Evidence-based recommendations are needed to guide the acute management of the bleeding trauma patient, which when implemented may improve patient outcomes.The multidisciplinary Task Force for Advanced Bleeding Care in Trauma was formed in 2005 with the aim of developing a guideline for the management of bleeding following severe injury. This document presents an updated version of the guideline published by the group in 2007. Recommendations were formulated using a nominal group process, the Grading of Recommendations Assessment, Development and Evaluation (GRADE) hierarchy of evidence and based on a systematic review of published literature.Key changes encompassed in this version of the guideline include new recommendations on coagulation support and monitoring and the appropriate use of local haemostatic measures, tourniquets, calcium and desmopressin in the bleeding trauma patient. The remaining recommendations have been reevaluated and graded based on literature published since the last edition of the guideline. Consideration was also given to changes in clinical practice that have taken place during this time period as a result of both new evidence and changes in the general availability of relevant agents and technologies.This guideline provides an evidence-based multidisciplinary approach to the management of critically injured bleeding trauma patients. Uncontrolled post-traumatic bleeding is the leading cause of potentially preventable death among trauma patients ,2. AboutThe early acute coagulopathy associated with traumatic injury has recently been recognised as a multifactorial primary condition that results from a combination of shock, tissue injury-related thrombin generation and the activation of anticoagulant and fibrinolytic pathways. The condition is influenced by environmental and therapeutic factors that contribute to acidaemia, hypothermia, dilution, hypoperfusion and haemostasis factor consumption ,4,8-11. In 2007, we published a European guideline for the management of bleeding following major trauma that included recommendations for specific interventions to identify and control bleeding sources using surgical, physiological and pharmacological strategies . The guiThese recommendations were formulated and graded according the Grading of Recommendations Assessment, Development and Evaluation (GRADE) hierarchy of evidence -16 summaBoolean operators and Medical Subject Heading (MeSH) thesaurus keywords were applied as a standardised use of language to unify differences in terminology into single concepts. Appropriate MeSH headings and subheadings for each question were selected and modified based on search results. The scientific questions posed that led to each recommendation and the MeSH headings applied to each search are listed in Additional file The selection of the scientific enquiries to be addressed in the guideline, screening and grading of the literature to be included and formulation of specific recommendations were performed by members of the Task Force for Advanced Bleeding Care in Trauma, a multidisciplinary, pan-European group of experts with specialties in surgery, anaesthesia, emergency medicine, intensive care medicine and haematology. The core group was formed in 2004 to produce educational material on the care of the bleeding trauma patient , on whicAs part of the guideline development process that led to the 2007 guideline, task force members participated in a workshop on the critical appraisal of medical literature. The nominal group process for the updated guideline included several remote (telephone and web-based) meetings and one face-to-face meeting supplemented by several Delphi rounds [In the GRADE system for assessing each recommendation, the letter attached to the grade of recommendation reflects the degree of literature support for the recommendation, whereas the number indicates the level of support for the recommendation assigned by the committee of experts. Recommendations are grouped by category and somewhat chronologically in the treatment decision-making process, but not by priority or hierarchy.We recommend that the time elapsed between injury and operation be minimised for patients in need of urgent surgical bleeding control (Grade 1A).Trauma patients in need of emergency surgery for ongoing haemorrhage have increased survival if the elapsed time between the traumatic injury and admission to the operating theatre is minimised. More than 50% of all trauma patients with a fatal outcome die within 24 hours of injury -23.In addition, studies that analyse trauma systems indirectly emphasise the importance of minimising the time between admission and surgical bleeding control in patients with traumatic haemorrhagic shock ,25. At pWe recommend adjunct tourniquet use to stop life-threatening bleeding from open extremity injuries in the pre-surgical setting (Grade 1C).Much discussion has been generated recently regarding the use of tourniquets for acute external haemorrhage control. Pressure bandages rather than tourniquets should be applied in the case of minor bleeding from open wounds in extremity injuries. When uncontrolled arterial bleeding occurs from mangled extremity injuries, including penetrating or blast injuries or traumatic amputations, a tourniquet represents a simple and efficient method to acutely control haemorrhage -31. SeveTourniquets should be left in place until surgical control of bleeding is achieved ,30; howeWe recommend that the physician clinically assess the extent of traumatic haemorrhage using a combination of mechanism of injury, patient physiology, anatomical injury pattern and the patient's response to initial resuscitation (Grade 1C).The mechanism of injury represents an important screening tool to identify patients at risk for significant traumatic haemorrhage. For example, the American College of Surgeons defined a threshold of 6 m 20 ft) as a 'critical falling height' associated with major injuries . Further0 ft as aWe recommend initial normoventilation of trauma patients if there are no signs of imminent cerebral herniation (Grade 1C).Ventilation can affect the outcome of severe trauma patients. There is a tendency for rescue personnel to hyperventilate patients during resuscitation ,39, and A high percentage of severely injured patients with ongoing bleeding have traumatic brain injury (TBI). Relevant experimental and clinical data have shown that routine hyperventilation is an important contributor to adverse outcomes in patients with head injuries; however, the effect of hyperventilation on outcome in patients with severe trauma but no TBI is still a matter of debate. A low partial pressure of arterial carbon dioxide on admission to the emergency room is associated with a worse outcome in trauma patients with TBI -43.There are several potential mechanisms for the adverse effects of hyperventilation and hypocapnia, including increased vasoconstriction with decreased cerebral blood flow and impaired tissue perfusion. In the setting of absolute or relative hypovolaemia, an excessive ventilation rate of positive-pressure ventilation may further compromise venous return and produce hypotension and even cardiovascular collapse ,42. It hVentilation with low tidal volume is recommended in patients with acute lung injury. In patients with normal lung function, the evidence is scarce, but some observational studies show that the use of a high tidal volume is an important risk factor for the development of lung injury ,47. The We recommend that patients presenting with haemorrhagic shock and an identified source of bleeding undergo an immediate bleeding control procedure unless initial resuscitation measures are successful (Grade 1B).The source of bleeding may be immediately obvious, and penetrating injuries are more likely to require surgical bleeding control. In a retrospective study of 106 abdominal vascular injuries, all 41 patients arriving in shock following gunshot wounds were candidates for rapid transfer to the operating theatre for surgical bleeding control . A similTraffic accidents are the leading cause of pelvic injury. Motor vehicle crashes cause approximately 60% of pelvic fractures followed by falls from great heights (23%). Most of the remainder result from motorbike collisions and vehicle-pedestrian accidents ,54. TherWe recommend that patients presenting with haemorrhagic shock and an unidentified source of bleeding undergo immediate further investigation (Grade 1B).A patient in haemorrhagic shock with an unidentified source of bleeding should undergo immediate further assessment of the chest, abdominal cavity and pelvic ring, which represent the major sources of acute blood loss in trauma. Aside from a clinical examination, X-rays of chest and pelvis in conjunction with focused abdominal sonography for trauma (FAST)  or diagnWe recommend early imaging (FAST or CT) for the detection of free fluid in patients with suspected torso trauma (Grade 1B).We recommend that patients with significant free intra-abdominal fluid and haemodynamic instability undergo urgent intervention (Grade 1A).We recommend further assessment using CT for haemodynamically stable patients who are either suspected of having torso bleeding or have a high-risk mechanism of injury (Grade 1B).Blunt abdominal trauma represents a major diagnostic challenge and an important source of internal bleeding. FAST has been established as a rapid and non-invasive diagnostic approach for the detection of intra-abdominal free fluid in the emergency room -65. LargThe role of CT scanning of acute trauma patients is well documented -81, and Some authors have shown the benefit of contrast medium enhanced CT scanning. Anderson and colleagues ,83 foundCompared with MSCT, all traditional techniques of diagnostic and imaging evaluation are associated with some limitations. The diagnostic accuracy, safety and effectiveness of immediate MSCT are dependent on sophisticated pre-hospital treatment by trained and experienced emergency personnel and short transportation times ,86. If aHypotensive patients (systolic blood pressure below 90 mmHg) presenting with free intra-abdominal fluid according to FAST or CT are potential candidates for early surgery if they cannot be stabilised by initiated fluid resuscitation -90. A reA number of patients who present with free intra-abdominal fluid according to FAST can safely undergo further investigation with MSCT. Under normal circumstances, adult patients need to be haemodynamically stable when MSCT is performed outside of the emergency room . HaemodyWe do not recommend the use of single Hct measurements as an isolated laboratory marker for bleeding (Grade 1B).Hct assays are part of the basic diagnostic work up for trauma patients. The diagnostic value of the Hct for detecting trauma patients with severe injury and occult bleeding sources has been a topic of debate in the past decade -94. A maWe recommend both serum lactate and base deficit measurements as sensitive tests to estimate and monitor the extent of bleeding and shock (Grade 1B).Serum lactate has been used as a diagnostic parameter and prognostic marker of haemorrhagic shock since the 1960s . The amoVincent and colleagues  showed tSimilar to the predictive value of lactate levels, the initial base deficit has been established as a potent independent predictor of mortality in patients with traumatic hemorrhagic shock . Davis aIn contrast to the data on lactate levels in haemorrhagic shock, reliable large-scale prospective studies on the correlation between base deficit and outcome are still lacking. Although both the base deficit and serum lactate levels are well correlated with shock and resuscitation, these two parameters do not strictly correlate with each other in severely injured patients . TherefoWe recommend that routine practice to detect post-traumatic coagulopathy include the measurement of international normalised ratio (INR), activated partial thromboplastin time (APTT), fibrinogen and platelets. INR and APTT alone should not be used to guide haemostatic therapy (Grade 1C). We suggest that thrombelastometry also be performed to assist in characterising the coagulopathy and in guiding haemostatic therapy (Grade 2C).Little evidence supports a recommendation for the best haemostatic monitoring tool(s). Standard monitoring comprises INR, APTT, platelets and fibrinogen, although there is little direct evidence for the efficacy of these measures. Increasing emphasis focuses on the importance of fibrinogen and platelet measurements.It is often assumed that the conventional coagulation screens (INR and APTT) monitor coagulation; however, these tests monitor only the initiation phase of blood coagulation and represent only the first 4% of thrombin production . It is tIt is theoretically possible that the pattern of change in measures of coagulation such as D-dimers may help to identify patients with ongoing bleeding. However, there are no publications relevant to this question, so traditional methods of detection for ongoing bleeding, such as serial clinical evaluation of radiology  should be used.We recommend that patients with pelvic ring disruption in haemorrhagic shock undergo immediate pelvic ring closure and stabilisation (Grade 1B).We recommend that patients with ongoing haemodynamic instability despite adequate pelvic ring stabilisation receive early preperitoneal packing, angiographic embolisation and/or surgical bleeding control (Grade 1B).The mortality rate of patients with severe pelvic ring disruptions and haemodynamic instability remains unacceptably high -122. TheThe initial therapy of pelvic fractures includes control of venous and/or cancellous bone bleeding by pelvic closure. Some institutions use primarily external fixators to control haemorrhage from pelvic fractures ,125 but Angiography and embolisation is currently accepted as a highly effective means with which to control arterial bleeding that cannot be controlled by fracture stabilisation ,131-140.We recommend that early bleeding control of the abdomen be achieved using packing, direct surgical bleeding control and the use of local haemostatic procedures. In the exsanguinating patient, aortic cross-clamping may be employed as an adjunct (Grade 1C).Abdominal resuscitative packing is an early part of the post-traumatic laparotomy to identify major injuries and sources of haemorrhage ,142. If We recommend that damage control surgery be employed in the severely injured patient presenting with deep haemorrhagic shock, signs of ongoing bleeding and coagulopathy. Additional factors that should trigger a damage control approach are hypothermia, acidosis, inaccessible major anatomical injury, a need for time-consuming procedures or concomitant major injury outside the abdomen (Grade 1C).The severely injured patient arriving to the hospital with continuous bleeding or deep haemorrhagic shock generally has a poor chance of survival unless early control of bleeding, proper resuscitation and blood transfusion are achieved. This is particularly true for patients who present with uncontrolled bleeding due to multiple penetrating injuries or patients with multiple injuries and unstable pelvic fractures with ongoing bleeding from fracture sites and retroperitoneal vessels. The common denominator in these patients is the exhaustion of physiological reserves with resulting profound acidosis, hypothermia and coagulopathy, also known as the 'bloody vicious cycle'. In 1983, Stone and colleagues described the techniques of abbreviated laparotomy, packing to control haemorrhage and of deferred definitive surgical repair until coagulation has been established . Since tThe same 'damage control' principles have been applied to orthopaedic injuries in severely injured patients ,163-166.We recommend the use of topical haemostatic agents in combination with other surgical measures or with packing for venous or moderate arterial bleeding associated with parenchymal injuries (Grade 1B).A wide range of local haemostatic agents are currently available for use as adjuncts to traditional surgical techniques to obtain haemorrhage control. These topical agents can be particularly useful when access to the bleeding area is difficult. Local haemostatic agents include collagen, gelatin or cellulose-based products, fibrin and synthetic glues or adhesives that can be used for both external and internal bleeding while polysaccharide-based and inorganic haemostatics are still mainly used and approved for external bleeding. The use of topical haemostatic agents should consider several factors such as the type of surgical procedure, cost, severity of bleeding, coagulation status and each agent's specific characteristics. Some of these agents should be avoided when autotransfusion is used and several other contraindications need to be considered ,168. TheThe different types of local haemostatics are briefly presented according to their basis and haemostatic capacity:i) Collagen-based agents trigger platelet aggregation resulting in clot formation when in contact with a bleeding surface. They are often combined with a procoagulant substance such as thrombin to enhance the haemostatic effect. A positive haemostatic effect has been shown in several human studies -172.ii) Gelatin-based products can be used alone or in combination with a procoagulant substance . Swelliniii) The effect of cellulose-based haemostatic agents on bleeding has been less well studied and only case reports that support their use are available.iv) Fibrin and synthetic glues or adhesives have both haemostatic and sealant properties and their significant effect on haemostasis have been shown in several human RCTs involving vascular, bone, skin and visceral surgery -178.v) Polysaccharide-based haemostatics can be divided into two broad categories : N-acetyvi) The inorganic haemostatics based on minerals such as zeolite or smectite have been used and studied mainly on external bleeding ,168.We recommend a target systolic blood pressure of 80 to 100 mmHg until major bleeding has been stopped in the initial phase following trauma without brain injury (Grade 1C).In order to maintain tissue oxygenation, traditional treatment of trauma patients uses early and aggressive fluid administration to restore blood volume. This approach may, however, increase the hydrostatic pressure on the wound, cause a dislodgement of blood clots, a dilution of coagulation factors and undesirable cooling of the patient. The concept of low-volume fluid resuscitation, so-called 'permissive hypotension', avoids the adverse effects of early aggressive resuscitation while maintaining a level of tissue perfusion that, although lower than normal, is adequate for short periods . A contrThe low-volume approach is contraindicated in TBI and spinal injuries, because an adequate perfusion pressure is crucial to ensure tissue oxygenation of the injured central nervous system. In addition, the concept of permissive hypotension should be carefully considered in the elderly patient and may be contraindicated if the patient suffers from chronic arterial hypertension.A recent analysis from an ongoing multi-centre prospective cohort study suggests that the early use of vasopressors for haemodynamic support after haemorrhagic shock in comparison to aggressive volume resuscitation may be deleterious and should be used cautiously . HoweverWe recommend that crystalloids be applied initially to treat the bleeding trauma patient (Grade 1B). We suggest that hypertonic solutions also be considered during initial treatment (Grade 2B). We suggest that the addition of colloids be considered within the prescribed limits for each solution in haemodynamically unstable patients (Grade 2C).P = 0.06) [It is still unclear what type of fluid should be employed in the initial treatment of the bleeding trauma patient. Although several meta-analyses have shown an increased risk of death in patients treated with colloids compared with patients treated with crystalloids -194 and  = 0.06) . Promisi = 0.06) . One stu = 0.06)  and a me = 0.06) . A clini = 0.06) . However = 0.06) . In conc = 0.06) .We recommend early application of measures to reduce heat loss and warm the hypothermic patient in order to achieve and maintain normothermia (Grade 1C).Hypothermia, defined as a core body temperature below 35°C, is associated with acidosis, hypotension and coagulopathy in severely injured patients. In a retrospective study with 122 patients, hypothermia was an ominous clinical sign, accompanied by high mortality and blood loss . The proHypothermia is associated with an increased risk of severe bleeding, and hypothermia in trauma patients represents an independent risk factor for bleeding and death . The effAnimal and human studies of controlled hypothermia in haemorrhage have shown some positive results compared with normothermia ,211. ConProlonged hypothermia may be considered in patients with isolated head trauma after haemorrhage has been arrested. If mild hypothermia is applied in TBI, cooling should take place within the first three hours following injury, preferably using selective brain cooling by cooling the head and neck, be maintained for at least 48 hours , rewarmiWe recommend a target haemoglobin (Hb) of 7 to 9 g/dl (Grade 1C).Erythrocytes contribute to haemostasis by influencing the biochemical and functional responsiveness of activated platelets via the rheological effect on platelet margination and by supporting thrombin generation ; howeverin vitro reduction of the Hct did not compromise blood coagulation as assessed by thrombelastometry [The effects of the Hct on blood coagulation have not been fully elucidated . An acutstometry .No prospective RCT has compared restrictive and liberal transfusion regimens in trauma, but 203 trauma patients from the Transfusion Requirements in Critical Care trial  were re-Despite the lack of high-level scientific evidence for a specific Hb transfusion trigger in patients with TBI, these patients are currently transfused in many centres to achieve an Hb of approximately 10 g/dl . This miIn an initial outcome study the lowest Hct was correlated with adverse neurological outcome and RBC transfusions were also found to be an independent factor predicting adverse neurological outcome . InteresWe recommend that monitoring and measures to support coagulation be initiated as early as possible (Grade 1C).Major trauma results not only in bleeding from anatomical sites but also frequently in coagulopathy, which is associated with a several-fold increase in mortality ,8,9,245.Early monitoring of coagulation is essential to detect trauma-induced coagulopathy and to define the main causes, including hyperfibrinolysis ,117. EarWe recommend that ionised calcium levels be monitored during massive transfusion (Grade 1C). We suggest that calcium chloride be administered during massive transfusion if ionised calcium levels are low or electrocardiographic changes suggest hypocalcaemia (Grade 2C).Calcium in the extracellular plasma exists either in a free ionised state (45%) or bound to proteins and other molecules in a biologically inactive state (55%). The normal concentration of the ionised form ranges from 1.1 to 1.3 mmol/l and is influenced by the pH. A 0.1 unit increase in pH decreases the ionised calcium concentration by approximately 0.05 mmol/l . The avaEarly hypocalcaemia following traumatic injury shows a significant correlation with the amount of infused colloids, but not with crystalloids, and may be attributable to colloid-induced haemodilution . Also, hWe recommend early treatment with thawed FFP in patients with massive bleeding (Grade 1B). The initial recommended dose is 10 to 15 ml/kg. Further doses will depend on coagulation monitoring and the amount of other blood products administered (Grade 1C).The clinical efficacy of FFP is largely unproven . NeverthThere are very few well-designed studies that explore massive transfusion strategy. The need for massive transfusion is relatively rare, occurring in less than 2% of civilian trauma patients, but higher (7%) in the military setting. Massive transfusion management has been based on the concept that coagulopathy associated with severe trauma was primarily consumptive due to the dilution of blood clotting factors and the consumption of haemostasis factors at the site of injury.FFP was recommended when PT or APTT was 1.5 times normal or after 10 RBC units had been transfused. Many massive transfusion protocols stipulated one unit of FFP for every four units of RBCs. In recent years, retrospective data from the US Army combat support hospitals have shown an association between survival and a higher ratio of transfused FFP and RBC units. These data show that casualties who received FFP and RBCs at a ratio of 1:4 or lower, had a three-fold higher mortality than those who received a massive transfusion with a 2:3 ratio. These data have induced many civilian trauma centres to modify their transfusion approach to incorporate the early use of thawed FFP in ratios approaching 1:1.Ten relevant studies addressing FFP:RBC ratio have been identified, all of which were retrospective studies, although some are based on data collected prospectively for other reasons. None of the studies were clinical RCTs. The majority of the authors used massive transfusion (10 RBC units within 24 hours) as the entry criterion; however, to limit bias due to FFP unavailability, one study  excludedSeven studies showed better outcomes using a high FFP:RBC ratio ,261-266 These combat data are retrospective, refer to young, previously healthy male patients with penetrating injuries and may be confounded to some extent by treatment biases. Because FFP requires a significant amount of time before it is thawed and available for transfusion and many trauma deaths occur soon after hospital admission, patients who die early may receive RBC units but die before FFP therapy has begun. These cases may therefore be included in the low ratio group even if a 1:1 strategy was intended. One further ground for criticism of many of these studies is that the number of RBCs units transfused is an indicator of severity of injury that cannot be completely adjusted for by regression analysis. All of these limitations must be kept in mind when analysing the available recent literature and emphasises the need for prospective trials.9/l (Grade 1C). We suggest maintenance of a platelet count above 100 × 109/l in patients with multiple trauma who are severely bleeding or have TBI (Grade 2C). We suggest an initial dose of four to eight platelet concentrates or one aphaeresis pack (Grade 2C).We recommend that platelets be administered to maintain a platelet count above 50 × 109/l, and platelet function decreases exponentially below this point [9/l or greater and agreed that platelet transfusion is appropriate to prevent or control bleeding associated with deficiencies in platelet number or function [9/l be maintained following injury.In medical conditions leading to thrombocytopaenia, haemorrhage does not often occur until the platelet count falls below 50 × 10is point -272. Thefunction ,274. The9/l, following injury. If a patient has increased fibrin degradation products due to disseminated intravascular coagulation and/or hyperfibrinolysis, this will interfere with platelet function and a higher threshold of 75 × 109/l has been suggested by consensus groups [9/l have been suggested for treatment of severe brain injury and massive haemorrhage, but the evidence for the higher threshold is weak [P = 0.001) [P = 0.03).An argument can be made for maintaining a higher level of platelets, perhaps up to 100 × 10s groups ,276. Mors groups . Transfu is weak ,276. One= 0.001) . Another= 0.001) . In this10 platelets on average and should increase the platelet count by 5 to 10 × 109/l in a 70 kg recipient. Aphaeresis platelet concentrates generally contain approximately 3 to 6 × 1011 platelets, depending on local collection practice, and physicians should be cognisant of the doses provided locally. A pool of four to eight platelet concentrates or a single-donor aphaeresis unit is usually sufficient to provide haemostasis in a thrombocytopaenic, bleeding patient. If required, the dose of platelets (× 109) can be calculated in more detail from the desired platelet increment, the patient's blood volume in litres , and a correction factor of 0.67 to allow for pooling of approximately 33% of transfused platelets in the spleen.When platelet transfusion was introduced in the 1950s, no clinical trials were employed to assess the utility of platelet therapy compared with placebo, and such trials today might be considered unethical. The appropriate dose of platelets is therefore uncertain. Platelet concentrate produced from a unit of whole blood contains 7.5 × 10We recommend treatment with fibrinogen concentrate or cryoprecipitate if significant bleeding is accompanied by thrombelastometric signs of a functional fibrinogen deficit or a plasma fibrinogen level of less than 1.5 to 2.0 g/l (Grade 1C). We suggest an initial fibrinogen concentrate dose of 3 to 4 g or 50 mg/kg of cryoprecipitate, which is approximately equivalent to 15 to 20 units in a 70 kg adult. Repeat doses may be guided by thrombelastometric monitoring and laboratory assessment of fibrinogen levels (Grade 2C).The formation of fibrin is a key step in blood coagulation ,279, andDuring serious perioperative bleeding, fibrinogen treatment  was associated with a reduction in allogeneic blood product transfusion . The fibFibrinogen administration using thrombelastometry as guidance may be preferable to measuring fibrinogen levels in the laboratory. Some methodological issues in the various laboratory methods to measure fibrinogen concentration remain ,287, andIt is not known whether the administration of fibrinogen via factor concentrate, cryoprecipitate or FFP is associated with a post-traumatic venous thrombotic risk. However, fibrinogen levels are expected to rise to a level of approximately 7 g/l after major surgery and trauma ,290 evenAn increasingly large body of evidence supports the use of antifibrinolytic agents for the management of bleeding in elective surgery and cardiac surgery patients. For the purpose of these guidelines, we have assumed that these effects are transferable to trauma patients, and our recommendations are based upon this unproven assumption. Since the last guidelines were written, aprotinin has been associated with patient safety issues, with an increased rate of renal disease and mortality when compared with the lysine analogues in a large clinical trial and is therefore no longer recommended -296.We suggest that antifibrinolytic agents be considered in the bleeding trauma patient (Grade 2C). We recommend monitoring of fibrinolysis in all patients and administration of antifibrinolytic agents in patients with established hyperfibrinolysis (Grade 1B). Suggested dosages are tranexamic acid 10 to 15 mg/kg followed by an infusion of 1 to 5 mg/kg per hour or ε-aminocaproic acid 100 to 150 mg/kg followed by 15 mg/kg/h. Antifibrinolytic therapy should be guided by thrombelastometric monitoring if possible and stopped once bleeding has been adequately controlled (Grade 2C).In vitro studies have suggested that a dose of 10 μg/ml is required to inhibit fibrinolysis [Tranexamic acid (trans-4-aminomethylcyclohexane-1-carboxylic acid) is a synthetic lysine analogue that is a competitive inhibitor of plasmin and plasminogen. Tranexamic acid is distributed throughout all tissues and the plasma half-life is 120 minutes. There is large variation in the dose employed. inolysis . Studiesinolysis  confirmeinolysis , shown tinolysis .ε-aminocaproic acid is also a synthetic lysine analogue that has a potency 10-fold weaker than that of tranexamic acid. It is therefore administered in a loading dose of 150 mg/kg followed by a continuous infusion of 15 mg/kg/h. The initial elimination half-life is 60 to 75 minutes and it must therefore be administered by continuous infusion in order to maintain therapeutic drug levels until the bleeding risk has diminished.The clear efficacy of antifibrinolytic agents in reducing bleeding in elective surgery and especially in cardiac surgery has been shown in numerous clinical trials -305. TheThe risk of precipitated thrombosis with the use of the lysine analogues tranexamic acid and ε-aminocaproic acid has been of major theoretical concern; however, the Cochrane review of antifibrinolytics cites studies that included more than 8,000 patients receiving lysine analogues and demonstrated no increased risk of either arterial or venous thrombotic events . The lysWe suggest that the use of recombinant activated coagulation factor VII (rFVIIa) be considered if major bleeding in blunt trauma persists despite standard attempts to control bleeding and best-practice use of blood components (Grade 2C).9/l and fibrinogen above 1.5 to 2.0 g/l), the use of antifibrinolytics and correction of severe acidosis, severe hypothermia and hypocalcaemia fail to control bleeding. Because rFVIIa acts on the patient's own coagulation system, adequate numbers of platelets and fibrinogen levels are needed to allow a thrombin burst to be induced by the pharmacological, supraphysiological doses of rFVIIa through direct binding to activated platelets [rFVIIa is not a first-line treatment for bleeding and will be effective only once sources of major bleeding have been controlled. Once major bleeding from damaged vessels has been stopped, rFVIIa may be helpful to induce coagulation in areas of diffuse small vessel coagulopathic bleeding. rFVIIa should be considered only if first-line treatment with a combination of surgical approaches, best-practice use of blood products  of rFVIIa is still under debate. Whereas the above dosing recommendation is based on the only published RCT available in trauma patients and is also recommended by a group of European experts , IsraeliIf rFVIIa is administered, the patient's next of kin should be informed that rFVIIa is being used outside the currently approved indications (off-label use), especially because the use of rFVIIa may increase the risk of thromboembolic complications . Recent We recommend the use of prothrombin complex concentrate for the emergency reversal of vitamin K-dependent oral anticoagulants (Grade 1B).Despite the increasing off-license use of PCC, there are no studies to support its use other than in haemophilia -323 or fBecause the use of PCC carries the theoretical increased risks of both venous and arterial thrombosis during the recovery period ,330, theWe do not suggest that desmopressin be used routinely in the bleeding trauma patient (Grade 2C). We suggest that desmopressin be considered in refractory microvascular bleeding if the patient has been treated with platelet-inhibiting drugs such as acetylsalicylsalicylic acid (Grade 2C).Desmopressin (1-deamino-8-D-arginine) enhances platelet adherence and platelet aggregate growth on human artery subendothelium and was originally licensed for use in von Willebrand disease , a diseaNo studies have investigated the effect of desmopressin in the trauma patient, and there is great uncertainty as to whether the results of studies involving non-trauma patients can be applied to bleeding following trauma. Therefore, only a weak recommendation can be made.We do not recommend the use of antithrombin concentrates in the treatment of the bleeding trauma patient (Grade 1C).Antithrombin concentrates are indicated in inherited and acquired antithrombin deficiency. Although antithrombin deficiency does occur in consumptive coagulopathy, this is not an isolated condition; all coagulation factors and physiological anticoagulants undergo consumption under these circumstances. The best replacement therapy is FFP. Clinical studies of antithrombin concentrate in severe blunt trauma and in critical care have shown no benefit ,339.This guideline for the management of the bleeding trauma patient is based on a critical appraisal of the published literature, a re-appraisal of the recommendations we published three years ago and a consideration of current clinical practice in areas in which RCTs will never be performed for practical or ethical reasons. In the process of generating this updated version of the guideline, we identified a number of scientific questions that have emerged or were not addressed previously and have developed recommendations to cover these issues. The new and revised recommendations included here reflect both newly available evidence and shifts in general clinical practice. As bedside testing, particularly thrombelastogram-based methodology, and multi-slice CT have become more established in the emergency department setting, we felt a need to update our guideline to discuss the use of these new technologies. We also include new recommendations on the use of tourniquets as an adjunct to halt life-threatening open extremity injuries, ionised calcium monitoring and treatment, and the use of local haemostatic agents and desmopressin in the bleeding trauma patient. Our recommendations have also been updated to reflect the recent removal of aprotinin as an antifibrinolytic agent from the market. The final draft of this document omitted a draft recommendation on the use of coagulation factor XIII because, although the author group feels that this agent may play a role in the haemostatic management of trauma patients in future, the present lack of evidence in trauma and means of monitoring the therapeutic effect of this compound in many hospitals precludes a specific recommendation at this time.Although the level of scientific evidence has improved in some areas, particulary those that have come under closer scruitiny in the context of ongoing military conflicts, other areas remain devoid of high-level evidence. Although evidence gathered in a military setting may or may not be readily transferable to the civilian setting, recent experience has shown that there is a need for uniform practices in the management of the traumatically injured patient . This obWe have excluded animal studies from the evidence considered here, and maintain our opinion that humans are the best subjects in whom to study human post-traumatic injury . We alsoAll of the recommendations presented here were formulated according to a consensus reached by the author group and the professional societies involved. Figure A multidisciplinary approach to management of the traumatically injured patient remains the cornerstone of optimal patient care, and we have made an effort to formulate this guideline in a manner that is widely applicable to a variety of settings in clinical practice. As the volume and level of evidence in this field accumulates, the current state-of-the-art as reflected in this guideline will need to evolve accordingly.• This clinical practice guideline provides evidence-based recommendations developed by a multidisciplinary task force with respect to the acute management of the bleeding trauma patient, which when implemented may improve patient outcomes.• Coagulation monitoring and measures to support coagulation should be implemented as early as possible following traumatic injury and used to guide haemostatic therapy.• A damage control approach to surgical procedures should guide patient management, including closure and stabilisation of pelvic ring disruptions, packing, embolisation and local haemostatic measures.• This guideline reviews appropriate physiological targets and suggested use and dosing of fluids, blood products and pharmacological agents in the bleeding trauma patient.• A multidisciplinary approach to management of the traumatically injured patient remains the cornerstone of optimal patient care.ACS: abdominal compartment syndrome; APTT: activated partial thromboplastin time; ARDS: acute respiratory distress syndrome; ATLS: Advanced Trauma Life Support; CT: computed tomography; DPL: diagnostic peritoneal lavage; FAST: focused abdominal sonography for trauma; FFP: fresh frozen plasma; GCS: Glasgow coma score; GRADE: Grading of Recommendations Assessment: Development and Evaluation; Hb: haemoglobin; Hct: haematocrit; INR: international normalised ratio; IQR: interquartile range; MeSH: medical subject heading; MSCT: multi-slice computed tomography; NIH: National Institutes of Health; PCC: prothrombin complex concentrate; PEEP: positive end-expiratory pressure; PT: prothrombin time; RBC: red blood cell; RCT: randomised controlled trial; rFVIIa: recombinant activated coagulation factor VII; TBI: traumatic brain injury.RR has received honoraria for consulting or lecturing from CSL Behring, Novo Nordisk, Bayer, Air Liquide and Eli Lilly and has received research grant funding from AGA-Linde, Air Liquide, Novo Nordisk, Eli Lilly and Glaxo Wellcome. BB has received honoraria for consulting or lecturing from Novo Nordisk, CSL Behring and Sangart. VC has received honoraria for consulting or lecturing from Fresenius (Czech Republic), Schering-Plough (Czech Republic), B. Braun (Czech Republic) and Novo Nordisk (Czech Republic). TJC has received research funding from the UK National Institute for Health Research, BOC Linde and the Mid Anglian GP Accident Service. He is a trustee of BRAKE (a road safety charity) and the College of Emergency Medicine. JD has received honoraria for consulting or lecturing from Novo Nordisk, LFB Biomédicaments and Hutchinson Technology. EF-M has has received honoraria for consulting or lecturing from Sangart and PULSION Medical Systems. BJH has received honoraria for consulting or lecturing from Bayer, Boehringer Ingelheim, Sanofi Aventis and Novo Nordisk. RK has no competing interests to declare. GN has received honoraria for consulting or lecturing from Novo Nordisk and Sangart and institutional research grant funding from Novo Nordisk. EN has received honoraria for consulting or lecturing from Biotest , Javelin Pharma , Novo Nordisk (Denmark), MSD Sharp & Dohme (Haar), Pfizer (Berlin), AstraZeneca (Wedel), B. Braun (Melsungen) and Bristol Myers Squibb  and has received institutional support from Mundipharma (Limburg), Cook Ltd. , QRX Pharma , Ethicon (Norderstedt), KCI  and Sanofi (Berlin). YO has received institutional support from LFB (Laboratoire français du Fractionnement et des Biotechnologies), Octapharma and Novo Nordisk. LR been involved in educational courses on bleeding control supported by Baxter. AS has no competing interests to declare. PFS has received honoraria for consulting or lecturing from Synthes, Stryker Spine and Novo Nordisk. JLV has received honoraria for consulting or lecturing from AstraZeneca, Edwards Lifesciences, Pfizer, Astellas, Eli Lilly, Ferring, GSK, the Medicines group amd Novo Nordisk and has received research grant funding from AM Pharma, Artisan, Astellas, Curacyte, Eli Lilly, Esai and Novo Nordisk. DRS has received honoraria or travel support for consulting or lecturing from Abbott AG  Alliance Pharmaceutical Corp.  AstraZeneca AG  Bayer (Schweiz) AG  B. Braun Melsungen AG , Boehringer Ingelheim (Schweiz) GmbH , CSL Behring GmbH , Curacyte AG  Fresenius SE , Galenica AG  Bern, Switzerland), GlaxoSmithKline GmbH & Co. KG , Janssen-Cilag AG , Novo Nordisk A/S , Octapharma AG , Organon AG , Oxygen Biotherapeutics , Pentapharm GmbH , Roche Pharma (Schweiz) AG  and Schering-Plough International, Inc. . His academic department currently receives grant support from the University of Zurich, the Research Award Center for Zurich Integrative Human Physiology, the Swiss National Science Foundation, the Swiss Foundation for Anesthesia Research, the European Society of Anaesthesiology (ESA), the Swiss Society of Anesthesiology and Reanimation (SGAR), the Gebert Ruef Foundation, the Swiss Life Foundation, the Olga Mayenfisch Foundation, Abbott AG Switzerland, B. Braun Switzerland, UBS Switzerland, Stiftung für Staublungenforschung, Switzerland.The ABC-T European medical education initiative is managed by Physicians World Europe GmbH  and supported by educational grants from Novo Nordisk.All of the authors participated in the formulation of questions to be addressed in the guideline, screening of abstracts and literature, face-to-face and remote consensus-finding processes, drafting, review, revision and approval of the manuscript.RR serves as chair of the Advanced Bleeding Care in Trauma (ABC-T) European medical education initiative. VC is a member of the ABC-T European medical education initiative faculty. TJC is a member of the ABC-T European medical education initiative faculty. JD is a member of the ABC-T European medical education initiative faculty. EF-M is a member of the ABC-T European medical education initiative faculty. PFS is a member of the ABC-T European medical education initiative faculty. RK represented the European Society of Trauma and Emergency Surgery (ESTES) on the ABC-T Task Force. YO represented the European Society of Intensive Care Medicine (ESICM) on the ABC-T Task Force. LR represented the European Society for Emergency Medicine (EuSEM) on the ABC-T Task Force. AS represented the European Shock Society (ESS) on the ABC-T Task Force. DRS serves as co-chair of the Advanced Bleeding Care in Trauma (ABC-T) European medical education initiative and represented the European Society of Anaesthesiology (ESA) on the ABC-T Task Force.MeSH terms and limits applied to address guideline literature queries - 2009. Word file containing MeSH terms and limits applied to address guideline literature queries.Click here for file


Bovine ISG15 (bISG15) is an interferon inducible ubiquitin-like protein that is responsible for the establishment of early pregnancy in ruminant, understanding the properties of bISG15 capable of being inducible in fetal bovine lung (FBL) cells upon infection of bovine immunodeficiency virus (BIV) is of significant importance. In this study, we investigated the expression of bISG15 in poly I:C treated FBL cells. The increased expression of bISG15 was observed, and the inhibition of BIV replication was also detected in FBL cells. Elimination of bISG15 expression by small interfering RNA reversed the bISG15 mediated inhibition of BIV replication. These findings demonstrate that bISG15 plays an important role in inhibition of the BIV replication in FBL cells. Furthermore, real-time PCR and western blot assay revealed that bISG15's expression can also be induced in BIV infected FBL cells. Taken together, bISG15 is an antiviral and inducible protein in BIV infected FBL cells. Type I IFNs' binding to cell type specific receptors can induce the synthesis of a plethora of proteins , many ofBovine ISG15 (bISG15) has 70% homology amino acid sequence to human ISG15 . The stuIn this study, FBL cells were isolated from fetal bovine lung issues. The primary FBL cells isolated from the lung issues of fetal calf were called passage"1". When FBL cells proliferated and separated, they were called passage "2" and so on. All the FBL cells used in the experiment were the passages between 10 and 20. BIV indicator cell line, designated as BIVL, has an integrated reporter plasmid which has a firefly luciferase reporter protein gene downstream of BIV LTR promoter . FBL celIn order to test the capacity indicative of BIV infection, different numbers of BIV-infected FBL cells were added to BIVL cells. To make the number of all FBL cells at equal level of 200 × 102, naive FBL cells free of BIV infection were added. Infected/naive FBL cells co-cultured with BIVL for 12 hours, and then the cells were taken luciferase assays. Naive FBL cells were added to the BIVL as negative control. As shown in Figure Poly I:C, which is the synthetic double-stranded RNA (dsRNA), can robustly induce synthesis of IFN or ISGs directly without the participation of IFNs . TLR3, cWe demonstrated that bISG15 played a role in inhibiting the replication of BIV in FBL cells. In addition, we found the expression of bISG15 in naive FBL cells is very low, it is intriguing to address the question, and how the expression of bISG15 change in FBL cells after being infected with BIV. In order to validate whether the level of the bISG15 was increased in FBL cells with infection of BIV, bISG15 was detected by western blot using the antiserum against bISG15. FBL cells treated with poly I:C or LPS served as a positive control. FBL cells treated with PBS or mock-infected with naive FBL cells were used as negative controls. After 6 h, cells were collected to perform western blot. It was found that free bISG15 was detected in treated or BIV infected FBL cells, even though the expression level of bISG15 was not strong. In contrast, bISG15 positive bands were not observed in untreated or mock-infected FBL cells, as shown in Figure This study demonstrates that bISG15 is an antiviral and inducible protein in BIV infected fetal bovine lung cells. BIV infection can induce the expression of bISG15 in FBL cells, but it is still unclear that the detail involved in this process. It was shown in Figure The authors declare that they have no competing interests.CL carried out all the experiments and drafted the manuscript. XL, XY, XK, WQ and YQ participated in the design of the study and revision of the manuscript. All authors read and approved the final manuscript.


Common variable immune deficiency (CVID), one of the most common primary immunodeficiency diseases presents in adults, whereas X-linked agammaglobulinemia (XLA), an inherited humoral immunodeficiency, is usually diagnosed early in life after maternal Igs have waned. However, there have been several reports in the world literature in which individuals have either had a delay in onset of symptoms or have been misdiagnosed with CVID and then later found to have mutations in Bruton's tyrosine kinase (BTK) yielding a reclassification as adult-onset variants of XLA. The typical finding of absent B cells should suggest XLA rather than CVID and may be a sensitive test to detect this condition, leading to the more specific test . Further confirmation may be by mutational analyses.The records of 2 patients were reviewed and appropriate clinical data collected. BTK mutational analysis was carried out to investigate the suspicion of adult-presentation of XLA. A review of the world literature on delayed diagnosis of XLA and mild or "leaky" phenotype was performed.+ B cells were reclassified as having a delayed diagnosis and adult-presentation of XLA. Patient 1, a 64 yr old male with recurrent sinobronchial infections had a low level of serum IgG of 360 mg/dl , IgA <27 mg/dl , and IgM <25 mg/dl . Patient 2, a 46 yr old male with recurrent sinopulmonary infections had low IgG of 260 mg/dl, low IgA <16 mg/dl, and normal IgM. Mutational analysis of BTK was carried out in both patients and confirmed the diagnosis of XLA2 patients previously diagnosed with CVID associated with virtual absence of CD19+ B cells. A diagnosis of XLA can have significant implications including family counseling, detecting female carriers, and early intervention and treatment of affected male descendents.These two cases represent an unusual adult-presentation of XLA, a humoral immunodeficiency usually diagnosed in childhood and the need to further investigate a suspicion of XLA in adult males with CVID particularly those associated with low to absent CD19 Primary humoral immune deficiency disorders are characterized by defects in antibody production leading to significantly weakened humoral immunity. These patients are highly susceptible to recurrent bacterial infections, bacteremia, and sepsis resulting in high mortality rates. Among these disorders there is heterogeneity of clinical manifestations and immunological defects observed. Differentiating one humoral immune deficiency syndrome from another requires thorough immunological evaluation in a timely fashion since early diagnosis and treatment are essential to patient outcomes and survival. In recent years, advances in genetic mutational analysis have allowed physicians to more accurately diagnose patients that present with recurrent infections and are suspected of having an underlying primary immune deficiency. X-linked agammaglobulinemia (XLA), although a disorder of infants and children, sometimes may be diagnosed late in life. In this instance, it may be easily confused for another disorder-common variable immune deficiency (CVID). Though the clinical and prognostic outcomes may be considered to be similar in the two disorders, the genetic basis is different, leading one to evaluate family transmission more aggressively or consider gene therapy as an option in one or more of these conditions.+ B lymphocytes and severely decreased levels of all isotypes of immunoglobulins, however wide variability in clinical presentation among families with XLA have been observed. In contrast to CVID, XLA is caused by a congenital defect of B cell development and most often presents during infancy after maternal Igs have dissipated, however, there have been several cases previously described in which individuals had late onset of chronic infection or were misdiagnosed with CVID and later found to have Btk mutations . . 12]. MoKornfeld et al described a case of extreme variation in a three-generation family in which the proband, a 51 year old male with recurrent sinusitis, was found to have atypical XLA as was a nephew whom was diagnosed upon contracting pseudomonas bacteremia at age 10 months and then died at age 8 from encephalitis . This ca+ CD19+ pro-B cells to mature B cells. Other cells express Btk including erythroid precursors, mast cells, monocytes, myeloid cells, megakaryocytes, and platelets, however its expression is absent in T and natural killer cells [XLA is caused by an arrest in B cell development associated with mutations in the Btk gene which has been mapped to Xq21.3-q22. BTK is a member of the Tec family of nonreceptor tyrosine kinases and is expressed throughout B-cell development from CD34The Btk genome sequence consist of 19 exons and mutations have been reported in all five of the domains of the Btk gene including the Pleckstrin Homology (PH), Tec homology (TH), Src homology 1 (SH1), Src homology 2 (SH2), and Src homology 3 (SH3). The PH domain is considered to be the most distinct and has crystal structure similar to many other signaling proteins. The PH domain consists of a positively charged ligand-binding pocket that binds phosphatidylinositol lipids. This binding is necessary to promote Btk translocation and localization to the cell membrane . The SH + B cells to survive long enough to produce sufficient amounts of immunoglobulins to avoid life-threatening infections in childhood. Some of the cases diagnosed as adults may represent "mild" phenotypes of XLA despite having significantly low to absent B cells. Noordzij et al suggest that these milder clinical phenotypes may be associated with BTK splice-site mutations which produce lower levels of wild-type BTK transcripts [According to Valiaho et al and their 2006 review of the online mutation database for Btk mutations, BTKbase, 1,111 patient entries have been compiled from 973 unrelated families with 602 unique molecular events. Of all these mutations 40% were missense mutations leading to amino acid substitutions, premature stop codons, and exon skipping, while the remainder consisted of 17% nonsense, 20% deletions, 7% insertions, and 16% splice-site mutations . The disKnowledge of the key characteristics of XLA and CVID may assist in the differentiation of these two clinically similar diseases Table . XLA and+ B cells should be suspected of having XLA. In atypical cases such as these two that we present, the virtual absence of CD19+ B cells may be a sensitive test to differentiate XLA from CVID, which may lead to the more specific genetic mutational analysis for Btk mutations.In conclusion, we present two males diagnosed in adulthood with CVID whom upon further investigation by BTK mutational analysis were found to have XLA. These two cases demonstrate delayed diagnosis and adult presentation of XLA which is usually diagnosed in the first decade of life in male children with recurrent bacterial infections. DNA sequencing of the BTK gene from each patient showed mutations in the PH domain of patient 1 and the SH1 domain of patient 2. Each of these mutations had been previously reported in the BTKbase online registry. Prior to these two patients, 16 other patients were reported in the world literature with adult presentation of XLA. Any male patient who presents with recurrent infections, hypogammaglobulinemia, and low to absent CD19The authors declare that they have no competing interests.JS carried out the literature review, drafted the manuscript, and configuration of tables and figures, EK assisted in data collection, design of tables, and literature review, GK supplied the manuscript outline and reviewed the manuscript for final submission.


REST is a well known repressor of neuronal gene expression. Genome-wide analysis of REST occupancy in different cell types now reveals new and cell-specific roles for REST in embryonic stem cells. For a cell to differentiate and adopt the identity of a specific cell lineage, transcriptional mechanisms must “switch on” a suite of genes that encode proteins characteristic of that cell type. Simultaneously, other genes characteristic for other cell types must reliably be “switched off.” As the proper regulation of gene expression is essential for cellular differentiation and normal development, understanding how differentiation programs regulate the correct number and types of cells in a developing organism is a fundamental issue in biology and medicine.Embryonic stem cells (ESCs) are believed to possess the innate capacity to differentiate into any of the multitude of cell types that make up the body, a capacity known as pluripotency (http://en.wikipedia.org/wiki/Transcription_(genetics)), including Mash1 , Math1 , and the Neurogenin family [http://en.wikipedia.org/wiki/Neurogenesis) by directly binding to the promoter of neurogenic genes such as Mash1, repressing the gene [Differentiation programs employ regulatory factors that both promote and repress transcription. For example, certain vertebrate activating transcription factors  in the promoter of certain neuronal genes, in 1995 Gail Mandel's and David Anderson's labs independently reported the discovery of a transcriptional repressor binding to this element, which they named REST (RE1 silencing transcription factor) and NRSF (neuron-restrictive silencing factor), respectively (from here referred to as REST) ,5. REST http://en.wikipedia.org/wiki/Chromatin), histones associated with DNA. Soon after the initial reports connecting transcriptional activation with histone acetylation (associated with “open” chromatin and accessible DNA) and transcriptional repression with histone deacetylation (associated with “compact” chromatin and less accessible DNA), the N-terminal repression domain of REST was shown to bind the transcriptional repressor Sin3A and associated histone deacetylases (HDACs) such as HDAC1 and HDAC2  Rett syndrome, Huntington disease, and recently with X-linked mental retardation [In association with the role for REST in repressing neuronal gene expression, it has also been implicated in neurological disorders. REST has been shown to regulate the neurotrophic factor BDNF (brain-derived neurotrophic factor) with implications for psychiatric disease. By interactions with various transcription factors, REST has also been associated with the neurodevelopmental disorder  for its ability to repress neuronal genes and thereby presumably repress differentiation of neuroectodermal tumor cells [The increased knowledge of the complex nature of the transcriptional regulation by REST has been accompanied by an increasingly more complicated view of the functional roles for REST in physiology and pathology . The peror cells .Concurrently with the first reports of REST as a tumor suppressor, it was demonstrated that the levels of REST protein in combination with how and where REST binds to the genome are critical parameters for the regulation of physiological neuronal differentiation in neural stem cells (NSCs) and also in ESCs ,18. It hhttp://en.wikipedia.org/wiki/Chip-Sequencing) in human T cells suggested that REST also interacted with gene networks involved in pancreatic islet ß cell development [Lately, it has been shown that REST may play additional roles in cellular events not necessarily associated with its repression of neuronal genes. Deletion of REST in ESCs was reported to result in loss of self-renewal and a markedly decreased expression of members of the transcriptional network required for the self-renewal of ESCs, i.e., Oct4, Sox2, and Nanog (see further discussion below), although any putative direct connection between REST and these factors was not noted . It has elopment . These oPLoS Biology, Johnson, Teh and colleagues report significant progress in the understanding of cell-specific roles for transcription factors in general and REST in particular by genome-wide studies of REST occupancy in different cell types, with particular focus on ESCs [In this issue of  on ESCs . To achi on ESCs . By usin on ESCs . This noThe next interesting finding was made when the authors analyzed the classes of genes regulated by REST with special emphasis on sites that showed enriched occupancy specifically in ESCs . WhereasThere are several reasons why this finding is noteworthy. Wnts constitute a major class of extracellular signal substances, and different Wnts affect specific intracellular signaling pathways with profound effects on cell proliferation, death, migration, polarity, progenitor expansion, differentiation, and maturation. Thus changes in Wnt signaling will induce a plethora of effects on several key aspects of the ESC phenotype. Moreover, this regulatory event provides a prime example of how modulation of a single transcription factor may have major secondary and tertiary bystander effects, with important implications for the specificity issue of drugs modulating transcriptional regulators.Arguably, the most timely of the observations made by Johnson, Teh and colleagues is that REST is a direct part of the Oct4/Sox2/Nanog transcriptional network in ESCs and that many REST targets are other transcription factors that are also regulated by the Oct4/Sox2/Nanog factors in ESCs . Oct4/SoThis study also raises many new questions. There are now several reports of the generation of induced pluripotent stem (iPS) cells achieved by introducing small sets of transcription factors such as Oct4, Sox2, and Nanog into differentiated cells, and it will be interesting to see whether regulation of REST is part of the dedifferentiation events. The very large amounts of data generated by the genome-wide studies by Johnson, The, et al. provide a vast dataset for deeper examination by the field, and it will be of immediate interest to study similarities and differences between epigenetic regulatory mechanisms at common and cell-specific REST sites. The cell-specific differences in REST occupancy serve to inspire further genome-wide investigations of the occupancy of other transcription factors as well as REST-associated chromatin modifying proteins in distinct cell types and different cellular contexts. Using these types of genome-wide studies, there is unlimited potential to shed new light on old problems concerning the influence of metabolic and environmental cues on physiological stem cell characteristics as well as pathological events such as tumorigenesis.


These results suggest that vesnarinone plus glucocorticoid might be useful in lung cancer therapy. © 1999 Cancer Research CampaignVesnarinone, an oral cardiotonic, inhibited the growth of several human non-small cell lung carcinoma cell lines, and its anti-proliferative effects in vitro and in vivo were greatly enhanced by combination with glucocorticoids, but not other steroids. Simultaneous treatment with vesnarinone and dexamethasone is the most effective to evoke the synergistic effect in the growth inhibition of lung carcinoma EBC-1 cells. Dexamethasone and other glucocorticoids induced morphological changes in EBC-1 cells and these agents together with vesnarinone induced alkaline phosphatase activity, which is a typical marker of type II pneumocyte maturation. This treatment arrested the growth of the cells at the G


In the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays.We developed three assays that measure the full set of TCAC enzymes. One assay relies on the sequential addition of reagents to measure succinyl-CoA ligase activity, followed by succinate dehydrogenase, fumarase and, finally, malate dehydrogenase. Another assay measures the activity of α-ketoglutarate dehydrogenase followed by aconitase and isocitrate dehydrogenase. The remaining assay measures citrate synthase activity using a standard procedure. We used these assays successfully on extracts of small numbers of human cells displaying various severe or partial TCAC deficiencies and on frozen heart homogenates from heterozygous mice harboring an SDHB gene deletion.This set of assays is rapid and simple to use and can immediately detect even partial defects, as the activity of each enzyme can be readily compared with one or more other activities measured in the same sample. Interest in assaying tricarboxylic acid cycle (TCAC) enzyme activities has been rekindled by evidence that deficiencies in these enzymes cause a variety of human diseases ,2, in coResidual activities associated with TCAC impairments in humans vary widely and may determine the magnitude of organic acid accumulation . OrganicThe ratios between TCAC enzymes are consistent for each mammalian tissues presumably reflecting their metabolic demand, as shown three decades ago in the seminal study by Pette and Hofer . This ecAt present, TCAC enzyme activities are measured using a series of independent assays that are both laborious and time consuming. We therefore developed a limited set of assays allowing both measurement of all TCAC enzyme activities and detection of abnormalities in enzyme activity ratios. We used these assays successfully to detect severe and partial isolated deficiencies in several TCAC enzymes.Given that TCAC enzyme activity ratios, because of their consistency , are imp+, allows estimation of NAD+-dependent GDH activity. Depending on the enzyme activity levels in the sample, it may be necessary at this point to add more DCPIP before performing the next assays (fumarase and MDH activity). Fumarase is assayed by adding a large fumarate excess, which is readily converted to malate by fumarase, this latter acid being used up by MDH to produce NADH and oxaloacetate. Owing to the presence of added aspartate aminotransferase and glutamate, oxaloacetate does not accumulate and, therefore, does not slow the MDH reaction. The last enzyme of the assay, MDH, is then measured by adding 10 mM malate.The first assay measures five enzymes sequentially in an individual sample Fig. . Importa+/NADP+) by KDH. This enzyme, one of the limiting steps of the TCAC, requires the presence of Ca++ ions, thiamine pyrophosphate, and coenzyme A to catalyze the oxidation of α-ketoglutarate. After KDH measurement, cis-aconitate is added for measurement of aconitase activity based on the formation of isocitrate, which, in the presence of IDH, is readily used up to reduce NAD+/NADP+. Finally, the maximal activity rate of IDH is determined after addition of a large isocitrate excess. Citrate synthase, the last TCAC enzyme to be measured, condenses acetyl-CoA and oxaloacetate into citrate while concomitantly releasing coenzyme A, whose thiol residue readily reacts with Ellman's reagent (dithionitrobenzene). It is measured using the standard procedure which, in the case of cultured skin fibroblasts, concomitantly allows the detection of mycoplasma [The second assay starts with measurement of the reduction of pyridine nucleotides  of fumarase activity. We studied a lymphoblastoid cell line from a human patient (P3) harboring a heterozygous mutation in the fumarase gene, previously shown to result in a nearly complete loss of activity when associated with a loss of the corresponding allele in tumors. Again, our assay proved capable of detecting the predictable partial (~40%) loss of fumarase activity in these cells, in terms of both the absolute activity and the activity relative to the other TCAC enzymes in the sample  were investigated , the supernatant discarded, and the pellet washed (5 min × 6000 g) with 1 ml of medium A devoid of digitonin and Percoll [g for 5 min).Fibroblasts derived from forearm biopsies taken with informed consent from healthy controls and patients with TCAC enzyme deficiencies were grown under standard conditions as described elsewhere  and froz Percoll . Lymphob2PO4 (pH 7.2) and 1 mg/ml BSA. The reduction of dichlorophenol indophenol (DCPIP) is measured using two wavelengths (600 nm and 750 nm) with various substrates and the electron acceptors decylubiquinone and phenazine methosulfate. The second assay measures α-ketoglutarate dehydrogenase (KDH), aconitase, and isocitrate dehydrogenase (IDG) activities. The same volume of the same medium is used, and pyridine nucleotide (NAD+/NADP+) reduction is measured with various substrates using wavelengths of 340 nm and 380 nm. In the third assay, citrate synthase is measured by monitoring dithionitrobenzene  reduction at wavelengths of 412 nm and 600 nm as previously described[The first assay measures succinyl-CoA ligase, SDH, glutamate dehydrogenase (GDH), fumarase, and malate dehydrogenase (MDH) ; TPP: thiamine pyrophosphate; Tx100: Triton X100.SG, JJB and PR designed the concept and experiments of the study. VP, ED and PB provided and studied the various cell types utilized, while JF and APGR provided and studied the SDH-mutant mouse. SG and PR analyzed the data. PR drafted the manuscript, helped by the other authors. All authors approved the final manuscript.


Gastroenteropancreatic neuroendocrine tumours (GEP NETs) are rare tumours that present many clinical features.They secrete peptides and neuroamines that cause distinct clinical syndromes, including carcinoid syndrome. However, many are clinically silent until late presentation with mass effects.In 2000 the WHO developed a new classification which gives a better description of the characteristics and biological behaviour of the tumour.Surgical resection is the treatment of first choice for a patient with a GEP NET. In metastatic disease multiple therapeutic approaches are possible. In these cases the goal is to improve quality of life and to extent survival.GEP NETs express somatostatin receptors (SSTRs), which are bound by somatostatin (SST) or its synthetic analogues, although the subtypes and number of SSTRs expressed is very variable.Somatostatin analogues are used frequently to control hormone-related symptoms while their anti-neoplastic activity, even if it has not been widely studied and the regarding data are discordant, seems to result prevalently in tumour stabilisation.A few patients who fail to respond or cease to respond to standard SST analogues treatment seem to have a response to higher doses of these drugs.The use of higher doses of somatostatin analogues or the development of new subtype selective agonists and chimaeric somatostatin analogues, or pan-somatostatin will probably improve the clinical management of these patients.This review provides an update on the use of somatostatin analogues in the management of GEP NETs and discusses novel clinical strategies based on SSTR 2 gene transfer therapy. Gastroenteropancreatic neuroendocrine tumours (GEP NETs) are an heterogeneous group of relatively rare tumours, whose yearly incidence is 1.2-3.0 cases/100,000 inhabitants  patients .The long-acting somatostatin analogue octreotide-LAR was administered in a patient with multiple type A gastric carcinoids for a period of 9 months with a normalisation of serum gastrin levels and permanent disappearance of the tumour . Fykse eet al have conducted a study regarding the effects of somatostatin analogues on cell proliferation in the rat-derived insulinoma cell line (INS1). Their preliminary data show that octreotide has a significant inhibitory effect on cell proliferation, as assessed by cell counting and MTS assay, and on phosphorylation states of a number of proteins in the PI3K/Akt/mTOR pathway DOTA-Tyr(3)-octreotate, a selective analogue of SSTRs 2. In spite of its favourable affinity profile, at its maximum tolerated dose, it is limited by toxic effects on the kidney and bone marrow. Nevertheless, the results seem encouraging compared with historical therapeutic data .et al obtained promising results using 177Lu DOTATATE [177Lu]DOTA-Tyr(3)-octreotate in 131 patients with NETs. A complete remission was observed in 2% of patients, a partial remission in 26%, a minor response in 19%, stable disease in 35%, and progressive disease in 18% of patients. Higher remission rates were positively correlated with high uptake on pre-therapy SSTRs imaging, whereas progressive disease was significantly more frequent in patients with extensive disease. Median time to progression was more than 36 months  [Recently, the high expression of SSTRs on gastrinomas has been considered as an opportunity to use radiolabeled somatostatin analogues, in order to achieve a cytotoxic effect [Novel strategies based on SSTRs 2 receptor gene transfer to target tumour growth and angiogenesis represents a new advance in the treatment of unresectable pancreatic tumours.Buscail et al initially demonstrated that in human pancreatic adenocarcinoma SSTR 2 expression was specifically los.Once gene defect corrected, cell growth as well as tumorigenicity, were significantly reduced in the absence of exogenous ligand . The synSeveral study conducted on pancreatic adenocarcinoma animal models demonstrated that intratumoural SSTR 2 gene transfer (using polyethylenimine synthetic vector) inhibited intratumoural production of somatostatin that was critical for the SSTR 2 antitumoral effect.Primary tumour growth and angiogenesis were highly decreased and associated with a reduction in microvessel density, inhibition of intratumoural production of VEGF and up-regulation of antiangiogenic SSTR 3 receptor expression in peripheral tumour vessels ,113,114.Neuroendocrine tumors of the gastroenteropancreatic (GEP NETs) system comprise a rare group of malignant neoplasms. The somatostatin analogues have been shown to be very useful for symptomatic and biochemical improvement in patients with these tumours while preclinical and clinical studies provide conflicting results on their antitumour effects. The mechanisms of these effects are unknown, but probably are in part due to direct effects on proliferative signalling pathways, activation of apoptosis, and effects on angiogenesis.Biological response to somatostatin analogs depends on distribution and level of expression of SSTRs subtypes in tumours, and the expression of selective somatostatin receptor-signaling pathway molecules.The high density of SSTR2 in endocrine tumours explains the use of SSTR 2 specific analogues in the diagnosis and treatment of these tumours. However, the role of SSTR1,3 and 5 appears to be of increasing interest.The development of new peptidic and non-peptidic somatostatin analogues, subtype selective agonists, chimaeric analogues, or pan-somatostatin analogues will probably improve the diagnosis and treatment of GEP NETs, which express somatostatin receptors other than SSTR 2.The combination of SSAs and IFN seems of benefit in patients where the treatment with somatostatin analogues alone failed to achieve a biochemical and symptomatic control while their synergistic effect on tumour growth is still unknown.The analysis of the SSTR status specifically for each patient, and studies of individual tumour biological behaviour, might be of therapeutic interest and could help to optimise treatment expecially in unresectable tumours.Peptide-receptor-targeted radiotherapy for advanced disease using radiolabeled octapeptide analogues appears to be a significant progress in the treatment of GEP NETs but data are limited, mainly about the best time for its administration, or what is the most appropriate radioligand/combination to be used for each patient, and if and how the doses should be fractionated.Novel strategies based on SSTR 2 receptor gene transfer to target tumor growth and angiogenesis might offer new prospectives of therapeutic interest mainly to treat unresectable tumours.Prospective studies including large number of patients regarding the optimal dosage and modes of administration of somatostatin analogues and the development of new slow release, SSTR subtype specific compounds are needed.We disclose any financial and personal relationships with other people or organisations that could inappropriately influence (bias) their work.MA and RB read and approval the final manuscript.


Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present “case-control” study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects ∼4 times as many males as females. Preliminary metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism. Autism spectrum disorders  are a group of pervasive neurodevelopmental disorders that are characterized by delayed or abnormal development and use of language, poor reciprocal social interactions, restricted interests and repetitive behaviors idiopathic autism. At present, genetic susceptibility loci have been identified on virtually every chromosome by a combination of whole genome scans, cytogenetics, and genetic linkage/association analyses de novo) are 10 times more frequent in the autistic population than in the general population Although ASD is the most heritable of all the psychiatric disorders based upon twin and family studies in silico to autism susceptibility regions on chromosomes that were previously identified by numerous genetic analyses Genomic methods involve simultaneous, large-scale expression analysis of thousands of genes on a cDNA (or oligonucleotide) microarray slide In this study, we postulate that differentially expressed genes in autistic vs. normal nonautistic siblings are at least in part responsible for the autism phenotype, as we had earlier shown for monozygotic twins discordant in diagnosis of autism The log2 ratios of relative gene expression from autistic and nonautistic siblings were analyzed by one-class SAM using 70% data filtering, which requires that at least 70% of the samples must have non-zero expression ratios in order for a given gene to be included in the statistical analysis. Significant differentially expressed genes with a log2 ratio≥±∼0.3 are shown in  and Quantitative RT-PCR (qRT-PCR) was used to confirm the differential expression of genes represented among the top biological functions , includiBased upon the qRT-PCR-confirmed differential expression of SCARB1 and SRD5A1 which are involved in cholesterol metabolism and steroid hormone biosynthesis, we constructed a multilevel biomolecular network representing the possible interactions and functions of the genes, gene products, and downstream metabolites Figure 5It is becoming increasingly clear that although the neurological symptoms of ASD are the most striking among the behavioral and functional manifestations of affected individuals, there are many associated peripheral physiological symptoms that have often gone unnoticed/ignored and clinically unaddressed. These include gastrointestinal disorders experienced by many on the spectrum (estimated at 50%) as well as immune disorders which have long been described in the literature on ASD Overall, our study of autistic-nonautistic sib pairs in which the autistic sibling has been subtyped according to severity of language impairment on the basis of cluster analysis of scores from the ADIR diagnostic interview , reveals altered expression of genes that participate in cholesterol metabolism and androgen biosynthesis. It is noteworthy that deficiency of 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme in cholesterol biosynthesis, is the genetic cause of Smith-Lemli-Opitz syndrome (SLOS) which is an autosomal recessive disorder characterized by pre- and post-natal growth retardation, distinct facial anomalies, microcephaly, and mental retardation both males and females The biological consequences of elevated testosterone on neurodevelopment and function are just beginning to be understood. While it has been known for more than 10 years that estrogens modulate synaptic plasticity in the hippocampus of female rats Pathway analyses using Pathway Studio 5 also implicated involvement of female hormones in that the estrogens were among the small molecule regulators of the differentially expressed genes Fig. 3. The schematic in Aside from genes involved in cholesterol metabolism and steroid hormone biosynthesis, we also confirmed the altered expression of several other novel genes that may play a role in the pathophysiology of autistic disorder . The useIn summary, gene expression profiling of LCL from autistic and nonautistic siblings reveals alteration of genes involved in both metabolic and signaling pathways in ASD that is consistent with the known pathophysiology of autism which includes inflammation as well as disturbances in axon guidance, neuronal survival, and differentiation, biological themes also implicated in our earlier study on monozygotic twins discordant in diagnosis and severity of autism 2. Lymphoblastoid cell lines (LCL) derived from lymphocytes of autistic and normal siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in RPMI 1640 with 15% fetal bovine serum and antibiotics in a humidified incubator under 5% COTo reduce the heterogeneity of the samples for gene expression analyses, we used a novel clustering procedure to identify phenotypically distinct groups of individuals on the basis of severity associated with 123 items on the Autism Diagnostic Interview-Revised scoresheets. This procedure, described in another manuscript (submitted for publication), resulted in separation of the autistic individuals into 4 phenotypic subgroups, as illustrated in www.tm4.org. Within MeV, the Significance Analysis of Microarray (SAM) module RNA was isolated from LCL 3 days after the last passage using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. Fluorescently labeled sample cDNA was obtained by incorporation of amino-allyl dUTP during first-strand synthesis, followed by coupling to the ester of Cyanine (Cy)-3 as previously described Select genes were confirmed by real time RT-PCR on an ABI Prism 7300 Sequence Detection System using Invitrogen's Platinum SYBR Green qPCR SuperMix-UDG with ROX. These included genes involved in cholesterol and steroid hormone metabolism as well as genes implicated in nervous system development that were enriched within the top biological functions identified by Ingenuity Pathway Analysis . Total RSupplemental Table S2.PCR primers for genes identified by microarray analysis as differentially expressed were selected for specificity by the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST) of the human genome, and amplicon specificity was verified by first-derivative melting curve analysis with the use of software provided by PerkinElmer  and Applied Biosystems. Sequences of primers used for the real-time RT-PCR are given in the Quantitative reverse transcriptase-PCR analyses (qRT-PCR) were performed on a representative set of 5 pairs of case-controls from the sib pair analyses, with quantification and normalization of relative gene expression using universal 18S rRNA primers, with samples normalized to their 18S rRNA standard curves. The qPCR reactions were done in triplicate. A one-sample t-test was used to determine significance of differential expression across the 5 paired samples.http://david.abcc.ncifcrf.gov) was also used for additional functional annotation The datasets of differentially expressed genes between autistic probands and unaffected siblings were analyzed using Ingenuity Pathway Analysis and Pathway Studio 5 to identify molecular and cellular processes, high level functions, disorders, and small molecules associated with the gene regulatory networks. DAVID Bioinformatics Resources .Metabolites were extracted from LCL using acetonitrile and analyzed by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry, a highly sensitive method which has been developed for the simultaneous determination of 11 steroids Table S1Demographic profile of subjects(0.02 MB XLS)Click here for additional data file.Table S2Primer sequences used for qRT-PCR(0.03 MB DOC)Click here for additional data file.


TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos.in vivo. Intriguingly, TBP, which in oocytes is undetectable at the protein level, can functionally replace TBP2 when ectopically expressed in oocytes, showing that switching of initiation factors can be driven by changes in their expression. Proteolytic degradation of TBP2 is not required for repression of transcription during meiotic maturation, suggesting a redundant role in this repression or a role in initiation factor switching between oocytes and embryos.We find that TBP2 is proteolytically degraded upon meiotic maturation; after germinal vesicle breakdown relatively low levels of TBP2 expression persist. Furthermore, TBP2 localizes to the transcriptionally active loops of lampbrush chromosomes and is recruited to a number of injected promoters in oocyte nuclei. Using an altered binding specificity mutant reporter system we show that TBP2 promotes RNA polymerase II transcription The expression and transcriptional activity of TBP2 in oocytes show that TBP2 is the predominant initiation factor in oocytes, which is substituted by TBP on a subset of promoters in embryos as a result of proteolytic degradation of TBP2 during meiotic maturation. A key regulatory step in eukaryotic transcription initiation is the assembly of basal transcription apparatus at the core promoter. This apparatus includes RNA polymerase II (pol-II) and a set of basal transcription factors. For a long time the basal transcriptional machinery was thought to be universal and mainly invariant across different promoters. However, a growing body of evidence points towards a more dynamic and regulatory role for this machinery (reviewed in -4). TATAin vitro. In addition, knockdown studies in fish and frogs showed that TBP2 is indispensable for embryonic development 21].Xenopus oocytes. Low levels of TBP are detected in egg extracts, whereas it is very abundant in embryos  and treated with progesterone. The injected synthetic TBP mRNA lacks its native 5' and 3'-UTR and is translated in oocytes, in contrast to the endogenous maternal TBP mRNA which is translationally masked . The kinXenopus embryos  of meiosis I. Transcriptional activity is accommodated by numerous laterally extended loops along the length of each of these chromosomes ,25. GV swdr42a) gene). Furthermore, TBP2 recruitment correlates with the occupancy of pol-II. TBP2 was also enriched on the endogenous amplified 5S rRNA loci which recruited very little pol-II, as expected for RNA polymerase-III promoters.To characterize the binding of TBP2 at a higher resolution, promoter reporter constructs were injected into oocyte nuclei and analyzed by ChIP. As a control for the activity of these promoters we also performed ChIP with a pol-II antibody. The results show that TBP2 is associated with these promoters Figure . AlthougTo further confirm the association of TBP2 with promoters in oocytes, we expressed an HA-tagged version of TBP2 in oocytes and tested the recruitment of TBP2 on these promoters with an anti-HA antibody. Oocytes injected with promoter constructs but lacking HA-TBP2 were used as a negative control. A similar pattern of recruitment was obtained Figure . TogetheGTAAAG) to which wild-type (wt) protein cannot bind. We introduced these altered binding specificity (abs) mutations in Xenopus TBP and TBP2. An EMSA was performed to assess the binding specificity of wt and abs-mutant TBP and TBP2. To this end, rabbit reticulocyte extracts containing in vitro-translated wt and abs-mutant versions of TBP or TBP2 were tested against regular and mutant TATA probes from the adenovirus major late promoter [In order to further assess the role of TBP2 in oocyte transcription functionally, we applied an altered binding specificity mutant reporter system . In thispromoter . Like TBXenopus oocytes and has a canonical TATA box upstream of its transcription start site [Next, we analyzed the behaviour of these proteins in oocyte transcription, for which we made use of the hsp70 promoter, which is known to be active in art site . The TATX. tropicalis ZFP36L2 promoter, which has a canonical TATA box positioned 27 bp upstream of the transcription start site. We generated the TGTA mutant of this promoter and used it to test the activity of TBP and TBP2 on this promoter. The TGTA mutation significantly reduces transcription from this promoter  and two TATA box-containing promoters . Oocytes injected with promoter constructs but lacking HA-TBP/2 were used as a negative control. Furthermore, a background control construct (intron) was injected. The results substantiate the notion that TBP, if ectopically expressed, can be recruited to these promoters in oocytes Figure .In addition to degrading TBP2, maturing oocytes globally repress transcription. This prompted us to ask whether the downregulation of TBP2 may be important for proper shut down of transcription as oocytes mature into eggs. This is conceivable because TBP levels are extremely rate-limiting for transcription in egg extracts, and precocious transcription can be induced in embryos before gene activation at the mid-blastula transition by simultaneous inhibition of chromatin assembly with competitor DNA and premature accumulation of TBP ,27. To tTo further examine this situation we compared the timing of TBP2 degradation and the shutdown of transcription. Due to the variability between individual oocytes to undergo GVBD, batches of oocytes were synchronized depending on the appearance of the maturation mark at GVBD, and the promoter association of pol-II and TBP2 was determined by ChIP at GVBD and 5 hr after GVBD Figure . The resXenopus oocytes lack any detectable TBP, whereas TBP2 is abundant in oocytes. In eggs and early embryos relatively low levels of TBP2 are present. TBP starts to accumulate after meiotic maturation and during cleavage stages of development , elevated levels of TBP2 compensate abnormally low levels of TBP at some but not all promoters [The altered binding specificity mutant of TBP, when expressed in the oocytes, could also promote transcription from the hsp-70 and ZFP36L2 promoters Figure . Earlier and DNA ,30,31. I and DNA . TBP pro and DNA . On a siromoters . This shA complicating matter is that both TBP and TBP2 are required for early development in fish and frog ,6,8,12, On the limited number of promoters tested in this study, TBP and TBP2 could replace each other depending on their relative expression levels, providing an experimental model for initiation factor switching. Our data suggest that the core transcription machinery is highly flexible and that changes in relative expression levels of TATA binding factors between oocytes and embryos can drive core factor switching. Based on these observations we propose a model Figure  accordinOur observations not only establish an important role of TBP2 in transcription of a highly specialized cell type which lacks any detectable TBP, but also provide evidence that the basal transcription machinery is highly flexible and can rapidly exchange factors depending on their expression. This leads to initiation factor switching on a subset of promoters that are active both in oocytes and embryos, whereas other promoters show factor selectivity, which accounts for their non-redundant function in early embryos .The authors declare that they have no competing interests.WA designed and carried out the experiments, and drafted the manuscript. GJCV conceived of the study and contributed to its design and to writing the manuscript. Both authors read and approved the final manuscript.


The                        adipocyte-derived hormone leptin inhibits appetite and bone mass accrual.                        To fulfill these two functions leptin requires the integrity of                        hypothalamic neurons but not the expression of its receptor, ObRb on these                        neurons. These results suggested that leptin acts first elsewhere in the                        brain to mediate these functions. However, this neuroanatomical site of                        leptin action in the brain remained elusive. Recent mouse genetic,                        electrophysiological and neuroanatomical studies provide evidence that                        leptin inhibits appetite and bone mass accrual through a two-step pathway:                        it decreases synthesis and the release by brainstem neurons of serotonin                        that in turn targets hypothalamic neurons to regulate appetite and bone                        mass accrual.                 Skeleton in vertebrates serves multiple mechanical,                        hematopoietic and endocrine functions ,2. In orTph genes: Tph1 and Tph2. Tph1 is expressed                        mostly in cells of the gut and is responsible for the production of peripheral                        serotonin [Tph2                        is expressed exclusively in neurons of the brainstem and is responsible for the                        production of serotonin in the brain [Serotonin (5-hydroxytryptamine) is a                        biogenic amine that functions both as a neurotransmitter in central nervous                        system and as a hormone in the periphery where most of it 95%) is produced ,8. Serot5% is prohe brain .       he brain . The   he brain ; and   he brain .       Tph2, the gene that catalyzes the rate-limiting step in the biosynthesis                        of BDS. The absence of serotonin in the brain resulted in a severe low bone                        mass phenotype affecting the axial (vertebrae) and appendicular (long bones)                        skeleton [Our studies with loss and gain of function mutations                        of low-density lipoprotein receptor-related protein 5 demonstrated that GDS is                        a powerful inhibitor of osteoblast proliferation and bone formation that does                        not affect bone resorption . Althougskeleton . This  skeleton . Hence, skeleton .       Tph2-deficient animals had a                        dramatic decrease in their adipose mass [Tph2-deficient                        animals i.e., decrease in bone mass and appetite, and an increase in the energy                        expenditure are a mirror image of what is observed in mice that lack leptin [While we were doing these studies we noticed, upon                        opening the abdominal cavities, that ose mass . This  ose mass . This  k leptin ,17.    ObRb either in Sf1-expressing neurons of the ventromedial                        hypothalamus (VMH) nuclei or in Pomc-expressing neurons of the arcuate                        (ARC) nuclei had normal sympathetic activity, bone remodeling parameters and                        bone mass; they also had normal appetite and energy expenditure, and when fed a                        normal diet, did not develop an obesity phenotype [ObRb in serotonin neurons (SERTObRb-/-)                        developed a high bone mass phenotype; they had a similar increase in appetite                        as ob/ob mice and had low energy expenditure. As a result, SERTObRb-/-                        mice, when fed a normal diet, developed an obesity phenotype. These genetic                        studies demonstrated that leptin signals in the serotonin neurons of the                        brainstem to regulate, to the most part, bone mass, appetite and energy                        expenditure  and a Rodan fellowship from IBMS (VKY).