The identification of new drugs exhibiting reduced adverse side-effects constitutes a great challenge for the next decade. Various steps are needed to screen for good ligand candidates and one of them is the evaluation of their binding properties. New strategies based on fluorescence measurement constitute excellent alternatives to the traditional radioactive assays. Less hazardous, faster and cheaper, these methods also exhibit very good sensitivity and can be used on various biological models such as heterologous expression systems or native tissues. These receptors, which share a common structural backbone composed of seven transmembrane domains, respond to diverse stimuli ranging from a single photon activating the rhodopsin receptor to large proteins binding and activating receptors such as luteinizing hormone receptor. The importance of this family of receptors is mirrored by the pharmaceutical research interest in them, with over 30% of drugs being directed against these targets .Moreover, it has been shown during the past two decades that receptors can couple to more than one signaling pathway and that some ligands, named biased agonists, have the ability to stimulate selectively only one of them . The emeIn this article we review the development of different methods based on fluorescent ligands as tracers to perform efficient ligand binding assays on GPCRs. Indeed, the synthesis of high affinity fluorescent ligands for various classes of GPCRs and the emergence of new concepts on GPCR activation have led to the development of various assays. The sensitivity of these methods is related to the biochemical properties of the fluorescent ligands and whether the receptors are expressed in a native context or in a heterologous expression system.2.Binding assays are the cornerstone of all pharmacological studies and are indispensable for the development of new drugs targeting specific receptors. Traditionally, assays based on radioactive probes  have been used successfully to determine ligand binding characteristics because of the high sensitivity of the techniques and the possibility to perform experiments on unmodified receptors expressed in native tissues and in transfected cells . However3.Fluorescence-based binding assays are promising alternatives to radioactive assays for several reasons. Firstly, the fluorescent probes needed for such tests are safe to use and they do not present the same restrictions of radioactive probes, resulting in easier to implement assays. Secondly, bright and stable probes have been developed over the past two decades and are now commercially available. Thirdly, because of the structure/activity relationship analyses, potent fluorescent ligands have been designed and synthesized for numerous receptors. Indeed, the addition of a fluorescent probe to a ligand is not as “harmless” as the tritiation of the ligand. It adds steric hindrance as well as hydrophobicity modifications which can significantly alter the pharmacological properties of the ligand, sometimes diminishing its affinity to such an extent that it can become useless in pharmacological studies.Finally, instruments needed to measure fluorescence are also becoming more sensitive and are very widespread as they are polyvalent and frequently used to perform common fluorescence or luminescence-based assays. Various experiment formats such as fluorescent intensity measurement, fluorescence polarization and energy transfer between probes can be used to evaluate ligand binding properties and they deeply impact the sensitivity of the test.4.This approach is probably the simplest one to perform and consists in the measurement of the fluorescent ligand bound fraction after separation from the free fraction. It therefore cannot be performed under homogeneous conditions. The sensitivity of this method is often questionable because of the auto-fluorescence of biological preparations. This issue can be addressed by temporally selecting the fluorescence of interest with strategies such as DELFIA . These a5.Fluorescence polarization techniques can be used to measure more precisely the amount of bound fluorescent ligand. These techniques are based on the excitation of the biological sample with polarized light. Constrained fluorophores will emit highly polarized fluorescence. On the other hand, freely moving fluorophores which can easily rotate during the short period between their excitation and the emission of fluorescence will scramble this polarization. Therefore, when exciting the sample with a polarized light, a fluorescent ligand freely diffusing in the medium with an important molecular mobility will emit a non polarized fluorescence, whereas upon binding a receptor, the molecular mobility of the fluorophore within the receptor-fluorescent ligand complex will frequently decrease, and a polarized fluorescence emission will be observed , leadingFluorescence polarization assays, like the ones previously mentioned, can be performed on receptors expressed either in native tissues or in transfected cells. Moreover, these assays can be performed in homogeneous conditions: fluorescent ligands are simply added to the biological sample and no washing steps are required to measure fluorescence polarization, making it very simple to adapt for high-throughput screening and allowing straightforward kinetics studies . HoweverAlthough the measures are very reproducible, the measurement window is very narrow, the polarization not being total or completely inexistent due to contamination by non-specific fluorescence and auto-fluorescence. Such a narrow window can be challenging when performing high-throughput screening as the variation between positive and negative results can be too small to guarantee simple interpretation and can lead to false negatives. This probably explains why fluorescent polarization assays which have been described as fast, sensitive and inexpensive  have not6.As mentioned above, the sensitivity of a binding assay is highly dependent on the non-specific binding which will decrease the signal-to-noise ratio and therefore the size of the measurement window, leading to less reliable and reproducible end results. Furthermore, a lack of selectivity (and not of affinity) of a ligand can be detrimental to evaluate the affinity of a ligand for the receptor of interest. Indeed, radioactive and fluorescence intensity or fluorescence polarization based assays cannot discriminate between the specific binding of a ligand on two different receptors.To overcome this challenge, binding assays based on fluorescence resonance energy transfer (FRET) have been developed . BrieflyAmong these parameters, the energy compatibility is probably the most crucial to perform FRET between a receptor and a ligand. The emission spectrum of the ‘donor’ should overlap the excitation spectrum of the ‘acceptor’ and the extent of overlap directly impacts the FRET efficacy. However looking for a large overlap to perform FRET is often associated with a poor separation between the excitation spectra of donor and acceptor and between their emission spectra. For the end user this leads to extensive calculations in order to differentiate the acceptor fluorophore engaged in a FRET from an acceptor directly excited and the bleed-through fluorescence of the excited donor.In contrast with ligands that are usually labeled by chemical approaches, labeling of the receptor can be achieved by various strategies. Non-covalent labeling can be performed with antibodies directed against the receptor to label it specifically. Although this approach is convenient for labelling receptors in a native context, obtaining high affinity and highly selective antibodies is difficult especially against subtypes of receptors of the same family. Consequently, it has been deemed easier to fuse an artificial tag at the N-terminal, extra-cellular end, of the GPCR and to label the receptor with an antibody directed against this tag .An alternative to the use of reversible antibody labeling of chimeric receptors can be the direct labeling of the receptor by fusing to its N-terminal extremity a fluorescent protein using molecular engineering, traditionally with proteins derived from the Green Fluorescent Protein (GFP)  Figure . This st7.To increase the sensitivity of FRET approaches, a new type of FRET was developed to address these issues implementing lanthanides as donor fluorophores . Whereas2+, Mg2+ and Ca2+), the interactions of lanthanide with cryptates are very stable leading to an easy interpretation of the data. Cryptates of europium generally exhibit an excitation peak between 300 and 350 nm and emission peaks around 585, 605, 620 and 700 nm. Due to the narrow bandwidth of the donor emission, near infra-red acceptors emitting around 665 nm can be used to selectively detect the delayed acceptor emission, with a minimum background from the donor emission. More recently, a new lanthanide cryptate using terbium instead of europium, Lumi4-Tb, has been developed and exhibits a brighter fluorescence. Moreover, due to its emission bands at 485 nm and 620 nm, it can be used to perform FRET experiments in association with both green or near infra-red emitting fluorophores. As a result, the increase in signal-to-noise ratio combined with the distinct separation of the donor and acceptor spectra render the use of TR-FRET very interesting for performing receptor binding studies with fluorescent ligands bound to a receptor labeled with a lanthanide cryptate.A second advantage of the use of lanthanides is that they are excited in the near-UV and have an important Stokes shift, making possible the selective excitation of only the donor and subsequent separate measurement of the donor or acceptor fluorescence . The excLabeling a receptor with lanthanide cryptates cannot be achieved by molecular engineering. Two methods can be currently used. The first labeling method implies the labeling of purified antibodies with lanthanide cryptates by biochemical approaches . If a hiSuch drawbacks have pushed the development of new techniques for a covalent receptor labeling with a tag smaller than antibodies and which can easily be derivatized with lanthanide cryptates. Labeling with a suicide enzyme can fulfill these criteria. The principle is to fuse a suicide enzyme to the receptor by molecular engineering. The fusion to the N-terminus is interesting since the enzyme fused to the GPCR becomes accessible to the substrate when receptors are targeted to the cell surface. Such techniques have recently been developed by Johnsson and colleagues -25. BrieThis technique has the advantage of allowing a complete and permanent labeling of surface receptors, maximizing the FRET signal between the receptor and the fluorescent ligand . MoreoveSimilar approaches have recently been developed: the covalent labeling of a protein can be performed using Acyl-Carrier protein (ACP) inserted in the sequence of the receptor and a fluorophore-conjugated phosphopantetheinyl moiety of coenzyme-A. This reaction is catalyzed by phosphopantetheinyl transferase (PPTase) added to the medium  Figure . This me8.An original strategy has been developed to perform TR-FRET experiments on receptors expressed in native tissues by taking advantage of the quaternary structure of GPCR complexes. Indeed it has been proposed that receptors can interact with each other and form homo- or hetero-oligomers. Considering the homo-oligomer model for which evidence have been provided both in heterologous expression systems and in native tissues , it woul50 obtained by competing the binding of the fluorescent ligands with oxytocin correlates very well with its known affinity for its receptor indicating the specificity of the FRET signal [Addition of competing unlabeled oxytocin induces a progressive decrease of the FRET signal which follows a sigmoidal curve . The IC5T signal . The FRE2 and dopamine D2 receptors expressed in heterologous expression system (Cos7 or CHO cell lines) [This method is based on the concept of GPCR oligomerization, and its sensitivity depends directly on the number of receptor oligomers present at the cell surface, as well as the availability of high affinity fluorescent ligands. Nevertheless, similar results have been obtained with different sets of fluorescent ligands on other class A GPCRs such as the vasopressin V1a, Vl lines)  proving 9.Development of fluorescence-based assays that propose a credible alternative to highly sensitive radioactivity-based assays constitutes a challenge. Among fluorescence-based tests, TR-FRET strategies represent original solutions since they yield a high signal-to-noise ratio. As previously mentioned, the high sensitivity is partly due to the reduction of short-lived fluorescence but also to the decrease of the non-specific binding signal because of the two levels of specificity. Indeed, only signals provided by fluorescent ligands in proximity of the labeled receptor are measured, reducing the importance of non-specific binding.In fact, fluorescent ligands reveal themselves to be extremely efficient tools when coupled with TR-FRET technology for performing binding assays and therefore may replace radioactive assays. These recently developed techniques have proven to be particularly robust and reproducible, allowing for a simple adaptation to high-throughput screening procedures . These aAlthough fluorescent ligands can evidently bring information through binding studies, their remarkable ability to specifically label receptors can also be a significant advantage for the study of GPCR dynamics, in heterologous expression systems such as cell lines as well as in native tissues. Although these fields require further development than the simple application of ligands to binding assays, information regarding the organization and the functioning of GPCRs in their native environment could prove to be the most important gain acquired with these fluorescent compounds.


Due to the advantages of its bone-conserving nature, hip resurface arthroplasty (HRA) has recently gained the interest of orthopedic surgeons for the treatment of young and active patients who have osteonerosis of the femoral head. However, in long-term follow-up studies after HRA, narrowing of the femoral neck has often been found, which may lead to fracture. This phenomenon has been attributed to the stress alteration (stress shielding). Studies addressing the effects of necrotic size and the orientation of the implant on stress alterations are lacking.Computed tomography images of a standard composite femur were used to create a three-dimensional finite-element (FE) intact femur model. Based on the intact model, FE models simulating four different levels of necrotic regions  and three different implant insertion angles  were created. The von Mises stress distributions and the displacement of the stem tip of each model were analyzed and compared for loading conditions that simulated a single-legged stance.Stress shielding occurred at the femoral neck after HRA. More severe stress shielding and an increased displacement of the stem tip were found for femoral heads that had a wider necrotic lesion. From a biomechanics perspective, the results were consistent with clinical evidence of femoral neck narrowing after HRA. In addition, a varus orientation of the implant resulted in a larger displacement of the stem tip, which could lead to an increased risk of implant loosening.A femoral head with a wide necrotic lesion combined with a varus orientation of the prosthesis increases the risk of femoral neck narrowing and implant loosening following HRA. Osteonecrosis of the femoral head (ONFH) is a devastating disease that typically affects patients who are in the third through fifth decades of life. The development of ONFH is usually progressive, and it often leads to the collapse of the femoral head and the subsequent destruction of the hip, requiring the joint to be reconstructed using a prosthetic replacement.Hip resurface arthroplasty (HRA) has several advantages compared with traditional total hip arthroplasty (THA), including a reduced rate of dislocation due to the larger diameter of femoral head, reduced wear of metal-on-metal interface, a larger range of motion and its bone-conserving nature -3. ConseIn the surgical procedure for HRA, the necrotic bone tissue is removed and replaced with cement. There are some concerns regarding this procedure. Previous studies ,12 have A commercially available synthetic femur  was used as the study model for HRA. 3-D Solid models of a standard composite femur were created using computed tomography (CT) images. CT images of the intact femur were obtained at 1.25 mm intervals in the transverse planes starting from the femoral head, using a GE Hi-speed scanner . The resolution for each CT image was 512 by 512 pixels, the field of view was 320 mm, and the pixel size was 0.625 mm/pixel. The obtained cross-sectional images of the femur were transferred to an automatic contouring program to detect the contours between the cortical and cancellous bone. The parallel stacked contours were then input into the Solidworks CAD software  to reconstruct a 3-D solid model of the intact femur. The solid model of the implant was generated based on the measurement of a commercially available product   and three different alignments of the femoral component  were created by modifying the intact model Figure . The necThe element type used for all materials in the FEA model was a 10-node, isoparametric tetrahedral element. A loading condition simulating single-legged stance was adopted from the literature  and applFor FEA validation, an experimental test of an intact synthetic femur was conducted on a MTS materials testing machine . A linear variable differential transformer  was attached vertically on the inferior aspect of the femoral head. The vertical displacement of the femoral head of the intact femur was measured under a 2000 N compressive load on the femoral head. The vertical displacement at the inferior aspect of the femoral head was also obtained from an intact FEA model that was subjected to an identical 2000 N compressive load. The experimental and analytical results were compared to validate the FEA model.The von Mises stress on the superior and inferior regions of the femoral neck intersected with the frontal plane for both the intact model and the models with various necrotic lesion sizes for HRA with a neutral orientation, as shown in Figure The displacement of the stem tip for femora with various necrotic lesion sizes resurfaced with 10° varus, neutral and 10° valgus aligned implants is shown in Figure The finite element method has become a useful tool in analyzing the stresses in structures of complex shapes, loading and material behavior. Numerous applications in orthopedics have been presented, and the models developed have successfully predicted the mechanical characteristics of skeletal components in interesting circumstances. There are also ample precedents for the use of this method in studying femoral head osteonecrosis and hip resurfacing arthroplasty. Liu et al.  designedAlthough several of the reports mentioned above showed good results in predicting the mechanical behavior of the femoral head with a certain necrotic lesion, FEM studies considering changes in the stress distribution due to changes in the necrotic lesion size along with variations in the orientation of the femoral component in femoral resurfacing have not been reported. Previous studies comparing the mechanical performance of different extents of necrosis and stem orientation accompanied with femoral resurfacing are lacking. A study focused on the basic principles of femoral resurfacing is still of paramount importance to avoid unnecessary complications.The validation and convergence of the FEM model must be taken into account before its interpretation. For model validation, the predicted displacement from FEA and the measured displacement from the experiment were 4.92 mm and 5.21 mm, respectively. The percentage error compared with the experimental result was small (5.57%). The convergence of the FEM models used in this study was justified by the total strain energy of the structure. Six models with different numbers of elements and nodes were created to perform the convergence test, and the results of the total strain energy for the six models were all within 5%. The model with the finest mesh was used in this study. The validity and convergence of the FEA model was thus demonstrated from the above procedures. The consistent results of the finite element and in vitro testing imply that the simulations are reliable.HRAs are appealing procedures because the potential for bone conservation and the non-violation of the femoral shaft make it a less invasive option. Therefore, HRA may be suitable for young and highly active patients with ONFH. Numerous reports -22 suppoThe etiology of femoral neck narrowing after HRA is unknown. It may be the result of avascular circulation to the femoral head and neck, impingement, an inflammatory response or bone remodeling due to stress shielding. Based on Wolff’s law of bone remodeling, if the loading on a bone decreases, it will become less dense and weaker, which can change the shape of the bone because there is no stimulus for the continual remodeling that is required to maintain bone mass . As obseThere are a number of limitations in the present study. First, the geometry and the linear, elastic, homogeneous material properties of a standard composite femur were used rather than the values for femurs from actual patients. One benefit of using a standard composite femur is that it eliminates the variations between subjects. However, the drawback is that this approach overlooks the effects of the nonlinear, inelastic, and non-homogeneous material properties of bone. Second, the only loading condition considered was the single-legged stance of gait. Further investigation on the effects of other loading conditions might be necessary in the future. Third, the interfaces on the implant/cement and cement/bone were assumed to be fully bonded, without considering the loosening of the implant. Therefore, the results from the FEA might only be interpreted for well-fixed conditions without implant loosening. Fourth, the necrotic lesion was arbitrarily designated to the most superior point of the femoral head. However, we demonstrated that better planning for femoral resurfacing could be achieved by predicting the stress distribution for different extents of necrosis. Last, the FE model was validated based on the intact condition without HRA, which may have an impact on the analytic results for the post-operative FE models. However, the boundary conditions including material properties, element types and element length are identical for FE models with or without HRA, and we believe that our results provide useful information to orthopedic surgeons performing HRA for patients with femoral head osteonecrosis.Necrotic lesion size and the orientation of the prosthesis have a considerable effect on the stress distribution in the femoral neck and on the postoperative stability of the implant after HRA. A femoral head with a wide necrotic lesion combined with a varus orientation of the prosthesis increases the risk of neck narrowing and implant loosening.CT: Computed tomography; FE: Finite element; HRA: Hip resurface arthroplasty; ONFH: Osteonecrosis of the femoral head; THA: Total hip arthroplasty.The authors declare that they have no competing interests.CLT participated in the design of the study, interpretation of the results and draft of the manuscript. PHH participated in the design of the study and helped with the analysis of data. YCC participated in carrying out the study and reviewing references. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/262/prepub


People heat treated silcrete during the Middle Stone Age (MSA) in southern Africa but the spatial and temporal variability of this practice remains poorly documented. This paucity of data in turn makes it difficult to interrogate the motive factors underlying the application of this technique. In this paper we present data on heat treatment of silcrete through the Howiesons Poort and post-Howiesons Poort of the rock shelter site Mertenhof, located in the Western Cape of South Africa. In contrast to other sites where heat treatment has been documented, distance to rock source at Mertenhof can be reasonably well estimated, and the site is known to contain high proportions of a diversity of fine grained rocks including silcrete, hornfels and chert at various points through the sequence. Our results suggest the prevalence of heat treatment is variable through the sequence but that it is largely unaffected by the relative abundance of silcrete prevalence. Instead there is a strong inverse correlation between frequency of heat treatment in silcrete and prevalence of chert in the assemblage, and a generally positive correlation with the proportion of locally available rock. While it is difficult to separate individual factors we suggest that, at Mertenhof at least, heat treatment may have been used to improve the fracture properties of silcrete at times when other finer grained rocks were less readily available. As such, heat treatment appears to have been a component of the MSA behavioural repertoire that was flexibly deployed in ways sensitive to other elements of technological organisation. Heat treatment of stone for knapping was one of the first fire-based transformative technologies used to alter the mechanical properties of natural materials. It was practiced from at least the second half of the Middle Stone Age (MSA) in southern Africa . MSA heaWhile heat treatment has been documented at several southern African sites, few data are available concerning its prevalence or the extent of variation within assemblages. At Pinnacle Point, on South Africa’s south coast, the majority of the silcrete from between 71 and 60 ka was heated . At BlomGiven the present paucity of data, factors governing the application of heat treatment and its temporal and spatial patterning remain relatively unknown. It has been argued that heat treatment was a necessary precondition for the use of silcrete, and that changing acquisition costs of fuel for heat treatment may have influenced the through-time prevalence of this rock type in archaeological assemblages . While tHow consistent is the relationship between the prevalence of heat treatment and the relative proportion of silcrete in an assemblage? Arguments suggesting that heat treatment was necessary for the use of silcrete would imply a fairly stable relationship between these two factors, with consistently high levels of heat treatment assumed to be associated with an abundance of silcrete.To what extent does the presence of other fine-grained silica rocks influence the use of heat treatment? If one of the effects of heat treatment is to improve silcrete’s fracture properties, the need for heat treatment may be offset by greater availability of finer-grained silica rocks such as chert.Is heat treatment influenced by different patterns of landuse? Allowing that variable mobility and provisioning through the MSA had the potential to constrain access to and transport of different rock types [ck types , 13, heaTo shed light on these issues, we investigated assemblages from a recently excavated site in South Africa’s eastern Cederberg region, Mertenhof Rock Shelter (MRS) . The sitMRS is located in a narrow canyon of the Biedouw River dominated by quartzitic sandstones of the Table Mountain Series, close to the point where the river emerges into the more open valley formed in Bokkeveld Series shales and sandstones . Though Of the main rock types in the MRS assemblage—quartzite, hornfels, silcrete, hydrothermal vein-quartz and chert—only quartzite and vein-quartz are known to be available within 20 km of the site. Quartzite of varying quality is ubiquitous throughout the region, occurring as massive bands including those of which MRS itself is formed. Vein-quartz  occurs in the vicinity of the site as small pebbles eroding out of conglomerate beds, though the distribution of these is variable and package size is heavily constrained . Hornfels is available as cobbles exceeding 100 mm maximum dimension in the Doring River, 20 km NE, but does not appear to occur west of the river, and, in any case, most of the cortex on hornfels in the MRS is of cobble form. Primary silcrete has been identified on ridges 25 km to the SE (Agtesfontein) and 25 km to the NE (Swartvlei), but closer sources cannot be precluded. Secondary silcrete cobbles occasionally occur in the Doring River but these are generally coarser-grained than those located in primary contexts and appear to have seen limited use . Chert cMRS has so far been excavated over four seasons from 2013–2015, revealing late Holocene, Pleistocene Later Stone Age, late MSA, post-HP, HP, Still Bay and early MSA components . The resExcavation was undertaken in a series of contexts, referring in this sense to a stratigraphically coherent aggregate. Most often these comprise small, distinct sedimentary units, though where sedimentary units exceed 30 mm in depth ‘context’ is used to refer to subdivisions of the unit (effectively spits following the last known slope).We analysed a series of contexts (n = 7) through the major stratigraphic unit BGG/WS, covering the start of the HP through to the early phases of the post-HP. It is in this part of the sequence that silcrete is most abundant, generally exceeding 25% of artefacts . To imprAs with other sites in the broader region, the basic characteristics of the HP at MRS vary through the sequence, including changes in raw material prevalence and in the abundance of backed and notched pieces , 17, 25 Heating patterns were evaluated as follows: For all contexts we analysed the silcrete artefacts for the presence/absence of heat treatment proxies according to the method described in Schmidt et al., . This meCompiling a meaningful reference collection for the Mertenhof assemblages is not straightforward because there is no silcrete source in the direct vicinity of the site. The closest known outcrops yield weathered blocks of yellow, sometimes grey silcrete that range from very coarse-grained to rather fine-grained. Larger pieces have knappable material below the brown weathering rind. We heat-treated a few blocks from these outcrops in a fire in order to produce a reference collection. The grey samples did not change colour but the yellow samples turned red . Both tyTwo of the characteristics setting MRS apart from most other MSA sites that lie in the Cape coastal zone are the relative degree of control available over raw material source locations see  and the Four varieties of chert can be identified at MRS. Red radiolarite is recognisable by its typical markers of tectonisation and the inclusion of radiolarian micro-fossils . The secTo evaluate the quantity of hornfels in the MRS assemblages, another material class must be taken into account, what we refer to as DWS (degraded white stone). DWS exhibits an inverse proportional distribution to hornfels through the sequence , and is Frequencies of all raw materials in the analysed contexts are summarized in Changes in raw material prevalence are to some extent matched by changes in provisioning, assessed here in terms of the relative frequencies of cores. In the lowest HP cores are proportionally abundant, occurring at the rate of 8.5 per hundred flakes (or one core per 11.7 flakes). Core proportions decline through the sequence reaching a minimum value in the post-HP. The two post-HP contexts analysed contained only one core between them, and a combined total of 341 flakes.Heat treatment proxies were observed on silcrete artefacts from all HP and post-HP contexts examined at MRS. This included evidence for flaking post thermal fracture (HINC) and also tentative evidence for artefacts with tempering residues following Schmidt et al. .Frequencies of heat treatment proxies are summarised by context in With respect to the questions posed at the start of this paper we can consider the relationships between heat treatment and the prevalence of different rock types by using both individual contexts and with respect to our industrial groupings and subgroupings. The latter approach provides more robust overall sample while the former obviates the assumption of validity in our industrial subgroupings.2 = 0.036, p = 0.682; by (sub)grouping: r2 = 0.203, p = 0.549) .First, we consider whether there is a relationship between the prevalence of silcrete in the assemblage and the frequency with which it is heated. At both the context and industrial (sub)grouping level there is no clear or statistically significant relationship between the relative abundance of silcrete and the frequency with which it was heated  and (sub)grouping data  .The second question we posed was whether there was a relationship between the prevalence of heat treatment and the frequency of non-silcrete fine-grained rocks such as chert in the assemblage . The und2 = 0.823; p = 0.005) and (sub)grouping data  . Extending this observation, we note that the relative abundance of cores through the sequence is also weakly negatively correlated with heat treatment of silcrete   .The general prevalence of heat treatment at MRS stands in contrast to analysed HP assemblages at other sites such as Diepkloof and Pinnacle Point where the majority of silcrete appears to have been heated , 8. At MOur data also allow some observations of the techniques or procedures used to heat silcrete at MRS: HINC fractures on the material indicate that, like at Diepkloof, silcrete exploded in the fire during heat treatment. Although the frequency of HINC-fractures in the middle HP layers at MRS is comparable to the intermediate HP at Diepkloof, indicating a procedure that involved fast heating, the lower percentages in the other layers might point toward slightly variable modes of heating through time. A residue, macroscopically similar to tempering residue  could alWe could not find support for the idea that the use of silcrete was contingent on the application of heat treatment, or that an increased use of silcrete would necessarily result in a greater frequency of heat treatment. In the HP layers at MRS, silcrete was often introduced to the site, flaked and discarded in an unheated state. The relationship between heat treatment and silcrete abundance is not simple or linear.2 = 0.927, p<0.001; Pearson’s r = -0.97, p<0.001). The further test conditions for this assertion are clear: we expect sites with high rates of both chert and silcrete to show lower frequencies of heat treatment than those in which silcrete is common but chert is not.Another potential factor in rates of heat treatment may be the relative prevalence of chert, assuming that heat treated silcrete is more chert-like in its knapping characteristics. As noted earlier, chert is not common at many Cape MSA sites, and MRS is an exception. In our analysed sample there was a clear inverse relationship between heat treatment of silcrete and proportion of chert, implying that an absence of the latter may have encouraged application of the former. While landuse effects may be confounding this relationship (see below), its plausibility is implied by the limited intermediate HP data we have available from units Frans and Frank at the site of Diepkloof (DRS). These two layers, which have very little chert , have abundant evidence for heat treatment of silcrete (93.7% and 96.7% respectively ). AdditiThe frequency of chert is not the only factor showing a strong relationship with the frequency of silcrete heat treatment in MRS; landuse and raw material provisioning are also implicated. From the early HP to the post-HP the prevalence of the non-local rocks chert and hornfels undergo steady decline, with most of the shortfall being taken up by an increase in the only consistently available local rock, quartzite. At the same time, cores were discarded less often, potentially reflecting less transportation of tool-making potential to the site. That this change is caused by either restrictions of raw material access or changing tool transport systems is highlighted by raw material selection patterns from the selected post-HP sample at Tweefontein published by Hallinan and Shaw , locatedAll of our discussion so far has focussed on specific factors for which we could make predictive statements. Some of these statements were consistent with the data from MRS, and others were not. We also noted the potential for confounding effects between inter-related variables, such as the effects of chert and mobility on heat treatment. One factor which we have not explicitly considered is the effect of time. Specifically, it could be that heat treatment is subject to a form of cumulative evolution, such that its relative proportion increases through time independent of changes in other variables. Heat treatment would thus be subject to its own evolutionary trajectory. Under such a scenario, statistically-significant relationships between heat treatment and other factors would be rendered largely coincidental. Given its implied insensitivity to economic considerations, we do not consider this possibility especially likely, however its test conditions are clear: increases in proportions of heat treatment from the SB through the HP into the post-HP should be a general feature of silcrete-rich lithic assemblages sharing those technological characteristics.The data we have presented allow us to draw a number of conclusions about the application of heat treatment during the MSA in southern Africa. The simplest of these is the extension of the cultural/technological contexts within which heat treatment has been observed. Though previously demonstrated in the HP , 15 and More substantially, our study of the MRS assemblages strongly indicate that, despite its prevalence at other sites like Diepkloof and Pinnacle Point, heat treatment was not a blanket strategy employed by MSA people whenever silcrete was accessed. In some horizons many tools were knapped from untransformed not heat-treated silcrete, documenting that the production of MSA tools does not generally require heat treatment and that heat treatment is not necessary to make silcrete functional as was suggested previously . Instead


Through the use of the wave reflection from any damage in a structure, a Hilbert spectral analysis-based rapid multi-damage identification (HSA-RMDI) technique with piezoelectric wafer sensor arrays (PWSA) is developed to monitor and identify the presence, location and severity of damage in carbon fiber composite structures. The capability of the rapid multi-damage identification technique to extract and estimate hidden significant information from the collected data and to provide a high-resolution energy-time spectrum can be employed to successfully interpret the Lamb waves interactions with single/multiple damage. Nevertheless, to accomplish the precise positioning and effective quantification of multiple damage in a composite structure, two functional metrics from the RMDI technique are proposed and used in damage identification, which are the energy density metric and the energy time-phase shift metric. In the designed damage experimental tests, invisible damage to the naked eyes, especially delaminations, were detected in the leftward propagating waves as well as in the selected sensor responses, where the time-phase shift spectra could locate the multiple damage whereas the energy density spectra were used to quantify the multiple damage. The increasing damage was shown to follow a linear trend calculated by the RMDI technique. All damage cases considered showed completely the developed RMDI technique potential as an effective online damage inspection and assessment tool. A reliable, effective and autonomous damage assessment technique for composite structures will be a very important factor in smart aerospace system design in the future. Damage generated in aerospace composite structures due to the various events and reasons may not be visible to surface inspection but still can cause significant loss of structural integrity. Especially, the delamination damage type arises in laminated composites due to a variety of reasons. Flaws in the manufacturing process, debonding or even impact damage can cause delaminations between the plies of laminated composites. Though not visible in the surface of composites, a delamination is still able to result in a loss in structural stiffness and strength, and further may conduct to catastrophic failure. Therefore, this provides the motivation for this study. et al.  was used to generate the different low-frequency probing waves from an actuator, where the maximum frequency of 200 kHz used is much below the boundary threshold of 300 kHz between the low frequency and high frequency. Actually, the factors that are the actuation frequency, the property and structure of a composite such as the layup and thickness can determine how many Lamb wave modes exist in the composite structure. Thus for a given composite with the certain stacking sequence, fiber direction and actuation frequency, there exists a finite number of propagation modes specified by their phase velocities.In that case, for the purpose of simplicity of analysis, it was considered that only the two fundamental modes y Cortes  with theθ is established by evaluating group velocity variation with different propagation directions. In order to obtain an actual distribution map of group velocities with regard to variable propagation angles, an experiment of the wave velocity measurements needs to be implemented. In the experiment, the propagation directions of ith propagation direction, the angle i = 0, 1, …, 23, ith sensing node can be assessed by adopting a general strategy that is given by Equation (1):ith sensing node for i. In order to precisely locate multiple delaminations in a composite panel, we need to be aware of the information regarding the angular dependence of group velocity of Lamb wave. By using the two parameters of wave propagation that are the group velocity of It is a challenge to select an effective estimation method of TOF, as the accurate extraction of the time of arrival of the instantaneous waveform is very difficult in complex and adverse environments such as recorded sensor signals having low signal-to-noise ratio . A methoOnce the group velocity is determined, the location of any damage can be inferred using the multi-damage index parameters—multi-functional multi-metrics. Accordingly, they are suitable for online structural health monitoring.The proposed rapid multi-damage index algorithm (MDIA) is composed of two metrics that are the energy density metric and the energy time-phase shift metric. The rapid MDIA is then used to process the signal data from the sensor dynamic responses, infer the presence of any damage, determine the locations of damage, and diagnose the extents of damage. Meanwhile, the rapid MDIA is also shown to trace increasing amount of damage based on the multi-functional multi-metrics. It therefore has the potential to promote the development of damage visualization techniques. In order to interpret the benefits of this rapid multi-damage index algorithm, the fundamental concepts and formula with respect to the Hilbert transform and corresponding analytical signal processing methods are introduced as follows.It supposes that a time domain signal The corresponding analytical signal versus time:The benefit of the analytical signal For the multi-damage identification purpose, a sensor signal The fast ensemble empirical mode decomposition is a new noise-assisted adaptive time-frequency data analysis method. It overcomes largely the scale (mode) mixing problem of the traditional empirical mode decomposition (EMD)  and provIn the decomposition procedure of FEEMD, the original sensor response data are decomposed into a collection of intrinsic mode functions (IMFs). For instance, an original sensor signal To interpret the advantage of the FEEMD, it is demonstrated that a case on non-damage and damage in an anisotropic composite panel structure. And the waveform from the excited diagnostic signal is made up of a 5-peaks narrow-band sine tone burst at 160 kHz modulated by a cosine Gaussian envelope. The decomposed IMFs resulting from the FEEMD processing on the selected sensor response signals are shown in n empirical modes. In other words, any sensor data from structural responses is mostly composed of different intrinsic modes of oscillation. As a result, each IMF is a frequency- and amplitude-modulated signal so that the IMFs have well-behaved Hilbert transformations. Then through the corresponding Hilbert transforming for the IMFs, any variation due to possible damage could be localized on the time as well as the frequency axis in the generated time-frequency spectrum. Meanwhile using the independent IMFs obtained from FEEMD decomposition, an evident full energy-time-frequency distribution for a given complex sensor data can be presented in the spectrum so that the local energy density and the instantaneous frequency can be obtained easily. In this way, with the property of time-dependent amplitudes and frequencies, each intrinsic mode function can therefore be treated as an individual signal, into which the Hilbert transform can be applied by:Through FEEMD decomposition processing, any measured sensor data in time domain can be decomposed into t. Thus, a time-frequency spectrum can be obtained by the integration of This approach of the FEEMD with Hilbert spectral analysis used in damage identifications is fundamentally different from the conventional Fourier transform method, of which the constant amplitude, frequency and other related information in frequency domain are derived from Fourier representations, as the instantaneous amplitude etc., a functional metric will become the key tool to give the necessary information about possible or diverse damaged situations. Thus, there are two defined multi-damage index parameters (MDIPs) used into the rapid multi-damage identification technique, which are the energy density metric and the energy time-phase shift metric. They are both robust and efficient functional metrics for multiple damage inspection in composite structures. The aim of the proposed multi-damage identification technique is to discover new damage index parameters, which can extract effectively and identify accurately complete multiple damage information. To realize an integrated damage inspection system, the different hierarchical goals of structural health monitoring (SHM) are classified, which include the presence of any damage, its location, its severity and the prediction of the remaining service life of a structure. From the measurement of a specific physical property of a monitored structure such as damping, stiffness, energy, A wave is an energy transport phenomenon. It travels through a medium such as a solid plate, and transports energy from one location (or its source) to another location without transporting matter. The amount of energy carried by a wave is thus related to the amplitude of the wave. Accordingly, the collected sensor signals are used to produce the corresponding energy time-frequency spectra. The energy time-frequency spectrum is defined as energy density metric that is obtained from the squared values of the instantaneous amplitudes. The amplitudes are deduced by the Hilbert transform of the high-energy IMFs. This metric is designated as a damage index parameter that can provide a high definition energy time-frequency representation. And also it can describe precisely the frequency content of any non-stationary or nonlinear signal based on the FEEMD process. Hence, the different features concealed in sensor signals can be revealed and better understood. In view of these reasons, an energy density spectrum needs to be found and used to identify and assess structural damage from the reflected wave energy. The energy density metric could not only quantify the extent of damage, but also map out the relationship between the released energy upon reflection due to damage and the defect growth through the corresponding energy time-frequency spectra from the sensor response data. Thus, in order to quantify the severity of any damage, a damage index parameter is introduced as a function of reflected energy in terms of the Hilbert time-frequency representation. The energy density is defined as a damage index parameter, which is expressed as Equation (9):In most damage detection schemes, the time of flight (TOF) is an important damage index parameter used often. The time of flight between the actuator and sensor on a structure is directly dependent on the layup structure of the composites, the properties of the standing (actuation) wave and the materials of the composites, which have been mentioned in The energy time-phase shift metric is proposed to locate any possible damage with an accurate determination of the time of flight, and to trace the severities of multiple damage by the phase shifts between the reflected waveforms. When a propagating wave encounters any damage, a part of the incident wavefront is reflected back while the rest diffracts through the damaged region. Actually, for the reflection wave, it is generated at the beginning of the damage. In this case, suppose that there are multiple damage with different sizes in a structure; the wave reflection from a bigger damage would be engendered sooner than that of a smaller damage located at the same position. Nevertheless, the scattered wave due to the existed damage can also be used to focus on the time of delay; thereby the different times between damaged and undamaged conditions are found and used in order to locate the damage.Therefore, the time of flight between an actuation waveform and a reflection waveform due to damage can be precisely extracted from an energy time spectrum provided by the FEEMD based Hilbert spectral analysis method. In the energy time spectrum obtained from the selected IMF components containing the highest energies, the peaks give the arrival times of interest for the waves. Hence, the locations of various damage can be determined through the basic formula (10), which is defined as the damage positioning function (DPF):If the time resolution of an energy time spectrum obtained from the HSA-FEEMD processing is distinct enough to recognize the small fluctuations from the different times of flight corresponding to the diverse damage dimensions, then the estimation for the severities of progressive damage would be feasible. In other words, it would be possible to trace damage increasing even if the dimension of an initial damage is unknown.To effectively verify the multi-damage inspection capability of the rapid multi-damage identification technique, another material patch that differed from the carbon fiber prepreg laminate was used to seed as the delamination in the tested CFRP specimens. Accordingly, every delamination was made up of a circular Teflon patch and was seeded in the middle of a composite panel, which is located between a 90° and a 0° ply. Nevertheless, three delamination patches with different diameters of 10, 19 and 30 mm were produced and respectively seeded with progressive growth in another composite specimen. An example of an instrumented composite specimen with a delamination of 19 mm diameter is demonstrated in The tested composite plates were fabricated from CFRP laminates (T700S/E022) with the stacking sequence of The actuation element creates a moment on the plate. This moment loading produces bending waves which are used for the multi-damage inspection. Most sensors of the designed PZT sensor array were bonded in the middle between the actuator and the delamination, and one sensor of the array was laid out in the behind of the delamination. The sensor array contained four circular sensor elements with a diameter of 6 mm for each. Because a sensor’s size is small enough, the influence of the sensors’ reflection can be considered negligible in the analysis and estimation of the collected signals. Meanwhile, the minimum size of which a probing wave is sensitive to a discontinuity is 10 mm. Thus, the propagation of an incident wave including wave velocity, propagation direction and so on isn’t affected by the sensors, most of the rightward propagation waves all diffract across the sensors, because a sensor’s size is much less than the minimal sensitivity size. Secondly, due to the delaminations seeded in the middle of the tested composite specimen, they didn’t emerge in the surface of the panel. As the laid sensors were affixed on the surface of the panel, wave propagation should not be affected much with the consideration of the boundary condition when the Lamb waves were actuated by the actuator. This will be proven by the experimental validations as well. A set of 5-peaks narrow-band sine tone burst signals with an actuation mode of the sweep frequencies from 60 kHz to 200 kHz and the amplitude of 50 V were used to excite the actuator. The sweep interval is 20 kHz. Meanwhile, the resulting response signals were observed and recorded at each sensor location. An instrumented composite specimen with the multiple delaminations that have separately 10, 19 and 30 mm diameters and another instrumented specimen with a single delamination of 19 mm diameter were examined by means of acousto-ultrasonics. The schematic diagrams of the experimental setup are illustrated in For the purpose of identifying complicated multi-damage, one needs to be aware of the angular dependence of group velocity in order to accurately estimate the locations of the multiple delaminations in the composite panel. Hence, a variation of group velocity in a map of propagation angles needs to be obtained by evaluating the time of flight of the Lamb wave propagating along different directions. The seeded delaminations that were simulated by Teflon patches and introduced between two adjacent plies were successfully detected and quantified by the developed multi-functional multi-metrics. This section presents a series of damage diagnosis results from the characterized multi-functional multi-metrics using the proposed RMDI technique to demonstrate a robust and efficient multi-damage inspection scheme. From the measurement of a specific physical property  of wave propagation in a structure, the energy density metric will give the necessary information to quantify the severities of various damage through the relevant energy density spectra. The energy time-phase shift metric is known as the key tool to infer the locations and sizes of multiple damage through the relevant energy-time spectra. Moreover, the increasing damage were shown to follow a linear trend obtained by the calculated prediction trend curves from the MFMMs, which can predict the extents of damage based on the released energies from the reflection waves. The details will be described in the following results and discussion. To diagnose damage presence, it can be determined rapidly through the two approaches from the proposed RMDI technique:(1) A new baseline method that is a direct appraisal approach based on the comparison results between the undamaged baseline and the various damage-lines that are all the intrinsic mode function components with the highest energies resulting from the fast EEMD process. The key advantage of the new baseline method is that it is more robust and discernible than the conventional baseline method ,24,25,26(2) The energy density metric is able to quantify the variations of energies released from the reflections due to damage, and damage presence can be thus certified by the energy density metric, of which the demonstration is presented in When a probing wave is excited, it propagates away from its origin. The energy carried by the wave will spread out in both space and time. Owing to dispersion and interaction with damage, the measured sensor signal is regarded as a combination of the excitation waveform versions that are scaled and shifted, as depicted in As mentioned in the EDM part in The energy increases monotonically with the diameter of damage as shown by the prediction trend in As shown in Accurate identification of localized events is required to determine the locations of various damages in a structure precisely and to quantify the defect growth. Then, in order to determine damage locations, this can be implemented rapidly by the damage positioning function Equation (10) based on the energy time-phase shift metric (ETPSM), where an estimation of the time interval between the actuation sensing wave and reflection waveform can be determined precisely from the high time resolution energy-time spectrum transformed. Hence, the importance of the determination of the time of flight between the actuation sensing wave and reflection waveform has been in evidence as described in the previous ETPSM part in The best method to obtain the interested time of flight is to look for the reflected energy peaks of damage for each IMF component containing the highest energy from the sensed signals’ FEEMD decompositions in the energy-time spectrum. In the case of three different delaminations, the energy-time spectrum for each IMF signal with the highest energy is plotted in The issue of the reflections from the left/right and top boundaries had been discussed and addressed in Once the relevant energy time-phase shift metric was computed and obtained using a new set of the sensed data measured from the damage tests, the amount of any new damage unknown can be inferred and its extent can be estimated further through the ETPSM combined with the TOF based DPTC. For instance, through the ETPSM obtained, the TOF of an unknown damage can be determined, of which is 0.0531 ms. Subsequently, the diameter of this unknown damage can be estimated by the TOF based DPTC presented in In For the TOF-based damage prediction trend curve, the P-value and the RMSE for the regression are estimated to be 0.0455 in the confidence range P < 0.05 and 0.0002 ms, respectively. Basically, the TOF-based DPTC does satisfy the demands of damage prediction. From the average estimation error ratio, the prediction accuracy of the ETPSM based DA method may be qualified to practical multi-damage quantification applications. The energy density metric from the RMDI technique can rapidly quantify various amounts of damage by its damage index parameter (DIP)—the energy density values.The energy time-phase shift metric from the RMDI technique can locate various damages by its DIP—the time of flight (TOF) from the high-time resolution spectra in real time mode.Furthermore, the RMDI technique also demonstrates its powerful competence to trace increasing damage. For various extents of damage in composite structures, the two metrics can both be used to track the progressive damage by their individual damage prediction trends. The energy density metric can track multiple damage by its damage-energy variation curve. The energy time-phase shift metric can trace increasing damage by its damage-time difference curve derived from the defined damage prediction function (DPF).In this paper, the combination of the CLPT theory and finite element modelling is adopted to model the dynamics of the laminated composites. Since Lamb waves are used to excite the structures, the wavenumber-frequency relationships of Lamb wave propagation in laminated composites are determined and computed. As a result, the wave group velocity of the To sum up, through all of the designed damage experiments, the rapid multi-damage identification technique based on the fast ensemble empirical mode decomposition with its associated Hilbert spectral analysis has shown promising results in the analysis of time-series data. Also, the rapid multi-damage identification technique evidently indicates a potential as an online onboard damage inspection and assessment tool. This great potential could be applied to facilitate the diagnosis and prognosis for the health or safety states of aerospace structures.


Understanding the aspects of the cell functionality that account for disease or drug action mechanisms is one of the main challenges in the analysis of genomic data and is on the basis of the future implementation of precision medicine.Here we propose a simple probabilistic model in which signaling pathways are separated into elementary sub-pathways or signal transmission circuits (which ultimately trigger cell functions) and then transforms gene expression measurements into probabilities of activation of such signal transmission circuits. Using this model, differential activation of such circuits between biological conditions can be estimated. Thus, circuit activation statuses can be interpreted as biomarkers that discriminate among the compared conditions. This type of mechanism-based biomarkers accounts for cell functional activities and can easily be associated to disease or drug action mechanisms. The accuracy of the proposed model is demonstrated with simulations and real datasets.The proposed model provides detailed information that enables the interpretation disease mechanisms as a consequence of the complex combinations of altered gene expression values. Moreover, it offers a framework for suggesting possible ways of therapeutic intervention in a pathologically perturbed system.The online version of this article (doi:10.1186/s12918-014-0121-3) contains supplementary material, which is available to authorized users. Precision medicine requires of better ways of defining diseases by introducing state-of-the-art genomic technologies into the diagnostic procedures. A more precise diagnostic of diseases, based on the description of their molecular mechanisms, is critical for creating innovative diagnostic, prognostic, and therapeutic strategies properly tailored to each patient’s necessities . ConsequNevertheless, functional modules with detailed information on their internal structures are available in several repositories such as KEGG , ReactomIn most of the methods described above individual values accounting for gene expression are combined, as defined by the internal structure of the pathway, into a global score for the entire pathway. That is, pathways are used as whole functional units in the interpretation of gene expression experiments. However, the biological consequence of the activity of a pathway is not a trivial concept. Stating that a pathway is activated (or deactivated) is not very informative by itself. In fact, partial activation (or deactivation) within the same pathway can have very different (and sometimes opposite) biological implications. For example, the apoptosis signaling pathway can lead to two opposite cell behaviors: apoptosis and degradation (cell death) or antiapoptosis  depending on the final protein that receives the signal (see hsa04210 pathway in the KEGG repository). Therefore, depending on their relative location within the pathway, changes in the activation state of one or several proteins can change (and even reverse) the cell behavior. Some of the PT-based methods use values of differential gene expression within the context of the pathways to calculate scores for pathway sub-structures -31,36. HFrom a completely different angle, other approaches have attempted to model protein signaling networks in detail -40. SuchThe approach proposed here offers a conceptually new way of understanding the functional consequences of changes in gene activity associated to any comparison of conditions by taking into account the internal structure of the pathways. This structure is used to split up the pathway into stimulus-response sub-pathways, or circuits, which trigger particular cell functionalities. In other words, the method enables the estimation of the probabilities of activation of these cell functionalities. This approach allows overtaking the simplistic view of a pathway as an entity that is either active or inactive as a whole. Here we show how the analysis of these signaling circuits results in a more detailed and realistic description of the functional consequences of gene up-and down-regulations within the context of each pathway. In many cases, the resulting activation/deactivation of individual functionalities within the pathway helps to suggest a causal explanation for disease mechanisms. We illustrate the application of the methodology with gene expression data obtained from various microarray experiments with different conditions and diseases.As previously commented pathways are multifunctional entities that can trigger a range of different cell functions and it is often meaningless to talk about the activation of a pathway as a whole or relating its global activity to a disease. Opposite to this view, we propose a different method which accounts for the change in the probabilities of signal transmission along the different signaling circuits defined within pathways. Briefly, we first use the values of gene expression to infer probabilities of gene product presence/absence . Then, we use such probabilities to calculate the probability of signal transmission along the circuit, from the receptor protein (that receives the stimulus) to the effector protein (that triggers the response). We take into account that interactions between proteins can be either activations or inhibitions . All the samples would represent pathways whose genes will have a similar activity state and, consequently, should have their signaling circuits in the same activity state. For this reason, no differences should be observed in any comparison. We produced 1000 datasets, of n = 10, 20, 50 and 100 samples with probabilities 0.1, 0.3, 0.5, 0.7 and 0.9 of being activated. Figure We have also used a large dataset of pediatric acute myeloid leukemia (AML) which contains gene expression microarray data of 237 children . In prinAdditionally, two microarray datasets containing cases and controls in a study of breast cancer (GSE27562)  and anotJak-STAT pathway  to illustrate this concept. A recent microarray (Affymetrix™ array HG-U133 Plus2.0) study has been used to systematically search for genes differentially expressed in early onset CRC . The stuWNT signaling pathway. This pathway has a canonical Wnt/β-catenin cascade and two non-canonical pathways named Wnt/Planar cell polarity (Wnt/PCP) pathway and Wnt/Ca2+ pathway, forming part of the complete WNT signaling pathway. The results of the test reveal a significantly increased activity for two out of the three circuits  whose expression is up-regulated in tumor cells by cyclooxygenase-2 (COX2), represented in the KEGG pathway in Additional file PTGS2 (prostaglandin G/H synthase and cyclooxygenase), and directly correlated to neoangiogenesis and clinical outcome [COX2 catalyzes the production of PGI2, and high levels of PGE2 have been reported in colorectal adenocarcinomas in comparison with adjacent normal tissue [PTGS2 (COX2), was significantly activated  population during aging contribute significantly to the manifestation of these age-associated hematopoietic pathologies in mice .AURKA, FOS, HOXA9, MYC, TRIM13, while MAFF and FPLT3 resulted to be age-down-regulated [FOS gene in the T-Cell receptor signaling pathway. This circuit triggers proliferation, differentiation and immune response , a disease in which signaling is known to play a relevant role. FA is a rare chromosome instability syndrome characterized by aplastic anemia as well as cancer and leukemia susceptibility . It has FANCC, is involved in Jak/STAT signaling and apoptotic signaling [Jak/STAT pathway specifically triggers one of the survival circuits of the apoptosis pathway that eventually results in the disease. Figure CIHS, leading to cell proliferation, is inhibited the activity of the circuit ending in AKT3 is significantly activated in FA  and other three circuits are significantly active . The consequences of the observed changes in the activation status of the circuits affected can easily be understood at the light of the functions they trigger  is inactivated in FA. Additionally, another two circuits triggering degradation (DFFA) and cleavage of caspase substrate (CASP6), respectively, are also deactivated. On the other hand, circuits ending in BCL2L1, BIRC2 and BCL2, that trigger survival, are activated. Thus, the final consequence is that the antiapoptotic pathway activity in FA increases with respect to a normal cell. This does not mean that apoptosis cannot occur in FA. Actually it does [BAX and/or TP53 -based methods of functional enrichment, even if they take into account the relationships between the components, fail in revealing details on the molecular mechanisms that mediate the pathologic behaviors of the apoptosis in FA. And, obviously, cannot account for the cross-talk between pathways.Cocaine addiction, African trypanosomiasis, Long-term depression, Salivary secretion, and many others completely unrelated to cancer are obvious false positives. Focusing only on cancer related pathways we consider VEGF, Jak-STAT, ERBB and WNT. In VEGF, clipper only detect a portion of the sub-pathway leading to the production of COX2 via PTGS2 (prostaglandin G/H synthase and cyclooxygenase), which is detected by our approach and is known to be activated in CRC [It is difficult to provide a reasonable comparison of the procedure proposed with other procedures because they test different aspects of the functionality of the pathway. A recent revision on pathway-based methods describe the evolution of the different algorithms proposed from those that only consider membership relationships between the genes of a pathway  to those, more sophisticated, that use the topology of the pathway (PT-Based) . Howeverd in CRC ,68. ClipAdditionally, two almost identical approaches that define sub-pathways in a similar manner to our signaling circuits but score them in a similar manner to SPIA  have recIn an apparent conceptual downgrade, the way in which new sub-pathway based methods check its validity implies checking whether the pathways in which they found significant sup-pathways are also significant in conventional FSC or PT-based methods or not. Although probably of limited value, we produced a similar comparison. We have used the CRC example  for the No main discrepancies were observed among the significant values reported by the SPIA method and the proposed method. In some cases, the behavior of SPIA is closer to the GSEA, probably because both methods return a global pathway score  However, our method is testing a different aspect of the activity of a pathway than SPIA and GSEA do. For example, a pathway known to be active in cancer is the VEGF signaling pathway, whose inhibition has been suggested as an anticancer therapy . SeveralBecause of the complexity of the cell, functional traits cannot be understood as the result of the action of only one or a few genes ,72. The Therefore, in a realistic scenario, the conventional paradigm that link disease to the failure of a particular gene needs to be changed for a systems-biology-oriented view -77, in wUnlike other conventional approaches, where either the activity of individual genes or the activity of the complete pathway was tested, this approach dissects the structure of the pathway and tests the activity of the stimulus-response signaling circuits. Actually, the notion of pathway is somehow an arbitrary concept based, in some cases, on historical or theoretical concepts that may include many different actual functional roles. The aim of the method proposed here is focusing on the actual elementary functionalities triggered by these circuits within the context of the pathway, whose failure  can be associated to the disease. There are only a few recently published methods that consider sub-pathways, however they present some problems that seriously limit its applicability and compromises the accuracy of the results provided -31. FirsThe analysis of type I and type II errors demonstrated the accuracy of the methodology proposed. The examples presented in this paper illustrate the use of the concept of the probability of stimulus-response circuit integrity for signal transmission in different scenarios including cross-talk between pathways.We have focused on gene expression data obtained from microarrays purely by practical reasons of availability: it is the most abundant genomic data publicly available. Obviously, gene expression values can be obtained by other methodologies, providing the data compared are in the same scale . In particular, it is likely that in a near future most of the gene expression measurements will be obtained from RNA-seq experiments . DiffereDetailed pathway models helps to understand the disease mechanism  as well affy library [limma library [All data used in this study was downloaded from the Gene Expression Omnibus (GEO), public repository of the NCBI . By the  library  of Bioco library  of Bioco library  platform library . The datGene expression data for the CRC analysis were retrieved from the GEO (GSE4107). Gene expression datasets for the Fanconi Anemia analysis were obtained from GEO (GSE16334) (Affymetrix HG-U133A Array). Gene expression datasets for the murine models of obesity were downloaded from GEO (GSE36669). Human bone marrow hematopoietic stem cells gene expression data were retrieved from GEO (GSE32719). Gene expression data of pediatric acute myeloid leukemia (AML) used in the validation of the methodology were retrieved from GEO (GSE17855). Gene expression data for breast cancer and data from the study of genes with increased expression in AML were also retrieved from GEO .The original GEO normalization of each dataset was used. Differential gene expression control versus case samples were carried out using the Limma library  from BioSignaling pathways offer an excellent framework for deriving models of biological activity because their functionality can be easily conceptualized: it simply consists on the potential of transmitting a signal from the protein that receives a stimulus  to the effector protein that triggers the action  in response to the stimulus. In practical terms, any node with no incoming interactions is defined as signal input node and any node with outgoing interaction is defined as a signal output node. Then, a sub-pathway is defined as a signal entry point and the collection of intermediate connecting nodes that links it to a signal output node. We will name circuits to these particular sub-pathways in order to distinguish them from any other possible sub-pathway that does not connect signal input to signal output nodes within the pathway. Thus, we recode each pathway into a collection of circuits. Any of these circuits represent a potential elementary functionality triggered by the corresponding signal output node. Some signal output nodes have specific annotations, included in the KEGG pathway definition, about the functionality triggered, although a number of them lack annotation. Figure Homo sapiens and 18 for Mus musculus belonging to the general categories Environmental Information Processing and Cellular Processes, which include important processes and systems such as Signal Transduction , Signaling Molecules and Interaction , Cell Growth and Death , Cell Communication (GAP junction and tight junction), Endocrine System  and Immune System , could be coded in this way.A total of 27 KEGG pathways for The information about the topology of each pathway was extracted from the corresponding KEGG’s XML files. The KEGG database provides information on the genes and their corresponding relationships, making it possible to reconstruct each pathway from its corresponding XML-formatted file (in KGML format - KEGG Markup Language). The Dijkstra's algorithm  is used Phosphorylation, Dephosphorylation, Indirect, Expression and Compound are initially considered activations, unless the contrary is specified. That is, if a relationship is labeled as phosphorylation, we consider that the signal is transmitted to the next node, however, if it is labeled as both, phosphorylation and inhibition, we consider that the relationship is a phosphorylation that is inhibiting the activity of the next node in the pathway. On the other hand, Ubiquitination, and Inhibition are considered inhibitions. When several proteins are linked by binding/association relationships, or are indicated as a group in the XML file, they are considered as a single node that need to be simultaneously activated to transmit the signal.Only two types of relationships are considered: activations and inhibitions. These are coded in the XML file, either directly or through a more detailed description of the mechanism by which the molecules perform the action. Thus, according to KEGG nomenclature, the labels Once a pathway is represented in this way, the probabilities of signal transmission from any entry point (stimulus receptor) in the input layer to any exit point (protein that triggers the response to the stimulus) in the output layer can be calculated, providing the activation state of the nodes connecting them are known. Moreover, changes in signal transmission probabilities when comparing different experimental situations can be tested.mixdist [As mentioned above, a node can be composed of only one protein, several alternative proteins or a complex of proteins. The probability of activity in any of these scenarios can be conceptually derived from the probability of activation of a protein. Commonly, the presence of the transcript(s) corresponding to a gene is taken as a proxy of the presence of the resultant gene product . We assumixdist , was usemixdist ,92). ForOnce such calculations are available for any of the probesets across all the microarrays studied, the distributions obtained can be used to estimate the probability of activation for this probeset in a different microarray ,92,93.The probability of a probe being activated given its expression level x can be calculated from the Bayes’ Theorem:where, according to the Law of total probability:That, in terms of our mixtures can be written as:0 = P(Inactive) and π1 = P(Active) are the proportions in the population for each distribution, that are calculated by the mixdist package, and p0(x) = p(x|Inactive) and p1(x) = p(x|Active) are the distributions that form the mixture.Where πAlthough the distributions derived from these particular datasets are only applicable to the particular microarray platforms used in this study , it is straightforward to produce similar distributions for the corresponding probesets of reference in other microarray platforms . Probesets mapping on multiple genes are discarded from the calculations.Given a microarray, the probabilities for each probeset of being activated can be used to derive the probabilities of gene activation (proxies for the corresponding protein activation probabilities) in the experiment studied. We use the 90% percentile of the distribution of the probeset activation probabilities as the value of probability of gene activation. The rationale behind this approach is to take the highest signal of hybridization of a probeset as an evidence of the existence of gene activity trying to avoid the most extreme values. In this way we choose a representative value for each gene  by meansNodes in the pathway are composed of one or more proteins. In the case of nodes of multiple proteins, these can be independent or can be part of a protein complex. When the node is composed by a single protein, the probability of node activity is the probability of activation of this protein. The second scenario corresponds to a node composed by more than one alternative protein. These are supposed to be redundant in its activity, meaning that the existence of only one of these proteins would be enough for the transmission of the signal. In this case, all the probes corresponding to all the genes in the node are taken together and the 90% percentile of the distribution of their activation probabilities is taken as the value of probability of node activation. The third scenario, corresponding to a complex of proteins, is slightly different because all the proteins are simultaneously necessary for its integrity. Unlike in the previous case, we consider that the activity of the node depends on the simultaneous presence of all their components. Consequently, we consider that the probability of having this type of node active is conditioned by the lowest probability of having any of the proteins of the complex active. The lowest of these probabilities will be the limiting factor for the integrity  of the node. Of course, the scenarios for the nodes are idealizations of a reality which may be much more complex, but they have demonstrated to represent a realistic enough scenario and to work in practical terms .a) having all the nodes connecting the input node to the output node active and b) having all the nodes that are inhibitors of nodes in the pathway in an activation state compatible with the transmission of the signal. When there are several possible ways (bi- or multi-furcations) to transmit the signal from the input node to the output node (linear sub-pathways), the formula of the probability of the union of several events can be used. Thus, once input and output nodes and the topology of the intermediate nodes has been defined for a particular circuit, and the probabilities of activation for each node in the pathway has been estimated (as described in the previous section), the probability of signal transmission across any circuit can be calculated as:As stated above, a signaling circuit is defined by an input node (the protein that receives the stimulus), an output node (the protein that triggers the response to the stimulus) and all the intermediate nodes connecting them. Thus, the probability of signal transmission along a circuit can be derived from the probability of n the number of linear paths conforming a circuit and being Ai  any of the paths in the circuit. Thus, the resulting probability, which can be assimilated to the probability of signal transmission across the circuit, is estimated as the probability of the union of all the linear paths that form this circuit.being Figure Circuits containing loops cannot be modeled under this simple approach and are not considered here. Specifically, this situation occurs in four pathways where we eliminated the following number of circuits .Since we are using gene expression values as proxies of protein activation statuses, the resulting probabilities of signal transmission across circuits at mRNA level might not have an exact correspondence with the protein level. In other words, the value of probability obtained for a circuit could not be by itself very informative about the real activation status of the circuit. However, the comparison of two conditions will render results with a clear biological interpretation: a significant change in the probability of signal transmission along a circuit is most probably accounting for a real change it the cellular response to any stimulus. In that case it can be anticipated that changes in mRNA levels causing significant changes in signal transmission circuit status will most likely be accompanied by the corresponding change at protein levels.wilcox.test function) to compare the difference of activation probability of each individual circuit in the pathway. Then we use a False Discovery Rate (FDR) [wilcox.test function returns the location parameter that indicates which is the activated condition when a circuit was found to be significantly activated. Depending on the topology of the pathway a few genes can produce a remarkable change in the number of circuits activated/deactivated or vice versa.This method seeks to assess the activation probability of every circuit, representing a canonical functionality within the pathway, given the status of the sample  In order to achieve that, we use the Wilcoxon test  (implemete (FDR)  control In order to compare the proposed approach to other pathway-based methods we have used different algorithms for functional analysis, namely singular enrichment analysis (SEA), gene set enrichment analysis (GSEA), and impact analysis . The FatIn order to test the sensitivity of the method for the estimation of the probabilities of circuit activation, we have used them as features to predict disease class. A low misclassification rate can be considered an appropriate proxy for a low type II error rate ,35. We hhttp://pathiways.babelomics.org. The results are displayed in a table that contains each circuit and its corresponding p-value, its FDR corrected p-value and its estimation of the location parameter. A graphical representation of the results in the pathway context of a KEGG-like map is also provided. This representation is obtained using the R package igraph [A web interface that implements the test described above has been developed  and is ae igraph . Nodes b


We present the computed tomography (CT) and magnetic resonance (MR) imaging findings of a 71-year-old woman with a cardiac extension of intravenous leiomyoma (IVL) that arose from the uterus, extended to the inferior vena cava (IVC), and reached the right ventricle through the right ovarian vein. Radiologic-pathologic correlation showed that the intravascular cord-like mass originating from the IVC and extending to the right ventricle was composed of degenerated smooth muscle cells with a number of large vessels that were regarded as arteries; moreover, the arteries within the cord-like mass appeared to be looping internally. Given the disappearance of the right ovarian venous wall around the IVL pathologically, extracting the tumor from the ovarian vein during an operation is considered to be impossible retrospectively. Also it was difficult to identify even the intravenous extension of the uterine leiomyoma histopathologically. Therefore, contrast-enhanced CT, in particular arterial phase imaging, provided important information that revealed the mass, range, and path of the lesion, ensuring that an appropriate operative plan could be drawn up and the tumor completely excised. Intravenous leiomyomatosis (IVL) is a rare neoplasm characterized by a smooth muscle cell tumor with a histologically benign appearance that grows within the uterine and pelvic venous system and inferior vena cava (IVC), even involving the heart. Although histologically benign, IVL can behave clinically in a malignant way, since sudden death can occur as a result of intracardiac leiomyomatosis with subsequent obstruction of venous return to the heart. Complete removal of the tumor is essential for the treatment of IVL. Therefore, precise information about the components of the uterine tumor and the complete path of extension of the lesion obtained with MRI and CT is required for surgical planning. However, no study has ever reported a detailed comparison between the imaging features of IVL and the histopathologic specimen. Here we present a case of intravenous leiomyoma that arose from the uterus, extended to the IVC, and reached the right ventricle through the right ovarian vein, including radiologic-pathologic correlation.A 71-year-old woman, gravida 6, para 4, with a two-week history of general malaise and no history of gynecological surgery, was referred to the department of cardiovascular medicine in our hospital since an echogenic mass in the right atrium was detected with cardiac ultrasonography. She had a history of hypertension, diabetes, and atrial fibrillation that had been well controlled by medication. Laboratory tests revealed normal results for tumor markers, liver and kidney function, and D-dimers, except for hemostatic abnormalities associated with anticoagulant drugs taken for atrial fibrillation.Dynamic contrast-enhanced computed tomography (DCE-CT) of the chest and abdomen was performed to determine the extent of the tumor. Arterial phase contrast-enhanced CT (AC-CT) images indicated the presence of a hypodense uterine mass and dilation of the right uterine artery (RUA). Although it was difficult to trace the tumor arteries using only axial plane images, multiplanar reconstruction images along the tumor arteries enabled tracing of the tumor arteries through the route of the right ovarian vein. Several small arteries originating from the RUA were considered to be tumor arteries because they extended to the IVC through the right ovarian vein with increasing diameter and then extended into the right atrium and the right ventricle. A plexus of small arteries, which comprised complex vascular formations originating from the right ovarian artery, the subcostal artery, and the right uterine artery , surrounMagnetic resonance (MR) imaging was performed to assess the characteristics of the uterine tumor. On MR images, the uterine tumor, approximately 13 × 7 × 6 cm in size, extensively involved the myometrium as a poorly demarcated diffuse lesion. On T1-weighted images, the lesion exhibited low signal intensity (SI) similar to that of the myometrium and included a number of hyperintense tiny foci that were suppressed on fat-suppressed T1-weighted images, reflecting a lipomatous tumor. On T2-weighted images, the lesion exhibited inhomogeneous high SI and involved a few hypointense nodules. On contrast-enhanced fat-suppressed T1-weighted images, the lesion demonstrated strong contrast enhancement but contained several poorly demarcated hypointense areas, reflecting degenerative or necrotic changes. On diffusion-weighted images and the apparent diffusion coefficient (ADC) map, the lesion showed slightly high SI . On the The presumptive diagnosis of intravenous leiomyoma (IVL) was made based on a combination of CT and MR findings, and single-stage surgery with deep hypothermic circulatory arrest was scheduled. The operation was performed with a team of gynecologists and cardiac surgeons. First, a skin incision was made from the pelvic symphysis to the xiphoid process via the left side of the navel, and the surgical wound was separated by a retractor (Lobster “Wishbone”-style retractor system). The uterine fundus was enlarged with smooth surface. Bilateral adnexa and omentum were inspected and found to be normal with the exception of mild omental adhesion to the anterior abdominal wall. The right infundibulopelvic (IP) ligament had thickened to 3 cm in diameter since plexiform arteries arising from the right uterine artery surrounded the ligament. Furthermore, the plexiform arteries formed a connection to IVC along the route of the right ovarian vein .Secondly, a thoracotomy was performed, and a cardiopulmonary bypass was established with ascending aortic arterial return and venous drainage through the right atrial appendage. After the circulation was interrupted, systemic temperature was reduced to 22°C. The IVC was then opened below the renal veins. The intracardiac mass was found to be floating freely, with the exception of being slightly adhered to the internal surface of the right ventricle. While the cardiac surgeons separated the adhesion, the gynecologists incised the IVC via a venotomy and cut off the intracaval tumor at its origin. The intracardiac and intracaval components of the tumor were drawn out upward through the right atrial incision. The uterus was found to be enlarged with an intramural tumor. Moreover, a continuous plexus of small arteries was observed along the right ovarian vein from the uterine tumor to the IVC. Subsequently, transabdominal hysterectomy and bilateral salpingo-oophorectomy were performed, with the right ovarian vein surrounded by the plexus of small arteries removed as well. Through these procedures, complete tumor resection was achieved. She was discharged on the 18th postoperative day without any complications. Clinical and radiologic assessment over a 7-month follow-up has shown no evidence of tumor recurrence. Histopathologically, the uterine lesion was composed of variable amounts of smooth muscle and fat cells with hydropic and hyaline degeneration. An intravenous component was not identified in the resected specimen. The intravascular mass originating from the IVC and extending to the right ventricle was composed of degenerated smooth muscle cells with a number of large vessels. On immunohistochemical staining, the nuclei of smooth muscle cells in the tumor were positive for estrogen receptor (ER) and progesterone receptor (PR) .Although the intravascular component around the uterine tumor could not be found histopathologically, AC-CT images revealed that the origin of the continuous mass extending into the IVC was a small nodule on the surface of the uterine tumor . On a phIVL is characterized by the presence of benign smooth muscle growth within the vascular spaces of venous and lymphatic spaces within the myometrium. Pathologic reports indicate that extrauterine involvement occurs in 30% to 80% of cases of IVL, and cardiac involvement occurs in 10% to 30% . To dateThe clinical characteristics of IVL mainly depend on the location and extent of the lesion. Even when the tumor extends intravenously as far as the IVC, patients may be asymptomatic until cardiac insufficiency develops due to intracardiac extension. More distal intravascular extension of the tumor can result in various cardiorespiratory symptoms such as shortness of breath, palpitation, and edema of the lower extremities.Although the precise histogenesis remains unclear, two theories exist about the etiology of IVL , 4. IVL The differential diagnosis of IVL by imaging features mainly includes intravenous leiomyosarcomatosis and low-grade endometrial stromal sarcoma (ESS) with cardiovascular involvement. Intravenous leiomyosarcomatosis consists of proliferating malignant smooth muscle cells originating from the walls of the uterine or pelvic veins . TypicalWith regard to the path of extension, AC-CT images demonstrated several small arteries emerging from the uterine tumor and extending to the IVC and the right heart via the right ovarian vein. However, HE staining did not show intravenous components composed of smooth muscle cells along the route of the right ovarian vein as no venous wall was confirmed around the plexiform arteries with a small amount of smooth muscle cells. It was therefore histopathologically impossible to prove intravenous extension of the uterine tumor based on anything other than the cord-like tumor extending from the IVC to the right ventricle. Thus, difficulty in identifying the intravenous extension of a uterine leiomyoma can result in misdiagnosis of the tumor histopathologically, leading to inadequate patient follow-up after hysterectomy and regrowth of residual intravenous tumor tissue. We assume that this is one of the reasons why IVL is usually diagnosed after an operation for uterine myoma. In actuality, 64% of women diagnosed with IVL had undergone a previous hysterectomy, with a range of 6 months to 20 years before presentation with the intravenous portion of the tumor .The cord-like mass extending from the IVC to the right ventricle was histopathologically composed of degenerated smooth muscle cells including plexiform vessels. When the number of vessels within the cord-like mass was compared to the number on AC-CT images at the same level, all vessels were regarded as arteries because the same number of arteries was detected on AC-CT images. However, it is a point of note that the cord-like mass only had feeding arteries without any drainage veins and, moreover, that the end of the tumor was not found to have clear arterial contact with the inner walls of the right ventricle during surgery as well as on AC-CT images. These results suggest that the arteries within the cord-like mass were looping internally.Complete removal of the tumor is the standard therapy for IVL to prevent recurrence. Given the disappearance of the right ovarian venous wall around the IVL pathologically, extracting the tumor from the ovarian vein during an operation is considered to be at risk of bleeding as well as impossible. Therefore, the removal of the tumor separately by cutting off the intracaval tumor at its origin was assumed to be a safe way to accomplish complete resection of the tumor. In premenopausal patients with IVL, a bilateral oophorectomy should also be performed since estrogen would stimulate the tumor to grow.In conclusion, IVL has some distinctive features on imaging. AC-CT should provide precise information to reveal the mass, the extent, and the complete path of the lesion, ensuring that an appropriate operative plan is created and that the tumor is completely excised. Furthermore, MRI can give additional detailed information on the components of the uterine tumor.