The principles of Virchov's triad appear to be operational in atherothrombosis or arterial thrombosis: local flow changes and particularly vacular wall damage are the main pathophysiological elements. Furthermore, alterations in arterial blood composition are also involved although the specific role and importance of blood coagulation is an ongoing matter of debate. In this review we provide support for the hypothesis that activated blood coagulation is an essential determinant of the risk of atherothrombotic complications. We distinguish two phases in atherosclerosis: In the first phase, atherosclerosis develops under influence of "classical" risk factors, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. Despite the presence of tissue factor – factor VII complex it is unlikely that all fibrin in the atherosclerotic plaque is the direct result from local clotting activity. The dominant effect of coagulation in this phase is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin.The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation as indicated by an elevated level of plasma C-reactive protein, the result of increased production influenced by interleukin-6. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin and endothelial cell protein C receptor (EPCR) expression. In this phase, the inflammatory drive leads to recurrent induction of tissue factor and assembly of catalytic complexes on aggregated cells and on microparticles, maintaining a certain level of thrombin production and fibrin formation. In advanced atherosclerosis systemic and vascular wall driven coagulation becomes more important and elevated levels of D-dimer fragments should be interpreted as markers of this hypercoagulability. The blood coagulation system comprises three basic elements: platelet adhesion, activation and aggregation, fibrin formation, and fibrinolysis. These elements interact with each other and with the blood vessel wall and under physiological conditions blood flow to tissues is unimpaired by clotting . Under pvenous thrombosis of the lower limbs, stasis, local inflammation on activated vascular endothelial cells induced by adhering leukocytes and platelets and in some cases direct vascular damage, promotes local thrombus formation. In a first episode of venous thrombosis the pre-existing composition of the blood is particularly important where congenital and acquired hypercoagulable factors such as factor V Leiden mutation and oral contraceptives, respectively, act in concert to accelerate clotting [In clotting .disseminated intravascular coagulation (DIC), widespread fibrin formation is the result of systemic inflammatory changes that induce cellular tissue factor dependent activated blood coagulation as well as local alterations in microcirculatory flow and enhanced activity and permeability of capillary endothelial cells [In al cells . Again, arterial thrombosis, local flow changes and particularly vascular wall damage are the main pathophysiological elements. Alterations in composition of the arterial blood are also involved but the specific role and importance of blood coagulation is an ongoing matter of debate [In f debate ,7. Whilef debate . In a ref debate . On the bystander), or a marker of a causal process, i.e. hypercoagulability. More specifically, the question remains whether hypercoagulability, here defined as an increased potential to produce fibrin in plasma (indicated by elevated thrombin production, fibrin production or both), as compared to individuals of similar age and sex, should be seen as a cause or merely consequence of atherosclerosis and thrombosis.In spite of the apparent controversies regarding this topic, observational studies focused on activity of coagulation and fibrinolysis in patients with arterial vascular disease continue to be published. As an example of a "clotting" marker, measurement of fibrin D-dimer fragments by one of many commercial assays, has been implicated as a risk indicator since more than 15 years, in a range of patient studies related to severity of atherosclerosis and/or risk of (recurrent) thrombotic complications -25. In gIn the majority of patients atherothrombotic complications develop on the basis of atherosclerosis in one or more coronary, cerebrovascular or peripheral arteries . AtherosActivation of blood coagulation occurs primary through interaction of platelets, vessel wall and plasma proteins . When injury to the blood vessel wall causes disruption of its endothelial layer, the underlying extracellular matrix is exposed. In this matrix, both von Willebrand factor (vWF) and collagen are present and after exposure, they will bind to specific receptors, glycoproteins (GP), present on the platelets. Dependent on the flow within the vessel other glycoproteins are involved in the adhesion of the platelets to the vessel wall. During low shear stress, GP Ia-IIa, GP VI and GP IV are the primary receptors for collagen, and during high shear forces, the primary indirect receptor for collagen is GP Ib-IX-V in a vWF dependent interaction. After adhesion of the platelets, they become deformed due to cytoskeletal changes, thereby exposing activated integrins and secreting ADP, serotonin etc. One of the integrins, GP IIb-IIIa, binds vWF (in high shear areas) or fibrinogen (in low shear areas) to mediate platelet aggregation under shear conditions. Also other platelet receptors and lipid products i.e. arachidonic acid, contribute to platelet aggregation. In this review however we will focus on secondary haemostasis, in which the interaction between circulating factor VII(a) to tissue factor, exposed by the damaged vessel wall, leads to activation of the coagulation cascade.prior to thrombosis is the principal issue of controversy.Several studies have shown that tissue factor is a prominent component of plaque lesions where it is localized in the outer membranes of infiltrating macrophages/foam cells and smooth muscle cells as well as on apoptotic cells and cell bodies Figure  30]. Un. Un30]. The involvement of coagulation in the pathological substrate of atherosclerosis is beyond dispute. For many years pathologists have noted the abundant presence of fibrin in advanced atherosclerosis and this finding has fueled part of the debate on the relevance of fibrin or fibrinogen for vessel wall lesions. Rokitansky and later Duguid proposed the encrustation theory as concept for the role of fibrin in atherosclerosis (reviewed in ). In thiearly event in atherosclerosis, i.e. a small amount of fibrinogen in a thickened intima was demonstrated in a 4 year old boy [-/- background did not have fewer arterial lesions ranging from early lesions to complex fibrous plaques, suggesting that fibrinogen is not an essential molecule for atherosclerosis [-/- x apo(a) crossbred animals [Autopsy data have indicated that fibrinogen accumulation in the vessel wall may be an  old boy . The depclerosis . However animals .The specific effect of fibrin and its split products in the vessel wall has also been studied. In general it appears that with increasing complexity of lesions there is an increase in the presence of intimal fibrinogen/fibrin and threads of fibrin, as well as an accumulation of various split products that may be involved in atherogenesis. The effect of fibrin and its split products on smooth muscle cells may be such that fibrin stimulates proliferation, while split products inhibit this process. Fibrin cleavage products may be detrimental for endothelial cell function, increasing permeability and promoting endothelial cell migration ,41,42. D-/-) results in markedly discrepant effects on atherosclerosis. While Plg-/- mice with an apoE-/- background showed an accelerated development and progression of intimal lesions, Plg-/- mice were protected against atherosclerosis in association with transplantation . The orAccumulating fibrin that polymerizes in the vessel wall triggers fibrinolysis. Fibrinolytic enzymes tissue plasminogen activating factor and urokinase plasminogen activating factor (tPA and uPA, respectively) are present in intima and are secreted by endothelial cells and likely play an important role in vascular remodeling -44. Howe-/- mice give important clues regarding the range of mechanisms that are influenced. Effective fibrinolysis may be important in limiting fibrin accumulation and atherosclerosis in the initial phases. However, upon stronger inflammatory stimulation the effect on cell trafficking into the vessel wall becomes more dominant and the outcome may reverse such that impaired fibrinolysis may limit atherosclerosis. The latter would imply that high levels of PAI-1 may even be protective against atherosclerosis under certain conditions. Consequently, high concentrations of D-dimers, reflecting active fibrinolysis, may indeed be regarded as a sign of progressive atherosclerosis under inflammatory conditions.The net effect on fibrin cleavage and progression of atherosclerosis cannot be estimated. The above mentioned experiments with PlgAll of these issues may have therapeutic consequences since several drugs that are routinely used in patients with atherosclerosis including statins, angiotensin converting enzyme inhibitors as well as angiotensin receptor blockers appear to influence the balance of coagulation and fibrinolysis, which may influence atherosclerosis on the long term by altering vascular properties .As mentioned above, a large number of clinical studies in different groups of patients with atherosclerotic disease have generally shown that increased levels of D-dimer fragments in plasma are associated with an increased risk of severe atherosclerosis and an increased risk of vascular complications. In population based studies the contributable risk of an increased D-dimer level is quite small but statistically significant. In specific cohorts of patients the risk association is more outspoken, but of course here selection bias may produce slightly stronger associations than may be found in "real life".Before addressing specific study findings a few general observations deserve attention.First, strong associations between age and sex on the one hand, and D-dimer levels on the other hand, are noted ,11,42. DSecond, D-dimer levels are oftentimes associated with markers of inflammation, i.e. CRP and Il-6 ,25,42,46Before addressing the mechanisms we will consider D-dimer as an independent entity, i.e. a marker of disease severity. The most striking associations with clinical disease come from patients with peripheral artery disease (PAD), a reflection of systemic and advanced atherosclerosis in the majority of individuals. The total risk of clinical complications or mortality reaches figures of up to 25% annually in patients with PAD 48). In patients with PAD, elevated D-dimer levels are independent predictors of complications and are associated with severity of atherosclerosis -14,16,25. In patiElevated levels of D-dimers are usually considered as a marker of increased clotting activity. This assumption is one of the key elements of the controversy regarding cause and consequence of hypercoagulability. Indeed, Herren et al observed increased levels of D-dimer in patients with PAD, correlating with severity of disease. They also noted an association between hypercoagulability (higher F1+2 and TAT) and occurrence of myocardial ischemia during exercise testing , suggestsystemic clotting activity then a specific anticoagulant intervention may theoretically be the preferred intervention. Clinical studies with anticoagulants in patients randomized or stratified on the basis of D-dimer levels have, however, not been carried out yet.In spite of the substantial observational data, application of D-dimer assays or other risk factor measurements such as for CRP have not gained acceptance in individual patients with PAD or other cardiovascular disease yet. Thus, secondary prevention of complications is not guided by any laboratory assay, but limited to general recommendations such as the advice to stop smoking and the prescription of a platelet inhibiting drug . Three rseverity of atherosclerosis such an intervention may be inappropriate and potentially harmful because of the avoidable risk of bleeding and calcification of the arterial vessel wall upon long-term administration (at least with vitamin K antagonists). Alternatively, if D-dimer levels merely reflect inflammation, than therapy should preferably consist of anti-inflammatory agents including higher doses of aspirin, statins or ACE inhibitors. Thus, the interpretation of elevated D-dimer levels is quite important in order to guide decisions about individual therapy.If however, D-dimers are a reflection of Atherosclerosis is a chronic inflammatory disease ,28. ThisStudies from the sepsis field including models of endotoxemia and sepsis in humans and primates, respectively, have shown that inflammatory stimulation leads to activation of blood coagulation ,53. TissIn this concept, there is an increased generation of thrombin and fibrin in the blood circulation due to increased presence of inflammatory cytokines and proteins, but this does not necessarily lead to increased free thrombin in plasma. One should realize that coagulation enzymes that are generated associate with any available "scavenger", which can be an inhibitor such as antithrombin, but could also be a protease activated receptor (PAR) on platelets or endothelial cells -56. ThusThe exact contribution of subendothelial fibrin formation and cleavage to D-dimer levels in blood cannot be estimated. While locally deposited tissue factor acts as a trigger of thrombin generation, it has not been shown that this is a source of ongoing subendothelial coagulation activity. The recent discovery of factor VII in plaque contents and the in vitro evidence for production of this Gla-protein by smooth muscle cells might form a basis for local thrombin production, but there is no indication yet that this might be quantitatively important as compared to hepatic production of factor VII .systemic hypercoagulability, a conclusion based on the above arguments and the experimental evidence indicating the intimate relationship between inflammation and coagulation [The point to make is that high D-dimer levels in blood from patients with atherosclerosis should be primarily viewed as an indication of gulation .Early studies from Rosendaal et al, suggested that thrombophilic traits including the prothrombin 20210 gene variant would be a risk factor for myocardial infarction in specific individuals such as heavy smoking young women . These dIn people of advanced age this situation may change considerably and the weight of risk factors may change over time. A study that specifically addressed this point is the Bruneck community study . In thisIn advanced atherosclerosis the influence of coagulation may indeed be more prominent than in early stages, but it should be realized that acquired rather than genetically determined forces are involved. In this regard, the similarities between arterial thrombosis and the risk of recurrent venous thrombosis was used by Reitsma to make the point that inflammation is a key player under such conditions, reducing the influence that genetic thrombophilic background might inflict. On the other hand the same argument could be used to illustrate that indeed inflammation plays a more prominent role in advanced atherosclerosis where it more strongly drives the risk of thrombosis.Let us consider this situation from the scope of venous thrombosis; this is not far edged because a recent study suggested similarities in risk factors between patients with previous venous thrombosis and atherosclerosis . Recent The influence of blood coagulation on atherosclerosis follows a two stage model in which variants may occur under exceptional conditions. In general in the first phase, roughly covering the first four decades of life, atherosclerosis develops under influence of "classical" risk factors, including hypercholesterolemia and smoking, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. The dominant effect of coagulation is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin. Defects in the protein C mechanism may indeed precipitate arterial thrombosis, but only under highly thrombogenic conditions. Fibrinolysis limits fibrin accumulation in the intima and herewith progression of plaque lesions. At this stage elevated PAI-1 levels may diminish fibrinolysis and may stimulate plaque progression, which may explain that in a large Japanese study the PAI 4G/5G polymorphisms appeared to be a risk factor for myocardial infarction in women .The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation and increased infiltration of fibrin in the arterial vessel wall, enforcing pro-inflammatory effects. The extensive interactions between inflammation and coagulation enzymes and inhibitors (in much greater detail than discussed here) amplify the chain of events that determine the risk of atherothrombosis. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) expression. In this phase, evidence of activated coagulation is measurable in peripheral blood reflecting both the extent of atherosclerotic burden and the systemic clotting tendency, which poses a direct risk of thrombotic complications. This point of view deviates from Tracy's viewpoint and provides a more constructive model for integrating coagulation in arterial disease.We would also propose that D-dimer and other, novel assays such as for endogenous thrombin generation   or for a


Recently, intracardiac echocardiography emerged as a useful tool in the electrophysiology laboratories for guiding transseptal left heart catheterizations, for avoiding thromboembolic and mechanical complications and assessing the ablation lesions characteristics. Although the value of ICE is well known, it is not a universal tool for achieving uncomplicated access to the left atrium. We present a case in which ICE led to interruption of a transseptal procedure because several risk factors for mechanical complications were revealed.A case of a patient with paroxysmal atrial fibrillation and atrial flutter, and distorted intracardiac anatomy is presented. Intracardiac echocardiography showed a small oval fossa abouting to an enlarged aorta anteriorly. A very small distance from the interatrial septum to the left atrial free wall was seen. The latter two conditions were predisposing to a complicated transseptal puncture. According to fluoroscopy the transseptal needle had a correct position, but the intracardiac echo image showed that it was actually pointing towards the aortic root and most importantly, that it was virtually impossible to stabilize it in the fossa itself. Based on intracardiac echo findings a decision was made to limit the procedure only to ablation of the cavotricuspid isthmus and not to proceed further so as to avoid complications.This case report illustrates the usefulness of the intracardiac echocardiography in preventing serious or even fatal complications in transseptal procedures when the cardiac anatomy is unusual or distorted. It also helps to understand the possible mechanisms of mechanical complications in cases where fluoroscopic images are apparently normal. Since the advent of ICE in the electrophysiology practice it proved its value in guiding transseptal procedures with providing an extra safety margin for the patients. The possibility to visualize the oval fossa, the LA free wall and the aortic root helps in preventing mechanical complications. ICE can visualize also intracardiac thrombus and spontaneous echocontrast, which is helpful in avoiding thromboembolic complications. Although the value of ICE is well known, it is rather hard to admit that it is a universal tool for achieving uncomplicated access to the left atrium. The aim of this case presentation is to show that ICE can lead to interruption of a transseptal procedure due to the presence of risk factors for mechanical complications when the fluoroscopic image is seemingly satisfying.A seventy-year-old male patient with paroxysmal atrial fibrillation and atrial flutter, and concomitant arterial hypertension was referred to our institution for LA circumferential ablation. The preprocedural TEE described an aneurysm of the IAS See . During In the last years ICE emerged as a useful adjunctive tool in the field of interventional electrophysiology. It serves not only scientific purposes but practical issues as well. Its value for achieving successful and uncomplicated transseptal access to the LA cavity is well known -4. HowevIn this case ICE showed that the aneurysm of the IAS observed during the TEE was actually the angulated continuity between the enlarged aortic root and the IAS. The echocardiographic orientations of ICE are sometimes clearly off axis in comparison to standard transesophageal echocardiographic views. Nevertheless, to our opinion ICE is superior in providing more detailed picture of the neighboring cardiac structures. Although useful in guiding transseptal catheterizations, TEE does not always provide complete avoidance of complications even in patients with normal hearts ,6. FurthEP procedures are relatively safe procedures and have low complication rate. One of the most frequent complications is related to cardiac wall perforation with consequent pericardial effusion and tamponade. ICE, especially the one with phased-array transducer (deeper penetration) allows continuous monitoring of the pericardial space during EP procedures. This permits prompt detection of a pericardial effusion and an immediate guidance of a therapeutic puncture. Phased-array transducers are equipped with Doppler capabilities allowing assessment of the pulmonary venous flow pattern after pulmonary vein ablation on top of diameter measurements to exclude pulmonary vein stenosis, which is the most important complication of this procedure. Monitoring microbubble formation during RF energy application allows prevention of pulmonary vein stenosis . ICE alsWe strongly believe that this case is a good illustration of the usefulness of the ICE in the electrophysiology field and further enhances its value as a tool for avoiding complications when the intracardiac anatomy is unusual or distorted. As the number of transseptal procedures in the electrophysiology laboratories all over the world is steeply growing ICE definitely has the potential to become a routine at least in those institutions with large volume of left atrial procedures.EP – electrophysiologicIAS – interatrial septumICE – intracardiac echocardiographyLA – left atrium; left atrialTEE – transesophageal echocardiographyPreprocedural transesophageal echocardiography. The irregular oval-shaped structure at the center of the screen is the aortic root. At a certain moment one can see at its upper part the ostium and the most proximal part of the right coronary artery. Below is situated the left atrium and to the left – the right atrium. The oval fossa is in between.Click here for fileIntracardiac echocardiography, showing distorted intracardiac anatomy. The oval shape in the center of the screen is the non-coronary sinus of Valsalva. Below is the right atrium at the bottom of which the transseptal needle is clearly visible. The prominent muscular structure in the left-hand part of the image is the terminal crest. The membrane of the oval fossa, adjacent to the right-hand part of the non-coronary aortic sinus shows bidirectional flapping motion. During the atrial contraction the cavity of the left atrium virtually disappears.Click here for fileIntracardiac echocardiography showing sliding of the needle towards the aorta. The transseptal needle is already in the oval fossa with its tip pointing to the aorta. This is especially clearly visible after a premature beat.Click here for file


Background to the debate: The US and Canadian task forces on preventive health recently declared that there is not enough evidence to recommend for or against routine universal screening of women for domestic violence. Yet some experts argue that routine enquiry is justified. Experts from both sides of the Atlantic debate whether screening can be justified based on the available clinical evidence Domestic violence is a misunderstood topic. The context of a trusted health professional talking to a woman is one that provides an important opportunity for providing information to counter misconceptions.asking all women about domestic violence and not in terms of screening women for domestic violence. It is not appropriate or helpful to regard enquiry about being abused as a form of screening. Domestic violence is not a disease present in the body of the person who experiences it—rather it is a health-related risk factor.I deliberately talk about this in terms of As such, knowledge of abuse puts health professionals in a position to respond better to the needs of women affected by it. Professionals can respond by providing information on specialist services—usually provided outside the health service—that women may access if they wish. By giving information to affected women, health professionals can also help to reduce women's sense of isolation and stigmatisation. Asking about experience of domestic violence can be seen as a routine part of history taking, just as health professionals regularly and repeatedly ask patients about their smoking behaviour, alcohol use, weight, and exercise.The prevalence of domestic violence among women is such that, even if it is not a personal issue for the woman concerned, it most likely will be for one or more of her relatives, friends, and neighbours . Since mMost women experiencing domestic violence report that the specialised services that exist to respond to their needs were difficult to find out about . The proStudies have examined women's views on being asked about domestic violence. These studies have shown that once they have experienced being asked, they are usually in favour of being asked. This is true both for those who have experienced or are experiencing abuse, and those who have not . It is oAsking about abuse should be done in a flexible fashion—the particular questions used should respond to the circumstances of the consultation. For example, it is appropriate to ask women about domestic violence as part of a health check in a Well Woman Clinic, but it would be completely inappropriate in a consultation where another adult or a child was present. By being flexible, health professionals can integrate their questioning within a variety of different encounters. Integrating questions about abuse into routine encounters provides for the maintenance of confidentiality and safety. In order to do this, health professionals require training on raising the issue and knowledge about local advice and support services.Committees on both sides of the Atlantic have rejected the notion of screening women for domestic violence, arguing that there is insufficient evidence of the effectiveness of interventions ,5. Part In one example of an excluded study, researchers used a randomised design to evaluate an advocacy service for women experiencing domestic violence ,8. WomenSystematic reviews have also excluded, or devalued, evidence from qualitative studies. For example, a study of 200 women who had used domestic violence outreach services found that about half were living in situations of domestic violence when they first contacted the service. All of these women reported that the outreach services had helped them to leave the abusive relationship—a valued outcome for them .Given the health impacts on women who experience domestic violence (not to mention their children) and the prevalence of the problem, routinely asking women about abuse should be seen as an important form of primary and secondary prevention for a wide range of health problems.Screening tools for domestic violence are abundant, and many are effective at identifying women experiencing abuse ,10. HoweGiven the morbidity and mortality associated with domestic violence, it is tempting to suggest that universal screening for abuse should be integrated into routine clinical care, such that all women, regardless of their reason for presenting to a clinical setting, should be “asked the question.” Some argue that this approach is justified by the need to increase awareness of domestic violence as a significant problem with serious health and social consequences, and to make abused women aware that they are not alone in their experience. These are important considerations.Certainly all women who disclose that they have been exposed to violence should be provided with options regarding seeking help . Good diwithout obvious signs and symptoms of domestic violence—such as a woman who comes to the clinic for assessment of an upper respiratory tract infection? Should such women be prompted to disclose whether they are being abused? The woman who is not being abused will answer to that effect, and the appointment can carry on. But for the woman who is experiencing violence, who has not volunteered this information, several factors must be considered. An important issue is whether she is ready—both psychologically and in terms of taking specific actions—to confront the issue. A number of excellent qualitative studies have examined the process that women undertake in acknowledging that they are “victims” of “abuse” and embarking on the often long and difficult journey to avoid, reduce, and ultimately stop the violence in their lives [However, what about women presenting ir lives ,17. GiveAny potential benefits of screening must then be weighed against its potential harms, including labelling women, prompting potentially premature disclosure, and triggering possible reprisal violence from the abuser if he discovers she has sought help. The last of these might be particularly exacerbated for the woman with the respiratory tract infection who was unprepared to disclose and did not take necessary precautions. Other potential harms include exposure to the ramifications of laws on mandatory child protection reporting, whereby health providers must report such disclosures to child protection authorities. This can lead to an investigation that potentially increases a woman's risk of exposure to violence, and in some cases of having her children placed in foster care. Research has shown that many of these potential harms are of concern to women when mandatory universal screening and/or reporting protocols are in place . FinallyGiven the lack of clear data on the benefits of screening and of the interventions to which women are referred, and the lack of data on potential harms, we and others have concluded the following ,19,20. UI agree entirely with Nadine Wathen and Harriet MacMillan that practice should be based on evidence. There are further areas of agreement. We agree that there is a lack of knowledge on effective interventions for abusers and on harm occurring as a result of enquiry, and that targeted case finding is important.all women about domestic violence. Underlying this difference is the issue about how much evidence we need, and of what type. My position is that the evidence that already exists is sufficient to justify the promotion of routine enquiry, aiming to ask all women about their experience of abuse. There is evidence of actual benefits to women—and their children—from interventions provided by specialised services for domestic violence and from brief discussions with health professionals [The key difference that exists between my viewpoint and theirs is the conclusion about whether health professionals should aim to ask ssionals .Aiming to ask all women has several advantages over targeted case finding . It contThe twin issues of women's safety and harm minimisation are extremely important, for both routine enquiry and targeted case finding. These issues are important reasons why training and protocols for enquiry are necessary. Standard principles of confidentiality should be reinforced in training and protocols, which need to be tailored to relevant legal requirements, such as when child protection issues are involved. Training and protocols also need to emphasise that the role of routine enquiry is to facilitate, and not force, disclosure. It must remain the woman's choice as to if, when, and to whom, she discloses.all women or only in situations where asking about it is part of a specific diagnostic assessment. As with screening for a disease, universal screening for domestic violence should not be implemented unless we are sure that interventions are available to help those identified via screening and that screening plus appropriate treatment will do more good than harm.We agree with Ann Taket that domestic violence is not a disease, and that the paradigm of “screening for disease” is problematic in this context. At issue, however, is the question of whether domestic violence should be “talked about” with Professor Taket outlines the importance of integrating discussions about abuse in consultations to raise community awareness. Unfortunately, there is no evidence that this type of consciousness-raising occurs, or if it does, what benefit it might have. Given the lack of effectiveness of educational campaigns in general, it is difficult to be optimistic about this approach.We disagree with her conclusion that existing systematic reviews have “excluded studies done outside the health service setting….” Our review included interventions such as the post-shelter advocacy counselling approach to which Professor Taket refers . This inFinally, we concur that qualitative studies are invaluable in understanding domestic violence. Such research has provided insight into the complex process that women undertake to address the violence in their lives. Until there is evidence that universal screening actually helps with this process, the focus should be on developing evidence-based approaches to assist women when they do disclose abuse and on training health professionals to respond appropriately to such disclosures.


The cellular signaling pathway (network) is one of the main topics of organismic investigations. The intracellular interactions between genes in a signaling pathway are considered as the foundation of functional genomics. Thus, what genes and how much they influence each other through transcriptional binding or physical interactions are essential problems. Under the synchronous measures of gene expression via a microarray chip, an amount of dynamic information is embedded and remains to be discovered. Using a systematically dynamic modeling approach, we explore the causal relationship among genes in cellular signaling pathways from the system biology approach.In this study, a second-order dynamic model is developed to describe the regulatory mechanism of a target gene from the upstream causality point of view. From the expression profile and dynamic model of a target gene, we can estimate its upstream regulatory function. According to this upstream regulatory function, we would deduce the upstream regulatory genes with their regulatory abilities and activation delays, and then link up a regulatory pathway. Iteratively, these regulatory genes are considered as target genes to trace back their upstream regulatory genes. Then we could construct the regulatory pathway (or network) to the genome wide. In short, we can infer the genetic regulatory pathways from gene-expression profiles quantitatively, which can confirm some doubted paths or seek some unknown paths in a regulatory pathway (network). Finally, the proposed approach is validated by randomly reshuffling the time order of microarray data.Arabidopsis thaliana and metabolic shift pathway from fermentation to respiration in yeast Saccharomyces cerevisiae, are reconstructed using microarray data to evaluate the performance of our proposed method. In the circadian regulatory pathway, we identified mainly the interactions between the biological clock and the photoperiodic genes consistent with the known regulatory mechanisms. We also discovered the now less-known regulations between crytochrome and phytochrome. In the metabolic shift pathway, the casual relationship of enzymatic genes could be detected properly.We focus our algorithm on the inference of regulatory abilities of the identified causal genes, and how much delay before they regulate the downstream genes. With this information, a regulatory pathway would be built up using microarray data. In the present study, two signaling pathways, i.e. circadian regulatory pathway in  Biological phenomena at different organismic levels have revealed some sophisticated systematic architectures of cellular and physiological activities implicitly. These architectures were built upon the biochemical processes before the emergence of proteome and transcriptome -3. Underin situ synthesized oligonucletide [In addition to northern blots and reverse transcription-polymerase chain reaction (RT-PCR), which study a small number of genes in a single assay, the transcriptome analysis has, via DNA microarray technology , manageducletide  chips, wucletide , Gene Exucletide  in NCBI,ucletide  in EBI. ucletide -17.Several analytic methods have been proposed to infer genetic interrelations from gene expression data. In the coarse-scale approach of clustering, the underlying conjecture is that co-expression is indicative of the co-regulation, thus clustering may identify genes that have similar functions or are involved in the related biological processes. The most widely used method is the unsupervised hierarchical clustering . This apA statistical model of Bayesian network  was propIn this study, the dynamic system approach could be employed to model how a target gene's expression profile is regulated by its upstream regulatory genes from the system causality point of view. Then, with the causal dynamic model, the upstream regulatory function can be extracted from the expression profile of the target gene by the optimal estimation method, i.e. maximum likelihood estimation. Since merely the second-order differential equation is employed to model the dynamic evolution of the target gene, only a few parameters need to be estimated. Furthermore, the derived regulatory function is closely related to the causal upstream information of the pathway and will create a basis for inferring the regulatory pathway from the system biology point of view.in silico, the defect of biological processes would be predicted and corrected before hand. Our aim is to construct signaling regulatory pathways quantitatively by the system inference approach with a dynamic model and microarray data.In either eukaryote or prokaryote, signaling regulatory pathways are considered as responses to the physiological activities or the deviation from homeostasis, which would affect the normal states of an organism. Among these signaling regulatory pathways, cell cycle  is one oIn this study, a second-order differential equation, which has been widely used to model many physical dynamic systems with good characteristics, is proposed to model the time-profile evolutional behavior of a target gene. The regulatory function is taken as the driving input of the dynamic equation of the target gene. Using the dynamic equation and microarray data, we first extract the regulatory function for each target gene. According to the extracted regulatory function, we deduce their upstream regulators to trace back upstream signaling pathways. Then, upstream regulatory genes are taken as target genes to trace back their upstream regulatory genes. Iteratively, we can construct the whole regulatory pathway to the genome wide using the dynamic regulatory model and microarray data from the system biology point of view. Finally, we give some independent validation of our approach by repeating the analysis with randomly reshuffling the time order of microarray data and see if the proposed pathways are destroyed.Arabidopsis thaliana [Saccharomyces cerevisiae [We have applied our dynamic system approach to two genetic regulatory pathways with microarray data sets publicly available on net ,30. One thaliana ,32, and revisiae . The cirrevisiae -38. Accorevisiae ,40, whicrevisiae  from therevisiae ,32,42-47The proposed methods in this study would be divided into four steps. In the first step, a dynamic model using the second-order differential equation is developed to describe the expression profile data as output and the regulatory function as input to denote the implicit characteristics of each gene with some parameters. With the help of the second-order dynamic model, we would then extract the upstream regulatory function from the expression profile of the target gene using the optimal estimation method. In the third step, the regulatory function estimated will help seek the correlative regulatory signals from the upstream paths. Iteratively, we can reconstruct the whole signaling regulatory pathway by linking up the upstream regulatory paths. Finally, some biological filters using available biological knowledge are employed to prune the constructed signaling regulatory pathway to improve the accuracy of the proposed method.Xi (t) denote the expression profile of the i-th gene at time point t. The following second-order differential equation is proposed to model the expression level of the i-th gene,The second-order differential equation is well used in the description of dynamic system evolved from the causality of gene regulatory function. Let Gi(t) is the upstream regulatory function to influence the expression profile Xi(t) of the i-th gene while ai, and biare the parameters that characterize the dynamic inherent property of the gene like degradation and oscillation, and εi(t) is the noise of current microarray data or the residue of the model. In general, the second-order differential equation has been widely used to model dynamic systems to characterize efficiently the dynamic properties of damping and resonance of systems in physics and engineering.where Gi(t). Thus, the first step is to detect the upstream regulatory function Gi(t) from both dynamic equation in (1.1) and microarray data. However, to detect the input regulatory function Gi(t) from both equation (1.1) and microarray data directly is not easy. In this situation, a Fourier decomposition technique is employed to decompose Gi(t) as a synthesis of some harmonic sinusoid functions so that the signal detection problem of Gi(t) is reduced to a simple parameter estimation problem.Obviously, the clue of upstream regulatory pathways is in Gi(t) by the following Fourier series,Accordingly, we can decompose Gi(t) becomes how to estimate the Fourier coefficients of αn and βn, which are the magnitudes of different harmonics of cos(nωt) and sin(nωt), for n = 0,..., N in equation (1.2), respectively. In science and engineering, the Fourier series has been widely employed to synthesize any continuous functions with finite energy. The estimation of αn, βn and the detection of Gi(t) in equation (1.2) are given in Methods in the sequel.Then the detection of  of regulatory function Gi(t) could be derived as follows,As a result of parameter estimation in Methods, the detection Gi(t) of a target gene is usually due to the transcriptional binding or some physical interactions from the upstream regulatory genes, in the following, we would trace back to the corresponding regulatory genes from input regulatory function  of the target gene.Since the input regulatory function Gi(t) in equation (1.1) contains the driving information for the target gene's expression from the upstream regulatory genes. The identified regulatory function  from equation (1.3) could be interpreted as the regulatory connectivity through transcriptional binding or protein-protein interaction imposed on the i-th target gene. Nevertheless, the expression data of protein type which should be considered directly in practice are by now unavailable and unreliable to trace back upstream regulatory genes. Instead, the expression data on mRNA level which is now widely available from microarray assays would make tracing back the upstream regulatory pathway possible under proper assumptions. All along the paper we assume that the expression levels of mRNA transcripts are proportional to the actual number of corresponding proteins in the cell. This assumption is indeed a strong approximation since post-transcription is known to play a very important role in down regulating the number of the transcription factor in the cell.Apparently the input regulatory function  shown below with the parameter set of θj = {γ, Mj, τj} is the sigmoid transformation of Xj(t), the expression profile of the j-th regulatory gene.Before the inference of upstream regulatory genes, it is reasonable to confine the effect of the regulatory genes on the regulated target gene. The saturated activity of expression level reveals that the regulatory ability cannot extend unlimitedly. The sigmoid function is often chosen to express the nonlinear saturation with proper parameters. Here, we apply the sigmoid transformation to represent the 'on' and 'off activities of the regulatory genes on binding or not to motifs of the target gene. So the regulatory signal γ is the transition rate, Mj is the mean expression of the j-th regulatory gene's profile, and τj is the corresponding signal transduction delay.where τj from the j-th regulatory gene to the target gene. The delay τj would be computed by statistical correlation between the regulatory signal  transformed from the j-th regulatory gene and the identified regulatory function  of the target gene. The delay τj is determined by the following maximum correlation criterion,The delay activity should be considered in order to describe the signal transduction delay rτ is the correlation between  and  under variable delay τ. If there are many τ to achieve the maximum correlation in (2.2), then only the smallest one is chosen.where Ri regulatory genes whose regulatory signals  are most correlated with the regulatory function  of the ith target gene, i.e. choose Ri genes with maximum correlation but with smaller τj in (2.2). The determination of number Ri will be discussed later. Then, we construct the regulatory pathway by tracing back Ri regulatory genes from the identified regulatory function  of the target gene as the following kinetic relationship,Using the correlation method, we trace back cij is the pathway kinetic parameters from the regulatory gene j to the target gene i, Ri are the searched upstream regulatory genes, the constant ci0 represents the basal level to denote the regulatory function other than upstream regulatory genes, and ei(t) is the residue of the model.where cij, equation (2.3) for m time points should be written in the following regression form,Furthermore, to estimate the pathway kinetic parameters , .where ei(tk), k = {1,..., m}, is an independent random variable with a normal distribution with zero mean and variance σ2. By maximum likelihood parameters estimation method (see Methods), the estimates of σ2 and Ωi are given as follows, which is solved asWe assume that each element in the error vector, andRi upstream regulatory genes in equation (2.3). For example, suppose the expression profile of gene j has a high correlation with regulation function Gi(t) of target gene i. However, if gene j is not a transcription factor, protein phosphorylation, post-transcription or specific enzyme of target gene i, it will be deleted from the candidates of Ri upstream regulatory genes because it may be only a co-expressed gene with the target gene corregulated by the other gene. On the contrary, a verified regulatory gene should be recruited into the candidates even with small correlation with Gi(t).It should be noted that with the combination of biological knowledge about the transcriptional factors, protein phosphorylation, post-transcriptional and specific enzyme regulation of target genes, lots of putative and verified genes correlated with the target genes are pruned by this biological filter for the more efficient and accurate searching of Ri of regulatory signal [Finally, we take the well-known Akaike Information Criterion (AIC) into account for determining the number y signal ,Ri is the number of regulatory genes. AIC includes both the estimated residual variance and model complexity in one statistic, which decreases as σ2 decreases and increases as Ri increases. AIC has been widely employed to determine the complexity of system modeling science and engineering [Ri of the upstream regulatory genes will be determined by the minimization of the AIC value in equation(2.7).The first term in AIC is the residual variance and the second term ineering . The optRi upstream regulatory genes by AIC in equation (2.7) after the biological filtering and determine their pathway kinetic parameters cij of regulatory signal by equation (2.6). After biological filter pruning, if the number of candidates of regulatory genes is still less than Ri determined by AIC, then some genes, which are highly correlative to Gi(t) but not of transcription factors or signaling proteins of target gene i, should be recruit into candidates to uncover regulatory relationships that were not suspected to be connected. After the combination of equations (2.3) and (1.1), the whole regulatory pathway is obtained asNow, for the selected target genes in the interesting pathway, we could search for the optimal i = {l, 2,..., L}, and L is the number of target genes in the pathway. The sub-paths related to the i-th target gene in the interesting pathway could be detected by the inference algorithm. Then, it is natural that the whole regulatory pathway would be constructed by the links of all the sub-paths. We also outline the whole flowchart of our dynamic inferring algorithm as shown in Figure for Arabidopsis thaliana and yeast Saccharomyces cerevisiae, have been well studied biologically and their microarray assays are abundant. Thus, we chose different types of pathways, one is the plant behavior under environmental variation and the other is the cellular metabolism in response to exhaustion of external source, as examples in this study. In other words, two signaling pathways, i.e. circadian regulatory pathway in Arabidopsis thaliana and metabolic shift pathway from fermentation to respiration in yeast Saccharomyces cerevisiae, are constructed from microarray data to confirm the accuracy of our proposed method.The two famous modeling organisms, Arabidopsis thaliana that are measured in replicate hybridization of 12 samples harvested every 4 hours over 2 days. With their investigation on the circadian regulatory system, Harmer et al. have provided an abundance of correlated genes for the regulatory pathway inference.For cells grown in the light/dark cycle according to circadian rhythm, Harmer and colleagues  used higSaccharomyces cerevisiae, is harvested at seven successive 2-hour intervals after an initial nine hours of growth under the diauxic shift. Adoption of the diauxic shift data set would make possible the inference of metabolic shift pathways.As for the metabolic pathway, an cDNA microarray assay from DeRisi et al. , containCry1 (CRYTOCHROME 1) and PhyA (PHYTOCHROME A) genes in the circadian regulatory pathway of Arabidopsis thaliana are interpolated by the cubic spline method among raw data points on the left-hand side. Similarly, Pgi1 (PHOSPHOGLUCOSE ISOMERASE 1) and Pgm2 (PHOSPHOGLUCOMUTASE 2) genes in the metabolic shift pathway of yeast Saccharomyces cerevisiae are on the right-hand side. After the expression profiles are smoothed by the cubic spline technique, we can obtain the data of the first derivative  and the second derivative  more accurately and abundantly.With the second-order equation and the optimal estimation method, the dynamic model should be developed first for the regulatory scheme of target genes in the signaling regulatory pathway. Because the raw microarray data sample of the biological assays that will be analyzed is small with less than 15 data points for an individual gene, the cubic spline method is used to interpolate the observed data to increase the data points of each gene's time-course microarray data. As shown in Figure  simultaneously as the scheme of Figure After data expansion by the cubic spline method, we would have enough data to estimate the parameters of the regulatory dynamic model of the target gene from equation 2.4). Following the dynamic model in equation 3.1), the parameters which characterize the dynamic regulatory mechanism are estimated successfully for each target gene in the pathway. By dynamic model fitting, the expression profiles of the mentioned genes in Figure .4. Follo.1, the p1~X13 and Y1~Y11) in two pathways of the circadian regulatory system in Arabidopsis thaliana and the metabolic shift pathway in yeast Saccharomyces cerevisiae to recognize their upstream regulatory genes, respectively. Their regulatory abilities with signal transduction delays are shown in the form of dynamic equation in Table For illustrations, the inferring strategy is applied to the selected core genes  and phytochrome (PhyA, PhyB, PhyD and PhyE), Cry1 [X6] and Cry2 [X10] are commonly regulated by Lhy [X3] (LATE ELONGATED HYPOCOTYL) in reciprocal ways with significant values (0.7569 in Eq.(6) and -1.8773, Eq.(10) of Table Lhy on crytochrome genes. In addition, from Eq.(10) in Table Ccal [X4] (CIRCADIAN CLOCK ASSOCIATED 1) has the greatest positive regulation (2.3465) on Cry2, meaning that Cry2 is jointly regulated by Lhy and Ccal. Because the binding sites of Lhy and Ccal found in the promoter regions of Cry2 [Cry2 affected by both Lhy and Ccal. In addition, the mutual activations of phosphorylation between Cry1 [X6] and PhyA [X7] in Eq.(6) and Eq.(7) of Table Cry2 [X10] is also positively regulated by PhyA [X7] with 0.5-hr activation delay similar to that in Cry1 (Eq.(6) in Table PhyA is considered as a post-transcriptional regulator of phosphorylation to crytochrome within 1.0-hr after transcription. On the other hand, PhyB [X11] down-regulates Cry2 with a significant effect (-0.7141) while Cry2 [X10] up-regulates PhyB (0.0511) weakly by feedback (see Eqs.(10), (11) in Table Cry2 and PhyB in nuclear speckles that are formed in a light-dependent fashion are also confirmed by Mas et al. [Cry1 and Cry2 are both negatively co-regulated by PhyD [X8] and PhyE [X12] significantly (see Eqs.(6), (10) in Table PhyA has apparently different behavior from PhyB, PhyD, and PhyE in activating crytochrome. This might suggest the mechanism that PhyA mediates the blue light by up-regulating Cry1 and Cry2, whilst PhyB, PhyD, and PhyE would mediate the red light by inhibiting blue photoreceptors [In the photo-transduction related genes Table , contain of Cry2  are conss et al. . Becauseeceptors ,54.(PhyA, PhyB, PhyD and PhyE) in Figure Lhy [X3] and Ccal [X4], well-known biological clock genes in the circadian system [Gi [X15] (GIGANTEA) in Figure Gi sequence lacks any motifs suggesting that it is a transcription factor of phytochromes [Gi might be a post-transcriptional regulatory factor. However, there is another gene Elf3 [X16] (EARLY FLOWERING 3) opposite to Gi on phytochrome, especially for PhyA, PhyB and PhyE (Eqs.(7), (11) and (12) in Table Lhy or Ccal, Elf3 might play the same role as Lhy and Ccal. Just as expected, Elf3 contains glutamine-rich motif suggesting that it is a transcription factor [In the mainly red-light photoreceptors of phytochrome n system ,46, are em [Gi X1  and PhyB [X11] (0.1223) (see Eq.(9) in Table Toc1 [X13] is transcriptionally regulated by Lhy [X3] (0.7009) and Ccal [X4] (-1.4704) whilst Pif3 [X16] (-0.1698) is presumably considered as the bridge between Toc1 and phytochrome (Eq.(13), Table Lhy and Ccal point of view, they are both positively affected simultaneously by Pif3 implying the regulation on the transcriptional level [Toc1 inhibits both Lhy and Ccal to form the structure of mutual transcriptional regulation (please compare Eqs.(3), (4) with Eq.(13) in Table Lhy and Ccal function as principal transcription factors.Before entrance of the biological oscillator of the circadian system formed by ] Figure  is mediaal level . In addiChs [X5] (CHALCONE SYNTHASE), Pap1 [X1], and Co [X2] (CONSTANS) in Figure Chs is known as correlated with UV-B protection. It seems that Ccal and Lhy have greater effect (2.7078, -0.7631, respectively) on Chs than Pap1 (-0.0455) as a transcription factor (Eq.(5) in Table Chs is regulated by Pap1 in a small scale with amplifying effect on the cis-regulatory level. Co is recognized as a pivotal gene of photoperiodic regulation of flowering. Indeed, strong regulations from Ccal and Lhy are identified to show that Co is regulated with a large-scale attenuation effect on the cis-regulatory level (Eq.(2) in Table We also infer some downstream pathways of Chs X , represented by black lines with more linking to upstream genes, the photo-transduction pathways are stabilized to provide oscillation. On the other hand, more blue lines of inhibitive interactions are revealed in the biological-clock regulatory pathways relevant to Co, Pap1, and Chs  underlying the anti-phase functional regulation between these output pathways and the oscillator. In addition, the essential signal transduction factors of Fkf1, Gi, Elf3, and Pif3  make some critical links between the functional blocks mentioned above in the circadian system[In the overview of the circadian system in Figure an system. Finallyan system but withSaccharomyces cerevisiae can switch from fermentatioon at high levels of glucose to respiration at low levels of glucose with major changes in metabolic activity (diauxic shift). In their experiment on the diauxic shift [Pyk1 to pgm2 and the partial fermentation from Pyk1 (PYRUVATE KINASE 1) to Adh1 and Adh2 (ETHANOL DEHYDROGENASE ISOZYME 1, 2) are unraveled as a result of the diauxic shift, so two sub-pathways in opposite directions are concluded.Sugars, such as glucose and sucrose, are excellent carbon sources for yeasts and almost all of the energy requirements of the cell can be satisfied by glycolysis -61,63-66ic shift , DeRisi Pykl [Y4] encoding an enzyme, which catalyzes PEP (Phosphoenolpyruvate) to pyruvate, is negatively regulated (-5.8763) by Pck1 [Y26] (Eq.(4) in Table Pck1 could be intepreted here as an indirect upstream transcription factor or regulatory gene for Pyk1 due to its function of decarboxylation and phosphorylation of oxalacetat in the presence of a nucleoside triphosphate and a divalent metal ion to yield PEP. Another Gcrl [Y15] gene is also identified as the strongest positive regulation (5.9829) to Pyk1  in Table Gcrl of Pdc1 [Y6] (PYRUVATE DECARBOXYLASE ISOZYME 1) plays a more essential role (-2.5615, Eq.(6) in Table Rap1 [Y17] (0.1164), and Pyk1 [Y4] is an upstream regulatory factor coding an enzyme with the most positive effect (3.1295) on Pdc1 according to the production of acetaldehyde from pyruvate. In the last kernel of the fermentation, Adh1 [Y7] and Adh2 [Y3] are involved in the ethanol metabolism of carbohydrate storage. Adh2 is implicated to up-regulate Adh1 (0.5145, Eq.(7) in Table Adh1 (1.0746, Eq.(3) in Table Gcr2 [Y16] and Sfp1 [Y30] with consistently dominant negative influences on Adh2 and Adh1 respectively would be at the transcriptional level presumably (see Eqs.(3), (7) in Table In the fermentation direction, by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)Eno2 [Y2] (ENOLASE ISOZYME 2) is regulated by Pck1 [Y26] (-0.7195) in the same way as Pyk1 while the main transcription factor is Stp2 [Y31] with significantly positive regulation (0.2147, see Eq.(2) in Table Eno2, Gpm1 [Y8] (PHOSPHOGLYCERATE MUTASE l), Tpi1 [Y10] (TRIOSE-P ISOMERASE 1), Fba1 [Y11] (ALDOLASE 1), and Pgi1 [Y9] (PHOSPHOGLUCOSE ISOMERASE 1) indicates the construction of a trunk of the glyconeogenesis (see Eqs.(8), (9), (10), and (11) in Table Rap1 [Y17] and Gcrl [Y15] are the common regulators of Gpm1, Pgi1, and Tpi1. This means that Rap1 and Gcrl might be the most important regulators in the glyconeogenesis pathway by the transcriptional binding. Finally, Pgm2 [Y5] (PHOSPHOGLUCOMUTASE 2) co-regulated by Glk1 [Y27] (GLUCOKINASE 1), Hxk1 [Y28], and Hxk2 [Y29] (HEXOKINASE 1, 2) significantly confers another pathway leading to the synthesis of UDP-GLU from Glucose-6-P (see Eq.(5) in Table In the sub-pathway of glyconeogenesis, Adh1 and Adh2 are apparent when compared with the obscure relationships in glyconeogenesis . Interestingly, three transcription factors Gcr1, Gcr2 and Rap1  appear to have very significant effects on the metabolic shift pathway. Finally, in order to validate the proposed method, an independent validation is also given by randomly reshuffling the time order of microarray experiment but with same choices of target gene and regulatory gene, as shown in Figure In the overview of the metabolic shift pathway in Figure Microarray expression analysis by the dynamic system approach offers an opportunity to generate functional regulation interpretation on the genome-wide scale. The crucial ontology behind using dynamic system techniques is that the causality between gene expression profiles could be identified according to the differential equation underlying a dynamic system. Therefore, because the microarray data were harvested with time progression, the simultaneously varied gene expressions implicated in a genetic regulatory system would be detected to infer the regulatory pathways in spite of the versatile interactions such as transcriptional control, protein phosphorylation, or specific enzyme regulation.The clustering method answers the problem of what is the functional catalogue of a specific gene by the identification of resembling patterns of gene expressions. Similarly, the co-regulations of upstream genes in our method also imply their concurrent functions. In contrast to the clustering algorithm, the causality of time-course data has been smoothly drawn by our dynamic method. The Bayesian networks were used merely for forward probabilistic estimation with time transition lacking in the feedback linkages. This unidirectional problem would not happen in our algorithm. Owing to the quantitative regulatory abilities of our model, we have a greater diversity of regulatory influence than the Boolean networks, which are deterministic with merely two states.In our dynamic system approach, we not only can link qualitatively the upstream genes to the downstream ones iteratively, but also indicate quantitatively their regulatory relationships, including the regulatory abilities and the activation delays. In terms of the regulatory abilities, the comparison between the upstream regulatory genes of a target gene can inspire us to ask which one is significant biologically and whether it is a positive or negative influence on the investigated gene. Moreover, the speculation of activation delays benefits the empirical reference by providing us when the upstream regulatory genes might interact with their target genes. Since any gene can be considered as a target gene to trace back its upstream regulators, these regulators are then considered as target genes to trace back their upstream regulators. Iteratively, the genetic regulatory pathway (or network) can be constructed to the genome-wide. According to the qualitative and quantitative features imbedded, two regulatory pathway examples are characterized as in Figure In the two pathways under investigation, we have a more detailed understanding about the regulatory interactions among phytochrome, crytochrome and biological clock in the circadian regulatory system. On the other hand, the sophisticated knowledge of the metabolic pathway after the diauxic shift can be unfolded properly in our analysis. Furthermore, the independent validation of our approach is also given by randomly reshuffling the time order of microarray experiment. We found that the proposed pathways in Figures There are some shortcomings in our study. First, although the time-course microarray data are available, its lower samplings will distort the real changes of gene expressions, especially for quick dynamic evolution. A more sampling experiment with respect to the intrinsic turnover rate is expected to have more precise analysis. Secondly, a regulatory gene with larger activation delay would not be recognized because the less activation delay criterion is used, but this might be overcome by properly relaxing the criterion. Thirdly, activation profiles under the proteome should be highly correlated with the transcriptional profiles to elevate the interpretation of our system model. In general, the synchronous time-course microarray assay is more suitable to underlie the transcriptional binding among causal genes, but an inference of physical interactions in the post-transcriptional level also has sufficient feasibility in our study.In the near future, the most pressing task is to investigate our presumed paths in the laboratory. As the pathway construction algorithms are further developed, we expect this system approach to have immense impact in elucidating the underlying molecular mechanisms of pathways in a variety of organisms, especially after the maturation of the protein chips. Ultimately, we envision that biologists will perform routine pathway inference to seek some novel regulations and to identify the evolutionarily conserved links.Gi(t) in equation (1.2), we substitute equation (1.2) into equation (1.1) to obtain the following dynamic equation for the expression profile of the i-th gene,After the decomposition of ai, bi, αn, and βn should be estimated by the time profile of microarray data of the i-th gene, i.e. these parameters should be specified so that the simulating output Xi(t) of the dynamic model in equation (3.1) should meet the empirical expression profile of the i-th gene. The least-squares estimation method is employed to solve this parameter estimation problem.In the above dynamic equation, parameters t = t1,…, tm and is then arranged in a vector differential form. Consequently, the vector differential form underlined in this equation is applied to m time points in order.To make the dynamic model effective, the dynamic equation in (3.1) should meet the expression profile at all time points , , and m denotes the number of time points.where In the next step, formula (3.2) can be translated into a differential matrix equation as follows,Yi = AiΦi + Ei     (3.3), Φi = [ai bi α0 β0 … αN βN]T, and  are in vector forms, while  is a matrix.where i, the least-squares method below is used to derive the optimal parameters estimation of ,To estimate the relevant unknown parameters in ΦEi as the noise of the gene-expression profile or of the microarray chips. So the consideration of modeling error makes equation (3.3) approach more the reality. By the way, in order to get accurate data of  and  from the expression profile of the target gene, the cubic spline should be employed to interpolate the time profile of the target gene. Furthermore, the choice of N is based on the tradeoff between the accuracy of approximation in (1.2) and the complexity of parameter estimation in (3.4). In this study N = 6 is chosen because these harmonics are enough to approximate regulation functions.Actually, the modeling error could be concluded into i in equation (2.4) is given as follows:Maximum likelihood method for Ωm data points is then described byThe log-likelihood function for given σ2 is , by which equation (2.5) is obtained.The necessary condition for the maximum likelihood estimation of variance Substituting equation (2.5) into equation (4.2) yields,i by minimizing the value of σ2 in equation(2.5). Then, the maximum likelihood estimate in equation (2.6) is obtained by  in equation(2.5).meaning that we can find the maximum likelihood estimation of ΩW.C. Chang and Chang-Wei Li carried out the computational studies and analysis. B.S. Chen gave the topic and suggestions. All authors read and approved the final manuscript.The raw data has been transform to .mat file, which is one of the Matlab files. This file has been divided into three parts: Name, Time, and Profile. see  Name denClick here for fileThe 'Shuffled_data' is the Shuffled raw data. see Click here for file


As anorexia and hypermetabolism are common in cirrhosis, leptin levels may be increased in this disease. In this study, we investigated the relation between the severity of disease and serum leptin levels in post-hepatitis cirrhosis and the role of body composition, gender and viral aetiology of cirrhosis in this association.Thirty-five cases with post-hepatitis cirrhosis and 15 healthy controls were enrolled in this study. Body composition including body mass index, body fat percentage and body fat mass were determined. Serum leptin levels were assayed.Leptin levels were significantly higher among cirrhotic patients independent of sex compared to controls (p = 0.001). Female patients in both groups have had higher leptin levels than males (in cirrhotics p = 0.029, in controls p = 0.02).Cirrhotic patients in each of A, B and C subgroups according to the Child- Pugh classification revealed significantly different levels compared to controls (p = 0.046, p = 0.004, p = 0.0001, respectively). Male cirrhotics in Child-Pugh Class B and C subgroups had significantly higher leptin levels compared to male controls (p = 0.006, p = 0.008). On the other hand, female patients only in Child Pugh class C subgroup have had higher levels of serum leptin compared to controls (p = 0.022).Child-Pugh classification has been found to be the sole discriminator in determination of leptin levels in cirrhotics by linear regression (beta: 0.435 p = 0.015).Serum leptin levels increase in advanced liver disease independently of gender, body composition in posthepatitic cirrhosis. The increase is more abundant among patients that belong to C subgroup according to the Child- Pugh classification. Leptin, a 16-kilodalton protein, is involved in the regulation of food intake and body composition  and has In normal humans, circulating level of leptin is higher in women than in men ,5. BesidMalnutrition is a common feature of cirrhotic patients . A negatIn this study, we investigated the relation between the severity of disease and serum leptin levels in post-hepatitis cirrhosis and the role of body composition, gender and viral aetiology of cirrhosis in this association.Thirty-five cases with post-hepatitis cirrhosis  which were diagnosed on the basis of the clinical, laboratory, radiological, and/or histopathological findings, and 15 healthy controls  were enrolled in this study. Cirrhotic cases were assigned into 3 groups on the basis of the Child-Pugh classification  as folloControl group consisted of healthy individuals with normal medical history, physical examination and blood biochemistry. None of them have had a restriction of diet for loosing weight during the last three months. Subjects who receive any medication have not been included into control group. The local human institutional review committee approved the study and written consents were received from all participants.th percentile [Body composition such as BMI, skin fold thickness, body fat percentage (BFP), and BFM analysis was performed in both cirrhotic cases and controls. To avoid incorrect BMI determination and body composition analysis, cirrhotic cases with ascite and edema had been put on sodium restricted diet of 51 mmol per day and they were received diuretics (spiranolactone 100–200 mg and, if necessary, furosemide 40–80 mg per day) until ascite and edema have been resolved. Cirrhotic cases with refractory ascite unresponsive to therapy impaired renal function following diuretic therapy, and triceps skinfold thickness less than 10rcentile  were exc2). Measurements of skin fold thickness were conducted at four different sites on the left side of the body (triceps, biceps, sub-scapular and supra-iliac) using a Holtain skinfold caliper (Holtain, Crosswell, Crymych, Dyfed, UK). All the measurements were made by the same physician (FB). Two measurements were made at each site and the average values were obtained. The BFP was calculated using the Jacksons' formula [BMI was determined as the actual body weight relative to the square of the body height  method by using Human Leptin IRMA DSL-23100 (Diagnostic Systems Laboratories, Inc. Texas, USA) kit. Following test procedures, test tubes were assessed with Gammabyt-CR gamma counter for one hour. Measurements for standards, controls and serums were repeated for confirmation. Sensitivity of the test was 0.10 ng/ml.Data were presented as median and range. Qualitative variables were assessed by Chi-square test. Between whole and sub-group comparisons were performed by non-parametric Kruskal-Wallis and Mann-Whitney U tests. A linear logistic regression analysis was performed with serum leptin levels as dependent variable and age, gender, BFM, aetiology of cirrhosis, Child-Pugh classification as independent variables in cirrhotics. A p value of <0.05 was considered statistically significant.Clinical and demographic characteristics of all and gender-based sub-groups were shown in table Serum leptin levels were significantly higher in cirrhotic group than controls p = 0.001) , 12.8 (4.2–18.8), 14.7 (8–41), respectively)] compared to controls  (p = 0.046, p = 0.004, p = 0.0001, respectively).Gender based serum leptin levels of controls and cirrhotic cases that were grouped according to Child Pugh Classification were as shown in Figure When age, gender, BFM, hepatitis B and C virus as etiologic factors of cirrhosis and child A, B and C as Child-Pugh classification were tested as independent variables for determination of serum leptin levels as dependent variable by linear logistic regression analysis in cirrhotic group, analysis result showed that Child-Pugh classification was the sole discriminator in determination of serum leptin levels in cirrhotic cases (beta: 0.435, p = 0.015) Table .Leptin regulates body weight by suppressing appetite and increasing energy expenditure ,3,4. AnoLeptin levels are higher in woman than in men ,6. McCulSince BMI and BFM values do not differ according to the sex and the presence or absence of cirrhosis, increased serum leptin levels could not be simply dedicated to BFM or malnutrition status in cirrhosis. In addition, linear regression test in the present study has shown that disease severity, which was determined by Child-Pugh classification, was the sole significant determinant of serum leptin levels in cirrhosis. In previous studies, association between the severity of cirrhosis and serum leptin levels is controversial -13. HenrIn an animal study, it has been shown that chronic ethanol consumption leads to increased serum concentrations of tumor necrosis factor and related cytokines such as leptin by inducing over production of these factors in the liver and peripheral adipose tissues . Leptin Serum leptin levels increase in advanced liver disease independently of gender, body composition and viral etiologic factor in post-hepatitis cirrhosis. The increase is more abundant among patients that belong to C subgroup according to the Child- Pugh classification.BMI, body mass index; BFP, body fat percentage; BFM, body fat massThe authors declare that they have no competing interests.Bolukbas FF conceived of the study, and participated in its design and coordination. Bolukbas FF, Bolukbas C, Erdogan M and Zeyrek F collected the samples and carried out the laboratory analysis. Bolukbas C conceived of the study and participated in the sequence alignment and drafted the manuscript. Horoz M participated in the design of the study, participated in the sequence alignment and drafted the manuscript. Gumus M collected the clinical data and performed the statistical analysis. Yayla A drafted the manuscript and revised it critically for important intellectual content. Ovunc O participated in study design and coordination and revised the manuscript critically for important intellectual content.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


When publishing large-scale microarray datasets, it is of great value to create supplemental websites where either the full data, or selected subsets corresponding to figures within the paper, can be browsed. We set out to create a CGI application containing many of the features of some of the existing standalone software for the visualization of clustered microarray data.We present GeneXplorer, a web application for interactive microarray data visualization and analysis in a web environment. GeneXplorer allows users to browse a microarray dataset in an intuitive fashion. It provides simple access to microarray data over the Internet and uses only HTML and JavaScript to display graphic and annotation information. It provides radar and zoom views of the data, allows display of the nearest neighbors to a gene expression vector based on their Pearson correlations and provides the ability to search gene annotation fields..The software is released under the permissive MIT Open Source license, and the complete documentation and the entire source code are freely available for download from CPAN  Microarray experiments produce vast amounts of data. The resulting datasets are highly complex and contain large matrices of expression measurements as well as sequence and experiment annotations that provide biological context to the data. To organize these different types of data in a way that allows intuitive exploration of the data, and provides the ability to gain important insights into relationships within a given dataset requires sophisticated visualization tools. Such visualization tools are of benefit not only to researchers analyzing and presenting or publishing their own data, but also to Model Organism Databases (MODs) for compiling and displaying microarray data for a given model organism.There are several excellent free tools available that allow an individual user to analyze their own data. These tools are either accessible on the web, or can be downloaded and used on a desktop machine. Examples include the EPCLUST , GEPAS ,3 and FGThe application was written using object oriented Perl following the Model-View-Controller (MVC) design paradigm . GeneXplThe Microarray::CdtDataset has two essential functions: during dataset creation (see below) it decomposes the data file into its constituent data parts and creates the files needed during data viewing (see below). During data viewing it provides the API for the viewer, and allows searching and retrieval of the data. Under the current model the dataset object itself is immutable. Microarray::CdtDataset was implemented as a client of the Microarray::DataMatrix module, which provides an API for accessing matrices of expression data. In the design of the classes certain compromises had to be made to accommodate the stateless client server environment in which the program operates. Specifically, to allow rapid responses, pre-generated images and correlation data are cached in a compact format on the web-server.There are two stages required to publish a microarray dataset on the web using GeneXplorer. The first stage (executed only once per dataset) involves creation of all the necessary files for GeneXplorer to use. The second stage uses these files to produce the display using the GeneXplorer web front-end.Dataset creation requires a file in the Clustered Data Table (CDT) format: a simple tab delimited text file format  that would allow us to do the updates via web services.The GeneXplorer package is provided as a typical Perl distribution on the Comprehensive Perl Archive Network (CPAN), and adheres to the usual installation mantra of perl modules. After unpacking the software, a user with administrative privileges merely needs to type:perl Makefile.PLmakemake testmake installThis will install the libraries and the executable files that are needed for dataset creation by GeneXplorer into the regular system locations, unless otherwise specified during the first step above. The example in Figure In addition to its use within SMD, GeneXplorer has been used by many publications to provide access to microarray datasets through their web supplements, that can be accessed through SMD's publication page , and wasWe are planning to further develop GeneXplorer to enable it to handle other data formats. Specifically, we would like it to be able to accept data files in MAGE-ML format , which iWe have developed a web-application, GeneXplorer, which allows the visualization of microarray datasets over the Internet using only a web browser. This application has been extremely useful in our experience, where it serves both SMD users during analysis of their data and the public while browsing published datasets.GeneXplorer is available at  under thSMD: Stanford Microarray Database.CAR designed and wrote the initial version of the GeneXplorer. This was extensively re-factored and modularized by JCM (library modules for dataset) and JD (for explorer). DB was involved in the guidance of the early stages of this project. GS wrote the correlations software and the DataMatrix classes, and guided the development of this project. All authors read and approved the final version of the manuscript.