was detected in a significantly higher level in two cells adjacent to anterior-posterior cone cells than in other cells at 18 h APF (GBE-Su(H)m8-lacZ expression was found in both PPCs and the difference in the level of lacZ expression between these cells became less obvious than earlier stages  was expressed in a single PPC in the eye using a FLP-out technique hbs in a PPC hbs transcription was activated upon activation of Notch, ICDN was expressed in a single IOC. Upon activation of Notch, an ectopic activity of the hbs reporter P[w+]36.1 was observed in the target IOC  was over-expressed in a single cone cell (either anterior or posterior), the Rst level was increased about 71% at the border between the adjacent PPC and its neighboring IOCs . When Dlhbs transcription, fa-gN mutant was used along with the hbs reporter P[w+]36.1. In the wild type eye, the hbs reporter was detected in emerging PPCs as well as in cone cells fa-gN mutants, the hbs reporter activity was retained in cone cells but lost in PPC precursors at 40 h APF  exhibited a kidney-shape seen in wild type PPCs. In contrast, when ICDN (activated Notch) was expressed in single IOCs in the same mutant, 100% of clones  exhibited kidney shape  was used to monitor the rst activity. Upon activation of Notch in a single IOC, F6-lacZrst was lost in the target cell  based on the hbs and rst reporters : eporters . Howevereporters . This iseporters . At 27 heporters . At 40 heporters . They weeporters . A similin trans (interactions between proteins from two adjacent cells or trans-interactions) stabilize the adhesion complex on the membrane cis-interaction). To assess the effect of cis-interaction, Hbs was mis-expressed in the cells that normally express the counterparts Rst and Kirre. Upon expression of Hbs in a single IOC, the level of Rst was reduced about 63% at the PPC-IOC border and the number of vesicles was increased significantly in the target IOC . Similarly, when Rst was mis-expressed in a single PPC that normally transcribes hbs and sns, the Hbs level on the membrane was reduced 93% and the number of vesicles increased markedly in the target PPC (hbs reporter P[w+]36.1 was unchanged in the clone (data not shown). These results suggest that, while heterophilic interactions between two groups of IRM adhesion molecules in trans stabilize both proteins on the membrane, interactions among these proteins in cis destabilize proteins on the membrane and promote turnover of these proteins.Hbs and Sns from the Nephrin group and Rst and Kirre from the Neph1 group co-localize at the border between PPCs and IOCs, and heterophilic interactions between these two groups of proteins rget IOC . Neverthrget PPC . Similarcis-interactions on pattern formation, rst was mis-expressed in all PPCs using spa-Gal4. Spa-Gal4 is known to drive expression of transgenes in cone cells and PPCs rst in cone cells and PPCs (spa>rst), the hexagonal pattern of the eye was severely disrupted. While spatial organization of cone cells was mildly affected, various numbers of PPCs  were found adjacent to cone cells. More strikingly, IOCs failed to sort into single file. As a result, 2–3 rows of IOCs scattered in between ommatidia across the eye and the eye was extremely rough  only led to mild defects in IOCs and cone cells with largely intact PPCs . In the 54>rst eye, IOCs were occasionally found in cluster with a bristle group. Nevertheless, the hexagonal pattern was only mildly affected  elongated and the interface between the target cell and IOCs expanded in a manner similar to a wild type PPC . Single PPCs mutant for both sns and hbs had a shorten PPC-IOC border and reduced apical surface , all IOCs that contact cone cells have access to Dl and express Hbs. However, asymmetric expression of Dl in cone cells creates a bias. IOCs that contact anterior-posterior cone cells receive a high level of Notch signaling . Results presented herein support a model that cis-interactions provide a mechanism for removing counterpart proteins from the same cells , they share one common feature: presence of one protein interferes with the function of the counterpart protein in the same cell. What structural elements are involved in cis-interactions between these proteins and how cis-interactions lead to a reduction of protein activity still remain questions for further investigation.Drosophila eye. First, hbs can restore the ‘kidney’ shape of PPCs at a lower frequency than Notch (or ICDN) in the fa-gN mutant. This observation suggests that adhesion genes may not represent all the function of Notch in recruiting PPCs. Notch is known to have a wide range of target genes. In particular, several transcription factors are known targets of Notch for the determination of PPC cell fate Notch signaling are also involved in conferring on PPCs the ability to enwrap cone cells. Second, this work suggests a positive feedback loop that promotes selection of PPC precursors in the developing eye . Other flies used: F6-lacZrstspa-Gal4UAS-N-cadherinZF1.4sns and UAS-sns (gift of Susan Abmayr), ICDUAS-N (gift of Cedric Wesley), ICD-lexAUAS-N (gift of Toby Lieber), P[w+]36.1 and 459hbs (gift of Mary Baylies), UAS-hbs (gift of Helen Sink), m8-lacZGBE-Su(H) (N-lacZ) Gal-54UAS-rst (gift of Karl-F. Fischbach), UAS-kirre/duf (gift of Marc Ruiz-Gomez), UAS-Dl (gift of Marek Mlodzik) and hsFLP MKRS (gift of Matthew Freeman).The Single cell clones for over-expressing a target gene were generated using a FLP-out technique as described previously ICD/Act5C>y+>Gal4 UAS-GFP; hsFLP MKRS/+UAS-N  of the selected region was recorded using ImageJ. The background integrated density (ID0) was recorded by moving the same selection box to a background region. The membrane protein level (I) reflected by integrated intensity per unit length was determined following I = 2*(ID1−ID0)/L, where L is the perimeter of the selected region in pixel. For each experiment, the average intensity from 6 neighboring wild type borders was calculated and used as a control.Levels of membrane proteins were quantified using ImageJ


Motivation: Data from metagenomics projects remain largely untapped for the analysis of transcriptional regulatory networks. Here, we provide proof-of-concept that metagenomic data can be effectively leveraged to analyze regulatory networks by characterizing the SOS meta-regulon in the human gut microbiome.Results: We combine well-established in silico and in vitro techniques to mine the human gut microbiome data and determine the relative composition of the SOS network in a natural setting. Our analysis highlights the importance of translesion synthesis as a primary function of the SOS response. We predict the association of this network with three novel protein clusters involved in cell wall biogenesis, chromosome partitioning and restriction modification, and we confirm binding of the SOS response transcriptional repressor to sites in the promoter of a cell wall biogenesis enzyme, a phage integrase and a death-on-curing protein. We discuss the implications of these findings and the potential for this approach for metagenome analysis.Contact:erill@umbc.eduSupplementary information:Supplementary data are available at Bioinformatics online. Metagenomics projects, therefore, offer a unique window into the genetic composition of microbial ecosystems . In wellEscherichia coli) aimed primarily at addressing DNA damage through the activation of repair enzymes, translesion synthesis polymerases and cell division inhibitors . Putative LexA-binding sites were located by scoring all metagenome sequences on both strands using the iR index as implemented in FITOM  across the bacteria domain were derived from the published literature  queries. Predicted protein-coding genes and their COG annotations were obtained from the MetaHit gene dataset (iR scores was computed by counting the number of filtered putative LexA-binding sites (Supplementary Table S3) above a given score threshold, using 1 bit intervals in the 12–20 bit score range. Prototypical SOS COGs were defined as those having a regression coefficient of determination (2R) >0.85 in the 12–20 bit range, at least one site with score above the mean (16 bits) and a minimum of 10 sites in the 12–20 bit range. The 2R cutoff value of 0.85 was set simply by ranking COGs according to 2R value and gradually decreasing the 2R threshold until the inclusion of additional COGs corresponding to known SOS genes (Supplementary Table S2) incurred the inclusion of a larger number of COGs not known to be associated with SOS genes.For all COGs reported in the MetaHit data, the cumulative distribution of site Bacillus subtilis lexA gene was overexpressed in a pET15b vector provided by Dr Jordi Barbé. The B.subtilis LexA protein was purified as described previously (Supplementary Table S4). Electrophoretic mobility shift assays (EMSA) experiments were performed as reported previously , 200-fold of either specific or non-specific non-labeled DNA was added to the binding mixture. In all cases, samples were loaded in 6% non-denaturing Tris-glycine poly-acrylamide gel. Digoxigenin-labeled DNA-protein complexes were detected following the manufacturer's protocol (Roche).The Supplementary Table S1) to search the combined 7.1 Gbp sequence of the human gut metagenome of 86 healthy individuals . We filtered the initial results by requiring that putative LexA-binding sites be located in such upstream regions  and that their associated genes mapped to Gram-positive bacteria. This two-pronged filtering step resulted in a final count of >40 000 sites associated with >38 000 genes with products assigned to COGs (Supplementary Table S3). We validated this approach by analyzing the distribution of gene counts per COG category as a function of the score threshold  and comparing it with a reference ensemble of 18 known SOS regulons (Supplementary Table S2). The fit of the inferred network with the reference ensemble improves with increasing score threshold, leading to a progressive enrichment of all canonical SOS categories  except for cell cycle control (D). This canonical SOS category (Supplementary Table S5) is not enriched because SOS-regulated cell division inhibitors in Gram-positive bacteria were described after the creation of the COG database and their associated COGs are assigned to several different categories (We used a position-specific scoring matrix derived from a multispecies Gram-positive bacteria motifs (lexA and recA) drawn from genomic sequences. For these reference genes, LexA-binding site scores follow a normal distribution with a mean of ∼16.2 bits . In its central range (12–20 bits), the cumulative distribution can be fit by a linear model, providing a simple means to define prototypical SOS COGs on the MetaHit data. Data for other well-known transcriptional regulators suggest that this is a general property of TF-binding site distributions upstream of regulated genes . We used the coefficient of determination (2R) of the linear regression for the cumulative distribution of site scores on each COG, together with the presence of at least one high-scoring site, as an indicator of the likelihood of a COG being a prototypical member of the SOS regulon. Using these criteria and a conservative cutoff, we identified 12 putative SOS COGs (lexA and umuD (COG1974) or the excision repair helicase uvrB (COG0556). The remaining three are promising candidate additions to the SOS regulatory network. COG0732 maps to HsdS, a type I restriction endonuclease linked to virulence in Streptococcus suis . COG0399, for instance, is linked with the biosynthesis of a phase-variable O antigen and the extracellular matrix of Vibrio cholerae (lexA), sensing of DNA-damage and stabilization of DNA (recA), translesion synthesis  and excision repair . In agreement with the experimental evidence uncovered in different phyla  in promoter regions. This trend is not seen outside promoter regions, where the percentage of high-scoring sites (>16 bits) for COGs mapping to known SOS genes is similar to that observed for all COGs (∼0.5%). To accurately identify prototypical SOS COGs, we first analyzed LexA-binding sites upstream of canonical SOS genes , a phage integrase (Pint) and a death-on-curing protein (rhuM) (Supplementary Table S4). We validated binding to these sites through EMSA using purified B.subtilis LexA protein . Our results confirm that these LexA-binding sites are bound by LexA in vitro, suggesting that they are functional sites in vivo regulating the activity of their associated genes. The potential regulation of the cell division protein MraZ by the SOS response coincides with recent reports of distinct cell division regulators under control of LexA in Gram-positive bacteria and gives credence to the idea that the link between the SOS response and control of cell division may have evolved multiple times in this clade  award and by US National Science Foundation [MCB-1158056]. Fundació Cellex (fellowship to N.S.A.). The UMBC High Performance Computing Facility is supported by the US National Science Foundation MRI  and SCREMS [DMS-0821311] programs and by the University of Maryland, Baltimore County (UMBC).Conflict of Interest: none declared.


The majority of previous heroin cue-reactivity functional magnetic resonance imaging (fMRI) studies focused on local function impairments, such as inhibitory control, decision-making and stress regulation. Our previous studies have demonstrated that these brain circuits also presented dysfunctional connectivity during the resting state. Yet few studies considered the relevance of resting state dysfunctional connectivity to task-related neural activity in the same chronic heroin user (CHU).We employed the method of graph theory analysis, which detected the abnormality of brain regions and dysregulation of brain connections at rest between 16 male abstinent chronic heroin users (CHUs) and 16 non-drug users (NDUs). Using a cue-reactivity task, we assessed the relationship between drug-related cue-induced craving activity and the abnormal topological properties of the CHUs' resting networks. Comparing NDUs' brain activity to that of CHUs, the intensity of functional connectivity of the medial frontal gyrus (meFG) in patients' resting state networks was prominently greater and positively correlated with the same region's neural activity in the heroin-related task; decreased functional connectivity intensity of the anterior cingulate cortex (ACC) in CHUs at rest was associated with more drug-related cue-induced craving activities.These results may indicate that there exist two brain systems interacting simultaneously in the heroin-addicted brain with regards to a cue-reactivity task. The current study may shed further light on the neural architecture that supports craving responses in heroin dependence. Heroin addiction is a complex disease of the brain, involving both affective and cognitive processes, characterized by a compulsive drive to take drugs despite serious negative consequences Recent studies have suggested that resting state activity had a specific impact upon subsequent task-induced activity and may be relevant to individual variability in behavioral and mental states Long-term heroin dependence impairs cortical and subcortical limbic/paralimbic brain regions involved in emotion, reward, motivation and impulse control To characterize the rest-task interaction in CHUs, we used the method of graph theory analysis (GTA), which has become a powerful tool to investigate resting brain networks All research procedures were approved by the Institutional Review Board of the Fourth Military University on Human Studies and were conducted in accordance with the Declaration of Helsinki.Sixteen abstinent heroin-dependent males  were recruited from a local methadone replacement therapy center  were composed of household objects and tasks. Trials had 2 sec heroin-related or neutral stimuli followed by a variable interval (4–12 sec) during which a crosshair was shown. Images were rear-projected to the center of the visual field via a mirror mounted on the scanner head coil. Stimuli were randomized in an event-related experimental design using E-prime software . Subjects were placed in the scanner in a supine position using a foam head holder to reduce motion artifacts. Earplugs were used to safely reduce scanner noise. The task was initiated with a 10 sec dummy scan followed by the image. Task duration was 8 min and 10 sec . The scan was at least 5–8 hrs after the last methadone dose; a time point when the methadone plasma level was stable with daily dosing Before and immediately after each imaging session, craving ratings were obtained. Craving was assessed by a 0–10 visual analog scale (VAS), in which participants marked a 0  to 10 (“extremely high”) in response to the question, “To what extent do you feel the urge to use heroin?”This experiment was carried out in a 3T GE scanner. Prior to the functional run, subjects underwent ‘mock scans’  in order to become familiar with the scanning environment (for 1 min). A gradient echo T2*-weighted sequence with in-plane resolution of 3.75 mm×3.75 mm  and a set of T1-weighted high-resolution structural images  were acquired. The resting run generated 150 whole-brain volumes, and 240 whole-brain volumes in each subject were acquired in the drug-related cue-reactivity run.http://www.fil.ion.ucl.ac.uk/spm). All datasets were initially slice time corrected with a reference to the first slice acquired, and then corrected for temporal offsets using sinc interpolation and head movement-related effects using a six-parameter spatial transformation Image preprocessing was carried out using SPM5  to reduce the effects of low-frequency drift and high-frequency noise. Then, the mean time courses from the deep white matter and ventricles were regressed out from the filtered time series. The mean time course from the deep white matter was obtained by averaging the voxel values within a sphere (8 mm radius) positioned in the anterior portion of the right centrum semiovale comprised solely of white matter voxels. The mean time course from the ventricles was obtained by averaging the voxel values within the ventricles using the ventricle mask produced by the WFU PickAtlas Tool We investigated the brain's topological properties by way of binarized graphs (G) in which each correlation matrix was thresholded and converted to the adjacency matrix. The adjacency matrix is a means of representing which nodes of the graph are adjacent to other vertices with 1 being the existing edge and 0 indicating the absence of an edge between the two nodes. A brain functional connection could be represented as an undirected edge if the correlation coefficient between the two nodes achieved a correlation threshold T. Since there is no definitive method in selecting a single threshold, we followed a processing stream that is widely used in threshold selection j, and j. The minimum path length Small-world models are useful for connectivity studies of nervous systems because they have high clustering and a short path length which confers the capability for both specialized or modular processing in local neighborhoods, as well as including distributed or integrated processing over the entire network Degree (D) is another important statistical parameter under the graph theoretical framework. The degree of a graph is the number of edges incident to the vertex, and it is defined as the number of nodes across the brain showing a strong correlation with the target node. The brain regions showing a high value of degree in the network were considered to be a cortical hub, which may play a critical role in integrating diverse informational sources and balancing the opposing pressure to evolve segregated, specialized pathways t-test. For the most significant between-group difference threshold value, the global efficiency of the CHUs' resting network was investigated by examining its association with the duration of heroin use while controlling for age. Since each node has a 3D voxel coordinate in the brain, every node in the voxel-based network could be located in particular anatomical areas t-test.In our study, we investigated the small-world properties of CHUs' and NDUs' resting networks over the entire range of threshold values, and statistical comparisons of C, L, Voxel-based network analyses have the advantage of localizing cortical hubs to particular anatomical areas since each node has a 3D voxel coordinate in the brain t-test to determine if the intensity of functional connectivity (connection weights) was significantly different between the two groups. Furthermore, we performed partial correlation analyses between the CHUs' resting state functional connectivity intensity and individual duration of heroin use across all subjects while controlling for age.The resting brain is organized into segregated systems of brain areas, and these subsystems interact in a flexible manner to support various cognitive functions The task datasets were filtered using a high pass filter and cut-off at 128 sec. The statistical evaluation was based on a least-square estimation using the general linear model for each run and across each subject. To construct the task model, a set of delta functions for each condition in the experiment was convolved with the hemodynamic response in SPM. For each subject, heroin-related images vs. neutral image contrasts were created and were then entered into a second-level random-effects group analysis.To test the hypothesis that abnormal resting state activity affected drug-related cues induced activity in the CHUs' brain networks, the degree value of voxels in the CHUs' voxel-based networks exhibiting dysregulated functional connectivity (compared with NDUs') was correlated with the same region's level of BOLD activation during the cue-reactivity task .p<0.001, paired t-test).The mean baseline subjective craving score prior to stimulus presentation was 0.6 (±1.4). The mean subjective craving score post-stimulus presentation was 5.5 (±3.2). There were significant differences in CHUs' craving scores before and after the experimental session  in the threshold range between 0.49 and 0.60 for the CHUs' and NDUs' resting networks. The most significant between-group difference was found at threshold T = 0.56   . Such a t-test to determine if the degree of the brain voxels was significantly different between the CHUs' and NDUs' resting networks . As can be seen from Based on each nodal degree, we performed a two-sample two-tailed t-test. In our results, many pairs of connections were prominently altered . Furthermore, these resting state functional connectivities between brain regions presented a significant correlation with the duration of heroin use. Specifically, ten connections showed a marked difference (CHUs>NDUs) and a positive correlation was present with dependence duration in the CHUs' networks, predominantly involving the PH-PCC, PH-PFC, striatum-PFC, striatum-ACC and a connection within the PFC (red lines in p<0.05). Sixteen connections exhibited decreased intensity in the CHUs' resting networks, including the HIP/PH-striatum, THA-PFC, ACC-INS and a connection within the striatum (blue lines in p<0.05).While voxel-based network visualization provided strong hints of dysregulated brain regions in the CHUs' networks, local spatial correlations could influence the results of our network analysis. In order to avoid local correlations, we chose abnormal brain regions as the ROIs from the voxel-based network analysis to reconstruct the resting network for a region-based network analysis. Thirty-eight brain regions were considered as network nodes in our analysis. The time course of each ROI was correlated to every other ROI to obtain a 38×38 matrix of correlation coefficients in the CHUs and NDUs groups respectively. A total of 703 connections were obtained in each group. Comparisons of functional connectivity between the two groups' region-based networks were made using a two-sample two-tailed p<0.01, FDR corrected, cluster size >20 voxels as shown in To detect the BOLD signal changes between heroin-related cues and neutral cues, a GLM model was calculated across each subject with regressors for the difference from these two conditions (p<0.05) in CHUs. The meFG  showed more functional connectivities  at rest in the heroin-addicted group and the degree value of the meFG was positively correlated to the heroin-addicted individual's craving responses in the meFG  (p<0.05)  . The deg(p<0.05) . Further(p<0.05) .Certain studies pointed out that the fundamental function of resting state networks could serve as an indicator for dysregulation in brain connectivity In the current study, we examined the patterns on how the CHUs' functional connectivity during the resting state interacted with their brain performance on an associative heroin cue-reactivity task. We found a cumulative effect in heroin-addicted individuals that the longer the heroin use, more functional abnormality existed in the resting networks and the greater the craving activity was related to drug cues. Specifically, in patients with heroin addiction, the small-world property and information processing efficiency of the resting networks were prominently disrupted on a whole-brain level . From a The major finding of our study is that the addicted individual differences in dysregulated functional connectivity of the PFC  and ACC at rest correlated with individual differences in the magnitude of the same region's drug-related craving activities and patients' self-report craving scores . The PFCHeroin addiction was characterized by uninhibited behavior and loss of self-control for drug seeking, which far exceeded the desire for other non-drug-related goals p>0.05). In the current study, abnormal topological properties were explored in the major parts of the striatum , PH, HIP and PCC/precuneus . Although the number of voxels within an anatomic brain region changed quantitatively for different thresholds, no additional brain regions were found. The sample size used was fairly minute and all abstinent heroin-dependents were male, which may have resulted in a non-significant correlation between the network parameter and the performance mentioned previously. A larger sample size is pertinent in future studies to be statistically significant in validating these results.In summary, drug addiction is characterized as a complex brain disease


Interspinous distraction devices (IPDD) are indicated as stand-alone devices for the treatment of spinal stenosis. The purpose of this study is to evaluate the results of patients undergoing surgery for spinal stenosis with a combination of unilateral microdecompression and interspinous distraction device insertion.This is a prospective clinical and radiological study of minimum 2 years follow-up. Twenty-two patients (average age 64.5 years) with low-back pain and unilateral sciatica underwent decompressive surgery for lumbar spinal stenosis. Visual Analogue Scale, Oswestry Disability Index and walking capacity plus radiologic measurements of posterior disc height of the involved level and lumbar lordosis Cobb angle were documented both preoperatively and postoperatively. One-sided posterior subarticular and foraminal decompression was conducted followed by dynamic stabilization of the diseased level with an IPDD (X-STOP).The average follow-up time was 27.4 months. Visual Analogue Scale and Oswestry Disability Index improved statistically significantly (p < 0.001) in the last follow-up exam. Also, the walking distance increased in all patients but two. Posterior intervertebral disc height of the diseased level widened average 1.8 mm in the postoperative radiograph compared to the preoperative. No major complication, including implant failure or spinous process breakage, has been observed.The described surgical technique using unilateral microdecompression and IPDD insertion is a clinically effective and radiologically viable treatment method for symptoms of spinal stenosis resistant to non-operative treatment. Lumbar spinal stenosis, refractory to nonoperative treatment, has been traditionally treated with surgical decompression achieving good and excellent results in almost 80% of the cases . In case“Dynamic stabilization” spinal system is defined as the system that alters favorably the biomechanics (movement and load transmission) of a spinal motion segment, without immobilization of the segment ,4. X-STOThis is the first usage of X-STOP  device, an FDA approved IPDD for patients with spinal stenosis , in conjThis a prospective study of 22 patients aged between 57–71 years (average 64.5 years) with unilateral sciatica and low-back pain, in the form of neurogenic intermittent claudication, who were treated by unilateral microdecompression and X-STOP insertion in our hospital between 2006–2007. All patients consented to participate in the study and ethics committee approval  for the study was obtained. The average T-score of bone density measurement (DEXA) studies of the aforementioned patients was −1.9. Patients’ activities involved mainly household duties, gardening and daily walking but the disease significantly affected their quality of life. All patients had plain x-rays in standing position, dynamic radiographs in flexion extension and MRI of the lumbar spine. No patient had osteoporosis (T-score>−2.5), more than grade I spondylolisthesis, dynamic instability, motor deficit or severe lumbar spinal stenosis (with or without kissing spinous processes)as these were exclusion criteria in our study and containdications for X-STOP insertion. All patients had both central and lateral spinal stenosis  with disc degeneration at the involved levels.The degree of spinal stenosis, according to radiologist reading of the MRI, was mild to moderate. For central stenosis, dural sac cross sectional area had to be between 100 and 130mm2 in the central canal area of transverse MRI. Less than 100mm2 was considered severe stenosis and these cases were excluded . Mild toIn all cases pathology was found at L4-L5 level except for four patients with stenosis both at L3-L4 and L4-L5 levels. Initially nonoperative treatment for at least 3 months was applied for all patients including non-steroidal anti-inflammatories, physical therapy, bracing and transforaminal epidural steroid injections/selective nerve root injections. When nonoperative treatment did not alleviate their symptoms, surgical treatment was performed. Microdecompression of the subarticular and foraminal stenosis was based on the responses from selective nerve root injections with anesthetic and steroids.The Oswestry Disability Index (ODI) and the Visual Analogue Scale (VAS) (for low-back pain and leg pain separately) scores were documented preoperatively, at the third week, six months and first year postoperatively and at the last follow-up. Self-rated walking distance preoperatively and after surgery at last follow up as well as satisfaction regarding overall result of back operation, according to questions of Zurich Questionnaire, were evaluated .Also, segmental lordosis of the involved levels and lumbar lordosis (upper plate of L1 to upper plate of S1) were measured by the Cobb angle in standing x-rays both preoperatively and at the last follow-up. Finally, radiographic posterior disc height of the involved levels was measured preoperatively and at the last follow-up. Radiographic manual measurements were done twice by the first two authors  and the average was recorded.Statistical analysis both for the functional and radiographic results was performed with the use SSPS 10.0. The comparison was calculated with Student’s paired t-test. Statistical significance was considered when p < 0.05.Under general anesthesia and prone position on a radiolucent table with padded support at the level of the iliac crests and sternum, slight flexion of hips and knees was preserved so that the subjects lie in a less lordotic position. After an appropriately small skin incision (approximately 4 centimeters), under fluoroscopic control, and subcutaneous tissue incision, the dissection went through the dorsolumbar fascia approximately 5–10 mm lateral to the midline towards the symptomatic side, preserving the supraspinous ligamentous attachment to the fascia. The multifidus was detached from the symptomatic side of the spinous processes and laminar attachments. Retractors were placed to keep the wound open and were being loosened regularly to avoid damage to the retracted muscles. The ligamentum flavum was detached with a freer elevator and then completely resected. The superior and inferior laminae were only partially resected. Decompression of both exiting and transversing nerve roots at that level were performed under microscope magnification. Partial facetectomies (mainly undercutting the superior facet) and foraminal decompressions were carried out with the aid of angled Kerrisson rongeurs of varying widths and lengths and/or a power drill. The adequacy of decompression was checked with foraminal probes. No removal of the central portion of laminae and no discectomy were performed.Next, the fascia was split longitudinally also to the other, non-decompression, side of midline and multifidus is detached only next to the interspinous area. It was of paramount importance for the stability of the segment to keep the supraspinous and part of the interspinous ligaments intact . A smallAfter completion of both thorough decompression and X-STOP insertion, the dorsolumbar fascia was sutured over a suction drain.The follow-up period ranged from 24 to 36 months and averaged 27.4 months. All patients were able to return to their daily activities within six weeks postoperatively. Both ODI and VAS low-back and leg pain scores improved significantly postoperatively p < 0.001) and this improvement deteriorated slightly (p > 0.05) in the last follow-up  declared very satisfied. In terms of walking distance, none deteriorated, six patients improved one grade and four improved two grades Figure .The interspinous implants used were mainly 10mm and 12mm height size and 8mm implant was inserted only in two levels.Radiographic analysis of standing x-rays of the lumbar spine showed a mean increase of 1.8 mm of the posterior intervertebral disc height of the operated levels and the postoperative height was significantly larger than preoperatively (p < 0.001) Figure . SpecifiIn a pilot study of our first patients, the postoperative MRI showed the expansion of the space available for the sac following the unilateral decompression Figure .There were no major complications in our series. No obvious by plain x-rays spinous process fracture was detected; however there was one asymptomatic case of gradual displacement of the superior to the implant spinous process which suggested a possible fracture. Two patients with radiculitis originating from the level above the decompressed level were treated successfully with anti-inflammatory medication five months postoperatively. In another two cases, the X-STOP device was not inserted anteriorly enough in the interspinous space but this did not cause symptoms.The rationale for this study was to evaluate if the combination of direct unilateral decompression and the indirect decompression with an IPDD device (X-STOP) was effective for the patients with neurogenic claudication due to spinal stenosis and, also, to compare our results with historical controls of other studies using only X-STOP device. This study intends to show the validity of the presented method of direct and indirect decompression of neural elements and not to show the superiority to other established treatment methods .The first IPDD implanted in patients with neurogenic intermittent claudicaton (NIC) due to spinal stenosis was the X-STOP (first FDA approved) . X-STOP X-STOP reduces facet load and prevents narrowing of the canal and foramen -20. As CIn cases with lateral spinal stenosis including subarticular and foraminal nerve root impingement, indirect decompression by X-STOP insertion only may not be as beneficial when there is long-term inflammation in the area. It is advantageous that a direct decompression is performed in lateral subarticular and foraminal area in order to free nerve roots from adhesions due to the chronic inflammation tissue. This combination of direct (mini-open) and indirect (by X-STOP insertion) decompression was performed in our patients. In a biomechanical cadaveric study of X-STOP device by Fuchs et al. , unilateThe addition of X-STOP device can distract the intervertebral space and preserve the nerve-occupying space without sacrificing the mobility of the motion segment -9.In Table In a randomized controlled, prospective, multicenter trial outcome study, patients suffering from neurogenic intermittent claudication treated with X-STOP were compared to outcomes of similargroup patients treated conservatively ,10. ThesLee et al . experie29% of the patients in Siddiqui et al. study  lost benIn our study elective microdecompression surgery was combined with X-STOP insertion. The patients included were suffering from mild to moderate severity central and unilateral lumbar spinal stenosis. Conservative treatment had been followed for at least 6 months with no relieving response. The procedure included partial unilateral laminotomy, subarticular and foraminal (undercutting) decompression with an effort to avoid complete facetectomies. After microdecompression, X-STOP was applied at the diseased levels. All the patients were evaluated before surgery and postoperatively at 3 weeks, 6 months, 1 year and 2 years follow-up. Our outcome tools were VAS, ODI and certain questions on walking capacity and patient satisfaction from Zurich Questionnaire, all of which improved significantly. Posterior disc height of the involved level and Cobb angle were documented pre- and postoperatively. Lumbar lordotic Cobb angle did not change statistically significantly even though segmental lordosis decreased. This means that lordosis increased in the adjacent motion segments to improve sagittal balance. In contrary to other studies, posterior intervertebral disc height at the involved levels increased in average 1.8 mm postoperatively compared to preoperatively in accordance to results from Sobottke et al . No majoThe anatomic advantage of direct decompression of neural tissues is obviously superior to indirect decompression. The financial cost of adding an IPDD in a microdecompression case may be exchanged by the lasting clinical improvement and the preservation of disc height and lordosis.Limitation to our study is the absence of a control patient group. A prospective randomized study comparing IPDD insertion only to IPDD insertion plus direct decompression is under way. This study provides level IV evidence and should be considered this way.Direct lateral microdecompression in addition to indirect decompression by an IPDD (X-STOP) insertion is a clinically effective and radiologically viable treatment option for patients with mild to moderate lumbar spinal stenosis.There is no personal or institutional financial support related to the study.AP conceived of the study, performed the statistical analysis and drafted the manuscript. PC, DK, IG, VV, AB participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Early screening for cancer is arguably one of the greatest public health advances over the last fifty years. However, many cancer screening tests are invasive , expensive  or both (colonoscopies). This has spurred growing interest in developing genomic signatures that can be used for cancer diagnosis and prognosis. However, progress has been slowed by heterogeneity in cancer profiles and the lack of effective computational prediction tools for this type of data.anti-profiles as a first step towards translating experimental findings suggesting that stochastic across-sample hyper-variability in the expression of specific genes is a stable and general property of cancer into predictive and diagnostic signatures. Using single-chip microarray normalization and quality assessment methods, we developed an anti-profile for colon cancer in tissue biopsy samples. To demonstrate the translational potential of our findings, we applied the signature developed in the tissue samples, without any further retraining or normalization, to screen patients for colon cancer based on genomic measurements from peripheral blood in an independent study (AUC of 0.89). This method achieved higher accuracy than the signature underlying commercially available peripheral blood screening tests for colon cancer (AUC of 0.81). We also confirmed the existence of hyper-variable genes across a range of cancer types and found that a significant proportion of tissue-specific genes are hyper-variable in cancer. Based on these observations, we developed a universal cancer anti-profile that accurately distinguishes cancer from normal regardless of tissue type .We developed anti-profiles as a new approach for developing cancer genomic signatures that specifically takes advantage of gene expression heterogeneity. We have demonstrated that anti-profiles can be successfully applied to develop peripheral-blood based diagnostics for cancer and used anti-profiles to develop a highly accurate universal cancer signature. By using single-chip normalization and quality assessment methods, no further retraining of signatures developed by the anti-profile approach would be required before their application in clinical settings. Our results suggest that anti-profiles may be used to develop inexpensive and non-invasive universal cancer screening tests.We have introduced  Early detection through mass screening remains one of the most effective approaches for reducing health care costs-4 and moanti-profile. We define an anti-profile score for a specific sample as the number of hyper-variable genes for which expression in that sample falls outside a defined range of normal expression (see Methods for details). We illustrate the technique on a colon cancer dataset, suggest its potential by predicting cancer in a peripheral blood dataset, and explore the possibility of a universal cancer predictor by simultaneously predicting outcome with data from 52 cancer types. All datasets were obtained from public repositories.Peripheral blood-based genomic signatures are a promising avenue for developing non-invasive cancer biomarkers-21. HoweWe complement our novel statistical approach with new biological insights related to cancer. For the colon cancer anti-profiles we incorporate the finding that consistent decreases in methylation are observed along large (5kb – 10Mb) genomic blocks. Specifianti-profile method we propose here is an application to the predictive setting of ideas in existing statistical methods developed to identify and model outliers in gene expression due to cancer[Gene expression variability and stochasticity have been studied previously in the context of normal populations,27, witho cancer,32. Hereanti-profile method as a simple and robust approach to define cancer genomic signatures by specifically taking advantage of heterogeneity in cancer. An important first step in our approach is to normalize raw gene expression data; an often-overlooked, but key issue in the development of genomic signatures based on microarray data. Standard microarray normalization methods cannot be used when developing clinical diagnostics since they require multiple samples and normalized values depend on which samples are normalized together[We developed the together,34. Thistogether. For alltogether and a sitogether. Since sTo illustrate our method we developed an expression anti-profile that distinguishes colon cancer from normal colon in tissue biopsies. We used two independent colon cancer studies, performed by different groups-40, as aTo determine the relationship between gene expression hyper-variability and CpG DNA methylation hyper-variability, we examined a publicly available DNA methylation dataset comparing colon cancer with matched normal colon tissue on the Illumina HumanMethylation 27k BeadChip array (see Methods). We found that there is significant overlap between genes with hyper-variable expression in colon cancer and promoter region CpG hyper-variable methylation . We then repeated the experiment on the two colon cancer expression datasets using CpG hyper-variable methylation to select anti-profile genes and observed worse prediction performance (AUC=.84 and AUC=.97). Enrichment of hyper-variable CpG DNA methylation in blocks of hypo-methylation for this dataset has been previously reported. Consideanti-profile signature (542 genes). We directly applied the anti-profile derived from colon tissue to publicly available peripheral blood samples that passed quality assessment (see Methods section for details) from cancer patients (n=15) and normal samples (n=15) without any retraining[anti-profile signature was equivalent to the training-set accuracy achieved by the 5-gene score developed by Han et al.[anti-profile for this task is possible by selecting the optimal number of genes based on performance on the peripheral blood samples themselves. For instance, a slightly larger anti-profile signature (650 genes) achieved an AUC of 0.93  from 59 tumor types and 102 normal tissue types across 176 different studies in the Gene Expression Omnibus . AdditiUsing these data we developed an anti-profile to predict cancer status regardless of tumor or tissue type. First, we confirmed that across-sample variability was a general characteristic of cancer  of universally hyper-variable genes in cancer are not consistently expressed in any of the normal tissues in our dataset . This implies that, for almost half of the universally hyper-variable genes in cancer, hyper-variability cannot be the result of a heterogeneous mixture of markers for different cellular subtypes since these genes are usually silenced in normal tissues. Also, while hyper-variable genes are enriched in the set of tissue-specific genes, we found that the majority of tissue-specific genes are not consistently hyper-variable (64%). The vast majority of tissue-specific genes show hyper-variability in a small number of cancer types  of hyper-variable genes in each of these cancers are not tissue-specific for the corresponding normal tissue  and found that 12% of the genes used in the peripheral blood colon cancer anti-profile fall under this category. Furthermore, lymphocyte-specific genes are enriched in the set of genes with hyper-variable expression in colon cancer inside colon cancer hypo-methylation blocks . This suggests that we cannot rule out that varying lymphocyte composition in the peripheral blood samples of colon cancer patients may drive the prediction performance of the peripheral blood anti-profile.While in the colon cancer anti-profile we restricted genes to be in the colon-cancer hypo-methylated blocks here we used our newly found biological insight: we restricted the anti-profile to tissue-specific genes defined as those genes that are expressed in at least 95% of samples for at most three tissues using the gene expression barcode method. With anWe used pathological tumor stage or grade annotation available for a subset of the samples used in the leave-one-tissue-out cross-validation experiment to determine if heterogeneity across samples in pathological tumor stage or grade may explain the increased gene expression variability observed in anti-profile genes used for prediction. For each of the leave-one-tissue-out experiments reported in Figure anti-profile signature in tissue samples from a colon cancer study and validated our signature in a second independent validation set, collected by a different experimental group. We have also applied this signature directly, without retraining, to classify patients with cancer from normals on the basis of genomic measurements in peripheral blood.We have introduced and developed gene expression anti-profiles for cancer biomarker discovery. Anti-profiles explicitly model increased gene expression variability in cancer to define robust and reproducible gene expression signatures capable of accurately distinguishing tumor samples from healthy controls. We have developed an anti-profile method classify samples based on deviation from a reference profile, there are two significant differences in the way Mammaprint and the anti-profile method achieve this: 1) Mammaprint uses tumor samples with good prognosis to determine the reference profile. Since these are tumor samples many of the genes used in the profile may exhibit high variability across the good prognosis group. Defining a stable and robust reference profile is essential to the success of this type of method. 2) Mammaprint uses correlation to measure how samples deviate from the reference profile. Our anti-profile method instead uses a robust measure where deviation is based on the number of the genes for which expression falls outside normal ranges of expression, which are themselves estimated using robust methods. It may be possible to improve on the accuracy of the Mammaprint test by adopting a more robust anti-profile based on the methods presented in this paper.We note that Mammaprint,47, one In this case we can use the anti-profile score, that is, the number of genes in the anti-profile where expression deviates from a normal range of expression obtained from normal breast tissue samples, to determine prognosis. Since this score is based on stable expression in normal tissues, it may be more robust than calculating correlation to a mean signature for tumors with good prognosis that would show high variability. This will require that more samples of both normal breast tissue and tumor are available on platforms for which robust, single-chip normalization methods exist.In addition to developing a peripheral blood signature for colon cancer, we have confirmed the existence of hyper-variable genes across 59 distinct cancer types. We also provide evidence of the close relationship between hyper-variability across cancer types and tissue-specific gene expression. Consistent with these observations on tissue-specificity, gene ontology category enrichment analysis found that categories involving development, organ morphogenesis and differentiation are enriched with hyper-variable genes and the remaining gene categories enriched with hyper-variable genes involved cellular interaction with extracellular matrix, e.g., adhesion, localization and collagen catabolic processing or in cell locomotion and cellular component movement. These results argue strongly against the observed hyper-variability being a consequence of sample heterogeneity in the cancer samples.anti-profiles.Incorporating this general result on tissue-specificity and hyper-variability we developed anti-profiles able to classify tissue samples across multiple tissue and cancer types, even when a specific cancer/tissue type is not included in the original training set. Our cross-validation results suggest that consistent hyper-variability of a small set of tissue-specific genes is a stable mark of cancer across tissue types. Our results also suggest the potential for developing peripheral blood signatures for cancer diagnostics on the basis of In the course of achieving these results we have used recently developed statistical preprocessing methods to remove potential artifacts in a way that is applicable to single clinical samples. This isfrma . CEL fifrma  single-scores . We remscores  to asserg = log 2(Sgc/Sgn)where sgc is the across-sample standard deviation of expression for probeset g among the colon tumor samples, and sgn is the across-sample standard deviation of expression for probeset g among the normal samples. The anti-profile includes probesets with rg>1 .We used the HGU133plus2 probeset annotation from Ensembl  to map probesets to genes and obtain each gene’s transcription start site. In the colon cancer anti-profile, we only consider probesets for genes with transcription start sites inside blocks of DNA methylation change . Methylation measurements were used with no further preprocessing. Differences in methylation variability were determined using an F-test and significance determined at 1% false discovery rate. For each probeset in our expression data we found the CpG inside it’s promoter region (defined as 1000bp upstream and 250bp downstream) nearest to the transcription start site. We determined significant expression hyper-variability using an F-test at 1% false discovery rate to determine overlap between expression hyper-variability and DNA methylation hyper-variability.We obtained peripheral blood Affymetrix HGU133plus2 samples from colon cancer patients and healthy controls  separately for each tissue t and cancer type c (sgc). We define the variance ratio statistic ug .To determine probesets that exhibit hypervariable expression in cancer we compute a variance ratio statistic across multiple tissues. We restrict this computation to tissues and cancer types with more than 10 samples in our dataset  along with median absolute deviation (madgt). The universal range is then defined as mg +/− 5 * madg where mg=mediant(mgt) and madg=mediant(madgt). The list of hyper-variable genes (ug>1) and associated median expression and median absolute deviation of expression are provided in Additional fileTo define the universal normal range of expression we use a similar method: we compute median expression for each gene g on each tissue t separately (mz-score greater than 2.54) for each tissue in our dataset with more than 10 normal samples. Tissue-specific genes were defined as those in which the gene is expressed in more than 95% of the samples of at most three tissues. Fisher’s exact test was used to determine enrichment of hyper-variable genes in the set of tissue-specific genes  and GO terms. We used the implementation in the Bioconductor GOstats package ([Gene ontology (GO) enrichment analysis was done using a hyper-geometric test for association between hyper-variable genes  selecting the top 100 genes based on the ratio statistic, and 4) computing the universal normal range of expression).We performed two types of cross-validation experiments to quantify the accuracy of universal cancer anti-profiles. The first was ten-fold cross validation, data was randomly split into 10 equal-sized subsets, retaining the proportion of normal and cancer samples from the full dataset in each subset. Each of the 10 subsets (or folds) was used sequentially as a test set, scored using an anti-profile trained on the remaining 90% of the data  filtering to include only tissue-specific probesets, 2) computing the universal variance ratio uno samples from the corresponding tissue  are included in the training set. Again, all steps required to train the anti-profile were done completely for each leave-one-tissue-out fold.The other type of cross-validation experiment was carried out on the 7 tissues for which we had at least 10 samples each of normal tissue and tumor. For each tissue type, we performed a leave-one-tissue-out experiment by using all samples  as test set and scored them using an anti-profile trained on the remaining data. This ensures that anti-profile score, are indicative of cancer. To develop a predictor for new samples, a cutoff must be defined on the number of genes outside the normal range. If the anti-profile score is less than the cutoff, the sample is classified as normal, if it is greater than cutoff then the sample is classified as cancer.To classify a new sample we count the number of anti-profile genes for which their expression fell outside their normal range Figure A. A largThe authors report no competing interests.HCB, JTL and RAI conceived, designed and performed experiments, analyzed data and drafted the manuscript; VP performed experiments and analyzed data; MM contributed reagents. All authors read and approved the final manuscript.Supplementary Material. This file contains supplementary Figures and Tables.Click here for fileTable S3. Colon cancer anti-profile. Contains Affymetrix probeset ids and normal expression median and median absolute deviation.Click here for fileTable S4. Universal cancer anti-profile. Contains Affymetrix probeset ids and normal expression median and median absolute deviation. Click here for file


MicroRNAs (miRNAs) are small 19–23 nucleotide regulatory RNAs that function by modulating mRNA translation and/or turnover in a sequence-specific fashion. In the nervous system, miRNAs regulate the production of numerous proteins involved in synaptic transmission. In turn, neuronal activity can regulate the production and turnover of miRNA through a variety of mechanisms. In this way, miRNAs and neuronal activity are in a reciprocal homeostatic relationship that balances neuronal function. The miRNA function is critical in pathological states related to overexcitation such as epilepsy and stroke, suggesting miRNA’s potential as a therapeutic target. We review the current literature relating the interplay of miRNA and neuronal activity and provide future directions for defining miRNA’s role in disease. MicroRNAs are endogenously encoded small RNAs that are processed sequentially into mature 19–23 nucleotide (nt) regulatory molecules . Once prThe mechanism by which miRNAs silence their mRNA targets remains highly controversial . There aThere are numerous ways in which miRNAs modulate the function of the nervous system. A variety of studies have shown how miRNA plays an important role in the development and in a wide array of disorders of the nervous system . The focFigure 1). Structural changes at the synapse are commonly associated with alterations in the cytoskeletal function, leading either to the establishment or dissolution of synapses  was identified as a miR-29a/b target in a screen for miRNAs involved in drugs of abuse-related plasticity . OverexpFunctional screening of synaptically enriched miRNA identified an interaction between miR-138 and the acyl protein thioesterase 1 (Apt1) mRNA . InhibitThe miRNA regulation of structural plasticity extends beyond the hippocampus and cortex. Auditory fear conditioning downregulates a number of miRNA in the amygdala . Bioinfoin vitro and by amphetamine and cocaine in various anatomical structures in vivo. Surface levels of GluR2 are reduced in neurons overexpressing miR-181a; however, only mEPSC frequency and not amplitude is affected. The authors suggest that AMPAR-dependent spine development might be effected, explaining reduced mEPSC frequency. However, the possibility remains that miR-181a may have additional relevant targets in hippocampal neurons.In addition to regulating the structural aspects of dendritic spines, a number of miRNAs have been shown to directly regulate components of the post-synaptic density. The synaptically enriched miR-181a can target the GluR2 subunit of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor (AMPAR) through a conserved binding site in its 3′UTR . InteresN-methyl-D-aspartate (NMDA) receptor 2A (NR2A) as a direct miR-125b target. Overexpression and knockdown of miR-125b had significant impact on the NMDAR currents, consistent with the observed up and downregulation of NR2A.A number of mRNAs encoding post-synaptic density proteins appear to be shared targets of miRNAs and the fragile-X mental retardation protein (FMRP). The FMRP negatively regulates mRNA translation by directly interacting with target mRNAs. In one study, the authors identified miRNA enriched in FMRP-bound RNA immunoprecipitation experiments . Of thesDrosophila have shown physical interaction between RISC and FMRP .The miRNA biogenesis is a multiple-step process that begins with the transcription of a primary miRNA transcript  and its mammalian homolog MOV10 are both implicated in the activity-dependent relief of miRNA repression . A fair amount of effort has been made toward understanding the global miRNA response to stroke in rodent models. These studies generally rely on comparisons of RNA samples from stroke and sham brains, and subject them to some type of high-throughput array. Not surprisingly, the resulting miRNA expression profiles between different experimental stroke conditions, different laboratories, and different array platforms show almost no overlap. Despite this lack of overlap, there is valuable information that can be gained from a brief review of these studies.Excessive neuronal activity is associated with neuronal cell death in a number of contexts. During stroke, neuronal depolarization leads to excess glutamate release that cannot be compensated for by normal reuptake mechanisms. The resulting excess glutamate results in glutamate receptor hyperactivation and excitotoxic cell death via excess calcium influx. As miRNAs are potent regulators of the cellular stress response  and neurOne of the best studies that highlights the complexity of the miRNA response to CNS injury was performed on both blood and brain tissue from rats after ischemic stroke, intracerebral hemorrhage, or kainic acid-induced excitotoxicity . In thisIn two related studies, the miRNA cortex of mice treated with middle cerebral artery occlusion (MCAO) model of stroke were profiled either by PCR 24 h post-stroke  or by arAnother approach toward finding potentially therapeutic miRNAs for treating stroke is to work backward from a known therapeutic target or pathway and identify miRNA interactors. GRP78  is a chaperone that is primarily localized to the ER and plays a key role in the ER-stress response. Our lab recently investigated the role of miR-223 in neuroprotection motivated by the disproportionate number of nervous system targets predicted to be miR-223 targets . Bioinfostatus epilepticus (SE) in rodents. A number of studies have profiled miRNA from the hippocampus of mice following establishment of SE. Two studies from different research groups using kainic acid , a chronic intractable form of epilepsy. TLE can be modeled in rodents by injection of pilocarpine, a muscarinic acetylcholine receptor agonist or injection of kainic acid, an agonist of the kainate-type glutamate receptor. Injection of either of these compounds results in establishment of nic acid  and pilonic acid  model, rnic acid , making in vivo, likely acting via Limk1. The long-lasting nature of the anti-miR-134 protection suggest that miR-134-based therapeutic may hold promise in treating intractable TLE.Two activity-regulated miRNAs, miR-132 and miR-134, are both upregulated in mouse models of TLE , 2012. IIn the relative short time since their discovery, the miRNA field has made a dramatic impact on our understanding of nervous system function. Insights concerning the mechanisms of synaptic plasticity have been of particular interest, potentially coupling activity-dependent synaptic plasticity and protein synthesis. The local translation of proteins has long been considered a mechanism for regulating the connections at individual synapses and miRNA may be a key regulator at this point . HoweverAs discussed above, individual miRNAs can have potentially hundreds of targets owing to the small amount of complementarity required to establish miRNA–mRNA interactions. In virtually all studies, there is a focus on a one miRNA–one target interaction. This is largely for practical purposes – requirements for publication dictate a laser-like focus on a making a bulletproof set of observations. While editors and reviewers require this type of focus, miRNAs do not have such single-minded focus . Having Virtually all the studies described in this review rely on antisense oligonucleotides or antagomirs to conduct loss-of-function experiments. While these reagents are widely used and proven effective under many circumstances, virtually nothing is known about their distribution and perdurance in the CNS following intracranial injection, making interpretation of how they work problematic. A reasonable complement to antagomir application would the use of traditional targeted mutations, which are becoming increasingly more available to the general scientific research world .Finally, the most successful candidate miRNAs for translation to the clinic have arisen from rigorous work in the basic sciences. Both miR-132 and miR-134 have a long track record of careful investigation in the basic synaptic plasticity field and have now been shown to have potential in the treatment of epilepsy. This cross-pollination of basic and translational science seems to be the most fruitful way forward for the future of miRNA-based therapeutics.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The sensitivity and specificity  were 100% (84.6% to 100%) and 100% (85.1% to 100%), respectively. Viral loads were significantly associated with a shorter course of illness but not with the use of ribavirin or steroids. CoV was consistently identified in the lungs of all patients who died of SARS but not in control patients, supporting a primary role for CoV in deaths.Efforts to contain severe acute respiratory syndrome (SARS) have been limited by the lack of a standardized, sensitive, and specific test for SARS-associated coronavirus (CoV). We used a standardized reverse transcription-polymerase chain reaction assay to detect SARS-CoV in lung samples obtained from well-characterized patients who died of SARS and from those who died of other reasons. SARS-CoV was detected in all 22 postmortem lung tissues (to 10 From its origins in November 2002 in Guangdong Province, China, severe acute respiratory syndrome (SARS) has become an emerging infectious disease that has spread to areas throughout the world, including Hong Kong, Vietnam, Singapore, Taiwan, and Canada The purpose of this study was to use a standardized, commercially available, RT-PCR assay to test for the presence of SARS-CoV RNA. Lung tissue obtained at autopsy from well-characterized patients with SARS who died during the outbreak in Canada were compared to lung samples obtained at autopsy from patients without SARS who died during the outbreak and lung samples from patients who died before the outbreak.,All patients who met the current World Health Organization case definition of probable SARS and who underwent a postmortem examination in Canada during the March–April 2003 outbreak were included in this study. Clinical details were extracted retrospectively from hospital records. Clinical descriptions of some of these cases have been published separately  with lung cancer were also included as negative controls. All samples collected at the time of autopsy were snap frozen in a mixture of absolute ethanol and dry ice and subsequently stored at –70°C until tested. The samples were coded and then processed, subjected to RT-PCR analysis, and interpreted before the identity of the samples was divulged. This study was approved by the research ethics boards at Mount Sinai Hospital and the University Health Network.http://www.who.int/csr/sars/primers/en/).Lung tissue samples were thawed and immediately homogenized in lysis buffer  with disposable tissue grinders . The homogenate was passed through QIAshredder columns (QIAGEN) before RNA isolation by using the RNeasy Mini Kit (QIAGEN). The sample was eluted in 30 μL of RNAse free water. The RT-PCR was carried out by using the RealArt HPA-Coronavirus LightCycler RT Reagents Assay  with a LightCycler real-time platform . The HPA-Coronavirus Master Mix contains reagents and enzymes for the specific amplification of an 80-bp region of the SARS-CoV polymerase gene from 5 μL of RNA with the primer pairs published by the Bernhard-Nocht Institute  as posted on the World Health Organization Web site  included in the RealArt HPA-Coronavirus LightCycler RT Reagents Assay kit  A and B.The clinical description and RT-PCR results for the 11 patients with probable SARS from whom postmortem lung tissue samples were examined are summarized in SARS-CoV was detected in all 22 postmortem lung tissue samples collected from all 11 patients who died with a diagnosis of probable SARS. All 13 postmortem lung samples from the seven non-SARS fatalities that occurred during the SARS outbreak were negative for SARS-CoV, as were all 10 lung-tissue samples collected from patients with lung cancer 5 years before the outbreak (Table 2)4 copies/g tissue to 3.8 x 109 copies/g tissue. Higher viral loads (>106 copies/g tissue) were associated with patients who had a shorter duration of illness (<21 days) . The use of ribavirin or steroids was not significantly associated with viral load levels (Table 2)The SARS-CoV viral load in postmortem lung tissue ranged from 2.7 x 10http://www.ncbi.nlm.nih.gov/BLAST/) search performed against the SARS-CoV genomes in GenBank, European Molecular Biology Laboratory, DNA Data Bank of Japan, and Protein Data Bank on the National Center for Biotechnology Information Web site (available from: URL: http://www.ncbi.nlm.nih.gov/), indicated that all amplicon samples contained SARS-CoV polymerase gene sequence.Twenty-five randomly selected amplicons from the sample pool were sequenced to assess specificity and possible cross-reactivity to other pathogens. A BLAST  the method of specimen collection, as well as the method of sample preservation may have substantial impact on the results obtained from a diagnostic test. The lower sensitivity of SARS-CoV detection reported by Peiris et al.


Pipelining or workflow tools allow for the Lego™-like, graphical assembly of I/O modules and algorithms into a complex workflow which can be easily deployed, modified and tested without the hassle of implementing it into a monolithic application.The CDK-Taverna project aims at building an open-source pipelining solution through combination of different open-source projects such as Taverna , the CheCurrent developments in CDK-Taverna refactor all workers as well as the complete setup on the basis of Taverna 2.2 and CDK 1.3.5 which themselves introduce major improvements to the whole platform. In addition the CDK is enhanced with specific functions and options for reaction enumeration based on a reaction template and corresponding reactant libraries. Reaction enumeration supports combinatorial chemistry approaches in the drug discovery process of the pharmaceutical industry. The CDK enhancements are applied and illustrated by corresponding CDK-Taverna-workflows.