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Background: Reactive oxygen species are known to contribute to platelet hyperactivation and thrombosis during aging; however, the mechanistic contribution of the specific oxidative pathway remains elusive., Objectives: We hypothesized that during aging, endogenous Nox2-NADPH oxidase contributes to platelet reactive oxygen species accumulation and that loss of Nox2 will protect from platelet activation and thrombosis., Methods: We studied littermates of Nox2 knockout (Nox2-KO) and -wild-type (Nox2-WT) mice at young (3-4 months) and old (18-20 months) age. Within platelets, we examined the expression of subunits of NADPH oxidase and enzyme activity, oxidant levels, activation markers, aggregation, and secretion. We also assessed susceptibility to in vivo thrombosis in 2 experimental models., Results: While aged Nox2-WT mice displayed increased mRNA levels for Nox2, aged Nox2-KO mice showed an increase in Nox4 mRNA. However, neither the protein levels of several subunits nor the activity of NADPH oxidase were found to be altered by age or genotype. Both aged Nox2-WT and aged Nox2-KO mice exhibited similar enhancement in levels of platelet oxidants, granule release, α IIb β 3 activation, annexin V binding, aggregation and secretion, and a greater susceptibility to platelet-induced pulmonary thrombosis compared with young mice. In a photochemical injury model, adoptive transfer of platelets from aged Nox2-WT or Nox2-KO mice to the aged host mice resulted in a similar time to develop occlusive thrombus in the carotid artery. These findings suggest that loss of endogenous Nox2 does not protect against age-related platelet activation and arterial thrombosis in mice., Conclusion: We conclude that Nox2 is not an essential mediator of prothrombotic effects associated with aging., (© 2024 The Author(s).)

Background: COVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state., Objectives: From the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis., Methods: We utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice., Results: Compared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo., Conclusion: We conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Published by Elsevier Inc.)

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field . This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks., (© 2024 The Author(s).)

Background: There is ongoing controversy concerning the indications and benefits of early postoperative intraperitoneal chemotherapy (EPIC) following cytoreductive surgery (CRS) and hyperthermic intra-peritoneal chemotherapy (HIPEC) in patients with pseudomyxoma peritonei. The main contra-indications preventing wide-spread use of EPIC are reports of an increased postoperative morbidity with no clear evidence of oncological benefit. This paper reports a single high volume institution experience with EPIC over a 25-year period., Methods: This is a retrospective analysis of a single institution prospective database of patients undergoing CRS and HIPEC for appendiceal neoplasms from March 1994 to December 2019. Sub group analysis of patients who received EPIC with 5FU 15mg/m2 is reported and compared with patients who did not receive EPIC., Results: Overall, 632/1564 (40%) received EPIC. Patients who received EPIC were younger (median age 55 (IQR 45-63) vs 59 (IQR 50-68)) with similar extent of disease to those who did not have EPIC. EPIC was more likely to be given after complete cytoreduction. The use of EPIC has reduced over the last 25 years from 78% of patients initially to 16% most recently. Length of stay in critical care and total hospital stay were longer in patients who received EPIC but surprisingly major morbidity (Clavien Dindo Grade >3) was lower (p = 0.001)., Conclusion: EPIC can be administered safely following CRS and HIPEC for PMP of appendix origin when used in carefully selected patients in a high-volume centre. Randomised trials are needed to establish impact on disease free and overall survival to optimize selection criteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Background: Coronavirus disease 2019 (COVID-19) is associated with coagulopathy but the optimal prophylactic anticoagulation therapy remains uncertain and may depend on COVID-19 severity., Objective: To compare outcomes in hospitalized adults with severe COVID-19 treated with standard prophylactic versus intermediate dose enoxaparin., Methods: We conducted a multi-center, open-label, randomized controlled trial comparing standard prophylactic dose versus intermediate dose enoxaparin in adults who were hospitalized with COVID-19 and admitted to an intensive care unit (ICU) and/or had laboratory evidence of coagulopathy. Patients were randomly assigned in a 1:1 ratio to receive standard prophylactic dose enoxaparin or intermediate weight-adjusted dose enoxaparin. The primary outcome was all-cause mortality at 30 days. Secondary outcomes included arterial or venous thromboembolism and major bleeding., Results: A total of 176 patients (99 males and 77 females) underwent randomization. In the intention-to-treat population, all-cause mortality at 30 days was 15% for intermediate dose enoxaparin and 21% for standard prophylactic dose enoxaparin (odds ratio, 0.66; 95% confidence interval, 0.30-1.45; P = .31 by Chi-square test). Unadjusted Cox proportional hazards modeling demonstrated no significant difference in mortality between intermediate and standard dose enoxaparin (hazard ratio, 0.67; 95% confidence interval, 0.33-1.37; P = .28). Arterial or venous thrombosis occurred in 13% of patients assigned to intermediate dose enoxaparin and 9% of patients assigned to standard dose enoxaparin. Major bleeding occurred in 2% of patients in each arm., Conclusion: In hospitalized adults with severe COVID-19, standard prophylactic dose and intermediate dose enoxaparin did not differ significantly in preventing death or thrombosis at 30 days., (© 2021 International Society on Thrombosis and Haemostasis.)

Background: Thromboembolism affects up to 30% of children undergoing treatment for acute lymphoblastic leukemia (ALL). Increased thrombin generation has been reported in ALL, but the mechanisms remain elusive., Objective: We aimed to show that extracellular traps and cell-free DNA (cfDNA) promote thrombin generation in pediatric ALL., Methods: In a longitudinal single-center study, we recruited 17 consecutive pediatric ALL patients. Serial blood samples were collected at diagnosis and weekly during the 4-week induction phase of antileukemic chemotherapy. Healthy children (n = 14) and children with deep vein thrombosis (DVT; n = 7) or sepsis (n = 5) were recruited as negative and positive controls, respectively. In plasma, we measured endogenous thrombin generation potential (ETP) and components of extracellular traps, including cfDNA., Results: In patients with ALL, ETP was increased at baseline and remained significantly elevated throughout the induction therapy. Plasma levels of cfDNA were increased at baseline and during the first 3 weeks of induction therapy. The extent of enhancement of ETP and plasma cfDNA in patients with ALL was similar to that seen in patients with DVT or sepsis. Treatment of plasma with DNase 1 lowered ETP in patients with ALL at each time point but did not affect ETP in healthy controls., Conclusion: We conclude that childhood ALL is associated with a prothrombotic milieu at the time of diagnosis that continues during induction chemotherapy, and cfDNA contributes to increased thrombogenic potential., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Background: Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy for pseudomyxoma peritonei (PMP) is associated with excessive bleeding and acquired fibrinogen deficiency. Maintaining plasma fibrinogen may support hemostasis., Objectives: To compare hemostatic efficacy and safety of human fibrinogen concentrate (HFC) vs cryoprecipitate as fibrinogen sources for bleeding patients with acquired fibrinogen deficiency undergoing PMP CRS., Methods: FORMA-05 was an off-label single-center, prospective, randomized, controlled phase 2 study. Patients undergoing PMP surgery with predicted intraoperative blood loss ≥2 L received human fibrinogen concentrate (HFC; 4 g) or cryoprecipitate (two pools of 5 units, containing approximately 4.0-4.6 g fibrinogen), repeated as needed. The primary endpoint was a composite of intraoperative and postoperative efficacy, graded using objective 4-point scales and adjudicated by an independent committee., Results: One hundred percent of patients receiving HFC (95% confidence interval: 83.9-100.0, n = 21) or cryoprecipitate (84.6-100.0, n = 22) achieved hemostatic success. HFC demonstrated noninferior efficacy (P = .0095; post hoc) and arrived in the operating room 46 minutes faster. There were significantly greater mean increases with HFC vs cryoprecipitate in plasma fibrinogen (0.78 vs 0.35 g/L; P < .0001) and FIBTEM A20 (3.33 vs 0.93 mm; P = .003). Factor XIII, factor VIII, and von Willebrand factor activity were maintained throughout surgery. Only red blood cells were transfused intraoperatively (median units: HFC group, 1.0; cryoprecipitate group, 0.5). Thromboembolic events were detected with cryoprecipitate only. Safety was otherwise comparable between groups., Conclusions: Human fibrinogen concentrate was hemostatically efficacious in patients undergoing major abdominal PMP surgery, with a favorable safety profile. These results are relevant to other surgical settings where bleeding and acquired fibrinogen deficiency occur., (© 2019 Octapharma. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Aim: Pseudomyxoma peritonei (PMP) is an uncommon malignancy, generally originating from a ruptured epithelial tumour of the appendix. Despite successful cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC), some patients recur. Currently there are no guidelines on the methods, frequency and intensity of follow-up., Methods: Between 1994 and 2016, 1070 patients underwent surgery for a perforated epithelial tumour of the appendix, predominantly with PMP. Overall (OS) and Disease Free Survival (DFS) were documented by annual CT scanning and evaluated according to the Kaplan-Meier method. The influence of histological differentiation was investigated., Results: Overall, 775/1070 (72%) had complete cytoreductive surgery (CCRS) and HIPEC. Histological classification was low grade PMP in 615 (79.4%), high grade PMP in 134 (17.3%) and adenocarcinoma in 26 (3.4%). DFS and OS were significantly worse for high grade disease, with the steepest decline for both in the first three years. DFS curves, for low as well as high grade PMP, levelled off at year 6 at approximately 60% and 20% respectively. Thereafter there were few recurrences in either group., Conclusion: Annual CT of the abdomen and pelvis in the first six years appears to be adequate follow-up for low grade PMP. In high grade PMP, additional imaging of the chest and more frequent surveillance, during the first three years postoperatively, may detect recurrent disease earlier. From year 6 on, reduced frequency of follow-up is proposed, independent of the histology. This long-term follow-up in a large number of patients gives insight into tumour behavior after CCRS and HIPEC for PMP and guides intensity of surveillance., (Copyright © 2018 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Pseudomyxoma peritonei (PMP) is a clinical syndrome characterized by gross mucinous ascites originating from a disseminated intraperitoneal neoplasm. Although typically confined to the abdomen, mortality is high if untreated. Biomarkers, including genetic mutation profiles, may aid treatment selection and decision making. We applied whole-exome sequencing to five patients diagnosed with low-grade appendiceal mucinous neoplasms, using paired tumor and germline samples identify biomarkers. Multiple bioinformatic approaches were applied to these data to assess both somatic mutation profiles and loss of heterozygosity events. Mutation profiles of the tumors were consistent with deamination of methylcytosine being the prevailing mechanism. Pathogenic mutations were identified in both KRAS and GNAS in all samples, and further mutations in genes implicated in PMP, namely FGFR2, APC, SMAD2, and FAT4. No TP53 somatic mutations were identified, matching expectations for low-grade tumors. Four of five samples exhibited clonal loss of heterozygosity; these regions were further examined and found to contain genes harboring pathogenic somatic mutations in some samples. RNF43 was hereby implicated in the pathogenesis of PMP of appendiceal origin, having previously been found to increase sensitivity to Wnt signaling and to have involvement in similar mucinous tumors. In conclusion, we have investigated the mutation profile of PMP of appendiceal origin and provided the first report of RNF43 involvement in its progression., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The modified Glasgow prognostic score (incorporating C-reactive protein and albumin) predicts survival in patients with gastro-intestinal tract cancer but has not been evaluated in patients with peritoneal malignancy. The aim was to evaluate the modified Glasgow score preoperatively in patients undergoing complete cytoreductive surgery (CCRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) for pseudomyxoma peritonei (PMP) of appendiceal origin., Methods: Prospectively collected data from patients with PMP of appendiceal origin following CCRS and HIPEC between January 2007 and December 2011 were analysed. The mGPS was calculated from preoperative C-reactive protein and albumin. Predicted overall survival (OS) and disease-free survival (DFS) for each mGPS score were calculated using the Kaplan-Meier model. In a separate analysis, a comparison was made between mGPS and Tumour Markers (TM)., Results: 260 patients were included in the study. The mGPS of 0, 1, and 2 were found in 111, 130, and 19 patients respectively. The median follow-up was 48 months. For mGPS-0, -1, and -2, the predicted OS was 82.2, 73.7, and 69.2 months and the DFS was 73.5, 62.9, and 54.4 months respectively. As mGPS increases, there is a reduction in long-term survival. There was no difference between mGPS and TM., Conclusion: Preoperative mGPS may be a cost effective prognostic tool for predicting OS and DFS in patients following complete CRS-HIPEC, and performs well compared to TM for predicting patients at high risk of recurrence., (Copyright © 2016 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Purpose: To report early and long term outcomes following cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) in 1000 patients with perforated appendiceal epithelial tumours, predominantly with pseudomyxoma peritonei (PMP)., Methods: Retrospective analysis of a prospective database of 1000 consecutive patients undergoing CRS and HIPEC for perforated appendiceal tumours between 1994 and 2014 in a UK National Peritoneal Malignancy unit., Results: Overall 1000/1444 (69.2%) patients treated for peritoneal malignancy had appendiceal primary tumours. Of these 738/1000 (73.8%) underwent complete cytoreductive surgery (CCRS), 242 (24.2%) had maximal tumour debulking (MTD) and 20 (2%) had laparotomy and biopsies only. Treatment related 30-day mortality was 0.8% in CCRS and 1.7% in MTD group with major postoperative morbidity rates of 15.2% (CCRS) and 14.5% (MTD). Five- and 10-year overall survival was 87.4% and 70.3% in the 738 patients who had CCRS compared with 39.2% and 8.1% respectively in the MTD group. On multivariate analysis, significant predictors of reduced overall survival were male gender (p = 0.022), elevated CEA (p = 0.001), elevated CA125 (p = 0.001) and high tumour grade or adenocarcinoma (p = 0.001)., Conclusions: Perforated epithelial appendiceal tumours are rare, though may be increasing in incidence and can present unexpectedly at elective or emergency abdominal surgery, often with PMP. CRS and HIPEC results in good long term outcomes in most patients., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Complete cytoreductive surgery (CRS) can achieve cure or long-term survival in selected patients with peritoneal malignancy. In selected patients, due to extensive disease, complete tumour removal is impossible and optimal strategy may be maximal tumour debulking (MTD). We analysed the stoma related outcome in a series of patients undergoing surgery in a National Peritoneal Malignancy Referral Centre., Methods: All patients who underwent CRS, with or without, intra-operative hyperthermic intraperitoneal chemotherapy (HIPEC) between 1994 and 2012 were included. Data was collected prospectively in an institutional database and analysed retrospectively., Results: CRS was performed in 958 patients (female: 595, male: 363) of whom 781 (81.5%) had a primary appendix tumour, 63 (6.6%) had a colorectal primary, 47 (4.9%) peritoneal mesothelioma, 38 (4%) an ovarian tumour and 29 patients (3%) other tumours. Complete CRS was achieved in 72% (693/958). Overall 352/958 (37%) had a stoma, which was permanent in 165/958 (17.2%). The median time interval from CRS to reversal of stoma was 4.4 months (range: 1.4-13.8). Stomas were created in 113/265 (42.6%) at MTD (permanent: n = 105 (93%), temporary: n = 8 (7%)), and 239/693 (34.5%) at complete CRS (permanent: n = 60 (25%), temporary: n = 179 (75%)) (p = 0.020). All temporary stomas in the 168/693 (24.4%) of patients who had complete CRS were subsequently reversed., Conclusion: To achieve complete CRS for peritoneal malignancy a stoma is often required and in a proportion this will be permanent. Overall over one third had a stoma at surgery with almost half subsequently reversed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Cytoreductive surgery (CRS) combined with Hyperthermic Intraperitoneal Chemotherapy (HIPEC) is the optimal treatment for Pseudomyxoma Peritonei (PMP). Despite treatment, disease often recurs and may not be amenable to further CRS. Clinical experience suggests a spectrum of disease which may correlate with tumour marker levels. The aim of this study was to analyse the influence of markers on recurrence and survival., Methods: The details of all patients undergoing surgery for PMP of appendiceal origin at a national centre for peritoneal malignancy were recorded in a dedicated prospective database. The data on all patients who had CRS and HIPEC between March 1994 and January 2012 was analysed and recurrence and survival correlated with pre-operative levels of CEA, CA-125 and CA19-9., Results: Overall, 519 (69%) of 752 consecutive patients, underwent complete CRS and HIPEC. The median (range) age was 56 (20-82) years with 342/519 (66%) females. The mean overall (OS) and disease free survival (DFS) in the 131/519 patients who had normal preoperative tumour markers was 168 (128-207) and 125 (114-136) months respectively, significantly higher when compared with the 109/519 (21%) who had all three tumour markers elevated (OS of 65 (42-88) and DFS of 55 (41-70) months respectively) (P = 0.002)., Conclusions: Elevated tumour markers predict an increased risk of recurrence and reduced survival after complete CRS. This may reflect cell biology in low grade tumours and is an independent prognostic feature. Further analysis may help to select patients for post-operative chemotherapy, second look procedures or stratification of follow up., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) is an established treatment modality for patients with pseudomyxoma peritonei. The majority of patients with pseudomyxoma who have complete tumour removal and HIPEC are cured. Over the last decade CRS for peritoneal metastasis of colorectal origin has emerged as an effective treatment strategy in carefully selected patients. Although convincing evidence is limited, available data shows promising results. The key to a successful outcome is appropriate selection of patients. In patients with extensive peritoneal disease, where complete cytoreduction is not achieved, surgical treatment may not be beneficial and might impair quality of life. In this paper we discuss the challenges of selecting patients with colorectal peritoneal metastases who are likely to benefit from CRS with HIPEC., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hyperhomocysteinemia confers a high risk for thrombotic vascular events, but homocysteine-lowering therapies have been ineffective in reducing the incidence of secondary vascular outcomes, raising questions regarding the role of homocysteine as a mediator of cardiovascular disease. Therefore, to determine the contribution of elevated homocysteine to thrombosis susceptibility, we studied Cbs(-/-) mice conditionally expressing a zinc-inducible mutated human CBS (I278T) transgene. Tg-I278T Cbs(-/-) mice exhibited severe hyperhomocysteinemia and endothelial dysfunction in cerebral arterioles. Surprisingly, however, these mice did not display increased susceptibility to arterial or venous thrombosis as measured by photochemical injury in the carotid artery, chemical injury in the carotid artery or mesenteric arterioles, or ligation of the inferior vena cava. A survey of hemostatic and hemodynamic parameters revealed no detectible differences between control and Tg-I278T Cbs(-/-) mice. Our data demonstrate that severe elevation in homocysteine leads to the development of vascular endothelial dysfunction but is not sufficient to promote thrombosis. These findings may provide insights into the failure of homocysteine-lowering trials in secondary prevention from thrombotic vascular events.

Hyperhomocysteinemia is a risk factor for thrombosis, but the mechanisms are not well defined. We tested the hypothesis that hyperhomocysteinemia accelerates arterial thrombosis in mice. Mice heterozygous for a targeted disruption of the cystathionine beta-synthase gene (Cbs+/-) and wild-type littermates (Cbs+/+) were fed either a control diet or a high methionine/low folate (HM/LF) diet for 6 to 8 months to produce graded hyperhomocysteinemia. The time to occlusion of the carotid artery after photochemical injury was shortened by more than 50% in Cbs+/+ or Cbs+/- mice fed the HM/LF diet (P < .001 versus control diet). Carotid artery thrombosis was not accelerated in mice deficient in endothelial nitric oxide synthase (Nos3), which suggests that decreased endothelium-derived nitric oxide is not a sufficient mechanism for enhancement of thrombosis. Cbs+/+ and Cbs+/- mice fed the HM/LF diet had elevated levels of reactive oxygen species in the carotid artery, increased aortic expression of the NADPH oxidase catalytic subunit, Nox4, and decreased activation of anticoagulant protein C in the aorta (P < .05 versus control diet). We conclude that hyperhomocysteinemia enhances susceptibility to arterial thrombosis through a mechanism that is not caused by loss of endothelium-derived nitric oxide but may involve oxidative stress and impairment of the protein C anticoagulant pathway.

During Strongylocentrotus purpuratus embryogenesis, aboral ectoderm-specific expression of spec2a relies on an upstream enhancer that confers its spatial specificity largely through repression. The purpose of this study was to determine how spec2a expression is repressed in endoderm and oral ectoderm territories. A 78-base pair DNA sequence within the enhancer contains five tightly spaced cis-regulatory elements including proximal (TAATCT) and distal (TAATCC) elements that bind to both SpOtx, a broadly distributed transcriptional activator, and SpGoosecoid (SpGsc), an oral ectoderm-restricted transcriptional repressor. We show here that these two seemingly redundant Otx/Gsc elements have distinct functions. The proximal element bound to SpGATA-E, an endomesoderm-specific transcription factor. Treatment with SpGATA-E and SpGsc morpholino antisense oligonucleotides (MASOs) resulted in enhanced transcriptional activity from the proximal element, suggesting that both factors functioned as repressors at this site. SpGATA-E MASO-treated embryos failed to express ectoderm markers, indicating a role for SpGATA-E in ectoderm differentiation. The spec2a proximal element was distinct from the corresponding element in the related spec1 enhancer, and swaps between spec1 and spec2a cis-regulatory elements indicated, that for optimal repression, the proximal element had to interact with a nearby CCAAT-binding factor element. Our results show that the recently evolved proximal element contributes to the repression of spec2a in endomesoderm and oral ectoderm territories.

The creation, preservation, and degeneration of cis-regulatory elements controlling developmental gene expression are fundamental genome-level evolutionary processes about which little is known. Here, we identify critical differences in cis-regulatory elements controlling the expression of the sea urchin aboral ectoderm-specific spec genes. We found multiple copies of a repetitive sequence element termed RSR in genomes of species within the Strongylocentrotidae family, but RSRs were not detected in genomes of species outside Strongylocentrotidae. spec genes in Strongylocentrotus purpuratus are invariably associated with RSRs, and the spec2a RSR functioned as a transcriptional enhancer and displayed greater activity than did spec1 or spec2c RSRs. Single-base pair differences at two cis-regulatory elements within the spec2a RSR increased the binding affinities of four transcription factors, SpCCAAT-binding factor at one element and SpOtx, SpGoosecoid, and SpGATA-E at another. The cis-regulatory elements to which these four factors bound were recent evolutionary acquisitions that acted to either activate or repress transcription, depending on the cell type. These elements were found in the spec2a RSR ortholog in Strongylocentrotus pallidus but not in RSR orthologs of Strongylocentrotus droebachiensis or Hemicentrotus pulcherrimus. Our results indicated that a dynamic pattern of cis-regulatory element evolution exists for spec genes despite their conserved aboral ectoderm expression.

Transcriptional enhancers are short stretches of DNA that function to achieve highly specific patterns of gene expression. To identify the mechanisms by which enhancers achieve their specificity, we made use of an enhancer from the aboral ectoderm-specific spec2a gene of the sea urchin Strongylocentrotus purpuratus. The spec2a enhancer contains five cis-regulatory elements within 78 base pairs that interact with five distinct DNA-binding proteins to confer aboral ectoderm expression. Here, we present an analysis of the sea urchin CCAAT binding factor (CBF), which binds to a CCAAT motif within the spec2a enhancer. S. purpuratus CBF and SpOtx, a ubiquitously expressed factor, act together at closely placed cis-regulatory elements to mediate spec2a transcription in the ectoderm. SpCBF was the sole factor that bound to the spec2a CCAAT element, and two of the three subunits that make up the CBF holoprotein were cloned and shown to have high sequence conservation with their vertebrate orthologs. Based on its involvement in the regulation of several other sea urchin genes, SpCBF appears to be a major transcription factor in the sea urchin embryo for positive regulation of ectoderm gene expression. In addition to its role in vertebrate cell growth and proliferation, our results indicate that CBF also functions at the early stages of germ layer formation, namely ectoderm differentiation.

Background/aims: Erythropoietin-independent endogenous growth of erythroid colony from bone marrow cells has been shown in many patients with Budd Chiari syndrome in earlier studies. In another report, increased megakaryocyte colony growth has also been documented in this disease. However, defects in granulocyte-macrophage cell lines in Budd Chiari syndrome have yet to be reported in the literature., Methods: Both in vitro erythroid and granulocyte-macrophage colony cultures from peripheral blood mononuclear cells with and without erythropoietin or granulocyte-macrophage colony stimulating factor, respectively, were studied in 32 patients with Budd Chiari syndrome, along with 20 normal healthy controls and ten patient controls with portal hypertension (five patients each with noncirrhotic portal fibrosis and liver cirrhosis). In 18 patients the occlusion was only in the inferior vena cava, in five patients only in hepatic veins, and in nine patients both inferior vena cava and hepatic veins were blocked., Result: Endogenous erythroid or granulocyte-macrophage colony growth was not observed in any of the normal healthy controls or in patient controls with portal hypertension. However, 22 of the 32 (68.8%) patients showed endogenous erythroid colony growth. Moreover, four of them also showed endogenous growth of granulocyte-macrophage colony, not previously reported in Budd Chiari syndrome., Conclusion: It may be inferred that stem cell defects affecting all three hemopoietic cell lines (erythroid, megakaryocyte and granulocyte-macrophage) occur in Budd Chiari syndrome, which may be the primary defect responsible for the pro-thrombotic state causing venous thrombosis in them.