The extracranial venous outflow routes in clinically defined multiple sclerosis (CDMS) have not previously been investigated.Sixty-five patients affected by CDMS, and 235 controls composed, respectively, of healthy subjects, healthy subjects older than CDMS patients, patients affected by other neurological diseases and older controls not affected by neurological diseases but scheduled for venography (HAV-C) blindly underwent a combined transcranial and extracranial colour-Doppler high-resolution examination (TCCS-ECD) aimed at detecting at least two of five parameters of anomalous venous outflow. According to the TCCS-ECD screening, patients and HAV-C further underwent selective venography of the azygous and jugular venous system with venous pressure measurement.CDMS and TCCS-ECD venous outflow anomalies were dramatically associated . Subsequently, venography demonstrated in CDMS, and not in controls, the presence of multiple severe extracranial stenosis, affecting the principal cerebrospinal venous segments; this provides a picture of chronic cerebrospinal venous insufficiency (CCSVI) with four different patterns of distribution of stenosis and substitute circle. Moreover, relapsing-remitting and secondary progressive courses were associated with CCSVI patterns significantly different from those of primary progressive (p<0.0001). Finally, the pressure gradient measured across the venous stenosies was slightly but significantly higher.CDMS is strongly associated with CCSVI, a scenario that has not previously been described, characterised by abnormal venous haemodynamics determined by extracranial multiple venous strictures of unknown origin. The location of venous obstructions plays a key role in determining the clinical course of the disease. The resulting gradient favours venous return to the right heart, which can be easily assessed with high-resolution echocolour Doppler (ECD) and transcranial colour-coded Doppler sonography (TCCS), which represent an ideal method by which to investigate the haemodynamics of cerebral venous return.9–8–12Posture and the mechanic movement of respiration play a fundamental role in ensuring the correct cerebrospinal venous outflow.the internal jugular vein (IJV) is the predominant pathway in the supine position, confirmed by an increased cross-sectional area of the internal jugular vein (CSA) related to increased blood volume in that posture; andredirection of venous flow to the vertebral veins (VVs) occurs in the upright position, with compliant reduction of the CSA of the IJV.In contrast, MR and selective injection venography are of course limited in evaluating cerebral venous haemodynamics under different postural and respiratory conditions. However, the latter especially provide excellent morphological but static details.We present the results of a study that evaluated the abnormalities of the cerebral venous outflow in patients with MS using ECD-TCCS and selective venography.We investigated 65 patients affected by clinically defined MS (CDMS) and diagnosed according to the revised McDonald criteria.As controls, we investigated 235 subjects subdivided as follows:60 healthy subjects matched for age and gender with MS patients (HM-C).82 healthy subjects older than the median age of onset of CDMS (HA-C);45 patients affected by other neurological diseases (OND) ; this gr48 other controls not affected by neurological diseases , but schWe excluded subjects who had Behçet disease, vasculitis, cerebral vascular malformations and congenital vascular malformations .Patients and controls underwent a non-invasive study of cerebrospinal venous return at the Vascular Lab; the ultrasound technicians and physicians interpreting the data were blinded to the patient diagnostic category.–Cerebrospinal venous return was examined with the subject positioned on a tilt bed by combining the extracranial ECD methodology for investigating the IJVs and VVs with that of the TCCS for studying the deep cerebral veins (DCVs).9reflux in the IJVs and/or VVs in sitting and supine posture;reflux in the DCVs;high-resolution B-mode evidence of IJV stenoses;flow not Doppler-detectable in the IJVs and/or VVs;reverted postural control of the main cerebral venous outflow pathways.–14In normal subjects, flow in the IJVs and VVs is directed toward the heart in any position of the head.8We assessed the presence of reflux with the body positioned respectively at 0° and +90°, in the four extracranial venous drainage pathways.19–19Physiological intracranial venous flow is monodirectional.162, never measured in normal subjects, was taken as reference value.15We assessed the presence of stenosing venous imaging by means of a complete ECD high resolution B-mode exploration of the cervical vessels, and measurement of the CSA of the IJV. A CSA ⩽0.3 cmWe assessed the lack of a Doppler detectable venous flow in the IJVs and/or VVs despite numerous deep inspirations, with the head positioned at 0° and +90° in the four extracranial venous drainage pathways. In normal subjects this finding was never observed with the head in any position,12–ΔCSA in the IJVs, obtained by subtracting the CSA measured in the supine from that in the sitting position, is a positive value in normal subjects.8Diagnosis of suspicious abnormal extracranial cerebral venous outflow required at least two of the five above listed criteria to be fulfilled, and was taken as an indication to continue the study using selective venography in all suspected subjects.This study was approved by the Ethics Committee of the Ferrara University Hospital. Our Ethics Committee approved the use of selective venography only in subjects (patients or controls) with abnormal venous ultrasonographic examination. An invasive investigation  was felt unnecessary when the ECD scan was negative at the level of the neck.We therefore used ultrasonography as a screening to venography. Finally, the Committee approved an additional venographic investigation to be carried out on patients without neurological disease, even if preoperative screening for venous return anomalies was negative but who, for other reasons, should have had a venographic examination (HAV-C group).–2O in the superior vena cava, in the azygous vein, and in both the IJVs.Sixty-five subjects with MS fulfilling the ECD-TCCS screening criteria and 48 controls of the HAV-C group underwent selective catheterism of the azygous and IJV system via the transfemoral route. Venography was performed, being aware of patients’ diagnoses. We considered a significant stenosis to be any venous lumen reduction greater than 50%.24Clinical and demographic data are expressed as the median and 25th–75th percentile, and venous pressure as the mean (SD). Differences among groups were tested for significance with the one-way ANOVA analysis of variance. The two-sided Fisher exact test followed by the determination of odds ratio (95% CI) was used for determining the associated risk of MS in case of positive ultrasonographic findings, by comparing the whole MS group with the control group.The two-sided Fisher exact test was also used for testing differences in the number of extracranial venous strictures between CDMS patients treated and not treated with drugs. Differences in venous pressure between patients and controls, as well as across the stenosies, were analysed with the Mann–Whitney test.2 test for independence was used for assessing clinical differences of MS patients among the different patterns of extracranial venous outflow obstruction.Finally, the χp Values up to 0.05 were considered statistically significant.Selective venography demonstrated that the detection of at least 2/5 TCCS-ECD criteria of suspected anomalous extracranial venous outflow (which never occurred in the control populations) was always related to multiple significant extracranial venous stenosis, localised at the cervical, thoracic and, less commonly, abdominal level of the principal cerebrospinal venous segments. In none of the HAV-C subjects who underwent venographic investigation with negative ultrasound were there any stenotic patterns in the IJVs, azygous and lumbar territory .x rays in In particular, the azygous vein in the MS group was affected in 86% of cases. Most cases involved membranous obstruction of the junction with the superior vena cava, twisting, or, less frequently, septum and atresia, as can be seen in the As for the jugular veins, they were found to be stenosed unilaterally or bilaterally in 59/65 patients (91%). The stenoses were frequently annulus and septum, followed by atresia; no twisting was observed .Finally, the number of extracranial venous wall stenoses did not differ significantly in patients treated with immunosuppressant/immunomodulator agents or in never-treated patients .2O higher as compared with the pressure measured in the adjacent superior vena cava of the same subjects ; equally, pressure in the stenotic IJVs was 1.8 cm/H2O higher with respect to the cava .Pressures measured in patients and controls respectively were not significantly different (Mann–Whitney) (superior vena cava 13 (SD 4) vs 13 (4), azygous 16 (7) vs 14 (4), IJVs 14 (4) vs 12 (5)). In contrast, the pressure gradient measured in CDMS across the stenosies was significantly different. For instance, pressure in the stenotic proximal azygous vein was 3.9 cm/HSelective venography enabled us to localise exactly not only the places of venous steno-obstruction, but also, by comparing the flow direction data collected by the ECD-TCCS method, to identify the pathways of venous reflux and substitute collateral circles. In this way it was possible to delineate a picture of chronic cerebrospinal venous insufficiency (CCSVI) associated with MS, for which we found four principal patterns, as shown in 2 test)  . In partIn this study we described the association between MS and the altered modality of venous return determined by extracranial multiple venous strictures. In our controls, venography resembled the normal imaging of extracranial cerebrospinal veins.The mechanism underlying this reflux differs from the reflux caused by incompetence of the jugular valve. In the latter case, valvular insufficiency tested with Valsalva can be related to a picture of transient global amnesia.Substitute circles are alternative pathways or vicarious venous shunts–19The ECD-TCCS protocol was performed once by a single team of investigators , thus not permitting the assessment of the intraobserver and interobserver variability coefficient. This is a limitation of our study because the assessment of the reproducibility of the proposed protocol, although beyond the aim of the present study, certainly deserves further investigation. However, it should be noted that our ultrasonic assessment can be easily performed in the clinical setting, and, despite the operator-dependency of ultrasounds, there is general agreement on the proposed technique.9Our results need to address two main questions:Does CCSVI influence the clinical course of MS?Are venous stenoses the cause or products of MS?First, we identified four main patterns of CCSVI, according to the location, number and association of venous stenosis, and the modality of collateral circulation.We also observed that the PP course was related to a CCSVI pattern significantly different as compared with RR and SP, suggesting that the location of venous obstruction plays a key role in determining the clinical course.–For instance, PP course, characterised by a slowly progressive syndrome with spastic paraparesis and MRI demonstration of MS plaques in the spinal cord,30In contrast, the RR course was significantly associated with type A and B patterns (83% of cases), and particularly to type A, a group in which three-quarters of patients were RR . The morSecond, regarding the causative role of CCSVI in MS, our review of the literature revealed descriptions of associations between the extracranial venous obstructive malformations described herein and disabling neurological pictures however, the latter were defined by these authors generically as myelopathies, without a precise diagnosis and any mention of MS.282O corresponds to a CSA reduction greater than 50%.2O . Currently, only longitudinal clinical and MRI observation in time and space is capable of establishing the possible conversion of a CIS into a CDMS.20


Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV) stage are considered essential for proper maturation or ‘programming’ of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication.We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO) and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation.Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level. Germinal vesicle (GV) breakdown is fundamental for maturation of fully grown, developmentally competent mammalian oocytes. Intercellular communication between oocytes and cumulus cells at GV stage is essential for proper maturation or ‘programming’ of oocytes, which is crucial for fertilization and embryonic development Published studies with mouse model show that cumulus cells play an important role in nutritional support of the developing oocyte in the form of pyruvate Proteins primarily determine cell phenotypes and here we used a shotgun proteomics approach that allows us to relatively quantify which proteins are actually expressed in the cell compartments . This is especially important in oocytes, where there is no linear correlation between amounts of mRNA and the proteins they encode Biological systems utilize highly complex, interrelated networks and pathways to function and in contrast to our previous work We identified 811 and 1,247 proteins in GV oocyte and cumulus cells, respectively . All proBos taurus, had no RefSeq database and so we had to derive a non-redundant protein database  using search terms ‘Bos’ and ‘taurus’. Together this means that any estimate of overlap based on GI or name will be an underestimate. Here we BLAST searched the protein sequences of the smaller current dataset against the larger previous dataset and found an overlap of 439 (26%) (It is complicated to directly compare between the data set in present study with our previous study 39 (26%) .Predicted and hypothetical proteins do not have any functional annotation associated with them and they represent ∼60% of total proteins in our oocyte and cumulus datasets. To compensate for this lack of functional annotation, and to provide the best foundation for biological modeling, we GO-annotated all proteins in our GV oocyte and cumulus data sets. We annotated all the proteins identified in GV oocyte and cumulus cells using GO and obtained annotation for 765 and 1159 proteins of GV oocyte and cumulus cell, respectively. Compared with our previous work, the GAQ score, which is measure of GO annotation quality We next focused on the 371 proteins differentially expressed between GV oocyte and cumulus cells. Application of the AgBase generic GO Slim Our GO based quantitative modeling also showed that GV stage oocytes are biased towards biological processes such as cell cycle regulation, signal transduction, DNA transcription, protein metabolism and modification, generation of precursor metabolites and energy, response to oxidative stress, protein amino acid phosphorylation, and cytoskeletol organization and biogenesis but biased against apoptosis .Analysis of protein-protein interactions as part of complexes, pathways and biological networks is complimentary to analysis of functional annotations and here we used canonical pathway analysis. Among the 811 proteins identified in GV oocytes, 727 proteins had information about their contribution in canonical networks and functions/pathways. We identified 30 canonical networks and 49 functions. Functions of proteins involved in the top five networks were related to protein synthesis, DNA replication, recombination and repair, cell-to-cell signaling and interaction, molecular transport, amino acid metabolism, reproductive system development and function, small molecule biochemistry, and cellular function and maintenance. Of the 1247 proteins identified in cumulus cells, 1114 had information about their contribution in canonical genetic networks and functions/pathways, respectively. We identified 46 networks and 50 functions. Functions of proteins involved in top five networks were related to cell-to-cell signaling and interaction, molecular transport, protein synthesis, nucleic acid metabolism, cellular function and maintenance, small molecule biochemistry, molecular transport, RNA trafficking and post translational modification. The top five networks , and their associated proteins are shown in Twenty-three and 39 canonical pathways were significantly represented in GV oocytes and cumulus cells, respectively. Oxidative phosphorylation is the top represented canonical pathway significant only in cumulus cells. In this pathway, expression of 21 proteins was significantly altered and all these proteins have higher expression in cumulus cells compared to GV oocyte. These proteins include 9 ATP (adenosine triphosphate) synthases , 3 cytochrome-c-oxidases , cytochrome C-1(CYC1), 6 NADH (reduced nicotinamide adenine dinucleotide) dehydrogenase (ubiquinone) complexes  and 2 ubiquinol cytochrome c reductase core proteins (UQCRC1 and UQCRC2) . GlycolyBidirectional communication between oocytes and cumulus cells is essential for the development and function of both cell types Here we provide a foundational, proteomics-based descriptive computational systems-biology modeling of oocyte and cumulus cell interaction at the GV stage. Bidirectional communication between oocytes and cumulus cells is considered essential for proper maturation or ‘programming’ of oocytes, which is crucial for normal fertilization and embryonic development In this study, we identified 811, and 1247 proteins of oocyte, and cumulus cells respectively, which is a significant increase in information compared to other female gametal proteomic studies This current work is based on a separate previous proteomics study Nalp5,), zona pellucid proteins-2, 3 and 4 , growth differentiation factor 9 (GDF 9), beta-actin (ACTB), heat shock protein-70 (HSP-70), peroxiredoxins 1 and 2 , DNA (cytosine-5) methyl transferase one (DNMT1), and fibroblast growth factor-8 (FGF-8) all of which are important to oogensis and early embryonic development Maternal effect proteins are the proteins that are present in the oocyte, remain expressed throughout oocyte maturation and early embryonic development until maternal-to-embryonic transition (MET) and they may also be required for successful activation of the embryonic genome. We identified 11 maternal effect proteins in bovine oocytes: Maternal Antigen that Embryos Require . By manual literature biocuration, we also captured the quantitative positive and negative regulatory roles that these GV oocyte proteins play in the various biological processes. Overall we could identify and quantify proteins agonistic or antagonistic to biological processes including signal transduction, cell cycle regulation, DNA transcription, apoptosis regulation, protein metabolism and modification, generation of precursor metabolites and energy, cytoskeleton organization and biogenesis, and response to stress in GV oocyte; all of which may have putative roles in oocyte competence and maturation. This type of quantitative modeling is not possible with the available pathway analysis tools. Furthermore, we have analyzed the protein datasets identified in this experiment in the context of protein interaction networks and pathways using Ingenuity Pathway Analysis. This pathway and network modeling also reveals signaling pathways associated with the proteins involved in cell-to-cell signaling biological process which may have implications in various reproductive processes such as oocyte development, and maturation.Our previous publication described an initial GO-based functional analysis of bovine GV oocyte and cumulus cells using ‘known proteins’ and focusing on the cellular component Gene Ontology to identify membrane and nuclear proteins. Here, we take a much more comprehensive approach to include not only ‘known’ but also ‘predicted’ and ‘hypothetical’ proteins. By definition, predicted and hypothetical proteins do not have any functional annotation associated with them and they represent ∼60% of total proteins in our oocyte and cumulus datasets. Hence we manually annotated all these predicted and hypothetical proteins based on sequence similarity and orthology from human, mouse and rat proteins. Overall our approach, while much more laborious and requiring recognized GO biocuration skills, provides more comprehensive information on the biological processes associated with bovine GV oocyte and cumulus cell function. Not only does this improved data quality allow a much better modeling, but we almost doubled the Gene ontology Annotation Quality (GAQ) score Our GO-based modeling identified that the percentage of proteins involved in translation in GV oocytes is twice as high as in cumulus cells and this is consistent with other's data showing that GV oocytes are transcriptionally and translationally active and also that proteins synthesized in this stage might be crucial to achieve maturation and successful subsequent development Cumulus cells provide a network of gap junction transmembrane channels facilitating two way communications for nutrient or paracrine factor exchange between oocytes and cumulus cells Cumulus cells are essential for protecting oocytes from oxidative stress-induced apoptosis and DNA damage and their communication with oocytes is essential for development of an oocyte competent to undergo fertilization and embryogenesis In addition to integrin and actin cytoskeleton signaling pathways, we also identified MAPK signaling, PI3K signaling, and ephrin receptor signaling pathways may involved in bidirectional oocyte-cumulus cell communication. MAPK and PI3K signaling are involved in activation of several membrane signaling molecules followed by sequential activation of protein kinases which involves highly regulated and modulated cascades of phosphorylation events We also observed expression of peroxiredoxins (PRDX's) involved in cell-to-cell signaling was significantly higher in oocytes compared to cumulus cells. PRDX's are peroxidases involved in antioxidant defence and intracellular signaling. Cumulus cells induce PRDX up regulation in oocytes In conclusion, our systems biology modeling of bovine oocyte and cumulus proteomes, in the context of gene ontology and canonical protein interaction networks identified ninty-one proteins involved in the cell-to-cell signaling biological process that may have role in bidirectional communication between oocytes and cumulus cells. Most of the proteins involved in the cell-to-cell signaling biological process are the components of integrin, actin cytoskeleton, MAPK and PI3K signaling pathways that may have implications in various reproductive processes such as oocyte development, and maturation. In addition, GO-based analysis of differentially expressed proteins facilitated the identification that compared to GV oocytes, cumulus cells have higher expression of proteins involved in the generation of precursor metabolites and energy, transport, and cell communication. This systems biology model of bovine oocytes and cumulus cell potential interacting proteomes not only provides a foundation for understanding signaling and cell physiology at the GV stage of oocyte development, but is also valuable for comparative studies of other stages of oocyte development at the molecular level. The proteomes and biological process identified in this study can also serve as reference points for further comparative studies on immature and abnormal oocyte and cumulus cells elucidating the underlying molecular mechanisms involved in normal and pathological oocyte-cumulus cell communication. Furthermore, some of the proteins involved in cell-to-cell signaling may have value as molecular biomarkers which could be useful for assessing oocyte quality.Separate from our previous work DDF sequentially extracts proteins from different cellular compartments using a series of detergents and provides information on cell location as well as increasing proteome coverage. Three replicates of five hundred GV oocytes and their surrounding CC were each subjected to DDF exactly as previously described Protein quantification and trypsin digestion of DDF fractions were done exactly as described Two-dimensional liquid chromatography (LC) analysis was accomplished by strong cation exchange (SCX) followed by reverse phase (RP) coupled directly in line with an electrospray ionization (ESI) ion trap tandem mass spectrometer  essentially as described in in silico trypsin-digested bovine database using TurboSEQUEST™ . The bovine database used in this study was different from the previous publication and here we used database of bovine Reference Sequence (RefSeq) proteins downloaded from the National Center for Biotechnology Institute . The Reference Sequence (RefSeq) collection aims to provide a comprehensive, integrated, non-redundant, and well-annotated set of sequences of proteins 2) mass tolerance was set to 1.0 Da. Rsp Value less than 5.Mass spectra and tandem mass spectra were searched against an As a primary filter we first limited our Sequest search output to include only peptides ≥6 amino acids long, with ΔCn ≥0.1 and Sequest cross correlation (Xcorr) scores of 1.9, 2.2, and 3.7 for +1, +2, and +3 charge states, respectively. We next used a decoy database search strategy ProtQuantProtQuant is a java based tool for label-free quantification that uses a spectral counting method with increased specificity . This increased specificity is achieved by incorporating the quantitative aspects of the Sequest cross correlation (XCorr) into the spectral counting method. ProtQuant also computes the statistical significance of differential expression by one-way ANOVA (α≤0.05).We used an isotope-free quantification method GORetriver to obtain all existing GO annotations available for known proteins in our datasets anna by manually checking the similarity between our proteins and orthologs GOSlim viewerstudent's t-test. Differences at p<0.05 were considered statistically significant.To identify the biological processes of all proteins in our datasets we used Gene Ontology annotations and the GO resources and tools available at AgBase GAQ) scores were calculated exactly as described GAQ score is a quantitative measure of the GO annotation of a set of gene products based on the number of GO annotations available, the level of detail of the annotation and the types of evidence used to make these GO annotations.GO annotation quality  terms defining the phenotype of oocyte competence and maturation. Although GOBP terms exists for gene products that effect and affect oocyte competence and maturation, and there is functional literature on these genes, the literature and GO are unconnected To gain insights into the biological pathways and networks that are statistically significantly represented in our proteomic datasets we used Ingenuity Pathways Analysis . We imported protein accessions from our GV oocyte and cumulus cell datasets into IPA. IPA selects “focus proteins” to be used for generating biological networks. Focus proteins are the proteins from our datasets that are mapped to corresponding gene objects in the Ingenuity Pathways Knowledgebase (IPKB) and are known to interact with other proteins based on published, peer reviewed content in the IPKB. Based on these interactions IPA builds networks with a size of no more than 35 genes or proteins. A p-value for each network and canonical pathway is calculated according to the fit of the user's set of significant genes/proteins. IPA computes a score for each network from the p-value that indicates the likelihood of the focus proteins in a network being found together due to chance. We selected only networks scoring ≥2, which have >99% confidence of not being generated by chance Figure S1Comparison of proteins identified in present study with previous published study by Memili et al., 2007.(0.01 MB PDF)Click here for additional data file.Table S1List of proteins identified in germinal vesicle (GV) stage oocyte and cumulus cells. Proteins identified by DDF-MudPIT and their distribution in GV oocyte and cumulus cells. GI numbers of the identified proteins (column A) and corresponding protein names . Protein distribution in GV oocyte or cumulus cells or common to both . Numbers of peptides and Sequest cross correlation scores (ΣXcorr) in columns D, F and E, G for oocyte and cumulus cells respectively.(0.29 MB XLS)Click here for additional data file.Table S2List of differentially expressed proteins in cumulus cells compared with GV oocytes. Gene name, numbers of peptides, Sequest cross correlation (ΣXcorr) score and P value for of each differentially expressed protein in GV oocyte and cumulus cells.(0.10 MB XLS)Click here for additional data file.Table S3Distribution of GV oocyte and cumulus cell proteins by subcellular compartments. The classification of the identified GV oocyte and cumulus proteins among subcellular compartments was performed using cellular component GO annotations.(0.02 MB XLS)Click here for additional data file.Table S4Proteins involved in cell-to-cell signaling biological process.(0.04 MB XLS)Click here for additional data file.


Hox genes are transcribed sequentially, in both time and space, following their relative positions within their genomic clusters. Analyses of animal genomes support the idea that Hox gene clustering is essential for coordinating the various times of gene activations. However, the eventual collinear ordering of the gene specific transcript domains in space does not always require genomic clustering. We analyzed these complex regulatory relationships by using mutant alleles at the mouse HoxD locus, including one that splits the cluster into two pieces. We show that both positive and negative regulatory influences, located on either side of the cluster, control an early phase of collinear expression in the trunk. Interestingly, this early phase does not systematically impact upon the subsequent expression patterns along the main body axis, indicating that the mechanism underlying temporal collinearity is distinct from those acting during the second phase. We discuss the potential functions and evolutionary origins of these mechanisms, as well as their relationship with similar processes at work during limb development.During development of the vertebrate body axis,  Hox genes encode proteins that control embryonic development along the head-to-tail axis. These genes are clustered in one site on the chromosome and their respective positions within the cluster determine their time and place of activation. Here, by using a large set of targeted mutations disturbing the integrity of the gene cluster, we show that the spatial organization of expression domains does not directly depend upon the timing of activation as was previously suggested. This uncoupling between space and time in the regulation of these Hox genes coincides with the existence of two major phases of regulation. The first is time-dependent and involves global regulatory influences, located outside the gene cluster, whereas the second relies upon more local regulatory elements, likely interspersed between the genes, inside the cluster. These results provide the bases for future analyses of collinear mechanisms and indicate that different types of collinearities are not necessarily related, neither in function, nor in their evolutionary histories. Hox genes play essential roles in patterning during the development of metazoans. In many species, they are found clustered in the genome, such as in vertebrates, which contain four Hox gene clusters (HoxA to HoxD), due to the additional two rounds of genome amplification that accompanied their emergence from early chordates. These genes are required to confer regional identities along the rostral to caudal body axis, a task that mostly depends upon particular combinations of HOX proteins found at a given anterior-posterior level, since genes of all four clusters are expressed in largely overlapping domains Hox gene activation during development follows the gene sequence, such that genes lying at one extremity of a cluster are activated earlier and more rostrally than genes located near the other extremity Hoxd genes thus become activated in the most posterior part of the embryo between late embryonic day 7.75 (E7.75) for Hoxd1 and early E9 for Hoxd13. This temporal progression was proposed to be a molecular clock (the ‘Hox clock’) controlling the proper timing of axial specification by coordinating the rostral-caudal positions of the various expression boundaries Hox gene expression in the incipient limb bud and the extent of expression along the anterior to posterior axis These regionalized expression domains are in part controlled at a transcriptional level, by using an intrinsic property of the gene clusters, conserved from insects to vertebrates and referred to as spatial collinearity Hox transgenes could be expressed with rather faithful anterior boundaries, yet not necessarily with the exact expression timing. Secondly, targeted Hox cluster modifications in vivo, which changed the timing of activation, induced patterning problems even without modification of the late expression boundaries Hox genes are not clustered such as the larvacean Oikopleura First, it was noticed early on Hox gene activation and their expression territories have been best documented in developing limbs  locus, about 3 megabases (Mb) centromeric to HoxD. The inversion separates the most ‘posterior’ part of the cluster  along with the adjacent 5′ region, from the rest (Hoxd10 to Hoxd1), thus allowing to evaluate the importance of regulatory influences associated with either the telomeric , where premature activation of al cells . As for e embryo , the mute embryo . We concHoxd9 and Hoxd11 was largely anteriorized, whenever the adjacent DNA was deleted up to the Hoxd4 locus (Hoxd9 was clearly anteriorized in the Del(i-8) when placed near Hoxd4 (Hoxd10 showed a wild type expression pattern in the same deletion (Hoxd11 was expressed too anteriorly in Del(i-10) mutant embryos, the ectopic domains recapitulating Hoxd4 specific domains  allele  but removed from Del(i-10), likely isolated Hoxd11 from enhancers located around Hoxd4. Interestingly, these two deleted alleles displayed similar timing of premature activations ) did not elicit the same response, even though the relative position of ) allele . In thisions see . Therefo tailbud . More im tailbud . Altogetvia a deletion were consistently down-regulated, as exemplified by Hoxd3, Hoxd4 and Hoxd9 (Hoxd3 in the Del(i-10) and Del(8-9)  . Repress noticed . Likewiscription  recalledHoxd4 in the mesoderm at later stages that became more pronounced the closer the gene was brought to the centromeric extremity  is (are) present, it may be one of the major constraints that kept Hox genes together. The analyses of additional animal species will be informative in this respect.The respective functional contribution of each phase of activation to the primary body axis is unclear. In the mouse embryo, while the necessity to establish correct expression boundaries has been largely documented through various genetic approaches, the function of the early temporal sequence of activation is less explicit. Because this temporal process has been thus far associated only with animals where (an) integral Hox gene(s) too early, a situation detrimental to embryonic development. This is suggested both by the early lethality associated with the inversion of the complete HoxD cluster, Hoxd13 becoming activated at the expected time for Hoxd1Hoxd10 and Hoxd9 in the split cluster (this work). Whichever mechanism evolved to prevent the most posterior gene(s) to be expressed too early may have incidentally generated a graded timescale for those genes located in between and hence this series of genes is transcribed following their genomic order, without any particular functional relevance in itself.Both instructive and restrictive contexts can be considered (non-exclusively) when looking for the ‘raison d'être’ of temporal collinearity. In the former, a need exists for a precise time-sequence in the transcriptional activation of these genes and important direct functional outputs of this process may occur, perhaps at a time and in a cellular population that have so far escaped our analyses. An example of such an early mechanism is the observed delay in ingression, during gastrulation, of epiblast cells containing abnormal combinations of HOX proteins vice versaHox expression domains found in animals having lost this developmental time sequence, such as in diptera, may have been progressively taken over by different regulatory mechanisms, disconnecting space from time (such as gap genes).The question as to which type of collinearity evolved first, i.e. whether the time-sequence preceded the spatial organization of the expression domains, or Hox gene cluster still show reminiscences of spatial collinearity, suggesting that the timing mechanism was built on the top of an already constrained gene cluster. Altogether, we consider it unlikely that an animal species will ever be found, which contains a broken Hox gene cluster, develops following a simultaneous segmentation process and implements temporal collinearity. Accordingly, any species displaying a clear time sequence in the ontogeny of its metameric aspect should have an intact Hox cluster, associated with a transcriptional time-sequence. Also, it should not be taken for granted that the ancestral Hox gene collinear function will still be found in extant animal species. Different collinear mechanisms can co-exist with one another and the implementation of a collinear regulation may have paved the way for its replacement by a more efficient strategy. For example, a mere distance effect to a remote enhancer could set up a time sequence in the appearance of transcripts encoded by contiguous genes, a situation selected due to a particular adaptive value. Once in place, this genomic topology may facilitate the evolution of yet a different progressive regulation, for example the spreading of chromatin modifications. Over time, the accumulation of such secondary mechanisms could take over the initial constraint for these genes to remain clustered, making it possible for an ancestral mechanism to turn into a fossil regulation and disappear from this particular phylogenetic branch.The evidence is compelling, however, that even animals containing an atomized Itga6-HoxDrVIII) was obtained by sequential targeted recombination , Del(4-13) and Del(1-13) were produced using loxP/Cre mediated site-specific recombination in ES cells in vivo using targeted meiotic recombination  allele, were described previously: The inversion allele Inv was performed according to standard protocols, with both mutant and control embryos processed in the same well to maintain identical conditions throughout the procedure. Probes were as before: Hoxd3Hoxd4Hoxd8Hoxd9Hoxd10 and Hoxd11Hoxd12Hoxd13Noon on the day of the vaginal plug was considered as E0.5. Embryos were dissected in PBS and fixed from 4 h to overnight in 4% PFA. Whole mount Figure S1HoxD cluster. Newborn animals were processed and stained for bone  and cartilage tissues . (A) Incidence of different lumbar vertebral formulae in wild-type, heterozygous and homozygous mutant animals. L5/6 and L6/7 indicate unilateral transformations of the first sacral vertebrae. (B) Complete transformation of the first sacral vertebra into a lumbar identity (S1>L7) in a homozygous mutant (right), as compared to the L6 formula observed in wild-type specimen (left) (C) Misalignment of the first rib to the sternum (#) and fusions of sternebrae four and five (*) in a homozygous mutant. (D) Seventh cervical vertebrae (C7) of heterozygous and homozygous animals showing ectopic bony material protruding from the transverse processes (arrowheads).Phenotypic alterations in the axial skeleton of mice with a split (1.45 MB TIF)Click here for additional data file.


Similarly, several other small-molecule inhibitors—including imatinib, dasatinib, and nilotinib—have been approved for the treatment of chronic myelogenous leukemia (cml). The combination of these targeted agents is an area of intense clinical investigation. Here, we describe a patient diagnosed with mrcc while on imatinib therapy for cml. Treatment of this patient with the combination of bevacizumab and imatinib led to a 6-month period of stable disease, with no treatment-related adverse events. More extensive clinical exploration of this combination of agents may therefore be warranted.Numerous therapeutic options have been introduced for metastatic renal cell carcinoma (m The patient subsequently underwent a right-sided nephrectomy, and pathology analysis of the surgical specimen revealed a 5-cm Fuhrman grade 2 clear-cell-type renal cell carcinoma. Imaging by ct of the chest, abdomen, and pelvis at that time showed no evidence of distant disease, and the patient was followed clinically by her urologist.Upon abdominal computed tomography (cml), and the patient was initiated on therapy with imatinib. The patient tolerated therapy with minimal adverse effects, and within 3 months of therapy initiation, she had mounted a complete cytogenetic response by peripheral blood Bcr–Abl mutation screening.Roughly 2 years later, the patient presented to her primary care practitioner with vague complaints of fatigue and lethargy. Initial workup included a complete blood count, remarkable for a pronounced leucocytosis . Subsequent bone marrow examination was consistent with a diagnosis of chronic myelogenous leukemia  represent two disorders whose therapies have been revolutionized by the advent of targeted agents. The natural history of cml has been drastically altered by the Bcr–Abl tyrosine kinase inhibitor (tki) imatinib. Use of imatinib has been validated with 5-year follow-up data from a study of 1106 patients randomized to either imatinib or interferon alfa plus cytarabine. In that study, a complete cytogenetic response was obtained in 69% of patients by 12 months and in 87% of patients by 60 monthsChronic myelogenous leukemia and metastatic renal cell carcinoma  of 13.1 monthsiii trial of sunitinib, an oral tki targeting the vascular endothelial growth factor receptor (vegfr) and platelet-derived growth factor receptor (pdgfr)rcc, an improvement in progression free survival (pfs) was observed with sunitinib , paralleled by improvements in quality of lifeEmerging data also suggest the utility of a targeted approach to the treatment of mtki with inhibitory activity against vegfr, pdgfr, Flt-3, and c-Kit) or placebo in such patients, a benefit in time to progression was observed with sorafenib pfs; moreover, temsirolimus is the first of the targeted therapies for mrcc to also improve os A distinct targeted approach has been used for patients refractory to standard therapy. In a trial using the agent sorafenib  therapy for mrcc has been evaluated in other recent studies. Bevacizumab, a mAb with specificity for vegfr, was assessed in a phase iiitrial comparing the combination of bevacizumab and interferon alfa with interferon alfa and placebo. In 649 randomized patients, a significant improvement in response rate was observed with the addition of bevacizumab , with most of responses being characterized as partial. Median pfs was improved to 10.2 months from 5.4 months with the addition of bevacizumab , and at the time of this (interim) analysis, median os in the bevacizumab-containing arm had not been reached 9.Beyond small-molecule ii trial of first-line treatment of mrcc with bevacizumab and erlotinib as compared with bevacizumab and placebo. In 104 patients, the combination of bevacizumab and erlotinib did not significantly improve pfs . Interestingly, median os was not reached in the bevacizumab monotherapy group, but a median os of 20 months was noted with bevacizumab and erlotinibThe combination of bevacizumab and imatinib exclusively has not been reported in the existing literature, but a recent trial tested these agents in combination with erlotinib. That study built on data from a randomized phase i/ii trial that assessed bevacizumab and erlotinib in combination with imatinib demonstrated a median pfs of 8.9 months and a median os of 20 months, thus mirroring the data obtained for single-agent therapy with bevacizumab. However, the addition of imatinib led to a marked increase in reported grades 3 and 4 toxicities, including diarrhea, rash, and fatigue A separate phase pdgfr, and c-Kitpdgfr is of particular relevance to rcc and is a demonstrated target of sunitinib (together with vegfr and Flt-3)The case described the present report represents a signal of activity for the combination of bevacizumab and imatinib exclusively. To date, no trials for this specific combination of agents are being conducted, although there are theoretical merits for the combination. Imatinib is an inhibitor of multiple tyrosine kinases, including Abl, Bcr–Abl, rcc suggested that immunohistochemical staining for pdgfr is a statistically significant prognostic factor for os on multivariate analysis rcc overproduces pdgfd, a recently discovered member of the pdgf family. Transfection of pdgfd in human rcc cell lines implanted in mice with severe combined immunodeficiency led to increased cellular proliferation and migration. Inhibition of pdgfr β activity by imatinib led to a decline in tumour growth, suggesting a therapeutic role of imatinib in rcc 13.A retrospective analysis of 64 patients undergoing partial or radical nephrectomy for vegf. In vitro and in vivo assays have both demonstrated the role of vegf as a mitogen and angiogenesis inducer; bevacizumab has been shown in experimental models to reduce the growth rate of tumours, with decreased vessel density in bevacizumab-treated specimens Imatinib may target the kinase domain of cell surface receptors, but bevacizumab acts in a unique manner—through specific binding to i/ii trial combining bevacizumab, erlotinib, and imatinib should not discourage further exploration of the combination of bevacizumab and imatinib exclusively. The excessive toxicity incurred with the addition of imatinib could be a result of overlapping downstream targets: although imatinib and erlotinib inactivate phosphorylation at distinct tkis, the result is inhibition of common mitogenic pathways such as the pi3k–Akt signalling cascade Results from the phase No research funding was applied toward the effort reported here, and this research has not been previously presented.


Evidence-based recommendations can be made with respect to many aspects of the acute management of the bleeding trauma patient, which when implemented may lead to improved patient outcomes.The multidisciplinary Task Force for Advanced Bleeding Care in Trauma was formed in 2005 with the aim of developing guidelines for the management of bleeding following severe injury. Recommendations were formulated using a nominal group process and the GRADE  hierarchy of evidence and were based on a systematic review of published literature.Key recommendations include the following: The time elapsed between injury and operation should be minimised for patients in need of urgent surgical bleeding control, and patients presenting with haemorrhagic shock and an identified source of bleeding should undergo immediate surgical bleeding control unless initial resuscitation measures are successful. A damage control surgical approach is essential in the severely injured patient. Pelvic ring disruptions should be closed and stabilised, followed by appropriate angiographic embolisation or surgical bleeding control, including packing. Patients presenting with haemorrhagic shock and an unidentified source of bleeding should undergo immediate further assessment as appropriate using focused sonography, computed tomography, serum lactate, and/or base deficit measurements. This guideline also reviews appropriate physiological targets and suggested use and dosing of blood products, pharmacological agents, and coagulation factor replacement in the bleeding trauma patient.A multidisciplinary approach to the management of the bleeding trauma patient will help create circumstances in which optimal care can be provided. By their very nature, these guidelines reflect the current state-of-the-art and will need to be updated and revised as important new evidence becomes available. Traumatic injury is the leading cause of death worldwide among persons between 5 and 44 years of age  and accoResuscitation of the trauma patient with uncontrolled bleeding requires the early identification of potential bleeding sources followed by prompt action to minimise blood loss, to restore tissue perfusion, and to achieve haemodynamic stability. Massive bleeding in trauma patients, defined here as the loss of one blood volume within 24 hours or the loss of 0.5 blood volumes within three hours, is often caused by a combination of vascular injury and coagulopathy. Contributing factors to traumatic haemorrhage include both surgical and non-surgical bleeding, prior medication, comorbidities, and acquired coagulopathy .Here, we describe early diagnostic measures to identify haemorrhage that should trigger surgical or radiological interventions in most cases. Specific interventions to manage bleeding associated with pelvic ring injuries and hypothermia are discussed, as well as recommendations for the optimal application of fluid, pharmacological, blood product, and coagulation factor therapy in trauma patients.These guidelines for the management of the bleeding trauma patient were developed by a multidisciplinary group of European experts and designated representatives from relevant professional societies to guide the clinician in the early phases of treatment. The recommendations presented here are based on a critical survey of the published literature and were formulated according to a consensus reached by the author group. Many of the critical issues faced by the treating physician have not been, and for ethical or practical reasons may never be, addressed by prospective randomised clinical studies, and therefore the formulation and grading of the recommendations presented here are weighted to reflect both this reality and the current state-of-the-art.These recommendations were formulated and graded according the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) hierarchy of evidence outlined by Guyatt and colleagues  and are Boolean operators and Medical Subject Heading (MeSH) thesaurus keywords were applied as a standardised use of language to unify differences in terminology into single concepts. Appropriate MeSH headings and subheadings for each question were selected and modified based on search results. The scientific questions posed that led to each recommendation and the MeSH headings applied to each search are listed in Additional file The selection of the scientific inquiries to be addressed in the guideline, screening, and grading of the literature to be included and formulation of specific recommendations were performed by members of the Task Force for Advanced Bleeding Care in Trauma, a multidisciplinary, pan-European group of experts with specialties in surgery, anaesthesia, emergency medicine, intensive care medicine, and haematology. The core group was formed in 2004 to produce educational material on care of the bleeding trauma patient , on whicIn the GRADE system for assessing each recommendation, the letter attached to the grade of recommendation reflects the degree of literature support for the recommendation, whereas the number indicates the level of support for the recommendation assigned by the committee of experts. Recommendations are grouped by category and somewhat chronologically in the treatment decision-making process, but not by priority or hierarchy.Evidence to support the initial phase of resuscitation and prevention of further bleeding is lacking, and there have been few studies on the effect of coagulopathy on outcome. Patients with a coagulopathic condition have worse outcomes than patients of the same injury severity without a clotting disturbance ,14, and There is no evidence as to whether the degree of initial bleeding affects coagulopathy. Coagulopathy is predicted by a systolic blood pressure of below 70 mm Hg , but thiThere is evidence to support expedient care for patients following traumatic injury; however, no study has examined the relationship between outcomes in patients transported to different types of hospital facilities and the amount of bleeding. Pre-hospital bleeding not controlled by compression and splintage requires rapid surgical or radiological intervention.We recommend that the time elapsed between injury and operation be minimised for patients in need of urgent surgical bleeding control (grade 1A).in extremis or with persistent haemodynamic instability also favoured early surgical intervention to stabilise a pelvic fracture or to surgically control bleeding.Trauma patients in need of emergency surgery for ongoing haemorrhage demonstrate better survival if the elapsed time between the traumatic injury and admission to the operating theatre is minimised -21. AlthIn addition, studies of different trauma systems indirectly emphasise the importance of minimising the time between initial care and surgery for those with signs of exsanguination or ongoing severe haemorrhage. Hill and colleagues  observedUpon patient arrival in the emergency room, an initial clinical assessment of the extent of bleeding should be employed to identify patients at risk of coagulopathy.We recommend that the extent of traumatic haemorrhage be clinically assessed using a grading system such as that established by the American College of Surgeons (ACS) (grade 1C).An evaluation of the mechanism of injury  is a useful tool for determining which patients are candidates for surgical bleeding control. Table We do not suggest hyperventilation or the use of excessive positive end-expiratory pressure (PEEP) when ventilating severely hypovolaemic trauma patients (grade 2C).There is a tendency for rescue personnel to hyperventilate patients during resuscitation ,28, and We recommend that patients presenting with haemorrhagic shock and an identified source of bleeding undergo an immediate bleeding control procedure unless initial resuscitation measures are successful (grade 1B).The source of bleeding may be immediately obvious, and penetrating injuries are more likely to require surgical bleeding control. In a retrospective study of 106 abdominal vascular injuries, all 41 patients arriving in shock following gunshot wounds were candidates for rapid transfer to the operating theatre for surgical bleeding control . A similIn blunt trauma, the mechanism of injury can determine to a certain extent whether the patient in haemorrhagic shock will be a candidate for surgical bleeding control. Only a few studies address the relationship between the mechanism of injury and the risk of bleeding, however, and none of these publications is a randomised prospective trial of high evidence. We have found no objective data describing the relationship between the risk of bleeding and the mechanism of injury of skeletal fractures in general or of long-bone fractures in particular.Traffic accidents are the leading cause of pelvic injury. Motor vehicle crashes cause approximately 60% of pelvic fractures followed by falls from great height (23%). Most of the remainder result from motorbike collisions and vehicle-pedestrian accidents ,33. TherWe recommend that patients presenting with haemorrhagic shock and an unidentified source of bleeding undergo immediate further assessment (grade 1B).A patient in haemorrhagic shock with an unidentified source of bleeding should undergo urgent clinical assessment of chest, abdominal cavity, and pelvic ring stability using focused abdominal sonography in trauma (FAST) assessment of thorax and abdomen and/or computerised tomography (CT) examination in the shock room.We recommend early FAST for the detection of free fluid in patients with suspected torso trauma (grade 1B).We recommend that patients with significant free intra-abdominal fluid according to sonography (FAST) and haemodynamic instability undergo urgent surgery (grade 1C).n = 2 with penetrating injuries) during a four year period. These findings were confirmed by Richards and co-workers [Blunt abdominal trauma represents a major diagnostic challenge and an important source of internal bleeding. FAST has been established as a rapid and non-invasive diagnostic approach for detection of intra-abdominal free fluid in the emergency room ,37. Larg-workers  in a fou-workers . Liu and-workers  conducte-workers .The hypotensive patient (systolic blood pressure below 90 mm Hg) presenting free intra-abdominal fluid according to FAST is a potential candidate for early surgery if he or she cannot be stabilised by initiated fluid resuscitation, according to a retrospective study of 138 patients by Farahmand and colleagues . A similA number of patients who present free intra-abdominal fluid according to FAST can safely undergo further investigation with multi-slice spiral computed tomography (MSCT). Under normal circumstances, adult patients need to be haemodynamically stable when MSCT is performed outside of the emergency room. In the retrospective study of 1,540 patients  who were assessed early with FAST, a successful non-operative management was achieved in 24 48%) of the 50 patients who were normotensive on admission and had true positive sonographic examinations. These results justified an MSCT scan of the abdomen rather than an immediate exploratory laparotomy % of the  also conWe recommend that haemodynamically stable patients with suspected head, chest, and/or abdominal bleeding following high-energy injuries undergo further assessment using CT (grade 1C).The increasing role of MSCT in the imaging concept of acute trauma patients is well documented -55. The Using modern 16-slice CT scanners, total whole-body scanning time amounts to approximately 120 seconds. Sixty-four-slice CT scanners may reduce scanning time to less than 30 seconds. In a retrospective study comparing 370 patients in two groups, Weninger and colleagues  showed tIf an MSCT is not available in the emergency room, the realisation of CT scanning implies transportation of the patient to the CT room, and therefore the clinician must evaluate the implications and potential risks and benefits of the procedure. According to established standards, such as those developed by the ACS, only the haemodynamically stable patient should be considered for CT scanning. During transport to the MSCT and imaging, all vital signs should be closely monitored and resuscitation measures continued.For those patients in whom haemodynamic stability is questionable, imaging techniques such as ultrasound and chest and pelvic radiography may be useful. Peritoneal lavage is rarely indicated if ultrasound or CT is available . TransfeWe do not recommend the use of single Hct measurements as an isolated laboratory marker for bleeding (grade 1B).Hct measurements are part of the basic diagnostic work-up for trauma patients. The diagnostic value of the Hct for detecting trauma patients with severe injury and occult bleeding sources has been a topic of debate in the past decade -61. A man = 21) compared to trauma patients without serious injuries (n = 39). Whereas a decrease in Hct of more than or equal to 6.5% at 15 and 30 minutes had a high specificity (0.93 to 1.0) for a serious injury, the sensitivity for detecting severely injured patients was very low (0.13 to 0.16) [Two prospective observational diagnostic studies determined the sensitivity of serial Hct measurements for detecting patients with severe injury ,60. Parato 0.16) . The autto 0.16) . ZehtabcWe recommend serum lactate measurement as a sensitive test to estimate and monitor the extent of bleeding and shock (grade 1B).n = 25) were excluded from the study. The remaining 76 patients were analysed with respect to the time of serum lactate normalisation compared between survivors and non-survivors who died after 48 hours [Serum lactate has been used as a diagnostic parameter and prognostic marker of haemorrhagic shock since the 1960s . The amo48 hours . Surviva48 hours . These f48 hours . The aut48 hours . Togethe48 hours ,72.We recommend base deficit as a sensitive test to estimate and monitor the extent of bleeding and shock (grade 1C).Base deficit values derived from arterial blood gas analysis provide an indirect estimation of global tissue acidosis due to impaired perfusion ,68. SiegAlthough both the base deficit and serum lactate levels are well correlated with shock and resuscitation, these two parameters do not strictly correlate with each other in severely injured patients . TherefoWe recommend that patients with pelvic ring disruption in haemorrhagic shock undergo immediate pelvic ring closure and stabilisation (grade 1B).We recommend that patients with ongoing haemodynamic instability despite adequate pelvic ring stabilisation receive early angiographic embolisation or surgical bleeding control, including packing (grade 1B).Markers of pelvic haemorrhage include anterior-posterior and vertical shear deformations, CT 'blush' , bladder compression pressure, pelvic haematoma volumes greater than 500 ml evident by CT, and ongoing haemodynamic instability despite adequate fracture stabilisation -85. InitResults of surgery to control pelvic haemorrhage via laparotomy have remained poor due to the existence of an extensive collateral circulation. However, in suboptimal situations , extraperitoneal packing of the pelvis may reduce the loss of blood. Extraperitoneal haemorrhage in patients with haemorrhagic shock and pelvic ring disruption may be attributed to ruptured veins, fracture surfaces, and/or arterial sources. The overall mortality rate of patients with severe pelvic ring disruptions and haemodynamic instability remains as high as 30% to 45% . AngioemWe recommend that early bleeding control be achieved by packing, direct surgical bleeding control, and the use of local haemostatic procedures. In the exsanguinating patient, aortic cross-clamping may be employed as an adjunct to achieve bleeding control (grade 1C).The choice of thoracic or abdominal aortic clamping should be determined according to the site of bleeding, available surgical skill, and speed. The patient in haemorrhagic shock in whom immediate aortic cross-clamping is warranted is characterised by an injury to the torso and the severity of the blood loss and shock. The hypotensive state will not respond to the intravenous resuscitation and may lead to cardiac arrest. The cause of injury is predominantly penetrating . Depending on the cause of injury, the mortality rate in these situations is extremely high ,19,95. HThe cross-clamping of the aorta should be considered as an adjunct to other initial haemorrhage control measures such as the evacuation of blood, direct surgical bleeding control, or packing of bleeding sources . When aoWe recommend that damage control surgery be employed in the severely injured patient presenting with deep haemorrhagic shock, signs of ongoing bleeding, and coagulopathy. Additional factors that should trigger a damage control approach are hypothermia, acidosis, inaccessible major anatomic injury, a need for time-consuming procedures, or concomitant major injury outside the abdomen (grade 1C).The severely injured patient arriving to the hospital with continuous bleeding or deep haemorrhagic shock generally has a poor chance of survival unless early control of bleeding, proper resuscitation, and blood transfusion are achieved. This is particularly true for patients who present with uncontrolled bleeding due to multiple penetrating injuries as well as patients with multiple injuries and unstable pelvic fractures with ongoing bleeding from fracture sites and retroperitoneal vessels. The common denominator in these patients is the exhaustion of physiological reserves with resulting profound acidosis, hypothermia, and coagulopathy. In the trauma community, this is also called the 'bloody vicious cycle' or the 'lethal triad.' In 1983, Stone and colleagues  describeDespite the lack of controlled randomised studies comparing damage control to traditional surgical management, a retrospective review by Stone and colleagues  presentsWe suggest a target systolic blood pressure of 80 to 100 mm Hg until major bleeding has been stopped in the initial phase following trauma without brain injury (grade 2C).To maintain tissue oxygenation, traditional treatment of trauma patients uses early and aggressive fluid administration to restore blood volume. However, this approach may increase the hydrostatic pressure on the wound and cause a dislodgement of blood clots, a dilution of coagulation factors, and undesirable cooling of the patient. The concept of low-volume fluid resuscitation, so-called 'permissive hypotension,' avoids the adverse effects of early aggressive resuscitation while maintaining a level of tissue perfusion that, although lower than normal, is adequate for short periods . Its gen2(mixed venous oxygen pressure) of less than 32 mm Hg [Red blood cell (RBC) transfusion enables the maintenance of oxygen transport in some patients. Early signs of inadequate circulation are relative tachycardia, relative hypotension, oxygen extraction greater than 50%, and PvO32 mm Hg -117. The32 mm Hg .We suggest that crystalloids be applied initially to treat the bleeding trauma patient. Colloids may be added within the prescribed limits for each solution (grade 2C).p = 0.06) [It is still unclear which type of fluid should be employed in the initial treatment of the bleeding trauma patient. Although several meta-analyses have shown an increased risk of death in patients treated with colloids compared with patients treated with crystalloids -123 and  = 0.06) . Promisi = 0.06) , and a m = 0.06) . A clini = 0.06) . However = 0.06)  found alWe recommend early application of measures to reduce heat loss and warm the hypothermic patient in order to achieve and maintain normothermia (grade 1C).Hypothermia, defined as a core body temperature of less than 35°C, is associated with acidosis, hypotension, and coagulopathy in severely injured patients. In a retrospective study with 122 patients, hypothermia was an ominous clinical sign, accompanied by high mortality and blood loss . The proHypothermia is associated with an increased risk of severe bleeding, and hypothermia in trauma patients represents an independent risk factor for bleeding and death . The effAnimal and human studies of controlled hypothermia in haemorrhage have shown some positive results compared with normothermia ,138. In If mild hypothermia is applied in traumatic brain injury, cooling should take place within the first 3 hours following injury and be maintained for approximately 48 hours, rewarming should last 24 hours, and the cerebral perfusion pressure should be maintained above 50 mm Hg (70 mm Hg). Patients most likely to benefit from hypothermia are those with a Glasgow Coma Scale of between 4 and 7 at admission . PossiblWe recommend a target Hb of 7 to 9 g/dl (grade 1C).There is experimental evidence that erythrocytes are involved in the biochemical and functional responsiveness of activated platelets, suggesting that erythrocytes contribute to haemostasis. In addition to the rheological effect on the margination of platelets, red cells support thrombin generation . Howeverin vitro reduction of the Hct did not compromise blood coagulation as assessed by thromboelastography [The specific effect of the Hct on blood coagulation is largely unknown . An acuttography .No prospective randomised trial has compared restrictive and liberal transfusion regimens in trauma, but 203 trauma patients from the Transfusion Requirements in Critical Care (TRICC) trial  were re-Despite the lack of high-level scientific evidence for a specific Hb transfusion trigger in patients with traumatic brain injury, these patients are currently transfused in many centres to achieve an Hb of approximately 10 g/dl . This maWe recommend treatment with thawed fresh frozen plasma (FFP) in patients with massive bleeding or significant bleeding complicated by coagulopathy (PT or aPTT more than 1.5 times control). The initial recommended dose is 10 to 15 ml/kg, but further doses may be required (grade 1C).The clinical efficacy of FFP is largely unproven . NeverthAs with all products derived from human blood, the risks associated with FFP treatment include circulatory overload, ABO incompatibility, transmission of infectious diseases (including the prion diseases), mild allergic reactions, and (particularly) transfusion-related acute lung injury (TRALI) ,160,161.9/l (grade 1C). We suggest maintenance of a platelet count above 100 × 109/l in patients with multiple trauma who are severely bleeding or have traumatic brain injury (grade 2C). We suggest an initial dose of 4 to 8 platelet concentrates or one aphaeresis pack (grade 2C).We recommend that platelets be administered to maintain a platelet count above 50 × 109/l, and platelet function decreases exponentially below this point [9/l or greater and agreed that platelet transfusion is appropriate to prevent or control bleeding associated with deficiencies in platelet number or function [9/l be maintained following injury.In medical conditions leading to thrombocytopaenia, haemorrhage does not often occur until the platelet count falls to less than 50 × 10is point -167. Thefunction ,169. The9/l, following injury. If a patient has increased fibrin degradation products , disseminated intravascular coagulation, or hyperfibrinolysis, this will interfere with platelet function and a higher threshold of 75 × 109/l has been suggested by consensus groups [9/l have been suggested for treatment of severe brain injury and massive haemorrhage, but the evidence for the higher threshold is weak [An argument can be made for maintaining a higher level of platelets, perhaps up to 100 × 10s groups ,171. Tra is weak ,171.10 platelets on average and should increase the platelet count by 5 to 10 × 109/l in a 70-kg recipient. Aphaeresis platelet concentrates generally contain approximately 3 to 6 × 1011 platelets, depending on local collection practice, and physicians should be cognizant of the doses provided locally. A pool of 4 to 8 platelet concentrates or a single-donor aphaeresis unit is usually sufficient to provide haemostasis in a thrombocytopaenic, bleeding patient.When platelet transfusion was introduced in the 1950s, no clinical trials were employed to assess the utility of platelet therapy compared to placebo, and such trials today might be considered unethical. The appropriate dose of platelets is therefore uncertain. Platelet concentrate produced from a unit of whole blood contains 7.5 × 109) can be calculated in more detail from the desired platelet increment, the patient's blood volume in litres , and a correction factor of 0.67 to allow for pooling of approximately 33% of transfused platelets in the spleen.If required, the dose of platelets .Cryoprecipitate or fibrinogen is used for the correction of both inherited and acquired hypofibrinogenaemia. Their use is based on the assumptions that low fibrinogen levels are associated with a risk of bleeding and that the achievement of higher levels of fibrinogen decreases that risk. The evidence for the clinical efficacy of cryoprecipitate and fibrinogen in trauma patients is limited; no clinical randomised studies have been performed to determine whether the administration of cryoprecipitate or fibrinogen improves clinical outcome in severely bleeding trauma patients. Only indirect observational studies are available, but this evidence suggests that clinically significant bleeding decreases in a variety of clinical scenarios following treatment with both agents. Hypofibrinogenaemia responds well to treatment with cryoprecipitate concentrate . AdminisThere are no specific risks related to administration of fibrinogen or cryoprecipitate other than the risks associated with other blood components and the increased risk associated with pooled versus single-donor blood products. Fibrinogen or cryoprecipitate can have unpredictable adverse effects. Of particular concern are allergic reactions and anaphylaxis. There are no reported specific adverse events related to administration of fibrinogen or cryoprecipitate in patients with hypofibrinogenaemia.A large body of evidence supports the use of antifibrinolytic agents for the management of bleeding in elective surgery and cardiac surgery patients. For the purpose of these guidelines, we have assumed that these effects are transferable to trauma patients, and our recommendation is based upon this unproven assumption.We suggest that antifibrinolytic agents be considered in the treatment of the bleeding trauma patient. Suggested dosages are tranexamic acid (trans-4-aminomethylcyclohexane-1-carboxylic acid) 10 to 15 mg/kg followed by an infusion of 1 to 5 mg/kg per hour, ε-aminocaproic acid 100 to 150 mg/kg followed by 15 mg/kg per hour, or (after a test dose) aprotinin 2 million kallikrein inhibitory units (KIU) immediately followed by 500,000 KIU/hour in an intravenous infusion. Antifibrinolytic therapy should be stopped once bleeding has been adequately controlled (grade 2C).In vitro studies have suggested that a dose of 10 μg/ml is required to inhibit fibrinolysis [Tranexamic acid is a synthetic lysine analogue that is a competitive inhibitor of plasmin and plasminogen. Tranexamic acid is distributed throughout all tissues and the plasma half-life is 120 minutes. There is large variation in the dose employed. inolysis . Studiesinolysis  confirmeinolysis , shown tinolysis .ε-Aminocaproic acid is also a synthetic lysine analogue that has a potency 10-fold weaker than that of tranexamic acid. It is therefore administered in a loading dose of 150 mg/kg followed by a continuous infusion of 15 mg/hour. The initial elimination half-life is 60 to 75 minutes and it must therefore be administered by continuous infusion in order to maintain therapeutic drug levels until the bleeding risk has diminished.Aprotinin is a broad-spectrum serine protease inhibitor isolated from bovine lung and forms irreversible inhibitory complexes with a number of serine proteases. In particular, it is a powerful antiplasmin agent, and the initial elimination of aprotinin is 1.5 to 2 hours . The 'hiThe clear efficacy of antifibrinolytic agents in elective surgery and especially in cardiac surgery has been shown in numerous clinical trials ,185. A lThe risk of precipitated thrombosis with the use of antifibrinolytic agents has been of major theoretical concern; however, the Cochrane review of antifibrinolytics cites studies that included more than 8,000 patients and demonstrated no increased risk of either arterial or venous thrombotic events . All antBecause aprotinin is a bovine protein with an associated risk of anaphylaxis, a test dose must be given. After high-dose aprotinin, as many as 50% of patients develop specific immunoglobulin G antibodies within three months of exposure. The manufacturer  estimates a 0.5% overall risk of anaphylactic reactions following aprotinin treatment, which may increase to 6% to 9% following re-exposure .An open study by Mangano and colleagues  suggesteWe suggest that the use of recombinant activated coagulation factor VII (rFVIIa) be considered if major bleeding in blunt trauma persists despite standard attempts to control bleeding and best-practice use of blood components. We suggest an initial dose of 200 μg/kg followed by two doses of 100 μg/kg administered at 1 and 3 hours following the first dose (grade 2C).9/l, and fibrinogenabove 0.5 to 1.0 g/l) and correction of severe acidosis, severe hypothermia, and hypocalcaemia  fail to control bleeding. Because rFVIIa acts on the patient's own clotting system, a sufficient number of platelets are needed to allow a thrombin burst to be induced by the pharmacological, supraphysiological doses of rFVIIa through direct binding to activated platelets [rFVIIa is not a first-line treatment for bleeding and will be effective only once sources of major bleeding have been controlled. Once major bleeding from damaged vessels has been stopped, rFVIIa may be helpful to induce coagulation in areas of diffuse small vessel coagulopathic bleeding. rFVIIa should be considered only if first-line treatment with a combination of surgical approaches, best-practice use of blood products  of rFVIIa is still under debate. Whereas the above dosing recommendation is based on the only published RCT available in trauma patients and is also recommended by a group of European experts , IsraeliWe recommend the use of PCC according to the manufacturer's instructions only for the emergency reversal of vitamin K-dependent oral anticoagulants (grade 1C).Despite the common use of PCC, there is no clear indication for its use in bleeding non-haemophilia patients. The evidence of clinical efficacy of PCC in patients without haemophilia is limited, and no clinical randomised studies have been performed to determine whether administration of PCC improves clinical outcome in severely bleeding trauma patients. PCC has been used to control bleeding in haemophilia patients -211 or tAdministration of PCC may carry the risk of venous and arterial thrombosis or disseminated intravascular coagulation ,216; howWe do not recommend the use of antithrombin III in the treatment of the bleeding trauma patient (grade 1C).Antithrombin concentrates are indicated in inherited and acquired antithrombin deficiency. Although antithrombin deficiency does occur in consumptive coagulopathy, this is not an isolated condition; all coagulation factors and physiological anticoagulants undergo consumption under these circumstances. The best replacement therapy is FFP. Clinical studies of antithrombin concentrate in severe blunt trauma and in critical care have shown no benefit ,220.These guidelines for the management of the bleeding trauma patient are based on a critical appraisal of the published literature and were formulated according to a consensus reached by the author group and the professional societies involved. We have attempted in an evidence-based manner to address a number of critical issues faced by the treating physician confronted with a critically bleeding patient. Figure Although the emphasis in these guidelines has been on the management of critical bleeding in the trauma patient, the high risk of venous thromboembolism in trauma patients should always be kept in mind and thromboprophylactic treatment considered once bleeding has been controlled . In addiWe have made an effort to consider a number of specific patient subgroups that may require treatment that has been adapted to their physiological condition. There is very little evidence, however, to support specific recommendations for these special patient groups. The physiology of elderly patients with respect to coagulopathy is probably not different than that of younger adults; however, the bleeding patient who has been treated with an anticoagulant or an antiplatelet agent may present with a greater risk of coagulopathic bleeding. We have also considered the management of bleeding following injury in children. A more conservative approach to the surgical management of bleeding in children is generally taken, and there is some evidence that commonly quoted age-related physiological norms are not applicable to the injured child. There is little evidence, however, for specific differences in bleeding and coagulation management in children; therefore, we suggest that these guidelines be applied to both adults and children until research data that are more specific become available.The apparent weakness of much of the published evidence cited in this work highlights the need for further clinical studies, and underlines the importance of future research that may lead to more clear-cut evidence-based clinical guidelines. The GRADE system employed in developing these guidelines  is approWe have made every effort to make these guidelines applicable in daily practice in a variety of clinical settings. We wish to emphasise our conviction that a multidisciplinary approach to the management of the bleeding trauma patient will help create circumstances in which optimal care can be provided. By their very nature, these guidelines reflect the current state-of-the-art and will need to be updated and revised regularly as important new evidence becomes available.• This guideline to clinical practice provides evidence-based recommendations that were developed by a multidisciplinary task force with respect to many aspects of the acute management of the bleeding trauma patient and that when implemented may lead to improved patient outcomes.• The time elapsed between injury and operation should be minimised for patients in need of urgent surgical bleeding control, and patients presenting with haemorrhagic shock and an identified source of bleeding should undergo immediate surgical bleeding control unless initial resuscitation measures are successful.• Patients presenting with haemorrhagic shock and an unidentified source of bleeding should undergo immediate further assessment as appropriate using focused sonography, CT, serum lactate, and/or base deficit measurements.• A damage control surgical approach is essential in the severely injured patient and may include the closure and stabilisation of pelvic ring disruptions, followed by appropriate angiographic embolisation or surgical bleeding control, including packing.• This guideline also reviews appropriate physiological targets and suggested use and dosing of blood products, pharmacological agents, and coagulation factor replacement in the bleeding trauma patient.ACS = American College of Surgeons; aPTT = activated partial thromboplastin time; CT = computerised tomography; DPL = diagnostic peritoneal lavage; FAST = focused abdominal sonography in trauma; FFP = fresh frozen plasma; GRADE = Grading of Recommendations Assessment, Development, and Evaluation; Hb = haemoglobin; Hct = haematocrit; ICU = intensive care unit; KIU = kallikrein inhibitory units; MeSH = Medical Subject Heading; MSCT = multi-slice spiral computed tomography; NIH = National Institutes of Health; PCC = prothrombin complex concentrate; PEEP = positive end-expiratory pressure; PT = prothrombin time; RBC = red blood cell; RCT = randomised controlled trial; rFVIIa = recombinant activated coagulation factor VII; TRALI = transfusion-related acute lung injury; TRICC = Transfusion Requirements in Critical Care.In the past 5 years, DRS has received honoraria for consulting or lecturing from the following companies: Abbott AG , Alliance Pharmaceutical Corp. , AstraZeneca , B. Braun Melsungen AG , Fresenius Kabi AG , GlaxoSmithKline , Janssen-Cilag AG , Novo Nordisk , Organon , Roche Pharma (Schweiz) AG  and CSLBehring . He serves as chair of the Advanced Bleeding Care (ABC) European medical education initiative and as co-chair of the ABC-Trauma (ABC-T) European medical education initiative, both of which are managed by Thomson Physicians World GmbH  and supported by educational grants from Novo Nordisk; he represented the European Society of Anaesthesiologists on the ABC-T Task Force. VC is a member of the ABC and ABC-T European medical education initiative faculties. TJC has received honoraria for consulting and lecturing for Novo Nordisk and is a member of the ABC-T European medical education initiative faculty. In the past 5 years, his research group has received research grant funding from Pfizer Inc , the Moulton Foundation , Novo Nordisk, Barts and the London Special Trustees, Boehringer Ingelheim GmbH , and the Anthony Hopkins Memorial Fund. JD is a member of the ABC-T European medical education initiative faculty. EF-M has received honoraria for consulting from PULSION Medical Systems AG  and is a member of the ABC-T European medical education initiative faculty. GG is a member of the ABC-T European medical education initiative faculty. PFS has received honoraria for lecturing for Novo Nordisk and is a member of the ABC-T European medical education initiative faculty. BJH has received educational grants or honoraria for lecturing from the following companies: AstraZeneca, GlaxoSmithKline, Novo Nordisk, and sanofi-aventis . RK represented the European Trauma Society on the ABC-T Task Force. EN has received honoraria for consulting or lecturing from the following companies in the past 5 years: Biotest , Bristol-Myers Squibb Company , Cook , Novo Nordisk, Pfizer Inc, and sanofi-aventis. He has received study grants from Bristol-Myers Squibb Company, Choice Medical Communications Ltd. , Mundipharma International Limited , and Novo Nordisk. In the past 5 years, YO has received institutional support from Bayer Pharma (France), Novo Nordisk, and LFB  for consulting or lecturing; he represented the European Society of Intensive Care Medicine on the ABC-T Task Force. LR represented the European Society for Emergency Medicine on the ABC-T Task Force. AS represented the European Shock Society on the ABC-T Task Force. J-LV has received honoraria from the following companies: Abbott Laboratories, AM-Pharma , ArisanPharma Inc. , AstraZeneca, Baxter , bioMérieux SA , Biosite Incorporated , Edwards Lifesciences LLC , Eli Lilly and Company , Eisai Inc. , Ferring , Novo Nordisk, Pfizer Inc, PULSION Medical Systems AG, Takeda Pharmaceutical Company Limited , Theravance, Inc. , and Wyeth . RR has received honoraria for consulting or lecturing from the following companies: Air Liquide , Bayer Pharma Leverkusen, Germany, AGA AB, Linde Gas Therapeutics, Lidingö, Sweden, Eli Lilly and Company, Messer Griesheim , Novo Nordisk, and ZLB Behring; he serves as the chair of the ABC-T European medical education initiative.All authors participated in the formulation of questions to be addressed in the guideline, screening of abstracts and literature, face-to-face and remote consensus-finding processes, and drafting, review, and revision of the manuscript. All authors read and approved the final manuscript.A Word document containing the MeSH terms and limits applied to address guideline literature queries.Click here for file


The identification of key signalling pathways controlled by this reversible phosphorylation opened a new means by which cell machinery could be activated or inhibited. As these signalling pathways were discovered to contribute in multiple ways to the oncogenic process, tyrosine kinases became key targets for clinical interventions. Today, it is routine to see cancer treatment regimens that include the use of kinase inhibitors such as anti-receptor antibodies or small molecules. Some examples include Herceptin , Gleevec , Iressa , and Tarceva (Genentech)The discovery nearly 30 years ago that v-ptks) for the first member of the family of protein-tyrosine phosphatases (ptps) to be identifiedptps have no structural similarity to serine threonine phosphatase may have contributed to the difficulties encountered in cloning those enzymes.Interestingly, it took more than 10 years after recognition of the protein-tyrosine kinases  are an excellent example of such functioning by a ptpptp superfamily act as typical oncogenesptp inhibitors and positive outcomes in clinical trials of PTPN1  antisense for treatment of type 2 diabetes may soon lead to the use of ptps as markers and novel targets in oncology.Because the ptp superfamily, describe three of the main members that are candidates with the most interesting potential for cancer treatments, and briefly survey the major hurdle standing in the way of the development of small-molecule ptp inhibitors for clinical applications.In the present paper, we comment on the diversity of the ptks, which phosphorylate their substrates on tyrosine residues, members of the ptp family remove phosphate from protein and from phospholipid substrates. The ptps are now recognized to constitute a large, structurally diverse family of tightly regulated enzymes with important regulatory roleset al.ptps encoded in the human genome. This superfamily of ptps can be divided into four major enzyme subfamilies. Enzymes from three of those families (classes i–iii) contain a catalytic oxidation-sensitive active cysteine residue embedded within a conserved signature motif (XHCSXGXGRXG), which carries out catalysis by executing a nucleophilic attack on its phospho substrateReversible tyrosine phosphorylation regulates many cellular functions, including cell proliferation, survival, adhesion, and migrationi is the largest ptp subfamily, comprising the 38 classical ptps and 61 dual-specificity phosphatases. Classical ptps show specificity for phosphotyrosine, and the dual-specificity phosphatases can dephosphorylate phosphotyrosine-, phosphoserine-, and phosphothreonine-containing substrates. Among the class i members, pten and the myotubularin subfamily are the enzymes that preferentially target phospholipids. Class ii has a single member, the so-called low molecular weight ptp (lm-ptp), and class iii encompasses the three mammalian Cdc25 proteins, which participate in cell-cycle regulation. Class iv is a newly identified aspartate-based ptp family related to the Eya family.Class PTEN (phosphatase and tensin homologue) gene on chromosome 10q23.31, also known as MMAC1 (mutated in multiple advanced cancers), encodes a tumour-suppressor protein that acts as an inhibitor of the pi3k-akt pathway. By removing the 3′ phosphate group of phosphatidylinositol--triphosphate, pten counteracts the activity of pi3k in promoting cellular growth, survival, and angiogenesisPTEN are responsible for Cowden syndrome and other autosomal dominant disorders such as Bannayan–Riley–Ruvacalba syndrome and the Proteus and Proteus-like syndromesThe pten inactivation has been identified in a series of sporadic human cancers, including glioblastomas of the central nervous system, endometrial carcinoma, prostatic adenocarcinoma, and melanomaPTEN mutations may lead to protein instability and rapid removal through protein ubiquitinationPTEN-related cancer, it may perhaps be possible to target the specific ubiquitin ligase involved in that process, thus blocking pten degradation and maintaining, or restoring, some phosphatase activity that could prevent akt pathway activation.Apart from its role in hereditary syndromes, PTPN11 gene in humans. It transduces mitogenic and pro-migratory signals from various receptor types via activation of the ras/erk cascadeShp2 is encoded by the PTPN11 are the major cause of sporadic juvenile myelomonocytic leukemia, accounting for about 35% of casesPTPN11 are uncommon in solid human tumours, but they have been detected in neuroblastoma, melanoma, lung adenocarcinomas, and colon cancerSomatic mutations of PTPN11 gene lead to expression of Shp2 variants with single aminoacid changes that enhance the protein’s activity. In addition to its well-established pro-oncogenic potential in various types of leukemia, Shp2 is also a key downstream target of other oncogenes shown to drive excessive mammary epithelial cell proliferation and a potentiator of neu-induced transformation in mouse modelsMost of the mutations in the ptps, ptpn1 has been implicated in multiple signalling pathways, including pathways triggered by growth factors, hormones, and cytokinesptpn1 was first thought to be a tumour suppressor. Yet surprisingly, PTPN1 knockout mice do not develop cancers.As the prototype for the superfamily of PTPN1 is located within 20q13, a region frequently amplified in ovarian and breast cancers and usually associated with a poor prognosisPTPN1 is overexpressed in 40% of human breast cancersptpn1 has been shown to be able to activate the oncogene srcras pathwayInterestingly, PTPN1 activity in mmtv-neu transgenic mice by breeding with PTPN1-deficient mice caused significant mammary tumour latency and resistance to lung metastasisPTPN1 inhibitor that protected the mmtv-neu transgenic mice from developing tumours to confirm those findingsPTPN1 is a true oncogene, because specific overexpression of PTPN1 in the mammary gland led to the development of spontaneous breast cancerPTPN1, previously recognized for its role in downregulating insulin signalling, has now been shown to function as a positive regulator of signalling events associated with breast tumorigenesis.Recently, our group and Dr. Benjamin Neel’s laboratory demonstrated that deletion of PTPN1 inhibition could have a significant benefit in type 2 diabetesPTPN1.Several years ago, our group, in collaboration with Brian Kennedy of Merck–Frosst, confirmed that ptp family and the general hydrophilicity of small molecules that bind to the ptp active pocket remain major hurdles in the development of specific inhibitors against PTPN1. It appears that the ptp field has now reached the stage occupied by the kinase drug development field more than 10 years ago. For example, Isis Pharmaceuticals developed and, in late phase ii clinical trials, successfully employed PTPN1 antisense inhibitors for the treatment of type 2 diabetesPTPN1 as a safe target in humans. In that context, the race for small-molecule inhibitors will certainly gain momentum. Other companies should soon emerge with their own versions of anti-PTPN1 small drugs. It stands to reason that these inhibitors should rapidly be tested in cancers in which PTPN1 is overexpressed.Unfortunately, the close similarities between the various members of the ptps have both inhibitory and stimulatory effects on cancer-associated signalling processes, and depending on their associated proteins and substrates, they act as oncogenes in multiple human cancers. Soon, several ongoing studies should validate ptps such as pten and ptpn1 as useful prognostic markers and potentially novel targets in cancer therapies.It is now clear that ptk inhibitors, chemical targeting of ptps remains largely unexplored. Despite major efforts by the pharmaceutical industry (given that these targets were identified more than 10 years after the tyrosine kinases), the development of small-molecule inhibitors of ptps is still in its early stages. Phosphatases represent 4% of the “drug-able” human genomeptp abnormalities—including cancer—has begun to elicit a growing interest in ptps as drug targets in oncology. The recent identification of PTPN1 as a potential oncogene in breast cancer may be key in focusing research efforts toward this relatively poorly known gene family.Although much current interest surrounds the clinical introduction of specific