Detection of circulating malignant cells (CMCs) through a reverse transcriptase-polymerase chain reaction (RT-PCR) assay seems to be a demonstration of systemic disease. We here evaluated the prognostic role of RT-PCR assays in serially-taken peripheral blood samples from patients with malignant melanoma (MM).One hundred forty-nine melanoma patients with disease stage ranging from I to III were consecutively collected in 1997. A multi-marker RT-PCR assay was used on peripheral blood samples obtained at time of diagnosis and every 6 months during the first two years of follow-up . Univariate and multivariate analyses were performed after 83 months of median follow-up.Detection of at least one circulating mRNA marker was considered a signal of the presence of CMC (referred to as PCR-positive assay). A significant correlation was found between the rate of recurrences and the increasing number of PCR-positive assays (P = 0.007). Presence of CMC in a high number (≥2) of analysed blood samples was significantly correlated with a poor clinical outcome . Multivariate analysis revealed that presence of a PCR-positive status does play a role as independent prognostic factors for overall survival in melanoma patients, adding precision to the predictive power of the disease stage.Our findings indicated that serial RT-PCR assay may identify a high risk subset of melanoma patients with occult cancer cells constantly detected in blood circulation. Prolonged presence of CMCs seems to act as a surrogate marker of disease progression or a sign of more aggressive disease. The poor prognosis of patients with malignant melanoma (MM) is mostly due to the high frequency of distant dissemination of the disease. Considering the small size of most primary melanomas, the metastatic potential of MM is considerably greater than that of most other solid tumours .The relationship between presence of circulating cancer cells and development of secondary disease is not fully understood. Detection of circulating malignant cells (CMCs) is actually aimed to the identification of either an earlier marker of tumour progression or a phenotypic feature of more aggressive disease that might change treatment options . DetectiReverse transcriptase-polymerase chain reaction (RT-PCR) can detect a single specific messenger RNA (mRNA) in a mixed cell population; thus, it can be a sensitive method for identification of circulating tumour cells -7. TyrosWe here investigated whether serial detection of circulating melanoma-associated markers by performing consecutive multi-marker RT-PCR assays on peripheral blood samples obtained during patients' follow-up may improve prognostic prediction, commonly based on pathological and clinical factors. Moreover, the current study was designed to examine the impact of longer duration of follow-up on the prognostic relevance of such a molecular assay in the assessment of clinical outcome among melanoma patients.The study was conducted with patients referring to the National Cancer Institute of Naples between January 9, 1997 and December 16, 1997. Patients were consecutively collected and considered eligible if they had a histological diagnosis of cutaneous malignant melanoma . Eight out of 157 enrolled patients were excluded from the series since they were early lost in follow-up (in a period ranging from 9 to 14 months).For the 149 patients included into the study, clinical stage was documented at the time of enrollment and prospectively followed-up. Patients with no clinical evidence of regional or metastatic disease underwent the sentinel lymph node (SLN) dissection. Histopathologic examination was performed on each formalin-fixed paraffin-embedded SLN using hematoxylin/eosin staining and immunohistochemistry (IHC) of adjacent sections. For IHC, 4-μm-thick sections were evaluated by using antibodies against HMB-45 and S-100 proteins. All stage III patients received the same adjuvant therapy with low dose interferon alpha (as we previously described ). DiseasFor collection of baseline peripheral blood samples , patients (stage I to III) were eligible if no more than two weeks had elapsed from the surgical treatment. All patients were visited every six months after the diagnosis. During the first two years of follow-up, peripheral blood samples were taken at 6, 12, 18, and 24 months after diagnosis  and addressed to the RT-PCR screening. Death before the third blood sample (within 12 months after diagnosis) was an exclusion criterion; no patients was excluded for such a criterion. No clinical decision was made based on the result of the RT-PCR assay.At each follow-up time, a clinical history, physical examination, cell blood count, and blood biochemistry were performed. Instrumental assessments were performed if clinically indicated. Patients were informed about aims and limits of the study; peripheral blood samples for RT-PCR analysis were obtained with their written consent. The study was reviewed and approved by the ethical review boards of both the National Tumor Institute of Naples and the University of Sassari.Nucleated cells from peripheral blood were processed in order to isolate total RNA, using standard procedures.In our previous report, a multiple-marker RT-PCR assay  was tested for its sensitivity and specificity in detecting circulating melanoma-associated mRNAs . AnalysiIntegrity of RNA was determined by performing parallel RT-PCR reaction using primers specific for the housekeeping gene GAPDH . SamplesTo assess whether the detection of CMC in peripheral blood samples serially taken from MM patients could have prognostic value, the following criteria were used. PCR-based amplification of at least one mRNA marker  in a peripheral blood specimen was considered a signal of the presence of CMC (referred to as PCR-positive assay). Presence of CMC was used as a binary (no/yes) explanatory variable.Univariate associations between PCR-based markers and clinico-pathological variables [AJCC stage (I to III), Breslow thickness of primary tumour , Clark lA consecutive cohort of 149 patients with histologically-proven diagnosis of MM from January 9, 1997 to December 16, 1997 was studied. Patients were prevalently females, with a median age of 48 years (range 16–85). According to the new AJCC stage classification, almost the half of the patients had stage I disease , and one-third had stage II . Overall, patients without lymph node metastases (stages I to II) accounted for 81% of the series  in a blood sample was considered a signal of the presence of CMC (referred to as PCR-positive assay). During the first two years after diagnosis, peripheral blood samples from MM patients undergoing follow-up visits every six month  were screened for CMC by RT-PCR assays. In Table in transit metastases), 6 visceral sites, 3 brain); 28 patients (19%) have died of melanoma. For the entire series of 149 patients included into the study, median follow-up was 83 months . Four patients recurred after 20 months; blood drawing at month 24 (sample 5) was anyhow performed and such patients were not excluded from the study. No patient in our series died within 24 months from diagnosis.As of December 2005, 49 patients (33%) have suffered progression  Table  or the mIn this paper, we showed that presence of circulating malignant cells (CMCs), assessed through a PCR-based detection of multiple mRNAs corresponding to melanoma-associated molecular markers (tyrosinase and MelanA/MART1), in a high fraction of peripheral blood samples serially obtained during follow-up visits, does play a role as independent predictive factor for clinical outcome.Taking into consideration both the raw mortality and the tumour specific mortality as a censoring, survival data analysis allowed to assess that patients with presence of melanoma cells in at least two out of the five analyzed peripheral blood samples (referred to as PCR-positive assays), collected every six months during the first two years of follow-up, had a worse clinical outcome with a significantly shorter overall survival over a median follow-up of about seven years (83 months). Multivariate analysis revealed that such a high level (≥2) of PCR-positive assays does correlate with the overall survival in melanoma patients, thus adding precision to the predictive power of the stage of disease  to provide clinically relevant information and define outcome for patients with occult melanoma metastases. Prediction of metastatic potential remains one of the main goals to be pursued in order to better assess the risk subgroups of patients with malignant melanoma.The author(s) declare that they have no competing interests.GP participated in the design of the study, performed the molecular analyses, and drafted the manuscript. SMRS performed the molecular analyses. MB performed the statistical analysis. AC performed the data management. FT participated in the analysis and interpretation of data. SC participated in the patients' collection. CC participated in the patients' collection. ES participated in the patients' collection. AD participated in the patients' collection. NM participated in the design of the study. GCo participated in the design of the study. GCa participated in the design of the study. PAA conceived of the study, and participated in its coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Peripheral blood from 67 patients with non-Hodgkin's lymphoma was examined at initial diagnosis for the presence of circulating lymphoma cells by DNA hybridisation using immunoglobulin and T-cell receptor gene probes. Clonal gene rearrangement was found in 31% (21/67) of patients and correlated with clinical stage, histological grade and bone marrow involvement. Clinical stage and the presence of lymphoma cells in peripheral blood were prognostic factors for progression-free survival in all patients on univariate analysis, but the detection of lymphoma cells was not independent of stage. It was also not a significant predictor for survival. In patients with intermediate- and high-grade lymphoma, the detection of lymphoma cells in peripheral blood was a significant prognostic factor for progression-free survival (PFS) and survival only on univariate analysis. The 3-year PFS was 17% in patients with circulating lymphoma cells compared with 75% if these were absent (P < 0.05). The presence of lymphoma cells in peripheral blood is associated with extensive disease and may be a biological marker of poor disease control. Sensitive techniques of detection should form part of large prospective studies in non-Hodgkin's lymphoma.


The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-γ (IFN-γ) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours. Both T- and B-cell immune responses to tumour-derived proteins have been identified in many cancer patients, however the responses are generally insufficient to result in tumour clearance. One of the challenges in cancer treatment is to enhance this anti-tumour immune response, perhaps by identifying novel tumour antigens with a higher immunogenic potential. Such antigens have the potential to be tumour biomarkers in serological testing and targets in anti-tumour vaccine development. Currently there are many serologically defined protein tumour markers known and in some cases the corresponding peptide sequences have been identified . PromisiTumour-derived heat shock protein (Hsp) preparations have been shown to elicit anti-tumour immune responses in both mice and man . In miceRecent studies have shown that chaperones such as heat shock proteins gp96, Hsp90, Hsp70 and calreticulin can be taken up by dendritic cells by receptor mediated endocytosis, where they enter the MHC class I antigen presentation pathway and are cross-presented to T-cells -19. Addiin vivo bio-panning in certain animal model systems to identify tissue-specific peptide ligands  relative to that observed when the DSP peptide is used in both the first and second stimulations [DSP (1) +DSP (2)]. When cells were stimulated with Hsp70, depleted of the associated peptides in the first stimulation and then challenged with iDCs loaded with the DSP peptide in the second stimulation, IFN-γ secretion is also observed however to a lesser degree than that observed with Hsp-70-PC [Hsp70 (1) +DSP (2)]. Reiterating the previous finding ], nor was IFN-γ produced if PBMCs were incubated with iDCs stimulated by LPS alone in the first stimulation and then challenged with iDCs loaded with the DSP peptide in the second stimulation (data not shown).CD14ing Fig. , a singl- PBMCs in vitro. Furthermore, the DSP peptide may structurally resemble peptides complexed with Hsp70.Taken together, our data suggest that the mimic peptide DSP resembles immunogenic peptides and/or protein components from both tumourigenic and non-tumourigenic breast cell lines and has the ability to stimulate human CD14Phage display libraries, since their discovery in 1990 , have beThe primary aim of this study was to test a "proof of principle", that functional synthetic mimics of native Hsp70-PCs could be identified by using two step bio-panning of random peptide phage display libraries. Previous studies have shown that Hsp-PCs from mice and humans can induce specific T-cell responses . The mimA variety of phages displaying unique peptides were identified by the bio-panning method. There appeared to be a high degree of enrichment of specific sequences following four rounds of bio-panning; certain peptides selected were common to at least two bio-pannings and one of the peptides (TMG) was recovered in all three bio-pannings. The diversity of peptides identified may be in part due to the complexity of the Hsp70-PCs fraction used as the bait. The peptide TMG was selected in three independent bio-panning experiments, suggesting that the motif recognised by TMG may be consistently abundant in the three pools of Hsp70-PCs.Three of the 'recogniser' peptides, identified in multiple bio-panning experiments, were then used in a reverse bio-panning experiment to identify sequences that interact with these peptides. Two of these recogniser peptides, NNY and IER, selected a wide variety of binders, one of which, SVS, was common between the two selected pools. Subsequent bio-panning experiments with an unrelated peptide bait, consistently selected the SVS peptide . Interestingly, this same phage peptide had been identified in two unrelated bio-pannings for peptides interacting with (i) murine cerebellar granular neurons and (ii), a Japanese encephalitis virus envelope protein neutralizing antibody ,41. The in vitro assay. The results show that lymphocytes were stimulated but required two consecutive iDC-mediated exposures to either the mimic peptide DSP or the recogniser peptide TMG. Unlike LPS, these peptides did not stimulate iDCs to produce IL-12. Therefore, we can conclude that (a) the activation of lymphocytes must be dependent upon the uptake of the peptide by iDCs and its representation to lymphocytes and (b) it is not the result of any adjuvant-like contaminant present in the peptide preparation. The DSP peptide showed lymphocyte stimulatory activity while, another peptide WHK produced no stimulation. Surprisingly, we did observe lymphocyte stimulation with the recogniser peptide TMG. The reason why lymphocyte activation was not limited to only mimics is currently unclear. The observed differences in effectiveness between the peptides may be reflective of the different proteolytic processing and/or the preference of the different HLA class I molecules for presentation of 9-mer peptides with specific amino acids in anchor positions. A search of the comprehensive database SYFPEITHI [The ability of the synthetic peptides to activate lymphocytes was examined using an YFPEITHI  for HLA YFPEITHI  as is itin vitro an in vivo [To determine if the DSP peptide represents a mimic of true tumour antigens present in tumour cells and in particular tumour antigens in the Hsp70-PCs pool, we employed an assay in which lymphocytes are stimulated in two consecutive rounds with different antigen pools. We find that tumour cell extracts contain certain antigens in common with those present in the Hsp70-PCs fraction as they successfully stimulate T-cells previously primed with Hsp70-PCs from the same tumour cells. Additionally, we observe that Hsp70 alone, in the absence of any associated peptides, can prime T-cells to respond to tumour cell extracts. Thus Hsp70 in addition to chaperoning peptides into the antigen processing pathway of iDC, may also trigger IFN-γ production in a similar way to that of LPS -49. Thes in vivo ,52. Ther in vivo ,53.Using the same technique, we also show that the DSP peptide resembles antigens present in total cell extracts from either tumourigenic (MDA-MB-231) or non-tumourigenic (MCF-12A) breast carcinoma cell lines. Thus, the DSP peptide may mimic a common antigen in both cell lines. The TMG peptide showed lower levels of lymphocyte stimulation following a second exposure to either extracts from MDA-MB-231 or MCF-12A cell lines. We also show that lymphocytes incubated initially with iDCs loaded with Hsp70-PCs can be re-stimulated with iDCs loaded the DSP peptide, again suggesting that this peptide resembles antigenic peptides associated with the Hsp70 in these cell lines.in vitro lymphocyte activation assay to validate their potential as tumour specific antigen mimics. Such peptides could be further modified or combined with other molecules to develop potential tumour vaccines. In this initial study we have identified two peptides with lymphocyte stimulatory activity. We envisage further adjustments to the bio-panning protocol such as pre-adsorbing the phage display library to a non-tumourigenic Hsp70-PC fraction to enrich for true tumour-specific recogniser peptides and/or the use of tandem mass spectroscopy to directly sequence and identify the peptides associated with Hsp70-PCs. In the latter case, their synthetic equivalents can be tested directly in T-cell stimulation. High throughput assays could be developed to relatively quickly screen large numbers of synthetic recogniser/mimic peptides identified through the strategies described here. Such mimics could be further modified to increase their immunological effectiveness either inherently at the sequence level or by using a cocktail of selected mimics for a given tumour based on their IFN-γ response. Furthermore, the essential concept of the screening strategy can be applied to many other potential drug and biomarker discovery applications.In conclusion, we have developed a bio-panning approach to enrich from phage display libraries potential peptide mimics of Hsp70-PCs and an The author(s) declare that they have no competing interests.BA and L M-E carried out the in vitro immuno-stimulatory assays. ST and DD contributed to the design and supervision of the immuno-stimulatory assays. TCJ and UB conceived and designed the methods for the isolation of the peptide mimics of Hsp-PCs and identified all of the peptides described in the manuscript.


An immunoliposome containing a 10B-compound has been examined as a selective drug delivery system in boron neutron-capture therapy. Liposomes, conjugated with monoclonal antibodies specific for carcinoembryonic antigen (CEA) were shown to bind selectively to cells bearing CEA on their surface. The immunoliposomes attached to tumour cells suppressed growth in vitro upon thermal neutron irradiation and suppression was dependent upon the concentration of the 10B-compound in the liposomes and on the density of antibody conjugated to the liposomes. The results suggest that immunoliposomes containing the 10B-compound could act as a selective and efficient carrier of 10B atoms to target tumour cells in boron neutron-capture therapy.


The dynamics of retroviral transgene repression were analyzed in several clones; repression was found to be slow and different genomic positions showed different dynamics. When gene expression varies unpredictably between genetically identical organisms, this is sometimes ascribed as stochastic. With the prevalence of retroviral vectors, stochastic repression is often observed and can complicate the interpretation of outcomes. But it may also faithfully reflect characteristics of sites in the genome.We created and identified several cell clones in which, within a given cell, retroviral transcription of a transgene was repressed heritably and essentially irreversibly. This repression was relatively slow; total repression in all cells took months. We observed the dynamics of repression and found that they were ergodic, that is, tending with a probability to a final state independent of previous conditions. Different positions of the transgene in the genome demonstrated different dynamics. At a position on mouse chromosome 9, repression abided by near perfect first-order kinetics and was highly reproducible, even under conditions where the number of cell generations per day varied.We propose that such a cell division independent 'off' mechanism could play a role in endogenous gene expression, potentially providing an epigenetically based timer for extended periods. Escerichia coli -TTCAAGTCCGCCATGCCCGAA- [TAMRA]-3'; beta-actin, 5'-AGGTCATCACTATTGGCAACGA-3', 5'-CACTTCATGATGGAATTGA-ATGTAGTT-3', 5'- [6-FAM]-TGCCACAGGATTCCATACCCAAGAAGG- [TAMRA]-3'.Expression of GFP and mouse beta-actin was measured using quantitative RT-PCR. RNA was extracted from cells and cDNA produced by standard application of reverse transcriptase and random hexamer primers. PCR was performed using the ABI PRISME. coli, and then sequenced. The final PCR product contained the virus-genome junction and the junction sequence was verified for each integration site.Integration sites of the vectors were PCR-amplified using Taq polymerase. The first amplification step of the PCR used a biotinylated primer homologous to the vector and a non-specific primer, degenerate at the 3' end and constant at the 5' end. Biotinylated PCR product was recovered by use of streptavidin-coupled magnetic beads (Invitrogen). The genomic DNA flanking the 3' long terminal repeat (LTR) of the provirus was then amplified from the biotinylated PCR product. This secondary PCR utilized a nested LTR-specific primer and a primer homologous to the constant section of the non-specific primer of the first PCR. PCR products were cloned into pCR2.1-TOPO, amplified in The following additional data are available with the online version of this paper. Additional data file Movie of GFP fluorescence of clone 5 over 84 days.Click here for fileMovie of GFP fluorescence of clone 6 over 84 days.Click here for fileMovie of GFP fluorescence of clone 18 over 84 days.Click here for fileExpression of GFP fluorescence as profiled by flow cytometry in clones 25 to 96. Clones 25 to 48 and 73 to 96 were transduced with GFP plus an adjacent Ig enhancer. Clones 1 to 24 Figure  and 49 tClick here for file


The transporter associated with antigen processing (TAP) is a critical component of the major histocompatibility complex (MHC) class I antigen processing and presentation pathway. TAP transports antigenic peptides into the endoplasmic reticulum where it loads them into the binding groove of MHC class I molecules. Because peptides must first be transported by TAP in order to be presented on MHC class I, TAP binding preferences should impact significantly on T-cell epitope selection.TAP is a computational system that predicts peptide binding to human TAP. It uses artificial neural networks and hidden Markov models as predictive engines. Extensive testing was performed to valid the prediction models. The results showed that PREDTAP was both sensitive and specific and had good predictive ability (area under the receiver operating characteristic curve Aroc>0.85).PREDTAP can be integrated with prediction systems for MHC class I binding peptides for improved performance of in silico prediction of T-cell epitopes. PREDTAP is available for public use at [PREDc use at . The major function of CTLs is recognition and destruction of infected , mutated (e.g. cancer), or foreign (e.g. transplants) cells. CTLs recognize short antigenic peptides (T-cell epitopes) presented by MHC class I molecules that mainly originate from degradation of cytosolic proteins. Intracellular antigen processing pathways determine the selectivity of peptides which are available for binding to MHC class I molecules and are thereby important targets of CTL responses . AmongsTAP, a computational system that predicts peptides binding to hTAP. It uses ANN and hidden Markov models (HMM) as predictive engines. Extensive testing was performed to validate the prediction models and ensure that PREDTAP is both sensitive and specific. PREDTAP is available for public use at .,15.12,15TAP server. The learning method was error backpropagation with a sigmoid activation function. The inputs to the ANN were the binary strings representing nonamer peptides. There are twenty naturally-occurring amino acids encoded by the standard genetic code. Each amino acid in a nonamer peptide can be encoded as a binary string of length 20 with a unique position set to "1" and other positions set to "0", resulting in a binary string of length 180 to represent the nonamer. For example the first two amino acids, by alphabetic order, alanine (A) and cysteine (C) are encoded by 10000000000000000000 and 01000000000000000000 respectively, and the last amino acid tyrosine (Y) is encoded by 00000000000000000001. The outputs were binding scores ranging from zero to ten. The higher the score, the higher the possibility of the peptide being a TAP binder. Two ANN architectures were used, 180-2-1 and 180-1-1. The maximum number of the ANN training cycles was set to 300. The training was repeated for four times, and four sets of weights were obtained. The value of momentum was 0.5 and of learning rate 0.2. The error threshold for stopping training was 0.01.3-layer backpropagation ANN models (in-house software) were used for the development of the PREDHMMs have been applied successfully in prediction of HLA class I-binding peptides ,27. An HCross-validation is a method for error rate estimation. It implements a simple idea: the dataset of size n samples is partitioned into two parts, the model parameters are estimated using one set and the goodness-of-fit criterion evaluated on the second set. The cross-validation estimates the goodness-of-fit criterion. Cross-validation tends to overfit when selecting a correct model – it may choos an overly-complex model for the given dataset. There is some evidence that for model selection multifold cross-validation, where more than one samples are deleted form the training set in each comparison, performs better than a simple leave-one-out cross-validation. In our The predictive performance of the models was evaluated by sensitivity (SE) and specificity (SP) measures. Sensitivity, SE = TP/(TP+FN), indicates percentage of correctly predicted binders, where TP stands for number of true positive predictions  and FN stands for number of false negative predictions . Specificity, SP = TN/(TN+FP), indicates percentage of correctly predicted non-binders, where TN stands for number of true negative predictions  and FP stands for number of false positive predictions . For the studied problem, we consider values of SP >0.8 useful in practice.The receiver operating characteristic (ROC) curve analysis provided a measure for overall prediction accuracies of prediction models . The ROCTAP(ANN & HMM) was compared with that of publicly available predictive systems, TAPPred (SVM & cascade SVM) [The prediction performance of PREDade SVM)  and SVMTade SVM) . Three pade SVM) , E7 (P03ade SVM)  peptidesade SVM) , were uset al. [Brusic et al.  showed tscoren = (score - scoremin) / (scoremax - scoremin)   × 10scoren denotes the normalized score, score denotes the raw prediction score, scoremin and scoremax denote the minimum and maximum values of the raw scores. The values for scoremin and scoremax were obtained using extensive simulation. More than 5000 randomly selected nonamer peptides were used for prediction using the ANN/HMM models. Since the testing data contains large number of nonamer peptides, the highest and lowest predicted score from the testing data were taken as reasonable maximum and minimum scores for normalization.TAP uses a set of Graphical User Interface forms. The interface was built using a combination of Perl, CGI and C programs. PREDTAP has been implemented in the SunOS 5.9 UNIX environment.The web interface of PREDAssessment of predictive accuracy was carried out for three subsets of peptide binders: 1) all binders including low, moderate and high binders were considered as positive samples, and all non-binders as negative samples (referred to as the LMH set); 2) moderate and high binders were considered as positive samples, all non-binder and low binders as negative samples (referred to as the MH set), and 3) only high binders were considered as positive samples, with all other peptides as negative samples (referred to as the H set). The Aroc values of ANN and HMM models are shown in Table vs. sensitivity plot of the ANN prediction model for prediction can be viewed at supplementary materials A [The specificity erials A . The speerials A .Sensitivities and specificities of ANN and HMM models at various thresholds  in 10-fold cross-validation experiments are shown in Figures To evaluate the predictive power of the methods, the dataset was partitioned into a training set containing two thirds of the data points randomly selected and a testing set containing the remaining one third of data points. The tests were conducted three times for each ANN and HMM methods. The Aroc values of ANN and HMM models are shown in Table TAP, TAPPred and SVMTAP were built using the same set of   training data [TAP (ANN & HMM) predicted similar sets of TAP-binding peptides while TAPPred (cascade SVM) predictions were different , but not by TAPPred (cascade SVM). Peptide 499–508, was selected by the four methods as a potential 16-mer, also as a 12-mer by PREDTAP (ANN), but not by TAPPred (cascade SVM). It was shown that some peptides are efficiently transported by TAP in their optimal size for MHC class I binding, while some peptides are transported as larger peptides that need further trimming in ER for MHC class I binding [Three naturally processed peptides from tumor antigen KM-HM-1, namely 196–204, 499–508, and 770–778, are naturally processed by HLA-24 . HLA-A24 binding . It is lTAP, the user needs to paste a protein sequence into the textbox and assign a name to the sequence. The sequence must contain between nine and 2000 amino acids. If the prediction is run with input sequence containing symbols other than 20 amino acid codes  or the total sequence length is outside 9–2000 amino acids range, an error message will be displayed and predictions will not be produced. The input can either be a contiguous protein sequence  or a list of peptides, one per line. The default selection on the webpage is "Protein sequence"  into a series of 9-mer peptides overlapping by eight amino acids. Individual 9-mer peptides are then submitted for prediction. Predicted binding scores for all 9-mers are displayed in the result tables  shows that consensus peptides can be selected by combining predictions. The examples suggested that individual predictions need to be taken with care and predictions may be improved by a consensus of multiple methods. A similar situation may be applicable to TAP predictions. Hence the combination of ANN and HMM predictions in PREDTAP should result in higher specificity  at the cost of slightly lower sensitivity. The predictions by TAPPred (cascade SVM) appear to be of a limited value.We have earlier compared four prediction servers for prediction of H-2Kpeptides . A 121-aTAP server provides for the prediction of peptide binding by TAP and can be used as a comparison method against other TAP-prediction servers.The combinatorial properties of molecular mechanisms involved in antigen processing and adaptive learning nature of the immune responses limit our ability to fully predict immune responses. Combining experimental and computational techniques improves our ability to decipher complex interactions of the immune system. Computer models are used to complement laboratory experiments and thereby speed up knowledge discovery in immunology. In particular, the number of large-scale laboratory experiments for T-cell epitope mapping can be minimised by the judicious use of experiments aimed at developing and validating computer models. These models can then be used to perform large-scale computer simulations rapidly and inexpensively. The hypotheses generated from these experiments can then be retested in the laboratory to confirm their applicability to real-life immunology. Further work will include both the refinement of computational models and scanning disease-related antigens for peptide sequences that show high probability of processing and presentation. Those peptides that are most likely to be produced by proteasomal cleavage, transported by TAP, and bound by HLA class I molecules are likely to be promising candidates for peptide-based CTL vaccines. The PRED


Sustained expression of fos is sufficient to induce cellular transformation in vitro and tumorigenesis in vivo. Fos functions as a component of the AP-1 transcription factor complex to regulate gene transcription and several differentially expressed genes have been identified in cells transformed by fos. We have extended these studies by constructing a cellular system for conditional transformation by v-fos. Using Affymetrix-based DNA microarray technology, we analyzed transcriptional changes over the course of transformation and reversion in an inducible v-fos system.The c-v-fos mediated cellular transformation and morphological reversion revealed a remarkably dynamic transcriptome. Of the more than 8000 genes analyzed in this study, 3766 genes were categorized into 18 gene-expression patterns by using self-organizing map analysis. By combining the analysis of gene expression profiles in stably transformed cells with the analysis of sequential expression patterns during conditional transformation, we identified a relatively small cohort of genes implicated in v-fos mediated cellular transformation.Microarray analyses of temporal gene expression during the process of fos.This approach defines a general conditional cell transformation system that can be used to study the endogenous transcription regulatory mechanisms involved in transformation and tumorigenesis. In addition, this study is the first reported analysis of dynamic changes in gene expression throughout experimentally controlled morphological transformation mediated by v- The c-Fgnalling . The tigfos as the oncogene carried by the Finkel-Biskis-Jenkins and Finkel-Biskis-Reilly murine osteosarcoma retroviruses contributed to the realization that tumorigenic retroviruses harbor viral versions of cellular genes and that these genes can elude the regulatory constraints imposed upon the endogenous gene [fos oncogenes contain point mutations and deletions that enhance their transforming potential [fos is sufficient to induce cellular transformation in vitro and tumorigenesis in vivo [fos-induced transformation and tumorigenesis is the consequence of inappropriate fos activity within susceptible cells rather than a gain-of-function mechanism specific to the viral fos oncogene.The identification of v-ous gene -5. The votential . However in vivo . Therefofos and AP-1, suggesting that inappropriate activation of c-fos contributes to various aspects of tumorigenesis. This contribution involves direct transcriptional regulation of AP-1 target genes and secondary mechanisms of transcriptional regulation. For example, increased expression and activity of Dnmt1, a DNA methyltransferase that methylates CpG dinucleotides [fos [fos-transformed cells [fos-mediated transformation, there is conditional deregulation of target gene expression dependent upon continual oncogene activity, in addition to long-term epigenetic reprogramming of gene expression that can persist even when the direct effects of oncogene activity are suppressed.Many signal transduction pathways implicated in tumorigenesis functionally converge on activation of c-leotides , is necedes [fos . CpG metdes [fos . A previed cells . Therefofos transformation and have yielded functional data that implicate differentially expressed genes in aspects of oncogenic transformation [fos) that allows control of v-fos expression and morphologic transformation. This approach refines the analysis of gene expression associated with fos transformation by distinguishing gene expression changes coincident with morphological transformation from those that are potentially associated with clonal variation or phenotypic changes that occur downstream of the transformation process. Comparisons of temporal gene expression patterns during conditional cellular transformation with transcriptome profiles of cells stably transformed by c-fos and v-fos revealed a cohort of genes likely to be critical for induction and maintenance of cellular transformation.Studies of stably transformed cell lines have found gene expression changes associated with ormation -14. In tfos cell system, the control of FBJ/R v-fos expression is dependent on the presence of isopropyl-b-D-thiogalactopryanoside (IPTG) in the cell culture medium [fos cells did not express v-Fos protein detectable by Western blot analysis ; from cells at 24, 48 and 72 hours following the removal of IPTG (transforming); and from cells at 24, 48 and 72 hours after the re-addition of 5 mM IPTG (reverting). Previous studies have demonstrated that the addition and removal of IPTG does not itself induce persistent changes in gene expression in the parental 208F rat fibroblast cell line [The ability to control both v-Fos expression and morphological transformation ell line ,11. RNA fos microarray data obtained at the seven time points throughout v-Fos transformation and reversion  analysis is a powerful tool that can be used to categorize gene expression data into groups that share common temporal expression profiles -17. To in Figure . The datn Figure . This grnscripts . ConversPatterns within the SOM that are consistent with v-Fos transformation and reversion were selected on the basis of the representative expression pattern of each SOM group. For candidate genes whose expression was upregulated, the standard deviation of the mean signal values at a minimum of two of the three time points in the absence of IPTG (transforming) had to be greater than that of the mean signal values at time zero . Also, the standard deviation of the mean signal values at a minimum of two of the three final time points in the presence of IPTG (reverting) had to be below that of the mean signal value of the final time point during transformation . For candidate genes whose expression was downregulated, the standard deviation of the mean signal values at a minimum of two of the three transforming time points had to be lower than that of the mean signal values at time zero, and the standard deviation of the mean signal values at a minimum of two of the three time points during reversion had to be higher than that of the final time point during transformation. On the basis of these criteria, patterns 7, 8, 14 and 15 (transforming) and patterns 5, 11, 12, 17 and 18 (reverting) were selected for further analysis. Each of these groups consisted of a large number of genes Figure . These gfos cells) or v-Fos (FBJ/R cells). Cells transformed by stable expression of c-Fos or v-Fos exhibit a large number of differentially expressed genes. For example, when differential expression was defined as a 2-fold increase or decrease based on a comparison with expression in 208F cells and when probe sets for expressed sequence tags (ESTs) were excluded, 70 probe sets were scored as upregulated and 104 were scored as downregulated in both CMVc-fos and FBJ/R cells [fos and FBJ/R cells to 208F cells, 63 (36%) were also categorized into LacIv-fos SOM patterns consistent with increased or decreased expression during conditional v-Fos transformation. Therefore, the majority of gene expression changes associated with stably Fos-transformed cell lines were not recapitulated during conditional v-Fos transformation and reversion. These findings emphasize the value of combining analyses of stable and conditional cellular transformation systems.To identify only the most promising candidate genes, we compared these datasets with gene expression profiles of the parental 208F cells and 208F cells transformed by stable expression of either c-Fos  demonstrated a reproducible temporal profile of gene expression in the independent LacIv-fos transformation and reversion time course experiment (TGF-β 3 and CAIII) had nearly identical patterns of gene expression when compared to microarray analysis [To address the reproducibility of gene expression changes during conditional v-Fos transformation, we performed microarray analysis using RNA samples isolated from LacIv-analysis . Therefofos, v-fos contains several deletions and point mutations that affect its oncogenic potential [fos is capable of inducing cellular transformation and tumorigenesis, v-fos is a much more potent oncogene [fos cells, revealed a dramatic increase in the number of transcriptionally upregulated genes in FBJ/R relative to CMVc-fos cells: 337 probe sets were scored as specifically upregulated in FBJ/R cells, whereas only 70 probe sets common to CMVc-fos and FBJ/R cells were considered as increased. In contrast, 61 probe sets were scored as downregulated specifically in FBJ/R cells but 104 probe sets in both CMVc-fos and FBJ/R cells were considered as downregulated [fos SOM and the results of the independent LacIv-fos transformation and reversion time course as described previously. These comparisons revealed an additional 38 upregulated and 29 downregulated probe sets that are differentially regulated in v-fos-transformed cells, but not in c-fos transformed cells . The initial interface of the database is the LacIv-M Figure . Users cThe process of cellular transformation involves complex alterations of gene expression regulation. This level of complexity raises the challenge of identifying the gene expression changes that are most relevant to mechanisms of tumorigenesis. The challenge is especially evident in studies of primary tumor specimens in which the analyzed material represents the end state of a progressive series of events rather than a snapshot of the process itself. Cell-based model systems of transformation and tumorigenesis offer an opportunity to experimentally simulate steps along the pathway of transformation; however, these systems are subject to their own set of caveats including gene expression changes resulting from variation inherent to cell culture conditions and clonal variation. In the study described here, we attempted to provide a more focused view of relevant gene expression changes associated with oncogenic cellular transformation by analyzing a conditional cell-based model system in which morphological transformation and reversion is experimentally manipulated.fos results in the differential expression of a large number of genes [fos cells and by arranging these patterns into SOMs, we have clustered differentially expressed genes into cohorts with common temporal patterns of expression. This type of clustering allows identification of groups of genes with expression patterns that are consistent with a potential functional role in the process of morphological transformation. For example, the exogenous v-fos transcript itself is clustered within the conditionally upregulated group 8. This group also includes the previously reported Fos-regulated target genes such as ezrin [Fra-1 [CD44 is more transiently induced (group 7); this characteristic is consistent with features of a gene whose expression is induced in response to serum stimulation. In contrast, ezrin expression is maintained at its maximal level throughout the 3-day morphological transformation period and rapidly returns to its initial expression levels upon repression of exogenous v-fos expression (group 8). These results demonstrate that coordinated yet distinct programs of gene expression regulation can be initiated to affect the expression of genes that function in common mechanisms of cellular transformation.Sustained expression of of genes ,9,11. Byas ezrin  and Fra-n [Fra-1 ,21 genesn [Fra-1 ,21. Inten [Fra-1  but the fos or v-fos, and it is conditionally upregulated during LacIv-fos transformation (group 8). PRL-1 promotes cell migration and invasion in vitro and its expression is elevated in a number of cancer cell lines. PRL-1 is also involved in regulation of progression through mitosis, possibly by modulating spindle dynamics [Our analysis has revealed additional conditionally regulated genes that have not been previously associated directly with Fos or AP-1. For example, PRL-1 is a protein tyrosine phosphatase originally identified as an immediate-early gene in liver regeneration . PRL-1 edynamics ,25.fos) with v-Fos transformed cells revealed additional differential gene regulation events in cells transformed by v-Fos relative to cells transformed by c-Fos. For example, expression signal values of both c-myc and the Ras GTPase, RalA, were slightly greater in CMVc-fos cells relative to 208F cells. However, expression of these genes was dramatically upregulated (>2fold) in FBJ/R cells. Likewise, PCPE and SPARC expression was slightly downregulated in CMVc-fos cells, but were dramatically repressed in FBJ/R cells. In contrast, two genes involved in enzymatic regulation of the extracellular matrix were differentially expressed only in FBJ/R cells. Expression of the matrix metalloproteinase MMP10 and the metalloproteinase inhibitor TIMP2 was specifically upregulated and downregulated in v-fos-transformed cells, respectively. Interestingly, MMP10 was previously identified as a gene whose expression was upregulated gene in cells stably transformed by FBR-v-fos [fos-transformed cells are currently unknown. Some of these genes may be direct targets of v-Fos, but not of c-Fos, or targets of secondary transcription regulatory factors whose expression is deregulated specifically in v-fos-transformed cells. Alternatively, the expression of some genes may be affected by both c-Fos and v-Fos, but to a higher degree in v-fos-transformed cells.Sustained expression of c-Fos is capable of initiating transformation ; howeverBR-v-fos . The mecjun-transformed primary rat embryo fibroblasts [un-transformed chick embryo fibroblasts, reduction in SPARC mRNA levels has been shown to be due in part to the formation of a DNA-Sp1/3-v-Jun chromatin-associated complex [in vitro invasiveness [v-fos in mediating gene silencing in the conditional system is not clear; however, our system represents a tool for identifying candidate genes that are subject to epigenetic modification during the process of oncogenesis, as well as a conditional cellular system that can be employed to investigate the temporal mechanisms underlying these regulatory events.The identification of 28 conditionally downregulated probe sets specific to v-Fos transformation raises the possibility that loss of expression of a subset of these genes may be due to chromatin modifying mechanisms leading to gene silencing. Included in our analysis are several genes previously implicated as targets for gene silencing via epigenetic repression. For example, the extracellular matrix molecule, SPARC, is downregulated in c-roblasts . Also, i complex . Tissue  complex . TIMP-2  complex . Interessiveness . The prein vitro cellular model systems of oncogenic transformation have provided a wealth of information relevant to both normal signal transduction pathways and tumorigenic mechanisms. However, the large number of genes differentially expressed in these model systems often complicates the identification of the most promising candidate genes for further study. Our web-accessible database of transcriptional changes detected in the conditional v-fos system provides a powerful tool to identify cohorts of gene candidates associated with specific cellular events during the process of transformation and reversion.Technological advances in the ability to analyze gene expression profiles on an increasingly global scale have contributed significantly to a more comprehensive view of the complex transcriptional networks that go awry in tumor cells ,33. Stud2. Establishment of the stably transformed cell lines, FBJ/R and CMVc-fos, was described previously [fos conditional cell line and the conditions required for the transformation/reversion time course were described previously [fos transformation and reversion time course experiment, total RNA was extracted from LacIv-fos cells cultured in the presence of 5 mM IPTG at time zero; 24, 48 and 72 hours after removal of IPTG (transforming); and 24, 48 and 72 hours after the re-addition of 5 mM IPTG (reverting). Total RNA was extracted from cells by using Tripure reagent according to the manufacturer's instructions (Roche). The integrity of all RNA samples was verified by using an Agilent 2100 Bioanalyzer.All cells were maintained in DMEM supplemented with 10% fetal calf serum, L-glutamine and penicillin/streptomycin at 37°C in the presence of 5% COeviously . Establieviously . BrieflyExpression of Fos protein during transformation and reversion was evaluated by Western blotting analysis using an antibody directed against Fos . Whole-cfos cells and FBJ/R cells relative to 208F cells had a signal log ratio ≥ 1 (increased) or ≤ -1 (decreased), and a change p-value < 0.001 (increased) or > 0.99 (decreased). For visualization of specific profiles of gene expression during the LacIv-fos conditional transformation and reversion time course, signal values obtained at each LacIv-fos time point were plotted by using Microsoft Excel.RNA samples were processed for hybridization without amplification and hybridized to Affymetrix Rat Genome U34A GeneChips that include probe sets representing approximately 7,000 annotated genes and 1,000 EST clones . GeneChiMicroarray data that were obtained from the LacIv-fos time course experiments and passed the variance filter were grouped into relative expression pattern bins by the self-organizing map (SOM) program in the GeneMaths software package . The SOM analysis was performed by using a 6 × 3 node format to allow optimal representation of gene expression patterns in a reasonably small number of independent bins.. The web application to query and manage the database is driven with a server-side scripting technology JSP. The entries of this database are generated from the tab-delimited output files of the SOM analysis. Data obtained from the SOM analysis can be queried by three approaches. Users can click on a graphical pattern key to view a list of all genes whose expression profiles match the particular self-organization pattern. Each entry in the list contains two parameters associated with the respective gene probe, i.e., the Affymetrix probe set ID number and a description of the gene. The detailed Affymetrix analysis data for a probe set can be obtained by clicking the link of the respective probe set ID number. The corresponding Affymetrix analysis page provides information about the number of oligonucleotide pairs, the signal magnitude, the detection status, and the detection p value at seven different time points derived from 2 independent experiments. Another search method is to query via the GenBank accession number. The query by accession number is based on the fact that each Affymetrix probe set ID number in our current experiments corresponds to a gene accession number. If an accession number in a query matches with any part within a probe set ID number, the entry containing the probe set ID number, its SOM pattern, and gene description is displayed. Hyperlinks are provided for both probe set ID numbers and SOM pattern. By following the link for a probe set ID number, the user can view the detailed Affymetrix analysis data for a selected gene probe; by following the link for an SOM pattern, the user can view the detailed Affymetrix analysis data for all the gene probes within the selected pattern. Also, a keyword-based text search is included in the database. A search for keywords in the paragraph of the gene description field is conducted in a case-insensitive manner. The search-by-keyword method is facilitated by the use of Boolean operations. The search can narrow or broaden quickly the results of the search by combining two or more keyword(s) with only one type of Boolean (and / or) operation.The SOM searchable database has been implemented on an Open Source MySQL 4.0.14 relational database management system. The database has a web interface at JMO prepared cells, purified RNA, and analyzed the microarray data, SDF performed immunofluoresence microscopy and Western blot analysis, and HR designed the web-accessible database. JMO, SDF, HR, and TC composed the manuscript.fos cells and in FBJ/R cells and conditionally regulated in LacIv-fos cells.Genes differentially expressed  in CMVc-Click here for file


We developed an IgG1 mouse monoclonal antibody (ONS-M21) directed against a cell surface antigen of medulloblastomas and gliomas in immunisation of mice with the ONS-76 medulloblastoma cell line. The antibody specifically reacted with medulloblastomas, supratentorial primitive neuroectodermal tumours (SPNETs) and gliomas, but not with other neuroectodermally derived tumours (neuroblastoma and melanoma) or with other kinds of tumours . No reactivity was identified with normal body tissues, including peripheral blood cells. Characterisation of the ONS-M21 antigen showed that it was a trypsin-sensitive glycoprotein with a molecular weight of 80 kDa on SDS-PAGE. The pattern of reactivity and the biochemical properties of this antigen were different from those of other markers of medulloblastoma. These results indicate that ONS-M21 detects a new tumour-associated cell surface antigen specifically expressed by medulloblastomas, SPNETs, and gliomas. This is the first report that medulloblastomas may share common cell surface antigens with gliomas, although most studies have concluded that medulloblastoma has a predominantly neuronal phenotype. The lack of reactivity with normal tissue implies that ONS-M21 has potential applications as both a diagnostic tool and a therapeutic agent.


The improved tumour to non-tumour ratios needed for effective tumour targeting with antibodies requires that blood background radioactivity is reduced. We investigated the effect of streptavidin as a clearing agent for 125I-labelled biotinylated anti-CEA antibodies in a human colon carcinoma xenograft model. By comparing the biodistribution of the monoclonal antibody A5B7 with four, nine or 22 biotins per antibody molecule, we investigated how the degree of biotinylation of the primary radiolabelled antibody affects its clearance with streptavidin. Limiting the degree of biotinylation limited blood clearance, whereas nine or 22 biotins per antibody molecule resulted in a 13- to 14-fold reduction in blood radioactivity, the streptavidin-biotinylated antibody complexes clearing rapidly via the liver and spleen. Although a reduction in tumour activity was also seen, a 6.6-fold improvement in the tumour to blood ratio was achieved. A comparative study of streptavidin versus second antibody clearance was carried out using the polyclonal antibody PK4S biotinylated with 12 biotins per antibody molecule. This study indicated that second antibody was superior for clearance of the polyclonal antibody, resulting in a larger and faster reduction in blood radioactivity and improved tumour to blood ratios. In this case the primary antibody was polyclonal, and therefore non-uniformity of biotinylation may affect complexation with streptavidin. Therefore, the degree of biotinylation and type of antibody must be carefully considered before the use of streptavidin clearance.


However, a detailed understanding of the action of ERK in neurons is made difficult by the lack of knowledge about its subcellular localization in response to physiological stimuli. To address this issue, we have studied the effect of visual stimulation Using immunogold electron microscopy, we show that phosphorylated ERK (pERK) is present in dendritic spines, both at synaptic and non-synaptic plasma membrane domains. Moreover, pERK is also detected in presynaptic axonal boutons forming connections with dendritic spines. Visual stimulation after dark rearing during the critical period causes a rapid increase in the number of pERK-labelled synapses in cortical layers I–II/III. This visually-induced activation of ERK at synaptic sites occurs in pre- and post-synaptic compartments and its temporal profile is identical to that of ERK activation in neuronal cell bodies.in vivo increases pERK expression at pre- and post-synaptic sites of axo-spinous junctions, suggesting that ERK plays an important role in the local modulation of synaptic function. The data presented here support a model in which pERK can have early and late actions both centrally in the cell nucleus and peripherally at synaptic contacts.Visual stimulation  The ERK/MAPK pathway has emerged as a central player in the signalling mechanisms underlying synaptic plasticity. Studies on the developing visual system have shown that ERK is strongly regulated by visual experience and that its activation is necessary for synaptic plasticity and ocular dominance plasticity It is presently believed that activated ERK translocates to the nucleus, where it targets several different regulators of gene expression, such as the transcription factors CREB and ELK-1 This wealth of biochemical and pharmacological information about the role of ERK in synaptic plasticity is not paralleled by a precise knowledge of the spatial and temporal regulation of ERK activity by physiological stimuli. This information is necessary to elaborate realistic models of the role of ERK in neuronal plasticity. To address this issue, we analyzed the spatio-temporal distribution of activated ERK in cortical neurons after visual stimulation by means of confocal and electron microscopy. We found that light exposure causes the activation of ERK both in the cell body of pyramidal cortical neurons and at the level of excitatory synapses, where phosphorylated ERK was found both in pre- and post-synaptic compartments. The kinetics of ERK activation were the same at the cell soma and at synaptic junctions. These data support a model in which pERK can have early and late actions at both nuclear and synaptic levels.Animals were used in accordance with protocols approved by the Italian Minister for Scientific Research. Control animals were exposed to a 12 hr light/dark cycle, with the light phase beginning at 6:00 A.M. All animals were killed at the same time of the day, between 8:00 and 9:00 A.M. Manipulations during dark rearing (DR) were performed in complete darkness using infrared viewers 2O2 for 4 min. The sections were mounted on gelatine-coated glass slides and observed either with a light microscope  equipped with a CCD camera  or imaged with a confocal microscope .Coronal vibratome sections (40 µm) were collected in phosphate buffered saline  and then treated free-floating. After a blocking step in a PBS solution containing 3% bovine serum albumin, 0.05% Triton X-100 and 10% normal goat serum (NGS), they were incubated overnight with a monoclonal antibody against the diphosphorylated form of ERK1/2  diluted 1∶500 in PBS with 3% NGS and 0.05 Triton X-100. This antibody was raised against an 11 amino acid peptide (HTGFLT(Pi)EY(Pi)VAT) corresponding to the phosphorylated form of the ERK-activation loop, and does not recognize the non-phosphorylated or monophosphorylated forms of ERK, as well as the diphosphorylated forms of ERK-related kinases 2O2 for 4 min. The DAB reaction product was silver intensified and gold toned as previously described Vibratome sections (90 µm) of the primary visual cortex were cryoprotected in 30% sucrose in 0.1 M PB and then freezed and thawed to optimize the penetration of immunoreagents. The anti-diphosphorylated ERK1/2 antibody was diluted in the same dilution media used for light microscopy, but Triton X-100 was omitted from all solutions. Free-floating sections were blocked in 10% NGS for 2 hr and incubated in the primary antibody for 3 d at 4°C. After several rinses in PBS (4×10 min), the sections processed for pre-embedding immunoperoxidase detection were incubated for 2 hr at room temperature in biotinylated secondary antibodies, rinsed again in PBS (4×10 min), and transferred to a solution containing a biotin-avidin complex (Vector Labs). Finally, the sections were reacted in 3,3′-diaminobenzidine  with 0.01% HTo test method specificity in the immunohistochemical procedures for both light and electron microscopy, the pERK antibody was omitted. Under these conditions, no immunoreactivity was observed. Moreover, intraperitoneal injection (100 ml/kg) of the specific inhibitor of the ERK kinase MEK1/2  before light exposure completely abolished pERK immunolabelling , thus coThe number of pERK-positive axo-spinous synaptic contacts in layers I and II/III of the rat primary visual cortex was counted in three distinct animals for each of the four different visual stimulation conditions . In each experiment, comparisons were made with littermates that had been reared together and the final observer was always blind to the treatment. Synapses were counted in thin sections cut at comparable depths (100–500 nm) from the surface of the labelled vibratome sections, in order to minimize false-negative results caused by limited penetration of the antibodies. We systematically sampled all asymmetric contacts in non-overlapping electron micrographs taken in layer I  and in layers II–III (n = 25), at a magnification of ×40.000. This led to an average of 190 asymmetric synapses analysed in each animal. The criteria for identifying asymmetric synapses were the presence of a presynaptic terminal containing synaptic vesicles, the parallel alignment of the pre- and post-synaptic membranes, and the presence of the postsynaptic density. A synapse was considered immunopositive when at least two gold particles were localized in either the pre- or the post-synaptic profile. The data were expressed as the percentage of pERK-positive profiles over the total number of synapses (mean±SEM of three rats). Statistical analysis was performed using a one-way ANOVA test with Origin 7 software . To determine the subsynaptic distribution of pERK in dendritic spine profiles, we divided the spine head profile into three distinct compartments: post-synaptic density (PSD), shell and core Although several studies have analysed the expression of pERK in the brain, no information about its subcellular localization in neurons is currently available. We used immuno-electron microscopy to study the distribution of activated ERK in the primary visual cortex of juvenile (P27–P28) rats with a monoclonal antibody that is specific for the active, dually-phosphorylated form of ERK . At the light-microscopic level, pERK immunoreactivity was evident in individual pyramidal neurons. A relevant number of labelled neurons were located in the more superficial cortical layers (II/III), whereas only a few pERK-positive neurons were found in the deeper layers. As shown in The ultrastructural analysis of immunoperoxidase labelling revealed strong pERK expression in the nucleus and in the perinuclear cytoplasm of layer II–III neurons , as wellERK can be activated in pyramidal cortical neurons by visual stimulation Visual stimulation caused a dramatic increase in the number of axo-spinous contacts positive for pERK in layer I and layer II–III of light-exposed rats  with resCytoplasmic proteins, membrane proteins and protein components of the PSD are potential substrates of activated ERK in dendritic spines in vivo analysis shows that the synaptic and somatic pools of ERK are activated with similar kinetics after physiological stimulation.It has been proposed that the early effects of ERK in synaptic plasticity are mediated by its action in the cell periphery, whereas the late effects involve the translocation of pERK into the nucleus In the present study we describe for the first time the ultrastructural localization of activated ERK in neurons. Using high-resolution immunolabelling, we show that visual stimulation evokes a robust activation of ERK at asymmetric synaptic contacts in layers I–II/III of the primary visual cortex. The activation of ERK at synapses is rapid and transient and occurs in both the pre- and post-synaptic compartment. This observation, together with previous findings showing that ERK inhibitors block visual cortical plasticity and cortical LTP Previous studies have shown that ERK plays a fundamental role in developmental plasticity of the visual cortex The contribution of ERK in synaptic plasticity extends to several other brain regions, including the hippocampus, amygdala and striatum What could be the role of the local activation of ERK at synaptic sites? In the cell periphery, ERK activity may target multiple substrates in the synaptic and dendritic compartment, and therefore contribute to the initial steps that lead to long-term synaptic changes + channel (Kv4.2), a regulator of dendritic propagation of action potentials v4.2 is often localized along the non-synaptic membrane of dendritic spines as well as in postsynaptic membranes of GABAergic synapses v4.2 channel function. ERK activation in dendritic spines could also be involved in regulating the insertion of AMPA receptors into the postsynaptic membrane. Recently, Malinow and colleagues (47) showed that ERK is directly involved in an increase of AMPA receptors-mediated synaptic transmission and in the synaptic delivery of AMPA receptors following LTP One interesting finding of our study is that pERK seems to be preferentially localized within the core and the shell of the spine, whereas it seems to be scarcely associated with the PSD. One of the potential postsynaptic targets of pERK is the voltage-dependent KIn addition to the postsynaptic activation, we found that visual stimulation leads to a very rapid increase of the number of pERK-immunoreactive axon terminals, suggesting that visually-induced activation of ERK might regulate synaptic transmission and plasticity at the presynaptic level. While the precise role of pERK in axonal boutons is still unclear, it has been shown that ERK may act to phosphorylate synapsin I, a phosphoprotein that regulates the binding of synaptic vescicles to the actin cytoskeleton in an activity-dependent manner in vivo physiological stimulation approach used in this study does not allow any control on the spatial specificity of the inputs. Thus, specific experiments in which the activated synapses can be analyzed at different times after stimulation are needed to address this possibility.Our study might also suggest that pERK could represent a mark of previously activated synapses, both at the pre- and at the post-synaptic level. However, the in vivo demonstration that ERK activation can be targeted to the RER, thus adding further evidence for a role of the Ras/ERK pathway in the translation of mRNA. In addition, the presence of activated ERK in the core of dendritic spines supports a role of this molecule as a key regulator in local protein synthesis. It has been shown that translation of mRNA residing in dendrites and at synaptic sites plays an important role in long-lasting forms of synaptic plasticity and memory in vivo synaptic activity may alter their distribution and function.It has been shown that ERK may enhance protein synthesis through phosphorylation of eIF-4E Many studies on the mechanisms underlying LTP have led to the idea that synaptic plasticity undergoes an initial phase that is independent from gene transcription. This early process is mediated by activity-dependent mechanisms that occur peripherally at the synaptic level and involve covalent modifications of pre-existing proteins and local protein synthesis Figure S1We controlled the specificity of pERK immunolabelling by analysing brain sections after SL327 injection. A and B show confocal images of pERK immunofluorescence in the visual cortex of a rat exposed to light for 2.5 minutes after 3 days of dark rearing and injected with SL327 (B), and of another rat that received the same visual stimulation and vehicle injection (DMSO) as a control. It is evident that in the SL327 injected animal pERK immunoreactivity is completely abolished. We further tested the distribution of pERK using a polyclonal antibody on visual cortical sections of a normally reared rat. As shown in C, p-ERK immunoreactivity is present in the soma and in the apical (arrow) and basal dendrites (arrowhead) of cortical pyramidal neurons that are localized in layer II/III of the primary visual cortex. This pattern of immunolabelling is virtually identical to the one that we observe using a monoclonal antibody against pERK see . Scale b(3.49 MB TIF)Click here for additional data file.Figure S2To assess the reliability of pERK immunogold localization at synapses, we analyzed consecutive thin sections of the primary visual cortex. Micrographs in A′-A′ illustrate the consistency of labeling in serial sections of an axo-spinous synapse. Immunogold particles decorate a presynaptic terminal (asterisks) in all three sections whereas the juxtaposed postsynaptic spine does not show any labeling. B′-B′ show another example of the reliability of pERK immunogold labeling. An unlabelled axo-spinous synapse (arrows) adjacent to a pERK-positive dendritic profile is shown in three serial thin sections cut through layer I of the visual cortex of a rat. Scale bars: 200 nm(2.99 MB TIF)Click here for additional data file.


Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix-loop-helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, we have investigated whether similar types of cells exist in the Drosophila antenna.The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila antenna. Mz317::GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional ~30 glial cells detected by GH146::GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. We denote these distinct populations of cells as Mz317-glia and GH146-glia respectively. In the adult, processes of GH146-glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317-glia form a peripheral layer. Ablation of GH146-glia does not result in any significant effects on the patterning of the olfactory receptor axons.We have used different P lines to drive Green Fluorescent Protein (GFP) in distinct populations of cells within the Drosophila antenna. GH146-glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca.We have demonstrated the presence of at least two distinct populations of glial cells within the  Odor information in animals is represented as a spatial pattern of activity among glomeruli in the olfactory lobe . This odDrosophila olfactory system have been reviewed recently [Down Syndrome Cell Adhesion Molecule (Dscam) gene [The cellular events occurring during development of the recently . Adult Orecently . Axon team) gene ,7.Drosophila olfactory system, peripheral glial cells have been shown to arise from sensory lineages and play a role in patterning ORNs within distinct fascicles as they exit the antenna [Manduca sexta, results in a failure in glomerular maturation and stabilization [Drosophila a group of about 200 neurons, determined by the basic helix-loop-helix (bHLH) transcription factor Atonal (Ato) are the first to enter the lobe and have been proposed to act as pioneers [Manduca an additional group of central glia, termed sorting glia, migrate to the base of the antennal nerve where ingrowing axons sort according to glomerular targets [In vivo and culture studies have shown that these glia induce morphological changes in growth cones of the ORNs suggestive of alterations in adhesion and cytoskeletal dynamics [There are a number of studies that demonstrate interdependence between neurons and glia during development ,9. In th antenna . A set o antenna . Ablatiolization ,12. Herelization . In Drospioneers . In thei targets . In vivodynamics ,17. ThisDrosophila, which share similarities with the sorting zone glia in the moth. The cells which are labeled with GH146::GFP become associated with the antennal nerve when it reaches the brain. The cells migrate into the third segment of the antenna where they ensheath axons of the ORNs as they project from the antenna to the brain. Although evidence that these glia influence the development or the function of the ORNs is lacking, their possible function is discussed with respect to findings in other insects.In this paper, we demonstrate the presence of a set of cells in Mz317-Gal4 (Mz317::GFP) or GH146-Gal4 (GH146::GFP). Mz317::GFP is known to mark peripheral, perineural and cell body glia [We stained pupal antenna with antibodies against the transcription factor Reversed Polarity (Repo) which serves as a marker for differentiated glial cells. Repo-positive cells are first detected at about 16 hours (hrs) after pupa formation (APF) when they closely associate with developing sensory axons ,14. At 3ody glia  and labeody glia ; see Figody glia . This maAt 36 hr APF, all the glial processes tightly ensheath fascicles of sensory axons as they exit the antenna p13) with anti-Repo . Pupal dissections exposing both antenna and brain Fig.  were staeyeless (ey) promoter (ey-FLP) generates a high frequency of large clones covering at least half of the antennal tissue [While these observations support the idea that GH146-glia migrate into the third segment along the olfactory neurons, it is possible that cells already located within the antenna turn on the GH146 enhancer in a dynamic fashion. We exploited the MARCM method  to furthl tissue ,26. In aData presented in this section, leads us to suggest that the GH146-glial cells arise in the brain and migrate along the nascent olfactory axons to line the fascicles within the third antennal segment. These cells are however, only a small subset of the nerve associated cells and both GFP  peripheral glia that arises from the antenna and ensheath olfactory axons [Drosophila, previous work has described the direct equivalents of peripheral antennal and the neuropil glia [Studies in ry axons ; ii) lobry axons ,12; iii)ry axons ,17. In Dpil glia . In thisManduca, Fasciclin II (FasII), the insect ortholog of N-CAM, is expressed in a subset of ORNs which are scattered throughout the antennal nerve. Upon reaching the glial rich sorting zone, these projections segregate from the non-expressing axons and terminate in distinct glomeruli [Drosophila, we were unable to detect the presence of FasII protein on the ORNs during axon projection using the available antibodies (unpublished observations). When the GH146-glia were completely absent within the third antennal segment, we could not detect significant abnormalities in ORN fasciculation within the antenna. The processes of the GH146-glia extend into the antennal nerve to ensheath axon bundles. This segregation is not based on Or gene type and its functional significance is obscure. A similar migration of glial cells from the centre into the periphery has been described during development of the Drosophila compound eye [Although these cells share many of the properties of sorting zone glia described in the moth, we were unable to implicate them in any guidance function. In lomeruli . In Drosound eye .Drosophila FGF receptors do not affect glial cell number in the antenna (our unpublished data).Cell migration in insect glia is well known to be mediated by signaling through the Fibroblast Growth Factor Receptors (FGFRs) . Our preWe suggest that GH146-glial migration is mediated by EGF signaling . The ligDrosophila. The cells originate centrally and use the newly targeted olfactory axons to migrate to the third segment of the antenna. The signal for migration appears to involve EGF signaling. The data in this paper support the idea that in Drosophila there is no discrete sorting region equivalent either to the sorting zone in Manduca or to the inner nerve layer in the vertebrate olfactory bulb.We have identified a new population of glial cells in the antenna of Mz137-Gal4 was kindly provided by Kei Ito and GH146-Gal4 by Reinhard Stocker. These lines were used to drive UAS-GFP (1010T2) thus labeling peripheral glia. The ato strains ato1/TM3 and Df(3R)p13/TM3 were obtained from Andrew Jarman [N17, UAS-Cdc42v12, UAS-Cdc42N17, UAS-P35, UAS-p21, yw;P [ry+t7.2-neoFRT}19A, Tub-Gal80 P[ry+t7.2-neoFRT}19A and ey-FLP were obtained from the Bloomington Stock Centre at Indiana, USA. Or22a-Gal4, Or47b-Gal4 and Or83b-Gal4 lines were kindly provided by Leslie Vosshall [Vosshall .All flies were reared at 25°C on standard cornmeal media containing yeast. For staging, white prepupae  were collected and allowed to develop on moist filter paper. This stage lasts for an hour; hence the error in staging is 30 minutes. Ages of pupae grown at other temperatures were normalized with respect to growth at 25°C. Wild type pupae take about 100 hrs to eclose when grown at 25°C in our laboratory.Pupal tissues were dissected in phosphate-buffered saline (PBS) and treated as described in . The priAntibody staining of the late pupal stages cannot be carried out because of the hardening of the cuticle. To visualize GFP fluorescence, antennae were removed, mounted in 70% glycerol and scanned on a Radiance 2000 confocal microscope immediately.Tub-Gal80 FRT19A; GH146-Gal4 UAS-GFP/+; ey-FLP/+ and FRT19A/Tub-Gal80 FRT19A; Or83b-Gal4 UAS-GFP/+; ey-FLP/+ were dissected and antennae and brains were stained with antibodies against GFP. Preparations were examined by confocal microscopy for GFP positive cells.Clones were generated using the mosaic analysis with repressible cell marker (MARCM) method described in . Pupae oAKS carried out the characterization of the Mz317 and GH146 glia in the antenna and studied the effect of DER and small GTpases signaling. CS established the migration of glia by following expression in dissected preparations and by clonal analysis. DJ carried out the initial observations on the GH146. VR conceived and designed the study, participated in drafting the manuscript. All authors have read and approved the final manuscript.


We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay.An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics) was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit  for quantitative performance, analytical sensitivity and precision.The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method.As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method. The severe acute respiratory syndrome (SARS), etiologically related to a newly emerged coronavirus (SARS-CoV) , caused The analytical protocol that had been developed involved the use of a manual RNA extraction method. As the demand for diagnostic testing at times of infectious disease outbreaks would be high, strategies that may enhance the throughput of analytical procedures would be advantageous. In this study, we assessed the feasibility of automating the RNA extraction procedure of the serum SARS-CoV RNA test by evaluating the performance of an automated extraction system.The aim of the study was to compare the efficacy of SARS-CoV RNA extraction based on the use of the QIAamp viral RNA mini kit  with protocols adapted for the MagNA Pure LC instrument . The former protocol is a manual column-based method based on silica-adsorption. On the other hand, the MagNA Pure LC instrument extracts nucleic acids from biological specimens based on magnetic bead separation. The principle and general setup of the instrument had been previously described . The maiAccording to the manufacturer's information, 2 kits, namely the MagNA Pure LC total nucleic acid isolation kit (Roche Diagnostics) and the MagNA Pure total nucleic acid large volume isolation kit (Roche Diagnostics), are recommended for use with the MagNA Pure LC instrument for the extraction of viral DNA or RNA from serum or plasma. The main differences between the two kits lie in the starting sample volume and whether an external lysis protocol is available. The former kit processes 200 μL of serum and is compatible with the use of an external lysis protocol preinstalled in the accompanying software  of the MagNA Pure LC instrument. The latter kit, however, processes 1000 μL of serum and no external lysis protocol had been predefined. External lysis is a processing step whereby lysis buffer can be added to clinical specimens manually prior to the transfer of the sample and buffer mixture to the automated instrument for further downstream processing. External lysis is a desirable step for the processing of potentially infectious specimens whereby the specimens could be processed according to the recommended biosafety precautions until the pathogens are lysed and the specimen rendered safe for further processing by the MagNA Pure LC instrument.8 copies/mL of the virus. Viral RNA was extracted from aliquots of this mixture in triplicate both according to the standard as well as modified protocols for the large volume kit. For the standard protocol, laboratory personnel were only involved with the initial transfer of 1000 μL of each specimen to individual sample cartridges placed inside the instrument after which proteinase K and buffers, including 450 μL of lysis buffer, were added by the instrument in a sequential and automated fashion. For the modified protocol, 310 μL of lysis buffer was first added to 690 μL of specimen in a biosafety cabinet to make up a final volume of 1000 μL. After vortexing, the mixture was transferred in ice to the sample cartridges on the MagNA Pure LC instrument. The instrument was then activated to run as per the standard protocol. The externally-added lysis buffer (310 μL) when mixed with the volume of lysis buffer pre-loaded on the MagNA Pure LC instrument (450 μL) amounts to a total of 860 μL of lysis buffer for 690 μL of specimen and thus contributes to a lysis buffer:sample ratio which largely resembles that adopted in the external lysis protocol of the total nucleic acid isolation kit. All viral RNA preparations extracted in this study were quantified using a real time quantitative one-step RT-PCR assay targeting the Nucleocapsid-gene of the SARS-CoV as previously described [Tth RNA PCR reagent set  on an Applied Biosystems 7700 Sequence Detector. 12 μL of extracted viral RNA was used for amplification in a reaction volume of 25 μL.However, the sensitive detection of SARS-CoV RNA from serum may be dependent on a large starting volume of serum. Therefore, we evaluated an in-house modification of the manufacturer's protocol for the "large volume" kit with the addition of an external lysis step. To minimise the infectious risk to the laboratory personnel, all analyses were performed using aliquots of a SARS-CoV culture isolate that had been inactivated by procedures previously described . Inactiv6 copies/mL of inactivated SARS-CoV. Quadruplicate extractions were performed. We also assessed the analytical sensitivities of both protocols by comparing the detection rates for aliquots of transport medium diluted to contain 10, 102 and 103 copies/mL of inactivated SARS-CoV, respectively. Five extractions were performed for each concentration. We then addressed the possibility of carry-over contamination within the MagNA Pure LC instrument by introducing nine aliquots of transport medium containing inactivated SARS-CoV ranging from 102 to 107 copies/mL alternating with aliquots of plain transport medium on the instrument and using the modified large volume protocol for extraction.The modified large volume protocol with the external lysis step was further compared with the external lysis protocol of the total nucleic acid isolation kit using a transport medium mixture containing 103 to 109 copies/mL. The pooled sera were first confirmed to be negative for SARS-CoV by the quantitative RT-PCR assay. Viral RNA was extracted from the serial samples by both the automated and manual methods and SARS-CoV RNA concentrations were determined by the quantitative RT-PCR test [The modified large volume protocol was selected for further comparison with the performance of the column-based manual method. The manual extraction method was performed according to the manufacturer's instructions. Transport medium and pooled sera were mixed with serially diluted aliquots of inactivated SARS-CoV to produce samples containing SARS-CoV with concentrations ranging from 10PCR test . Viral R2 and 103 copies/mL of inactivated SARS-CoV were extracted by both methods. Ten replicate extractions were performed for each concentration. The detection rate at each concentration was compared amongst the two extraction methods. Lastly, we assessed the reproducibility of both protocols by performing replicate analyses (n = 10) of two pooled sera containing 103 and 105 copies/mL of inactivated SARS-CoV, respectively. The mean and coefficient of variation were determined and compared.We next compared the effects of the RNA extraction methods on the overall assay sensitivity. Pooled sera spiked with 10, 10Statistical analysis was performed using the MedCalc software version 8.0.8 copies/mL inactivated SARS-CoV by the standard protocol of the large volume kit yielded SARS-CoV RNA concentrations of 3.11 × 108, 3.14 × 108 and 3.17 × 108 copies/mL. By incorporating an external lysis procedural step, the modified protocol yielded 4.92 × 108, 5.20 × 108 and 5.48 × 108 copies/mL. As the modified large volume protocol yielded results comparable to that of the standard large volume protocol, the former was used for further comparison with the external lysis protocol of the "total nucleic acid" kit which uses a starting sample volume of 200 μL. Quadruplicate analysis of the transport medium mixture containing 106 copies/mL SARS-CoV yielded 6.57 × 106, 7.41 × 106, 1.34 × 107 and 6.17 × 107 copies/mL when the large volume kit was used. When the total nucleic acid kit was used, the results were 8.20 × 106, 8.40 × 106, 1.05 × 107 and 6.80 × 107 copies/mL.Triplicate viral RNA extractions of the transport medium mixture containing 103 copies/mL SARS-CoV. However, when the sample contained 102 copies/mL SARS-CoV, the large volume kit yielded positive detection in all five replicates, while SARS-CoV was only detected from three replicates when extracted by the total nucleic acid kit. Furthermore, when the sample contained 10 copies/mL SARS-CoV, the large volume kit yielded positive detection in four replicates, while viral RNA extractions from the total nucleic acid kit was only positive in one replicate. These data suggest that the modified protocol of the large volume kit has comparable performance with the total nucleic acid kit for the extraction of samples containing high SARS-CoV concentration, but enabled more sensitive detection when samples containing low levels of SARS-CoV were extracted. By aligning samples positive and negative for SARS-CoV in an alternating manner for extraction by the modified protocol of the large volume kit, there was no evidence of carry-over contamination. All the negative samples were indeed tested negative regardless of the magnitude of the SARS-CoV concentrations (ranging from 102 to 107 copies/mL) in the adjacent wells. Thus, the modified protocol of the large volume kit was selected for further evaluation for SARS-CoV RNA extraction from serum.The analytical sensitivities contributed by the two kits were next compared. Viral RNA extracted by both kits was detectable in all five replicates when the sample contained 10Serially diluted inactivated SARS-CoV isolate in transport medium was extracted by both the column-based manual method and the MagNA Pure LC instrument using the modified large volume protocol with external lysis. The SARS-CoV concentrations from both series of viral RNA extracts were compared using the Passing-Bablok regression method . The PasA similar comparison was performed for serially diluted SARS-CoV mixture in pooled sera. Figure 3 copies/mL SARS-CoV were extracted by either methods. For serum containing 102 copies/mL SARS-CoV, nine and seven of the replicates were tested positive when viral RNA was extracted by the manual and automated methods, respectively. For serum containing 10 copies/mL SARS-CoV, four of the replicates were positive when extracted by the manual method and only one was positive when the replicates were extracted by the automated method.The effect of the automated and manual methods on the overall assay sensitivity was next compared. Results for this part of the study are summarized in Table 3 and 105 copies/mL. Results for this part of the study are summarized in Table 3 copies/mL, the overall assay coefficient of variation (standard deviation/mean × 100%) was 45.3% and 76.9% when RNA extractions from the manual and automated methods were quantified, respectively. For serum SARS-CoV concentration at 105 copies/mL, the assay coefficient of variation was 47.4% for the manual RNA extraction and 51.0% when automated RNA extraction was used.To assess the effects of the two extraction protocols on the precision or reproducibility of the quantitative SARS-CoV RT-PCR assay, RNA extractions by each protocol were repeated 10 times for serum aliquots containing SARS-CoV concentration well above the detection limit of the assay, namely 10In an attempt to increase the throughput of a previously developed quantitative serum SARS-CoV RNA RT-PCR assay ,5, we evThe automated extraction method involving the use of the large volume kit with the external lysis procedure was further compared with the quantitative performance of a previously described manual viral RNA extraction method based on the use of the QIAamp viral RNA mini kit (Qiagen). Viral RNA extracted from the automated method led to SARS-CoV concentrations that were consistently lower than that extracted by the manual method across a wide range of SARS-CoV concentrations in both transport medium and serum. Furthermore, better detection rates were observed for serum containing low concentrations of SARS-CoV when extracted by the manual method in comparison with the automated method. The manual method also contributed to better overall analytical precision as evident by the lower coefficients of variation.We have developed a modified protocol based on the use of the MagNA Pure LC large volume kit (Roche Diagnostics) which is more sensitive than the predefined external lysis protocol of the MagNA Pure LC total nucleic acid kit (Roche Diagnostics). Albeit the convenience and potential improvement in throughput offered by an automated protocol, our evaluation revealed that the automated viral RNA extraction protocol is less sensitive, less precise and produced quantitative results that were consistently lower than those of the column-based manual extraction method. Though the reasons for the observed differences in kit performance is not known at present, we recommend the use of the column-based manual RNA extraction method as the diagnostic performance of the serum SARS-CoV RNA quantitative RT-PCR test ,5 is criSARS, severe acute respiratory syndrome; SARS-CoV, SARS-coronavirus, RT-PCR, reverse transcriptase polymerase chain reaction.Patent applications covering aspects of the serum SARS-CoV RNA quantitative RT-PCR test have been filed by The Chinese University of Hong Kong.RWKC and YMDL designed the study. RWKC interpreted the data and drafted the manuscript. YJ, GTYC and WBL performed the molecular and data analyses. WL provided the inactivated viral material. ATCC provided the expertise for the use of the MagNA Pure LC instrument. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: