N-ethylmaleimide-sensitive fusion protein attachment receptors (SNAREs), vesicle associated membrane protein/synaptobrevin, synaptosome associated protein of 25 kDa, or syntaxin. As these substrate proteins are core components of the vesicular membrane fusion apparatus, cleavage of any of the substrate molecules results in the blockade of neurotransmitter release. This review summarizes the present knowledge about the molecular basis of the specific substrate recognition and cleavage mechanism and assesses the feasibility of reengineering catalytic domains to hydrolyze non-substrate members of the three SNARE families in order to expand the therapeutic application of botulinum neurotoxins.The clostridial neurotoxin family consists of tetanus neurotoxin and seven distinct botulinum neurotoxins which cause the diseases tetanus and botulism. The extreme potency of these toxins primarily relies not only on their ability to specifically enter motoneurons but also on the activity their catalytic domains display inside presynaptic motoneuronal terminals. Subsequent to neurotoxin binding and endocytosis the catalytic domains become translocated across endosomal membranes and proteolyze unique peptide bonds of one of three soluble  N-terminal ~50 kDa light chain (LC) and a C-terminal ~100 kDa heavy chain (HC). Both chains remain attached via a single disulfide bond formed between cysteines at the C-terminus of the LC and the N-terminus of the HC, a peptide loop formed by the N-terminal ~55 amino acids of HC that wraps around the LC and further non-covalent interactions , a family of closely related bacterial protein toxins, consist of seven antigenically distinguishable botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT). Each CNT is synthesized as a ~150 kDa single chain protein, but subsequently cleaved by specific clostridial or host proteases. Cleavage results in an ractions . The HC main HCC .via the recycling pathway of synaptic vesicles (SV), they first bind to complex gangliosides at the surface of nonmyelinated nerve terminals [CC as demonstrated for TeNT and several BoNTs. In contrast, the role of the HCN‑domain is still unresolved, although low affinity binding to lipidic membrane components was recently reported for BoNT/A HCN [N and the formation of a cation selective channel. This is probably coupled to partial unfolding of the LC, its entry into and transit through the HN channel followed by its refolding in the cytosol [versus TeNT. BoNTs act inside the motoneuron and thus cause flaccid paralysis, which can result in respiratory failure and death. TeNT poisons inhibitory interneurons to cause just opposite symptoms, spastic paralysis [Poisoning by BoNTs mainly occurs upon oral ingestion and toxin uptake at intestinal epithelial cells, whereas TeNT is produced and released into the circulation by bacteria that germinate in infected tissue lesions. Eventually CNTs reach cholinergic motor nerve terminals, where a four step process is initiated that ultimately renders a synapse inoperative. In order that they can be taken up erminals . Requisierminals ,3,4,5 anerminals ,7,8, anderminals ,10. ProtNT/A HCN . Intrace cytosol ,13. Subsaralysis .CNTs are regarded as the most hazardous natural substances known. The 50% lethal doses for susceptible mammals including man amount to approximately one nanogram per kg of body weight . Due to 2+ binding motif, His-Glu-X-X-His, in the primary sequence of TeNT [via the demonstration of zinc binding for BoNTs [C-terminal transmembrane domain . A common feature of all SNAREs is the presence of one (or as an exception two) α-helical domain(s) of ~70 amino acids, the SNARE motif, endowed with the capacity to form coiled-coils. Sets of SNAREs generally build the core for the individual intracellular membrane fusion processes and zippering of such sets of SNAREs via their SNARE motifs into parallel four-helix coiled-coil bundles is regarded to provide the driving force for membrane fusion [The CNT substrate proteins are members of larger protein families that are collectively termed SNAREs . Suscep2+-coordination classifies the LCs of CNTs into the gluzincin superfamily among the clan MA of metalloproteases [2+ is coordinated by two histidine residues of the His-Glu-X-X-His motif, a water molecule, which is bonded to the glutamate residue of the motif, and another glutamic acid residue located about 35 residues downstream in the primary structure. Searches for proteins exhibiting a related tertiary structure revealed that the central portion is structurally similar to a portion of the zinc metalloprotease thermolysin [via side-chain side-chain interactions. Substrate specificity is thus imparted by distinct amino- and carboxyl-terminal structural elements flanking the core, which appear to be structurally unique to the CNTs [The mode of Znroteases ,47. As armolysin . These sthe CNTs , as theythe CNTs ).via its interaction with Zn2+ and the glutamic acid carboxylate group of the His-Glu-X-X-His-motif and can thereby nucleophilically attack the carbonyl carbon of the scissile peptide bond to form an oxyanion. Simultaneously, a proton abstracted from the attacking water is shuttled via the carboxyl group of the glutamate to the scissile peptide bond nitrogen, and the glutamate may then stabilize the tetrahedral intermediate by forming a salt bridge with the positively charged amide nitrogen. The negative charge that develops on the carbonyl oxygen atom in the tetrahedral transition state is stabilized by hydrogen bonding interactions with a protonated histidine (position 231) and the hydroxyl group of a tyrosine (position 157). It is assumed that His-231 is retained in proper position and protonated state through a hydrogen bonding interaction with an aspartate (position 226). The protonated amide nitrogen then facilitates C-N-bond disruption and may subsequently receive a second proton derived from the water, possibly again mediated by the glutamic acid carboxylate group [As thermolysin represents the well characterized prototypical member of the gluzincin metalloprotease superfamily, its mode of peptide bond cleavage is presumably to a large extent similar to that of CNTs. In case of thermolysin, catalysis follows a general base-type mechanism . It was te group ,52.The available data on CNTs support a similar mechanism . In facti.e., a glutamate, an arginine, and a tyrosine  lines up in close proximity to the scissile peptide bond ,56 and maffected ,59. Thisaffected  and was affected . Substitaffected ,61. The affected .cat, while KM remained unaffected [vs. wild-type LCA as well as the guanidino group positioning in the vicinity of the scissile peptide bond led to the suggestion that this residue might also be involved in oxyanion binding in the transition state [The counterpart of the thermolysin His-231 might be the above mentioned strictly conserved arginine . Its replacement in BoNT/A by various residues and by alanine in BoNT/E generally led to an approx. 100-fold decrease in kaffected ,59,61. Ton state ,60. Alteon state ,56.2+ coordinating histidines at the active site [CNTs presumably also possess an analog to Asp-226 of thermolysin. A glutamate, the third of the above mentioned strictly conserved residues in the vicinity of the zinc coordinating residues, occupies a similar position in all CNTs . Removalive site ,61.versus CNTs  of SNAP-25 . The co-ive site . A compaive site . These dThe co-crystal structure of LCA bound to SNAP-25 does not allow for the definition of the LC pockets involved in the binding of substrate residues near the scissile bond, as a double mutant of the active site was used to render the enzyme inactive . A numbeN-terminal direction and forms major interactions with LCA (Structure data about the mode of VAMP/synaptobrevin-2 recognition by LCF have recently also been achieved . In thisSuperposition of the substrate-free LCF structure onto the LCF-VAMP/synaptobrevin-2 structure led to the detection of major side chain rotamer variation around the active site. These facilitate proper substrate binding by opening the active site and allow for salt bridges with the P3 and P2 residues . The steTwenty years ago BoNTs became an approved therapeutic for the treatment of the neurological disorders strabismus, blepharospasm, and hemifacial spasm. Since then their application range steadily increased and includes today further medical conditions like axillary hyperhydrosis or even cosmetic use. However, the clinical application of BoNTs is restricted to disorders that rely on hyperactivity of cholinergic neurons, due to the toxins’ tropism for these neurons and the preferential cleavage of neuronal SNAREs.C. For example, the Erythrina cristagalli lectin, selectively recognizing galactose containing carbohydrates as present on nociceptive afferents, was chemically conjugated to purified L-HN of BoNT/A. This conjugate was shown to affect nociceptive neurons, which opens up the possibility to develop retargeted BoNTs for the treatment of chronic pain [N of BoNT/C and the epidermal growth factor was designed. This construct was reported to inhibit mucin secretion from the human respiratory epithelial cell line A549 and might thus have the potential to treat mucus hypersecretion in asthma and chronic obstructive pulmonary disease [These limitations can now probably be bypassed, owing to the elucidation of the molecular structure of BoNTs and the increasing information about structure-function relationships. Consequently, attempts were undertaken to retarget BoNT activity to specific neurons or non-neuronal cells by replacing their cell binding subunit, Hnic pain ,83. Furt disease . The latIn vitro cleavage assays showed that the mutated LCE exhibited a hydrolysis rate in the range of SNAP-25 cleavage by wild-type LCE versus the otherwise non-cleavable human SNAP-23; KM/kcat was nine-fold reduced [Such a re-targeting has recently been achieved for LCE. Its enzymatic activity could be extended to human SNAP-23 by a single amino acid replacement . Within  reduced . Further reduced .versus other BoNTs and the existing practice of its handling. It appears feasible that LCA could also be re-targeted to human SNAP-23. Promising starting points are an enlargement of or/and introduction of a negatively charged side group into the S3’ pocket to accommodate the SNAP-23 P3’ Lys206, or to adapt the hydrophobic S5’ pocket for the exchange of SNAP-25 Met202 by Leu208 in SNAP-23 (BoNT/A is currently the most attractive CNT serotype in terms of medical application, due to its pronounced longevity The non-cleavable TI-VAMP ; also ca. Enlargeversus the original substrate, in case that the original and the new substrate are co-expressed in the desired target cell. Therefore, in addition to mutations which enable cleavage of the new substrate, LC amino acids must be identified and replaced whose mutations exclusively impair cleavage of the original substrate.If LC re-targeting succeeded one may wish that the re-targeted LC loses its activity


Glioma is the most common and fatal primary brain tumour with poor prognosis; however, the functional roles of miRNAs in glioma malignant progression are insufficiently understood. Here, we used an integrated approach to identify miRNA functional targets during glioma malignant progression by combining the paired expression profiles of miRNAs and mRNAs across 160 Chinese glioma patients, and further constructed the functional miRNA–mRNA regulatory network. As a result, most tumour-suppressive miRNAs in glioma progression were newly discovered, whose functions were widely involved in gliomagenesis. Moreover, three miRNA signatures, with different combinations of hub miRNAs (regulations≥30) were constructed, which could independently predict the survival of patients with all gliomas, high-grade glioma and glioblastoma. Our network-based method increased the ability to identify the prognostic biomarkers, when compared with the traditional method and random conditions. Hsa-miR-524-5p and hsa-miR-628-5p, shared by these three signatures, acted as protective factors and their expression decreased gradually during glioma progression. Functional analysis of these miRNA signatures highlighted their critical roles in cell cycle and cell proliferation in glioblastoma malignant progression, especially hsa-miR-524-5p and hsa-miR-628-5p exhibited dominant regulatory activities. Therefore, network-based biomarkers are expected to be more effective and provide deep insights into the molecular mechanism of glioma malignant progression. Glioma is the most common and malignant brain cancer, and ∼10 000 new cases of high-grade or malignant glioma occur each year . It is aet al.  are small non-coding RNAs that regulate gene expression post-transcriptionally and play important roles in regulating diverse biological processes . During et al.  identifi  associated with glioma malignant progression. Here, we expected that a gene is regulated by a miRNA, only if the miRNA has predicted binding sites in this gene and that their expressions are negatively correlated. Then, we uncovered that several key miRNAs regulating most targets in the FMRN not only play important roles in glioma malignant progression, but also can independently predict the survival of patients with different glioma grades. The integrative analyses provided novel insights into regulatory mechanisms at the miRNA level during the progression of glioma, and both the method and predictions generated here could serve as important resources for future experimental dissection of miRNA functions in glioma.Supplementary Table S1 and Supplementary materials. All these samples were histologically graded according to current WHO classification of tumours of the nervous systems, including 63 WHO grade II patients, 33 grade III patients and 64 GBM patients. Written informed consent was obtained from all donors. Clinical investigations were conducted after approval by the local research ethics committee and in accordance with the ethical principles.A retrospective series of 160 glioma patients received from Beijing Tiantan Hospital was considered, and the extended demographics are provided in Supplementary materials). All data were deposited in Chinese Glioma Genome Atlas (CGGA), a publicly available database that focuses on glioma. The expression values for each gene were background-subtracted, quantile-normalized and log2-transformed, whereas the miRNA expressions were also log-transformed. The miRNA (or gene) probes were filtered to remove non-specific probes and probes lacking association with miRBase entry IDs (or Entrez Gene IDs) (Matched genome-wide miRNA and mRNA expression profiling was successfully obtained from these 160 glioma samples by using human v2.0 miRNA expression BeadChip and Agilent Whole Human Genome Array (see ene IDs) , and, inSupplementary materials). Here, we built on previous results and particularly on the conclusion common to multiple studies that had used the combination of the predictions from these algorithms rather than the intersection, which provides a more promising strategy to select true targets  was controlled by Benjamini and Hochberg algorithm   associated with the malignant progression of glioma by respectively comparing the miRNA or gene expressions in grade III or grade IV gliomas with those in grade II gliomas. An miRNA or gene was defined to be associated with the malignant progression of glioma, only if it showed differential expression in both conditions. Differential expression was detected by unpaired Student’s esholds) .Supplementary Figure S1A). Specifically, the functional regulations were detected using the subset of miRNAs and genes characterized by (i) both miRNAs and genes associated with glioma malignant progression, (ii) a regulatory relationship according to computational predictions, and (iii) expression profiles strongly anti-correlated. Indeed, as miRNAs tend to down-regulate target mRNAs, the expression profiles of genuinely interacting pairs were expected to be anti-correlated. Thus, the Pearson correlation coefficients were calculated for each predicted miRNA–mRNA pair, whose threshold was set to <−0.4 and corresponding P value was set to <0.05. Finally, after assembling all functional miRNA–target gene pairs, we generated the FMRN, which is associated with glioma malignant progression. This network is a bipartite graph containing two sets of vertices (nodes) corresponding to miRNAs and target genes. A directed edge (connection) from a miRNA to one of its targets exists if their relationship is functionally regulated. To facilitate analysing the topological structure of the FMRN and identifying miRNAs playing central roles in the FMRN by the well-used measure—degree, the weights of all edges were set to 1.To identify the functional regulations from miRNAs to mRNAs, we combined both the computational target predictions at the sequence level and the inverse expression relationships between miRNAs and mRNAs in the context of glioma progression , a mathematical formula for survival prediction was constructed, taking into account both the strength and positive or negative association of each miRNA with survival. More specifically, we assigned a risk score to each patient according to a linear combination of the miRNA-expression values weighted by the regression coefficients from the aforementioned univariate Cox regression analysis. The risk score for each patient was calculated as follows:i in the training set, and n is the number of miRNAs that are significantly associated with survival. All patients in the training data set were thus dichotomized into high-risk and low-risk groups using the median risk score as the cut-off point. Patients having higher risk scores were expected to have poor survival outcomes. The coefficient and threshold values derived from three training sets were directly applied to miRNA expression data of the corresponding test sets to divide the patients in the test sets into high-risk and low-risk groups. The Kaplan–Meier method was further used to estimate the overall survival time for the two subgroups. The differences in the survival times were analysed using the log rank test. The workflow was shown in Supplementary Figure S1B.We aimed to identify three clinically significant prognostic miRNA signatures from three training sets and tested them using the corresponding internal validation sets. The miRNAs playing central roles in the FMRN, miRNA hubs, were considered as the candidate members of the signatures. The hubs were commonly defined as the top 15% of the nodes by degree , in thisP values of log-rank tests were respectively calculated in the training and test data sets, and the negative log-transformed P values were used as evaluation indexes. The higher these two scores, the better was the performance of a miRNA signature. We repeated each procedure 1000 times, and the significance was defined as the proportion of times in which in random conditions, the P values for training and test sets were both lower than the real ones.To evaluate whether our integrated approach in identifying the survival-associated miRNA signatures is better than the traditional approach only based on miRNA expression, we firstly ranked miRNAs based on the FDR in an ascending order, and chose the top 21 miRNAs with the most differential expression as candidate miRNAs to evaluate their prognostic performance in the training and test samples. Furthermore, to determine the significance of each miRNA signature, we performed three different randomization tests by randomly choosing miRNAs from three different backgrounds: non-hub miRNAs in the FMRN, miRNAs differentially expressed in grade III and IV, when compared with grade II glioma, and miRNAs assayed in microarray. The first type of randomization considered miRNA topological feature in the FMRN. For each kind of randomization test, we randomly selected 21 miRNA candidates from the background at a time, the miRNA signature was detected and its effectiveness for clinical outcome prediction in the training and test data set were computed based on the aforementioned risk scoring system. The corresponding N genes, of which K were involved in the function category under investigation, and the set of interesting target genes for analysis had a total of M genes, of which x were involved in the same function category, then the P value can be calculated to evaluate the enrichment significance for that function category as follows:To explore the functional roles of miRNAs in two disparate subnetworks of the FMRN as well as the identified miRNA prognostic signatures in high-grade glioma and GBM, we respectively used their regulatory gene subsets to perform functional enrichment. This process was realized by the tool WebGestalt , using tP values were adjusted by Benjamini and Hochberg method and finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (or GO terms) with adjusted P values < 0.01 and including at least two interesting genes were considered.Then, the enriched significance Supplementary Table S2). Most of these regulations connected together and formed two big subnetworks , showing over-expression during glioma progression. In contrast, the left subnetwork of the FMRN includes relatively more miRNAs that were not found to be involved in glioma, which might be considered as novel candidate disease ones. Their dysregulation directions during progression imply their suppressive progression potential. However, their functions in glioma are still largely unknown, let alone glioma progression. Fortunately, enrichment analysis of the target genes of these miRNAs in the FMRN could give us a global clue of their functional roles in the progression of glioma. Functional enrichment analysis was respectively carried out for the target genes of miRNAs in each subnetwork of the FMRN. As a result, miRNAs in two subnetworks significantly participated in several biological pathways directly relevant to gliomas and neurodevelopment . Calcium signal is the most common way of transmitting signals from cell environment to the cytoplasmic calcium binding effectors, and has broad effect on cell behavior. Being a crucial player in neuronal transmission, increasing studies have shown that it is important for glia physiology  or a test set (n = 80), we found that the clinical characteristics had no significant difference between these two sets (Supplementary Table S3). By using the miRNA risk scoring system as described in the method section, patients in the training set were further divided into high- and low-risk groups based on their risk scores. Consequently, patients with high-risk scores had shorter median survival than those with low-risk scores (P = 1.03e-8); the median survival time of high-risk group was 13.78 months and that of low-risk group was 29.25 months. Next, we validated the miRNA signature in the test set where samples were also classified into the high-risk group or low-risk group using the same cut-off point as in the training set. Similarly, patients with high-risk score also had shorter median overall survival than patients with low-risk score (P = 6.43e-7). To further ascertain whether the risk signature comprising of hub miRNAs is an independent predictor of glioma patients’ survival, the prognostic association between our newly identified hub miRNA signature and other known clinical and pathologic risk factors for glioma progression was assessed by univariate and multivariate analyses. As expected, in addition to patient age and IDH1 mutation status, which are already well-known risk factors, the 21-hub miRNA signature was a significant risk factor for survival in univariate analysis (Supplementary Table S4). Multivariate analysis further revealed that the miRNA signature remained an independent prognostic risk factor for glioma patient survival , although isocitrate dehydrogenase (IDH) mutation status had weak significance (P = 0.063).It is known that hubs (nodes mainly making links in a network) are a central part of network, important to the stability of the biological system. Likewise, miRNAs regulating more targets in the FMRN might play more important roles in glioma malignant progression and influence more critical functions. Thus, it is interesting to study the efficiency of hub miRNAs as prognostic signatures. Hubs are commonly defined as the top 15% of the nodes by degree ,26,28, ctwo sets . Next, wSupplementary Table S5). Then, a set of five miRNAs  was identified to be correlated with patient survival in the training set (see details in Supplementary Table S6). Four of these five miRNAs were protective factors and the other one acted as a risky factor (Supplementary Table S6). Moreover, patients in high-risk group had a significantly shorter median survival than those with low-risk score in both training and test sets (P = 7.36e-6), IDH1 mutation status was also associated with the survival of high-grade glioma patients in univariate analysis (P = 0.026). However, in multivariate analysis, only the signature comprising of five hub miRNAs was detected as an independent predictor . These results suggest that these five hub miRNAs can effectively stratify high-grade glioma patients into different risk groups, and may play important roles in high-grade glioma.Next, we further explored which hub miRNAs can be effectively used as a prognosis signature for high-grade gliomas (III and IV). Likewise, high-grade glioma patients were divided into training and test subsets, and there were no significant difference in clinical characteristics (est sets A and B. Supplementary Table S8), a five-miRNA signature was identified in the training set by univariate Cox regression analysis. Of these five miRNAs, hsa-miR-628-5p and hsa-miR-524-5p were found to be protective miRNAs and other three  were noted to act as risky factors (Supplementary Table S9). Then, the samples in training set were segregated into high- and low-risk groups, and these two groups exhibited significantly different survival times (P = 0.0056); the median survival time of high-risk group was 9.68 months and that of low-risk group was 17.77 months. Moreover, the potential of the miRNA signature was independently validated in the test set (P = 0.008). In addition, in univariate Cox regression analysis, only the miRNA signature could independently predict the survival of GBM (P = 3.02e-7), and clinical (age and sex) and genetic variables (IDH1 mutation status) were not identified as risky factors (Supplementary Table S10). Furthermore, we also used receiver operating characteristics (ROC) analysis to compare the sensitivity and specificity for the prediction of survival by the miRNA signature, age, sex and IDH mutation status. As a result, the five-miRNA signature also showed better prediction of survival than age, sex and IDH1 mutation status with regard to overall survival in the combined GBM patients .To reconfirm our miRNA signature as an independent predictor, we used the independent cohort of The Cancer Gene Atlas (TCGA), which provides the miRNA expression levels in a total of 371 GBMs. In the miRNA expression profiles obtained from TCGA, three of five miRNAs were assayed . To evaluate the performance of our miRNA signature, we first performed Z-score transformation on expression levels for each of the three miRNAs and then summarized the Z-scores of these three miRNAs as the risk score for each patient. Using the median value of these scores as the threshold, the GBM patients were divided into a high-risk group and a low-risk group. Likewise, Kaplan–Meier analysis indicated that our miRNA signature significantly classified the patients into a high-risk group and a low-risk group . In addition, one miRNA (hsa-miR-384) was detected in the training set of GBM; however, it could not stratify GBMs either in the training or test data sets . We took these results as evidence that the network-based nature of the signature detection leads to the hub miRNA signatures with higher performance than non network-based methods.Moreover, three comparisons were additionally performed to evaluate the prognostic performance of the miRNA signatures identified based on the FMRN, which were realized by three different randomization tests by randomly choosing miRNAs from three different backgrounds: non-hub miRNAs in the FMRN, all miRNAs differentially expressed in grade III and IV compared with grade II glioma and miRNAs assayed in microarray. As shown in In the above-mentioned section, a five-hub miRNA signature was found to be an independent predictor of high-grade glioma patient’s survival, in which four miRNAs  were protective factors and the other one (hsa-miR-15a) acted as a risky factor. We further found that the expressions of four protective miRNAs were down-regulated not only in high-grade glioma and GBM compared with that in grade II glioma, but also in the progression from grade III glioma to GBM A, indicaSupplementary Table S11). Meanwhile, hsa-miR-15a was significantly negatively associated with other four protective miRNAs (Supplementary Table S11). These results indicated that these miRNAs might have potential functional association. Functional enrichment analysis was also performed for target genes regulated by these five miRNAs, which showed that these genes were significantly enriched in many critical biological pathways, such as ‘cell cycle’, ‘extracellular matrixc (ECM)–receptor interaction’, ‘focal adhesion’ and ‘P53 signalling pathway,’ etc. , Top2a was regulated by hsa-miR-139-5p and hsa-miR-628-5p, and several other important cell cycle-related genes were regulated by hsa-miR-524-5p, including Wee1, Cdk2 etc. Ttk has been demonstrated to play important roles in cell cycle and cell proliferation. It has been found to be a critical mitotic checkpoint protein for accurate segregation of chromosomes during mitosis. Tumourigenesis may occur when this protein fails to degrade and produces excess centrosomes resulting in aberrant mitotic spindles. Cdk2 also plays important roles in cell cycle to regulate progression from G1 into S phase , suggesting their oncogenesis roles in the progression of gliomas. Another important function, ECM–receptor interaction, is thought to affect cell migration and also play critical roles in the progression of cancers. Six genes were enriched in this pathway, mainly including several collagen genes , 83.33% of which were targeted by hsa-miR-628-5p and hsa-miR-524-5p. During cancer progression, tumour cells eventually invade the surrounding collagen-rich extracellular matrix. Collagen is a major component of the interstitial extracellular matrix, and previous reports showed that some were dysregulated in various types of cancers. Col4a1 has been shown to be over-expressed in glioma  were found to act as risky ones. As described earlier, hsa-miR-524-5p and hsa-miR-628-5p were the protective members of the signature of high-grade glioma. Here, we further found that these two miRNAs have the same roles in GBM, especially, the average expression of hsa-miR-524-5p in GBMs reduced to 8.40% of the expression level of type II samples. We further found that the expressions of the other three miRNAs were also gradually down-regulated following the progression of glioma A, and thR = 0.41, P = 8.84e-4), meanwhile the other three risky miRNAs also exhibited expression correlations (Supplementary Table S12). Functional enrichment analysis was performed for target genes regulated by these five miRNAs, which also revealed that a number of these targets were implicated in pathways that have important roles in direct tumour growth and metastasis , indicating their crucial roles in the progression of gliomas, such as hsa-miR-524-5p and hsa-miR-628-5p. Indeed, we found that the signatures identified in high-grade glioma and GBM are both involved in many tumour-related functions. More importantly, a striking proportion of target genes were implicated in cell cycle, and tended to be regulated by hsa-miR-524-5p and hsa-miR-628-5p. Typically, Ttk, Cdk2 and Wee1, the critical members of cell cycle, were not only regulated by the miRNA signatures, but also relatively strictly regulated by miRNAs in the FMRN. Moreover, these target genes were all repressed by hsa-miR-524-5p in microarray analysis. In addition, their expressions were dominantly reduced after transfection of hsa-miR-524-5p. Here, we also found that hsa-miR-524-5p has multiple co-regulations with other members within the signatures, forming connected subnetworks. It has been noted that hsa-miR-524-5p is a brain-specific miRNA, and is associated with the pathological grade and overall survival of gliomas. These results provided models of the molecular mechanisms underlying complex diseases, and can broaden our understanding about the molecular regulatory mechanism of hsa-miR-524-5p. In addition, hsa-miR-628-5p and hsa-miR-15a are the other two examples related to glioma progression, which are worthy to be further studied. Thus, the miRNA signatures identified in our study could assist in the exploration of the functional mechanism behind the prognostic value of miRNAs in glioma, and might generate potential molecular targets for development of anticancer therapy.The FMRN was constructed by considering the differential expression of miRNAs and genes and active miRNA–target relationships in the progression of glioma. Active miRNA–target relationships are needed to characterize the functions of miRNAs in a specific context of disease, which can help in identifying the dysfunctional miRNAs associated with glioma progression. The miRNA signatures identified in our study not only have prognostic value in glioma, but also regulate more genes in the FMRN, indicating their important functional roles in tumourigenesis and progression. We found that some miRNAs were all identified in three miRNA signatures . In addition, in the process of identification of hub miRNAs, we also tried to consider the regulatory strength between a miRNA and its targets, and used the summation of the absolute correlation coefficients of expression levels as an alternative measure to weigh the central roles of miRNAs in the FMRN. The top 15% miRNAs with highest summary scores were the same as hub miRNAs based on degree in the text. Thus, our network-based method was robust in the identification of the miRNA prognostic signatures. In addition, although the IDH1 gene has been discovered as a predictor of glioma survival in previous studies , suggesting the underlying genetic heterogeneity among different populations. Fortunately, a five-miRNA signature was effective to predict the survival in the cohort of Chinese GBM patients. Moreover, the miRNA signature still showed a better prediction of survival in the non-Asian patients. However, additional independent samples will be needed to further confirm our findings before the miRNA signatures identified in the present can be used in clinic globally.Prognosis assessment is crucial for making appropriate treatment choices. In the present study, hub miRNAs were found to act as independent risk predictors to effectively distinguish the glioma patients with distinct survival, even in high-grade glioma and GBM. Their good performances were assessed in both training and test samples. In addition, when compared with the traditional method based on differentially expressed miRNAs to identify the prognostic signatures, our network-based method exhibited better performance. To investigate the influence of hub miRNA candidates on the performance of our proposed method, another two commonly used thresholds to define hubs (top 10 and 20%) were considered. As a result, we found that the network-based method also exhibited better performances than the traditional method . This result indicated that a large proportion of miRNAs involved in glioma progression might also play important roles in GBMs. Next, we particularly analysed the three signatures identified in our study. First, for the 21 hub miRNAs composed of the prognosis signature for glioma, 66.67% were reported to be expression changes in GBMs. Here, our analysis suggested that they are also associated with the progression of glioma, extending our understanding about their functions in glioma. Second, all the five hub members of the miRNA signature for high-grade glioma were reported in previous studies; especially, hsa-miR-544 has been identified to exhibit a progression-associated down-regulation in glioma tumours, whose expression decreased significantly in anaplastic gliomas or GBM, when compared with low-grade gliomas. Another example was hsa-miR-139-5p, exhibiting consistently down-regulated in GBM, which was found to be involved in the mesenchymal mode of migration and invasion, demonstrating the importance of miRNAs in the context of the cellular niche. Finally, for the five hub miRNAs of the GBM signature, hsa-miR-524-5p and hsa-miR-628-5p were previously reported. In addition, when compared with the results reported by Mark D. Johnson and colleagues (Many efforts have been made to explore the molecular mechanism of GBMs, such as the REMBRANDT  and TCGAlleagues , we founet al. (Although there are a couple of miRNAs that were previously reported to play important roles in GBMs, we found that there are also several miRNAs not differentially expressed in the progression of glioma. This result might suggest that they were not involved in the malignant progression of glioma, or be explained by the differential experiment designs and the underlying genetic heterogeneity among different populations. For example, 4 out of 10 miRNA signature detected in GBMs by Srinivasan et al.  were difet al.  and hsa-et al. .P value of survival analysis, in-degree is significantly positively associated with the power for survival analysis. These results suggest that the in-degree may be a good indicator for identifying the genes involved in the progression of gliomas, extending the application of the integrative method.In this study, we focused on the identification of the miRNA signatures associated with the glioma progression by considering their topological central roles in the FMRN. Similarly, ‘in-degree’ for genes also could measure their central roles in the FMRN. By computing the correlation between in-degree of gene in the FMRN and the negative log-transformed In summary, by integrative analyses, we not only identified the miRNA predictors of survival, but also analysed the functional roles of miRNAs in the context of glioma progression. In conclusion, systematic analysis of the FMRN provides new insights into post-transcriptional gene regulation in the progression of glioma.Supplementary Data are available at NAR Online, including [55–57].Graduate Students of Heilongjiang province [YJSCX2012-196HLJ]. Funding for open access charge: Funds for Creative Research Groups of The National Natural Science Foundation of China [81121003]; National Natural Science Foundation of China ; Specialized Research Fund for the Doctoral Program of Higher Education of China [20102307110022]; China Postdoctoral Science Foundation [2012M520764]; Heilongjiang Postdoctoral Fund [LBH-Z12214]; The Innovation Research Fund for Graduate Students of Heilongjiang province [YJSCX2012-196HLJ].The Innovation Research Fund for Conflict of interest statement. None declared.


Particularly vulnerable are health care workers who do not use personal protective equipment and persons who do not wash hands after traditional burial practices. Active Nipah virus encephalitis surveillance identified an encephalitis cluster and sporadic cases in Faridpur, Bangladesh, in January 2010. We identified 16 case-patients; 14 of these patients died. For 1 case-patient, the only known exposure was hugging a deceased patient with a probable case, while another case-patient’s exposure involved preparing the same corpse for burial by removing oral secretions and anogenital excreta with a cloth and bare hands. Among 7 persons with confirmed sporadic cases, 6 died, including a physician who had physically examined encephalitis patients without gloves or a mask. Nipah virus–infected patients were more likely than community-based controls to report drinking raw date palm sap and to have had physical contact with an encephalitis patient . Efforts to prevent transmission should focus on reducing caregivers’ exposure to infected patients’ bodily secretions during care and traditional burial practices. Pteropus spp. fruit bats, has been identified as a vehicle for transmission of NiV to humans in Bangladesh  infection in humans were identified from 2001 through 2008; 98 73%) were fatal , altered mental status, new onset of seizures, or a new neurologic deficit, either diffuse or localized to the brain . The phyWe defined possible case-patients as persons with acute onset of fever living in the outbreak area with onset of illness from January 7 through January 28, 2010. We identified possible case-patients by conducting door-to-door visits of all homes and contacting local physicians in the affected villages. We defined probable case-patients as persons who met the possible case definition and who had new onset of altered mental status or new onset of breathing difficulty. We defined case-patients with laboratory-confirmed NiV infection as those with detectable serum IgM against NiV. To assess asymptomatic NiV infection in the outbreak community, we asked community members who had close physical contact or had shared date palm sap from the same pot with probable or confirmed case-patients within the preceding month, to provide a blood specimen for serologic testing.We obtained a clinical history and information about exposures during the month before illness from each probable and confirmed case-patient. Friends, relatives, and neighbors of deceased or unconscious case-patients served as proxy informants for interviews.The investigation team collected acute-phase and subsequent whole blood specimens from possible case-patients. Samples were centrifuged in the local government community clinic, and the separated serum was stored and transported to the IEDCR laboratory in liquid nitrogen. Samples were stored at −70°C.We tested serum samples at IEDCR with an IgM capture enzyme immunoassay that detects NiV IgM . We shipWe conducted in-depth interviews with families of case-patients to explore history of illness and exposures of case-patients. We also had informal discussions with neighbors of case-patients to explore possible modes of transmission.We conducted a matched case–control study to identify risk factors for transmission of NiV. Persons with probable and confirmed cases from the outbreak and with sporadic cases identified from surveillance were considered to be case-patients. The field team selected 4 neighborhood controls for each case-patient, starting from the fourth closest courtyard to the case-patient’s residence. The courtyards where other case-patients resided were excluded. In each courtyard, only the household closest to the main entry was selected. The age of every person in the selected household was recorded. Then a control (only 1 from each courtyard), whose age was closest to the case-patient’s age, was selected. If that control was absent during the first visit, the team tried 3 times to reach the control. If the team was unsuccessful, no control was selected from that household. This process was repeated at the next closest courtyard household until we had selected the required number of controls.All case-patients, except 1, were either too sick or too confused to respond or had died, so the field team selected multiple appropriate proxy respondents for interview. We used standardized, structured questionnaires in the Bengali language.To estimate the association between each exposure and NiV infection, we calculated the matched odds ratio (mOR). We used conditional logistic regression and considered any association to be statistically significant if the p value was <0.05. We analyzed data in STATA 10 .Legal guardians of study participants and healthy adult participants provided informed verbal consent for participation in this investigation. The Ethical Review Committee of icddr,b reviewed and approved the protocol for NiV surveillance and outbreak investigation.During January–April 2010, surveillance physicians from the 6 hospitals reported 328 meningoencephalitis case-patients. Of these, 106 (32%) were reported by surveillance physicians in Faridpur, including 4 outbreak case-patients. Ninety-seven (92%) serum samples were collected at the Faridpur surveillance site. Of these, 12 had IgM against NiV (12%), including 4 from the outbreak area.The outbreak investigation team identified 100 possible NiV-infected persons with febrile illness in the outbreak area during January 5–28, 2010. Of these, 68 persons gave a blood sample for NiV laboratory diagnosis. One person’s sample was positive for IgM against NiV and that person was classified as a confirmed NiV-infected case-patient. We collected 4 blood samples positive for IgM against NiV from 7 probable case-patients. The remaining 3 case-patients died before blood samples were collected. We defined these persons as probable NiV-infected case-patients. All but 1 case-patient had altered mental status, and two thirds of case-patients had breathing difficulty . Seven .Among 7 patients with sporadic cases of NiV infection, 6 of whom died, 5 had a history of drinking raw date palm sap in the month preceding illness onset, and 2 of these 5 were harvesters of date palm sap. One case-patient attended to a family member who was hospitalized at FMCH with an illness unrelated to encephalitis for 3 days, 4 days before the onset of illness. While staying in the hospital, he spent the days talking with and caring for other patients and slept alongside them.Another case-patient was a physician in training at FMCH. From January 1 through February 28, 2010, he worked in the adult medicine and pediatric wards and clinically managed cases of meningoencephalitis in 14 patients, including 3 with confirmed NiV infection and other severely ill febrile patients who had not been enrolled in NiV surveillance. He performed physical examination, intravenous canalization, and nasogastric intubation for 1 confirmed case-patient in the adult medicine unit >1 month before the onset of his illness. He also similarly treated the condition of 2 confirmed NiV case-patients in the pediatric unit 4 days before his onset of illness. A handwashing station for physicians did not exist in either the adult medicine or pediatric wards, and his colleagues reported that he did not use gloves or a mask during patient care or wash his hands after patient care. His illness began on February 28, 2010, with fever and myalgia. Subsequently, he experienced coughing, respiratory distress, convulsions, altered mental status, and loss of consciousness. He died on March 6, 2010. The duration between onset of illness of the physician and his contact with 2 known NiV-infected case-patients in the adult medicine ward was 32 days, and the time from his contact with the 2 children with NiV infection to illness was 2–4 days. Two NiV genomic sequences, obtained from 2 of 14 meningoencephalitis patients that the physician had been in contact with, did not match the NiV genomic sequence of the physician’s isolates , bought and drank raw date palm sap from the village date palm sap harvester. All became ill on January 9, within hours of each other. None had any history of contact with any person who had symptoms similar to encephalitis. Probable case-patients A and B died on January 13; confirmed case-patients C and D died on January 16 and 17 .Among those with second-generation cases, case-patient E (confirmed case) was the wife of case-patient A and was involved in feeding, comforting, and transporting her husband to FMCH and in bringing his dead body back home on January 13. She cleaned saliva her husband using her bare hands and did not wash her hands afterwards. She also drank raw date palm sap with case-patient A in last week of December and did not get ill. On January 15, 2010, a fever developed, but she did not have altered mental status. She recovered on the fourth day of illness and was a confirmed case-patient who survived. Case-patient F (confirmed case) was a friend of case-patient A, who frequently visited case-patient A during case-patient A’s illness. When case-patient A’s condition deteriorated, case-patients E and F took him to the hospital. Case-patient A had to be supported by case-patient F, and the distance between the faces of case-patients A and F was <1 foot while case-patient A was coughing, salivating, and having difficulty breathing. While bringing the dead body back from hospital by bus, case-patient A’s head rested on the thigh of case-patient F. Case-patient G (confirmed case) was the uncle of case-patient A who lived in the same village, and he never visited case-patient A during his illness. He had no contact with any other encephalitis case-patient. He arrived only after case-patient A died and had caressed the head of the corpse before the ritual corpse bathing. Case-patient H (probable case), was a neighbor of case-patient A, who did not come into contact with case-patient A during his illness. He carried out the Muslim practice of ritual purification by cleaning the body and washing it. He used pieces of cloth to clean the body orifices  with an ungloved hand. He then washed the entire corpse with water. He took a bath 1 hour after the ritual cleansing. Case-patient G came in contact with the corpse of case-patient A 3 hours after case-patient A’s death, and case-patient H came in contact with the corpse of case-patient A 6 hours after case-patient A’s death. None of these 3 case-patients , who cared for case-patient A before or after his death, had drunk raw date palm sap in the preceding month. Their illnesses began 8–12 days after contact with case-patient A, they had symptoms similar to those of case-patient A, and they died on January 27 or January 28, 2010, after 4–8 days of illness.The field team enrolled 15 case-patients and 58 controls . The meaDuring 2010, we identified an outbreak and several sporadic cases of encephalitis caused by NiV infection. Two case-patients from the outbreak and 3 patients with sporadic cases had IgM against NiV in serum and NiV RNA in oropharyngeal swab samples by conventional and real-time reverse transcription PCR . We coulPteropus bats, an exposure that has been linked to NiV infection in previous outbreaks (The first 4 case-patients of the initial phase of the outbreak, and 6 of 8 of the patients with sporadic cases, apparently contracted NiV infection by drinking raw date palm sap contaminated with NiV by utbreaks ,17.The remaining 4 case-patients from the outbreak probably acquired NiV infection from physical contact with the source case-patient. Such person-to-person transmission has been observed in prior NiV outbreaks in Bangladesh –10. The The dead bodies of all NiV-infected patients who are Muslim in Bangladesh have undergone the same process of ritual bathing, but to our knowledge, corpse-to-human transmission has not been previously. In other NiV outbreaks when NiV infection developed in family members, many persons had contact with the source case-patient during illness and when preparing the corpse, so we were unable to separately assess corpse-to-person transmission. This investigation suggests that occasional NiV transmission could occur during the Muslim ritual purification of a corpse before burial.This study also documents the death of a physician in Bangladesh from NiV encephalitis after he cared for NiV-infected patients with encephalitis in the surveillance hospital. The physician’s colleagues and roommates did not report any history of his drinking raw date palm sap during the month preceding onset of illness. Although the physician had contact with oral secretions of several meningoencephalitis patients during the outbreak, the genetic sequence of NiV found in the physician was distinct from those of 2 hospitalized NiV-infected case-patients who were positive for NiV by reverse transcription PCR . Indeed,During 2001, health care workers were infected by NiV in Siliguri, India. Among 66 infected persons, 45 case-patients were hospital staff or family caregivers attending to the patients, and 11 patients were infected from an unidentified, hospitalized index case-patient . HoweverBecause NiV infection is not the major cause of acute meningoencephalitis in Bangladesh, and because most persons who contract NiV infection have died by the time a diagnosis is made, it is difficult to identify a strategy to prevent person-to-person transmission that could be consistently applied to NiV-infected case-patients. Strategies to reduce care providers’ exposure to respiratory secretions could prevent a broad array of saliva-transmitted infections, including NiV encephalitis. Prevention approaches to reduce corpse-to-person transmission of NiV and other potentially fatal respiratory secretion-transmitted viruses should focus on minimizing exposure to saliva and other bodily fluids from the body of a person who died of severe febrile illness. Wearing gloves and a mask during the handling and washing of a dead body before burial would not be feasible in low-income communities, where the annual total per capita spending on health is US $12 per person per year . ResearcThis report of nosocomial transmission of NiV to a health care worker in Bangladesh after caring for NiV-infected patients highlights the risk of working without PPE. Barriers to developing an appropriate prevention strategy for nosocomial transmission of NiV in hospitals in Bangladesh include the following: inadequate supplies of PPE for hospital staff, absence of isolation wards, absence of handwashing facilities in hospital wards and physicians’ rooms, and inadequate training and monitoring for infection control . Because


The mortality benefit of whole-body computed tomography (CT) in early trauma management remains controversial and poorly understood. The objective of this study was to assess the impact of whole-body CT compared with selective CT on mortality and management of patients with severe blunt trauma.The FIRST (French Intensive care Recorded in Severe Trauma) study is a multicenter cohort study on consecutive patients with severe blunt trauma requiring admission to intensive care units from university hospital trauma centers within the first 72 hours. Initial data were combined to construct a propensity score to receive whole-body CT and selective CT used in multivariable logistic regression models, and to calculate the probability of survival according to the Trauma and Injury Severity Score (TRISS) for 1,950 patients. The main endpoint was 30-day mortality.In total, 1,696 patients out of 1,950 (87%) were given whole-body CT. The crude 30-day mortality rates were 16% among whole-body CT patients and 22% among selective CT patients (p = 0.02). A significant reduction in the mortality risk was observed among whole-body CT patients whatever the adjustment method . Compared to the TRISS predicted survival, survival significantly improved for whole-body CT patients but not for selective CT patients. The pattern of early surgical and medical procedures significantly differed between the two groups.Diagnostic whole-body CT was associated with a significant reduction in 30-day mortality among patients with severe blunt trauma. Its use may be a global indicator of better management. The availability of high-performance diagnostic imaging methods is a key element in the early diagnostic work-up of patients with severe blunt trauma. In the last two decades, the introduction of whole-body computed tomography (CT) has largely modified clinical practice in the management of patients with severe trauma and may influence surgical decisions. Recent technological advances related to the introduction of multislice CT led to increasing use of whole-body CT thanks to the reduction in data acquisition time and improvement in the quality of imaging data. However, the importance of this technology in early trauma management remains controversial. Besides its cost and the risk of radiation exposure, whole-body CT raises safety concerns about time delays due to patient transportation from the emergency room to the CT room and scanning -3.To our knowledge, few studies have examined the benefit of whole-body CT on mortality in patients with major trauma and these yielded conflicting results -6. One oThe FIRST (French Intensive care Recorded in Severe Trauma) study is a French observational prospective study that aimed at studying the impact of emergency care on hospital mortality of patients with severe blunt trauma. The collection of information about pre-hospital and hospital care, including diagnostic work-up, gave us the opportunity to examine the impact of whole-body CT compared with selective CT on blunt trauma mortality and to compare the global hospital management of the two studied groups.This analysis is an ancillary study of the FIRST epidemiological study which was conceived in order to prospectively gather pre-hospital and hospital data about patients with severe blunt trauma . Accordin = 502), leading to a FIRST study sample of 2,703 patients.As previously described, consecutive patients were recruited between December 2004 and March 2007 if they were at least 18 years old and had a severe blunt trauma defined as trauma requiring admission into an ICU within 72 hours after injury or, in the case of early death before ICU admission, trauma managed by a mobile ICU (MICU). Exclusion criteria were (a) penetrating traumas and (b) deaths occurring before the implementation of any advanced life-sustaining treatment. A total of 3,205 patients were eligible for inclusion in the FIRST study. Patients with incomplete or poor-quality data regarding hospital of first admission, ISS, pre-hospital management, or vital status were secondarily excluded  patients' characteristics; (b) data about accident circumstances; (c) hospital units involved in the early care of patients before admission to the ICU; (d) clinical and biological data on the pre-hospital phase if available, upon hospital admission, and 24 and 72 hours after the trauma; and (e) a summary of clinical variables at patient discharge or death.During the pre-hospital phase, the following data were recorded: initial physiological variables , pupil status, Glasgow Coma Scale [GCS] score, and life-sustaining treatments .Information on physiological variables and life-sustaining treatments was also collected upon arrival at the first hospital and 24 and 72 hours after the accident. The first available measurement, either at the pre-hospital phase or upon hospital admission, was used to describe the initial physiological status of the patient. At patient discharge from the ICU or death (within 30 days), anatomic injury diagnoses with corresponding Abbreviated Injury Scale (AIS) codes and the ISS were recorded from medical records. The AIS was coded according to the 1998 updated classification  by localSeveral variables regarding the accident circumstances or the initial medical trauma assessment of the patient were important to take into consideration because they could influence imaging strategy. Two variables dealing with either the severity of accident or the suspected severity of trauma were constructed ,12. The All surgical procedures received by patients until ICU discharge were recorded and coded by physicians at the coordination center. Hemostatic procedures included arterial embolization and hemostatic thoracotomy or abdominal laparotomy. Orthopedic procedures included all types of bone fixation of upper and lower limbs.Whole-body CT was an unenhanced CT of the head followed by contrast-enhanced CT of the chest, abdomen, pelvis, and complete spine. Information about whole-body and selective CT to one or more of these body regions was recorded in the first clinical department where the patient was admitted  and, if needed, in subsequent departments that received the patient until his or her admission to an ICU. All other imaging procedures were recorded in a similar way. The main outcome measurement was the vital status at 30 days or at ICU discharge if discharge occurred within the first 30 days.Comparisons of patients given a whole-body CT with those given a selective CT were performed by using chi-squared tests or, if needed, Fisher exact tests. To address selection and confounding biases that could not be totally controlled by the exclusion of patients who did not have any kind of CT and to assess the mortality reduction risk by using the initial whole-body CT, we constructed a propensity score. This approach is based on the idea that the probability of undergoing either whole-body or selective CT may depend on the patient's age, sex, study center, accident circumstances, initial medical assessment, and physiological status as well as on the administration of life-sustaining treatments during the pre-hospital phase or at hospital admission. We computed a non-parsimonious logistic regression model that included 24 potentially relevant covariates regarding the use of either whole-body or selective CT  associated with mortality at a significance level of less than 0.20 in bivariate analysis were introduced and selected through a backward procedure as described by Hosmer and Lemeshow . Second,To compare our results with those obtained in a previous study , we alsoP value of less than 0.05 was considered significant, and all P values were two-tailed.Data were expressed as mean with standard deviation, median with interquartile (25th to 75th) range, or percentage. We performed the statistical analyses by using SAS™ version 9.1 (SAS Institute Inc.) and STATA version 11  software. A P = 0.15). There were significant differences in baseline characteristics between the two groups  had a whole-body CT and 254 (13%) had a selective CT. Among patients with selective CT, the body regions were head in 202 patients (80%), abdomen/pelvis in 105 (41%), cervical spine in 89 (35%), and thorax in 59 (23%). The proportions of patients who received Focussed Assessment Sonography for Trauma (FAST) imaging  were not significantly different in patients with selective CT and in those with whole-body CT . Furthermore, all baseline characteristics that were significantly related to the use of whole-body versus selective CT in the univariate analysis were no longer significant after adjustment for the propensity score Table .P = 0.02). There was no significant difference between the two groups in the mortality rate at 24 hours (6.0% in the former group versus 8.3% in the latter). The adjusted impact of whole-body CT on 30-day mortality according to the various adjustment methods is summarized in Figure P = 0.54).At day 30, 277 patients (16%) in the whole-body CT group and 56 (22%) in the selective CT group were deceased  of 1% to 11%; P = 0.016). The other main causes of death - hemorrhagic shock (14% versus 13%) and multivisceral organ failure (8% versus 2%) - were similar in the two groups.Cerebral death was the main cause of death and was significantly less frequent in the whole-body CT group than in the selective CT group  among whole-body CT patients, indicating a significantly better survival in these patients than that predicted by the TRISS method. Corresponding Ws and Zs scores among selective CT patients were, respectively, 0.3 (95% CI -2.9 to 3.5) and 0.17 (P = 0.44), indicating the lack of significant difference between the observed and the TRISS-predicted survival in the latter group.The TRISS method was applied to 1,864 patients with an available revised trauma score. The TRISS-predicted mortality rates were 30% for the 1,635 patients who received whole-body CT and 22.7% for the 229 patients who received only selective CT. After standardization according to the case mix of injury severity of the US prediction database, the Ws and Zs scores were, respectively, 3.3 (95% CI 1.9 to 4.6) and 4.81 (P < 0.001) and spinal areas (P = 0.05) as well as for the lower limbs (P < 0.001)  Table . SimilarP < 0.02) and until ICU discharge . The main differences between groups in surgical procedures used within the first 24 hours were the higher percentage of patients with hemostatic or orthopedic surgery in the whole-body CT group than in the selective CT group . In contrast, more patients had undergone intracranial surgery at 24 hours in the selective CT group than in the whole-body CT group (P < 0.001). Furthermore, early medical management, as reflected by the use of intubation, blood transfusion, and catecholamines, was significantly more aggressive in patients given whole-body CT. A similar pattern was observed in surgical procedures until discharge, with significantly more hemostatic (P = 0.02) and orthopedic (P < 0.001) surgery and less intracranial surgery (P < 0.001) for patients given whole-body CT than in those given selective CT. Furthermore, spinal surgery (P = 0.01) and the use of a thoracic drain (P < 0.001) were significantly more frequent in the whole-body CT group.As shown in Table The main finding of this observational study was the significant reduction in 30-day mortality among patients who had a whole-body CT for early assessment of blunt trauma in comparison with patients who had only selective CT. According to the adjustment method, the relative risk reduction ranged from 0.42 to 0.45.a priori characteristics that may influence both the probability of receiving whole-body versus selective CT and the risk of death but also a posteriori variables  related to the risk of death. Interestingly, we obtained very consistent results using either a classic multivariate logistic regression model (the best model according to c statistics and the Hosmer-Lemeshow test) or logistic regression models based on propensity score considered as a continuous variable, a variable categorized according to score quintiles or a matching variable.The main strength of this prospective study was to deal carefully with potential indication biases of whole-body CT. First, we excluded patients who did not survive long enough to undergo a whole-body CT. This precaution was taken because whole-body CT may be more time-consuming than selective CT or other diagnostic methods used in the emergency room (or both) and thus may not be proposed to the most severely injured patients ,20. SecoOur study supports and extends the results of the German Trauma Registry-based study . Using aa priori factors likely to influence the choice of the imaging methods, we were able to highlight a pronounced reduction in 30-day mortality for patients given whole-body CT.The interpretation of the association between whole-body CT and ISS is uncertain. Clearly, patients given whole-body CT had higher ISS than patients given selective CT. The first explanation may be that patients presumed to have more severe injuries at admission were more likely to receive whole-body CT. If this was the case, ISS should have been included either in the propensity score or in adjustment variables. However, we did not find any association between the use of whole-body CT and the suspected severity of trauma. The second explanation is that whole-body CT led to a better detection of lesions than selective CT and thus to higher ISS. In that case, adjustment for ISS, which is strongly related to the risk of death, may result in an overestimation of the beneficial impact of whole-body CT. This reason led us to judge the TRISS method inappropriate in the study context and to decide to exclude ISS from the propensity score and adjustment variables. Nevertheless, using a conservative strategy based solely on The reasons why the use of whole-body CT may induce a reduction in 30-day mortality in patients with severe trauma are difficult to unravel in an observational study. Our study revealed more intensive pre-hospital management as reflected by more frequent on-scene intubation and higher fluid loading and continuous intravenous catecholamine infusion in the whole-body CT group in comparison with the selective CT group. Early management of hypoxemia and hypotension can reduce the risk of early fibrinolysis and prevent patients from being admitted with clinical coagulopathy ,26. On tWe did not find any significant difference between groups in the early mortality rate, suggesting no major differences in their initial clinical status but rather a later deterioration of patients from the selective CT group. This may be due to unrecognized injuries or delayed in-hospital management or both. In our study, hospital surgical strategies were available within the first 24 hours and at discharge. During the first 24 hours, whole-body CT patients benefited from more frequent surgical management and more intensive life-sustaining treatments characterized by more frequent transfusion, intubation, and catecholamine infusion. After a whole-body CT, this overall dynamic therapeutic approach may reduce preventable deaths. Indeed, many deaths are due mainly to incomplete or poor assessment of organ injuries, delayed decision of surgical operation, delayed hemostasis intervention, or errors in resuscitation procedures -30. OverAnother explanation lies in the higher proportion of cerebral death in the selective CT group than in the whole-body CT group. Eighty percent of selective CT patients underwent head CT. These patients tended to have more severe cerebral lesions (AIS score of at least 4) and had significantly more frequent early neurosurgical intervention. Head injury is known to be the single largest contributor to trauma center deaths . Other sOur study also presents several limitations. This was an observational cohort, so that, despite our careful adjustment strategy, we cannot rule out residual confounding effects and thus a causality link cannot be definitely demonstrated. In particular, we have no information about scanning protocols or type of scanners used, so that possible variations in CT protocols between centers cannot be excluded. We have no reason to suspect major between-center differences in whole-body CT indications, since, in France, whole-body CT is systematically recommended unless severe trauma patients present an unstable hemodynamic status or severe isolated head injury or both. In addition, in all trauma centers, scans are first interpreted by radiologists and further reviewed by clinicians in charge of the patients (emergency physicians or surgeons or both). Because the FIRST study was not specifically designed to address this topic, we have no details about the time elapsed between admission in a university hospital trauma center and diagnostic imaging work-up. However, since more than 80% of the patients received whole-body or selective CT in the first unit of admission , we can hypothesize that all patients were examined within the first 24 hours and that the majority of them were examined in the first two hours after their admission. Furthermore, because the quality of data regarding diagnostic imaging was uncertain for patients initially admitted in general hospitals before their transfer to university hospital trauma centers, these patients were excluded from the present analysis. Thus, our results can be extrapolated only to severe trauma patients admitted directly to university hospital trauma centers. Lastly, the design of the FIRST study did not take into account patients with penetrating trauma or pediatric trauma patients.Our prospective study showed that initial whole-body CT was associated with a significant 30-day mortality reduction that could be related to higher detection of traumatic lesions and higher use of surgical treatment. However, our study stressed the important contribution of severe head injury for explaining the lower mortality in patients who received selective CT. Alternatively, whole-body CT may be only an overall indicator of better pre-hospital and hospital management of patients with severe blunt trauma. Clearly, only a randomized controlled trial could solve the issue but its feasibility is highly questionable in the present state of diagnostic practices in severe trauma.• Initial whole-body computed tomography (CT) is used for a large majority of patients with severe blunt trauma.• Whole-body CT is associated with a significant reduction in 30-day mortality. This reduction is due mainly to a lower proportion of cerebral death.• The beneficial impact of whole-body CT on mortality is independent of the initial physiological status.• Surgical management was more frequent among patients with whole-body CT. Whether this could explain the reduction in mortality remains unclear.AIS: Abbreviated Injury Scale; CI: confidence interval; CT: computed tomography; FIRST: French Intensive care Recorded in Severe Trauma; GCS: Glasgow Coma Scale; ICU: intensive care unit; ISS: Injury Severity Score; MICU: mobile intensive care unit; ROC: receiver operating characteristic; TRISS: Trauma and Injury Severity Score.The authors declare that they have no competing interests.J-MY and AY conceived of this study with considerable help from CB-K and MF for sequence alignment. BR gave support for proofreading of this paper, was involved in the initiation and design of the study, participated in the acquisition of data, and contributed to the interpretation of data and final revision of the manuscript. J-MY was involved in the study design and in the acquisition, analysis, and interpretation of data and wrote the first draft of the manuscript. AY participated in the conception of this analysis, the interpretation of data, and draft writing. DG, CJ, and CM participated in the design of the study, the acquisition of data, and the final revision of the manuscript. CB participated in the design of the study and performed the statistical analysis. CB-K was responsible for the logistic coordination of the study; was involved in the design of the study, statistical analysis; and interpretation of data, and helped to draft the manuscript. MF initiated and coordinated the FIRST study and was involved at all steps of the study. All authors read and approved the final manuscript.


Mucopolysaccharidosis type I (MPS I) is traditionally divided into three phenotypes: the severe Hurler (MPS I-H) phenotype, the intermediate Hurler-Scheie (MPS I-H/S) phenotype and the attenuated Scheie (MPS I-S) phenotype. However, there are no clear criteria for delineating the different phenotypes. Because decisions about optimal treatment (enzyme replacement therapy or hematopoietic stem cell transplantation) need to be made quickly and depend on the presumed phenotype, an assessment of phenotypic severity should be performed soon after diagnosis. Therefore, a numerical severity scale for classifying different MPS I phenotypes at diagnosis based on clinical signs and symptoms was developed.A consensus procedure based on a combined modified Delphi method and a nominal group technique was undertaken. It consisted of two written rounds and a face-to-face meeting. Sixteen MPS I experts participated in the process. The main goal was to identify the most important indicators of phenotypic severity and include these in a numerical severity scale. The correlation between the median subjective expert MPS I rating and the scores derived from this severity scale was used as an indicator of validity.Full consensus was reached on six key clinical items for assessing severity: age of onset of signs and symptoms, developmental delay, joint stiffness/arthropathy/contractures, kyphosis, cardiomyopathy and large head/frontal bossing. Due to the remarkably large variability in the expert MPS I assessments, however, a reliable numerical scale could not be constructed. Because of this variability, such a scale would always result in patients whose calculated severity score differed unacceptably from the median expert severity score, which was considered to be the 'gold standard'.Although consensus was reached on the six key items for assessing phenotypic severity in MPS I, expert opinion on phenotypic severity at diagnosis proved to be highly variable. This subjectivity emphasizes the need for validated biomarkers and improved genotype-phenotype correlations that can be incorporated into phenotypic severity assessments at diagnosis. IDUA gene have been described [Mucopolysaccharidosis type I  is a rare autosomal recessive lysosomal storage disorder caused by a deficiency in the alpha-L-iduronidase (IDUA) enzyme, which is involved in the breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate . The resescribed . The birescribed ,5.MPS I is traditionally divided into three phenotypes: the severe Hurler (MPS I-H) phenotype, the intermediate Hurler-Scheie (MPS I-H/S) phenotype, and the attenuated Scheie (MPS I/S) phenotype. In reality, however, MPS I presents with a continuous spectrum of phenotypic severity ,6,7.nd or 3rd decade in the absence of treatment [MPS I-H patients have marked cognitive delay, coarse facial features, corneal clouding, hearing impairment, ear-nose-throat infections, hepatosplenomegaly, umbilical and inguinal hernias, restricted joint mobility and orthopedic, cardiac and respiratory problems in early childhood. Without appropriate treatment, their life expectancy is severely limited. At the other end of the spectrum, MPS I-S patients have normal intelligence and near-normal life expectancy but experience significant morbidity as a consequence of restricted joint mobility, carpal tunnel syndrome, skeletal dysplasia, cardiac disorders and pulmonary dysfunction. Patients with the intermediate MPS I-H/S phenotype are generally described as having no or only mild cognitive impairment and relatively severe somatic symptoms that limit life expectancy to the 2reatment ,6-9.Two therapeutic options are currently available: hematopoietic stem cell transplantation (HSCT) and intravenous enzyme replacement therapy (ERT). HSCT can stabilize neurocognitive function, prevent fatal cardio-pulmonary complications, and improve overall survival . HoweverWhen an MPS I diagnosis is made, the phenotype needs to be assessed as soon as possible so that the optimal treatment strategy can be quickly determined. The outcome of HSCT in MPS I-H is less favorable when there is a longer delay between diagnosis and transplant ,20. If EThe aim of this study was to develop a consensus scale for phenotypically classifying MPS I patients at diagnosis based on clinical signs and symptoms.The process for constructing a diagnostic disease severity scale included two written rounds, a consensus meeting and a combined modified Delphi method and nominal group technique ,23. All In the second written round, 20 MPS I case descriptions were sent to the experts. The case descriptions were based on clinical information available at the time of diagnosis and were retrieved from two participating centers (Amsterdam and Manchester). The cases were selected to represent a wide range of MPS I phenotypes. The experts rated each case description for severity on an 11-point scale . If one of the experts was involved in treating one of the specific cases, that expert's rating for that case was excluded from the analyses. The median expert rating for a case was considered to be the 'gold standard' severity score.All 20 case descriptions were scored by one investigator (QGAT) according to the presence or absence of the 39 criteria in Table A face-to-face consensus meeting was subsequently held in Amsterdam in May 2008). To investigate the intra-observer reproducibility , all 20 008. To iFor the three items in which the severity definition or grading was complicated, working groups of 3-5 experts were asked to make a proposal for each item based on the best evidence.After the meeting, the scores of the items in the final consensus list that were present in each patient description were equally weighted and summed to obtain an objective MPS I phenotypic severity score at diagnosis. The correlation between the scores on this 'MPS I severity scale' and the median expert scores ('gold standard') for the 20 case descriptions was calculated.To assess the validity of this scale, a set of 18 new MPS I case descriptions, again representing the full MPS I phenotypic spectrum, was compiled. Care was taken to include information on all six of the items in the final consensus list Table  in each U test (SPSS 18.0). To quantify the intra- and inter-rater reproducibility of the expert scores, the variance components were calculated using a three-way random effects analysis of variance model with the scores as the dependent variable. The following variance components were estimated using the model: between patients (σP2), between experts (σE2), between rounds (σR2), the patients × experts interaction (σPE2), the patients x rounds interaction (σPR2), the experts × rounds interaction (σER2), and the residual (σPER2). Because the absolute values of the expert scores were not of interest, intraclass correlation coefficients (ICCs) were calculated to assess consistency [The association between the presence or absence of each item and the median expert severity scores for the individual cases was investigated using the Mann-Whitney sistency .consistency was calculated using the following formula:The intra-rater ICCconsistency was calculated as follows:The inter-rater ICCThere was no 'rounds' factor in the inter-rater reproducibility of the subjective expert scores of the 18 patient descriptions, which therefore use the following formula:Spearman correlation coefficients were used to investigate correlations between the median expert scores and the objective phenotypic severity scores. Numerical weights were assigned to the items used in the objective score according to the experts' assessment of their clinical relevance. The goal was to maximize the correlation with the median subjective expert score and thus achieving the best phenotypic differentiation. The level of statistical significance was set at p < 0.05.Of the 30 items included in the first-round questionnaire Table , 7 were Of the 39 items identified in the 'first written round' Table , 12 wereThe 24 items proposed by the individual experts are shown in Table The median expert scores of the 18 case descriptions used in the validation phase ranged from 2 to 8.5. The inter-observer ICC of the expert scores was 0.71. Again, there was considerable variation in the expert severity assessments of the case descriptions  techniques based on measuring IDUA activity and/or immune-quantification of the IDUA protein in dried blood spots -28. A seBecause clinical signs and symptoms appear to be insufficiently reliable to assess phenotypic severity at diagnosis, other methods should be vigorously investigated. Combined genotyping and biomarker analysis in plasma and/or urine, such as the recently reported plasma heparin cofactor II-thrombin (HCII-T) complex and the urinary dermatan sulfate:chondroitin sulfate (DS:CS) ratio, promises to be a good strategy for determining disease severity in newly diagnosed MPS I patients ,3,29,30Our study has several limitations. First, the experts differed with respect to the age, phenotype and ethnic background of their patient experiences. These observations may have influenced their opinion of disease severity. Second, the patient information used to write the case descriptions was gathered retrospectively. Thus, some follow-up results were known for most of the patients, which may have biased the data retrieval and the description of the cases. Moreover, the information that had been recorded in the patient files may have been influenced by knowledge of which interventions had (or had not) been performed. Finally, assessing phenotypic severity is hampered by the subjective rating of certain items, e.g., the presence or absence of kyphosis and frontal bossing on clinical examination, the parents' report of the age of symptom onset and the influence of decreased range of motion due to joint disease on performing activities of daily living.This robust and transparent consensus procedure did not result in a reliable and validated numerical MPS I severity scale. However, the process did produce a list of six items rated by the experts as being most important for phenotypic classification, which may be useful for classifying newly diagnosed MPS I patients. Further studies into the possible roles of genotypes and biomarkers as indicators of disease severity are necessary to optimize clinical diagnosis and decision-making in MPS I patients.ERT: Enzyme replacement therapy; GAG: Glycosaminoglycan; HSCT: Hematopoietic stem cell transplantation; ICC: Intraclass correlation coefficient; IDUA: Alpha-L-iduronidase; MPS I: Mucopolysaccharidosis type I; NBS: Newborn screening; OSAS: Obstructive sleep apnea syndrome.The authors declare that they have no competing interests.FAW, CEH, JHL, JEW and QGAT were part of the planning committee, were involved in the methodological process, and wrote the first draft of the manuscript. MHdR was involved in the validation phase and wrote later drafts of the manuscript. JHL performed the statistical analysis. ATS, CEH, EPT, FAW, GMP, JAR, JEW, JZ, LAC, MB, MS, MVMR, OAB, and SPL participated in written rounds 1 and 2 and the face-to-face consensus meeting. All of the authors reviewed and corrected earlier versions of the manuscript and participated in the creation of its final form.


While a network of cortical regions contribute to face processing, the lesions in acquired prosopagnosia are highly variable, and likely result in different combinations of spared and affected regions of this network. To assess the residual functional sensitivities of spared regions in prosopagnosia, we designed a rapid event-related functional magnetic resonance imaging (fMRI) experiment that included pairs of faces with same or different identities and same or different expressions. By measuring the release from adaptation to these facial changes we determined the residual sensitivity of face-selective regions-of-interest. We tested three patients with acquired prosopagnosia, and all three of these patients demonstrated residual sensitivity for facial identity changes in surviving fusiform and occipital face areas of either the right or left hemisphere, but not in the right posterior superior temporal sulcus. The patients also showed some residual capabilities for facial discrimination with normal performance on the Benton Facial Recognition Test, but impaired performance on more complex tasks of facial discrimination. We conclude that fMRI can demonstrate residual processing of facial identity in acquired prosopagnosia, that this adaptation can occur in the same structures that show similar processing in healthy subjects, and further, that this adaptation may be related to behavioral indices of face perception. Prosopagnosia is a neurological syndrome characterized by the failure to recognize familiar faces in the absence of more pervasive dysfunction of vision or memory Barton, . PatientOne question of interest is the residual function of spared regions of the face network in prosopagnosia. Identifying surviving face-selective regions in acquired prosopagnosia with a standard contrast between viewing faces and viewing objects  had or had not survived their lesion, and then with our adaptation paradigm to determine the residual sensitivity to identity and expression changes in these surviving regions. Given current models, we hypothesized that we would find residual sensitivity for identity changes in the right FFA, and for expression changes in the right pSTS. In addition, we hypothesized that residual sensitivity in the fMRI experiment may be indicative of a residual ability of prosopagnosic subjects to discriminate the structural properties of faces, as determined by our own experimental tests and standard neuropsychological instruments such as the Benton Face Recognition Test  is a 24 year-old right-handed male who had herpes simplex encephalitis three years prior , is a 24 year-old right-handed female. One year prior to testing she had a selective right amygdalohippocampectomy for epilepsy , is a 49 year-old left-handed male who twelve years prior had suffered an occipital cerebral hemorrhage from rupture of an arteriovenous malformation Figure ] and on Face images were selected from the Karolinska Database of Emotional Faces , which were different from the faces used in the experimental conditions, displaying 3 different expressions  were selected and formatted in a gray oval as described above. Upright and inverted versions of these six faces were created. Two face pairs were formed from each of the six identities; upright-inverted and inverted-upright. These 12 pairs became target trials in the fMRI adaptation experiment.TR = 2 s). The first face was presented for 500 ms and followed by a 300 ms inter-stimulus-interval (ISI). This was followed by a 500 ms presentation of the second face and a 700 ms inter-trial-interval (ITI). In order to avoid retinal adaptation image location randomly varied from image to image within a region of 50 by 50 pixels.An experimental trial consisted of a pair of faces presented within each repetition time  from these quartets were presented during the scan. This resulted in 32 experimental trials per condition (from the 32 randomly selected face quartets) and 96 trials total. In addition to these experimental trials 10 of the 12 target trials  were randomly selected and included. Participants were asked to respond to the inverted face in these target trials with a keypress, which acted as a means to ensure subjects attended to the faces. Finally, 48 fixation trials, in which the face images were replaced by a fixation cross, were randomly interspersed among the experimental and target trials, producing the jittering required for rapid event-related experimental designs  were acquired using an interleaved ascending EPI sequence, consisting of 36 axial slices of 3 mm thickness (1 mm gap) with an in-plane resolution of 1.875 mm × 1.875 mm (FOV = 240).Structural and functional MRIs were performed on all participants. All scans were acquired in a 3.0 Tesla Philips scanner. Stimuli were presented using Presentation 9.81 software and were rear-projected onto a mirror mounted on the head coil. Whole brain anatomical scans were acquired using a T1-weighted echoplanar imaging (EPI) sequence, consisting of 170 axial slices of 1 mm thickness (1 mm gap) with an in-plane resolution of 1 mm × 1 mm (FOV = 256). T2-weighted functional scans . Anatomical scans were not preprocessed, but were standardized to Talairach space , we identified the core regions of face perception, bilaterally, within each participant  and faces (F) as predictors, and a F > O contrast was overlaid on the whole brain. Using a False-Discovery-Rate of Experimental MRI scans were analyzed using a deconvolution analysis that accounts for non-linear summation of the blood oxygen level dependent (BOLD) response in rapid event-related designs. The deconvolution analysis samples BOLD activity at trial onset (time = 0 s) and a further 9 times in 2 s intervals, resulting in an unbiased model of the hemodynamic response (HDR). The inverted target trials were included as a separate condition in the deconvolution analysis, to account for all non-fixation trials, but were not included in subsequent analyses.same-identity/same-expression, different-identity/same-expression, and same-identity/different-expression) as functions within the GLM (BrainVoyager). While one cannot determine the significance of differences in a single scan in a single subject, averaging across multiple scans enables the assessment of statistical significance in the single subject. Significant adaptation of the HDR may take a number of forms including a reduced HDR-peak due to neural fatigue or a narrowing of the full-HDR due to a facilitated neural response . The HDR-peak was defined as the time point exhibiting a maximal increase in BOLD activity, or the average of this time point and adjacent time points that did not significantly differ . Using these definitions, the values of the full-HDR and HDR-peak were then determined for each of the three experimental conditions. Contrasts of the different-identity/same-expression > same-identity/same-expression and the same-identity/different-expression > same-identity/same-expression were performed, using the multi-study GLM, to assess identity and expression adaptation, respectively. Significant release from adaptation in the different conditions was set at α < 0.05, and would indicate sensitivity of the ROI to changes in identity or expression. Only positive release from adaptation values indicate sensitivity to the varied stimulus; negative values would suggest priming of an ROI to the presented stimulus and are not discussed herein (in fact only one control demonstrated a negative release from adaptation in the L-FFA). The difference values resulting from these two contrasts are presented graphically. As all effects in the full-HDR condition were replicated in the HDR-peak condition, but were stronger in the latter, we only present the results of the HDR-peak analyses  and left  OFA (Figure B-AT1 has extensive bilateral damage to the anterior temporal lobes, which extends to the inferior surface of the middle temporal lobe Figure . Releasep < 0.05; Figure p < 0.005; Figure R-AT1 has a small lesion in the anterior right temporal lobe that affects the anterior hippocampus, amygdala, and overlying temporal cortex Figure . Releasep < 0.005; Figure p < 0.005; Figure R-IOT1 has a unilateral right lesion affecting both the occipital and posterior temporal cortex Figure . ReleaseA surviving right FFA was found in two prosopagnosic patients (B-AT1 and R-AT1; Figure The left FFA did not show sensitivity to identity changes in either patient B-AT1 or R-AT1, but significant sensitivity was observed in the left FFA of R-IOT1, who differs from the others in that he is strongly left-handed Figure . This raBeyond the FFA, we also found identity adaptation in the OFA of our three patients Figure . The rigWe did not find any identity adaptation in the right pSTS of any patient. Residual processing of identity in the STS has been suggested by some as a possible compensatory mechanism in prosopagnosia, particular by those who promote a dissociated dorsal route of face processing as an explanation for covert recognition (Tranel et al., Another possibility for the failure to identify adaptation to facial expression within the current design may be the restriction of our analysis to predefined ROIs. A recent study by Mur et al.  demonstrIt is interesting to compare the patient's residual ability to discriminate faces of different identities and parallel these findings with the fMRI adaptation results for identity. B-AT1, R-AT1, and R-IOT1 all performed normally on the Benton Face Recognition Test and had mild to moderate deficits on the morph discrimination test for identity; on the fMRI experiment all showed identity adaptation effects in at least one face-selective region. In contrast, a prosopagnosic patient in another study, PS, was significantly impaired on the Benton Facial Recognition Test and showed no identity adaptation effects in the FFA (Rossion et al., In conclusion, we devised an fMRI adaptation protocol which can reveal significant adaptation to facial identity in the single subject. In three acquired prosopagnosics with a variety of lesions, we found residual sensitivity to identity in the spared right FFA of two right handed prosopagnosic patients with anterior temporal damage, and in the spared left FFA of one left-handed prosopagnosic patient who had loss of the right FFA and OFA. We also observed sensitivity to identity within the left OFA of these three patients, which may reflect either normal sensitivity to facial structure or a compensatory enhancement following damage to the face processing network. The presence of adaptation effects for identity paralleled residual ability to discriminate between different faces, as measured by the Benton Facial Recognition Test but not the more difficult morphed-face discrimination test. Further study in a larger cohort of subjects with either acquired or congenital prosopagnosia would be of interest.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.