Assessing Oral Epithelial Dysplasia Risk for Transformation to Cancer: Comparison Between Histologic Grading Systems Versus S100A7 Immunohistochemical Signature-based Grading.

While a 3-tier oral epithelial dysplasia grading system has been utilized for decades, it is widely recognized as a suboptimal risk indicator for transformation to cancer. A 2-tier grading system has been proposed, although not yet validated. In this study, the 3-tier and 2-tier dysplasia grading systems, and an S100A7 immunohistochemical signature-based grading system were compared to assess prediction of risk of transformation to oral cancer. Formalin-fixed, paraffin-embedded biopsy specimens with known clinical outcomes were obtained retrospectively from a cohort of 48 patients. Hematoxylin and eosin-stained slides were used for the 2- and 3-tier dysplasia grading, while S100A7 for biomarker signature-based assessment was based on immunohistochemistry. Inter-observer variability was determined using Cohen's kappa ( K ) statistic with Cox regression disease free survival analysis used to determine if any of the methods were a predictor of transformation to oral squamous cell carcinoma. Both the 2- and 3-tier dysplasia grading systems ranged from slight to substantial inter-observer agreement ( Kw between 0.093 to 0.624), with neither system a good predictor of transformation to cancer (at least P =0.231; ( P >>>0.05). In contrast, the S100A7 immunohistochemical signature-based grading system showed almost perfect inter-observer agreement ( Kw =0.892) and was a good indicator of transformation to cancer ( P =0.047 and 0.030). The inherent grading challenges with oral epithelial dysplasia grading systems and the lack of meaningful prediction of transformation to carcinoma highlights the significant need for a more objective, quantitative, and reproducible risk assessment tool such as the S100A7 immunohistochemical signature-based system.

Clinical utility of purgative bowel preparation before capsule endoscopy: a multicenter, blinded, randomized controlled trial.

BACKGROUND AND AIMS: Optimal bowel preparation before capsule endoscopy (CE) is currently unknown. In this multicenter, blinded, randomized controlled trial, we assessed clinical effectiveness of 2 types of purgative regimen and a control arm of clear fluid only. METHODS: Patients with suspected small intestinal bleeding were randomized into 3 arms: arm A, clear fluids only for 18 hours before CE and simethicone 200 mg in 150 mL water immediately before CE; arm B, same as A + 2 L of polyethylene glycol (PEG) 12 hours before CE; and arm C, same as A + 1 L PEG + sodium ascorbate 3 hours before CE. To assess diagnostic yield, lesions were classified either as highly relevant (P2) or less relevant (P0 or P1) lesions. Small-bowel visualization quality (SBVQ) was assessed using the Brotz score. Patient tolerability was assessed using the visual analog scale (0-10, with lower scores indicating better tolerability). RESULTS: Two hundred twenty-nine patients completed the study. The mean age was 58.7 years (95% confidence interval, 29.3-87.9), and 47.2% were men. There was no significant difference in diagnosis of P2 lesions in arms A, B, and C (48.7%, 48.0%, and 45.9%, respectively; P = .94). Overall SBVQ and distal SBVQ were similar across the 3 arms (P = .94 and P = .68, respectively). Patients reported better tolerability in arm A (mean score, 1.5) compared with arms B and C (mean score, 3.5 and 2.6, respectively; P < .001). CONCLUSIONS: The use of a purgative bowel preparation before CE does not improve diagnostic yield or small-bowel visualization and is associated with lower patient tolerance. (Clinical trial registration number: ACTRN 12614000883617.).

The effect of tracheostomy on extracorporeal membrane oxygenation outcomes.

INTRODUCTION: The optimal timing for pursuing tracheostomy in patients with prolonged mechanical ventilation with either veno-arterial (VA) or veno-venous (VV) extracorporeal membrane oxygenation (ECMO) is a discussion of risk versus benefit. Depending on the etiology, cardiothoracic surgical patients carry some of the highest risk for respiratory failure postprocedure. Given that patients with end-stage cardiopulmonary status may be fraught with substantial comorbidities, it is critically important to manage the risk-benefit profile of performing a tracheostomy procedure on a patient requiring ECMO support. These cohorts have risk factors that may depend on each patient's inflammatory state, lung de-recruitment peri-procedure and postprocedure and bleeding requiring transfusions to name a few. We provide a descriptive analysis of ECMO patients on both VA and VV configurations who survived to hospital discharge receiving tracheostomy either during or after their ECMO course. METHODS: A retrospective single-institutional study collected all consecutive patients age 18 and above who received any form of ECMO between 2016 and 2020. Five hundred forty-five patients were screened based on having received ECMO. Patients with mixed EMCO modality were excluded due to heterogeneity of disease process. A total of 521 patients received either VV or VA ECMO. A total of 54 patients received tracheostomy and had sufficiently clean data for analysis. Tracheostomy patients were compared based on survival to discharge, tracheostomy surgical complications, ECMO duration, ECMO configuration, inotrope and vasopressor use, transfusion rates, total ventilator days, total days on intravenous sedation, and history of cardiotomy or heart transplant were assessed. Baseline characteristics of race, age, gender, and body mass index (BMI) were also collected. RESULTS: A total of 54 patients received tracheostomy. Twenty-nine of those patients received tracheostomy during the course of their ECMO, of whom 13 were on VV ECMO, 16 on VA ECMO. Another 25 patients underwent tracheostomy after successful ECMO explant; 8 of those were VV ECMO with the remaining 17 were on VA ECMO before explantation, with mean delay to tracheostomy, 10 and 19 days after explant between both modalities, respectively. A statistically significantly greater proportion of VV ECMO patients received a tracheostomy at any point versus VA ECMO patients (25.93% vs. 8.35%, p ≤ .0001). No statistically significant difference was noted in timing of tracheostomy when stratified by EMCO modality (VA 51.51% after explant vs. VV 38.10% after explant, p = .33). There was a greater frequency of minor tracheostomy complications in patients who were on ECMO at the time of their tracheostomy (p = .014) than in those who received their tracheostomy after being explanted. However, these minor complications did not contribute to a change in survival to hospital discharge (p = .58). Similarly, the small number of major complications (n = 13) did not impair survival to hospital discharge (p = .84). Finally, mean duration of ECMO was longer in those who received tracheostomy during ECMO versus after ECMO. (488.45 vs. 259.72 h, p < .01). CONCLUSIONS: Tracheostomy is known to increase patient mobility, clinical participation, and overall decrease in sedation use. Pursuing tracheostomy during ECMO is feasible, does not result in major bleeding, and is associated with only minor complications that overall do not decrease survival. While there is an increased duration of ECMO support in the tracheostomy cohort, this may be due to existing patient conditions, and may not be causal. Research is needed to further determine the external patient factors and specific timing to optimize both VV and VA ECMO courses. CLINICAL IMPLICATIONS: We hope that our analysis will pave the initial pathway for an evidence-based guideline on optimal timing of tracheostomy in ECMO patients, whether initiated during or after ECMO and taking into consideration ECMO configuration, its expected duration, and patient comorbidities.

Bell-state tomography in a silicon many-electron artificial molecule.

An error-corrected quantum processor will require millions of qubits, accentuating the advantage of nanoscale devices with small footprints, such as silicon quantum dots. However, as for every device with nanoscale dimensions, disorder at the atomic level is detrimental to quantum dot uniformity. Here we investigate two spin qubits confined in a silicon double quantum dot artificial molecule. Each quantum dot has a robust shell structure and, when operated at an occupancy of 5 or 13 electrons, has single spin-[Formula: see text] valence electron in its p- or d-orbital, respectively. These higher electron occupancies screen static electric fields arising from atomic-level disorder. The larger multielectron wavefunctions also enable significant overlap between neighbouring qubit electrons, while making space for an interstitial exchange-gate electrode. We implement a universal gate set using the magnetic field gradient of a micromagnet for electrically driven single qubit gates, and a gate-voltage-controlled inter-dot barrier to perform two-qubit gates by pulsed exchange coupling. We use this gate set to demonstrate a Bell state preparation between multielectron qubits with fidelity 90.3%, confirmed by two-qubit state tomography using spin parity measurements.

Oral Potentially Malignant Disorders (OPMD): What is the clinical utility of dysplasia grade?

INTRODUCTION: Oral epithelial dysplasia is considered a potential histologic precursor of subsequent squamous cell cancer. As standard clinical practice, pathologists grade dysplasia to assess risk for progression to malignancy. Except for the most advanced grade, severe dysplasia, dysplasia grading has failed to correlate well with the risk to develop invasive cancer. The questions of what process dysplasia grading best represents and what clinical utility dysplasia grading may have are explored. AREAS COVERED: This narrative review is based on PubMed search with emphasis on papers since 2010. Epithelial dysplasia as a precursor lesion of cancer and dysplasia grading as a risk assessment tool for progression to cancer are discussed. The close clinical association of dysplasia with known carcinogens, alcohol, and tobacco products is presented. EXPERT OPINION: Oral epithelial dysplasia is often, associated with prolonged exposure to tobacco and alcohol products. With reduction of carcinogen exposure, dysplasia is known to regress in some cases. It is proposed that histologic dysplasia grade together with macroscopic images of dysplastic clinical lesions be used as an educational tool to incentivize patients to reduce their known carcinogen exposure. This strategy has the potential to reduce lesion progression thereby reducing the disease burden of oral cancer.

Exchange Coupling in a Linear Chain of Three Quantum-Dot Spin Qubits in Silicon.

Quantum gates between spin qubits can be implemented leveraging the natural Heisenberg exchange interaction between two electrons in contact with each other. This interaction is controllable by electrically tailoring the overlap between electronic wave functions in quantum dot systems, as long as they occupy neighboring dots. An alternative route is the exploration of superexchange-the coupling between remote spins mediated by a third idle electron that bridges the distance between quantum dots. We experimentally demonstrate direct exchange coupling and provide evidence for second neighbor mediated superexchange in a linear array of three single-electron spin qubits in silicon, inferred from the electron spin resonance frequency spectra. We confirm theoretically, through atomistic modeling, that the device geometry only allows for sizable direct exchange coupling for neighboring dots, while next-nearest neighbor coupling cannot stem from the vanishingly small tail of the electronic wave function of the remote dots, and is only possible if mediated.

Diagnostic and therapeutic considerations in biopsy-proven type 2 autoimmune pancreatitis: comparative analysis with biopsy-proven type 1 autoimmune pancreatitis.

BACKGROUND: Autoimmune processes are now an increasingly recognized cause of acute and chronic pancreatitis. Autoimmune pancreatitis is a rare, benign pathology with two distinct clinicopathologic subtypes. The aim of this study was to compare the presentation, diagnostic considerations and outcomes of patients with biopsy-proven type 1 and 2 autoimmune pancreatitis (AIP). METHODS: A retrospective review of the Queensland Health pathology database of histologically proven AIP was conducted. Parameters compared included demographics, diagnostic criterion and post-treatment outcomes. RESULTS: Twenty-three patients had a confirmed histological diagnosis of AIP (type 1 = 13, type 2 = 10). Patients with type 2 AIP were younger (median age 49 versus 59 years, P < 0.05). There was no significant difference in gender distribution of disease at presentation. Type 2 AIP presented with significant increased focal pancreatic changes on cross-sectional imaging (80% versus 54%, P < 0.05). Serum IgG4 levels were raised (>1.40 g/L) in 69% of patients with type 1 AIP and not detected in type 2 (P < 0.01). Concurrent underlying inflammatory bowel disease was present in a higher proportion of type 2 AIP (40% versus 15%, P < 0.05). A significantly increased proportion of patients with type 2 AIP underwent surgical resection (70% versus 30%, P < 0.05). Conservative management was utilized in more patients with type 1 disease (54% versus 30%). On follow-up, two patients have experienced symptomatic relapse at 6-18 months. CONCLUSIONS: Diagnostic challenges do exist and clinicians must suspect 2 type AIP in young, serum IgG4-negative inflammatory bowel disease patients with recurrent pancreatitis.

Use of Transcarotid Artery Revascularization to Treat Symptomatic Carotid Artery Stenosis Associated with Free-Floating Thrombus.

BACKGROUND: Free-floating thrombus in the internal carotid artery (ICA) has traditionally been treated via an open surgical approach through a longitudinal incision and exposure similar to that for carotid endarterectomy (CEA). In this case report, we present a novel use of transcarotid artery revascularization (TCAR) for the treatment of recurrent carotid stenosis associated with free-floating ICA thrombus. CASE DESCRIPTION: We describe a 67-year-old female who presented with a diagnosis of right hemispheric stroke in evolution and prior history of right CEA and a mechanical mitral valve. Imaging confirmed high-grade recurrent stenosis of the right ICA with free-floating thrombus. TCAR was utilized to repair both the recurrent stenosis and the thrombus. CONCLUSIONS: Redo CEA in the face of recurrent stenosis is a challenging clinical scenario, which in this instance, was further complicated by the presence of free-floating thrombus and active anticoagulation due to a mechanical mitral valve. This case report describes the successful management of ICA thrombus and restenosis with the novel use of TCAR.

A Complicated Entity: Acute Celiac Artery Dissection with Resultant Pancreatitis, Duodenitis, and Cholecystitis.

Arterial dissection is a well-recognized pathology often seen in Vascular Surgery offices and Emergency Departments alike; however, visceral arterial dissection is an extremely rare, small subset of this entity. With that, an isolated celiac artery dissection as presented within this report is an exceptionally unique pathology that has scarcely been reported, and due to this, management guidelines are undefined. Given the viscera supplied by the celiac artery, many intra-abdominal structures are at risk for ischemia when damage to the celiac artery occurs, potentially witnessed by this report. Due to the exclusivity of this pathology, we are compelled to report the case of a 71-year-old male who presented with complaints of abdominal pain and was found to have an acute celiac artery dissection, which likely resulted in severe ischemic duodenitis, as well as possibly acute pancreatitis, and questionable influence on cholecystitis.

RETRACTED: Straticyte demonstrates prognostic value over oral epithelial dysplasia grade for oral potentially malignant lesion assessment.

OBJECTIVES: Straticyte™ was previously shown to be a more effective prognostic assessment than the current standard of care, histopathological dysplasia grading, to assess progression risk of oral epithelial dysplasia to invasive cancer [Hwang JT, Gu YR, Shen M, Ralhan R, Walfish PG, Pritzker KP, et al. Individualized five-year risk assessment for oral premalignant lesion progression to cancer. Oral Surg Oral Med Oral Pathol Oral Radiol. 2017;123:374-81]. In this follow-up study, our aim is to confirm the prognostic value of Straticyte using an independent cohort of oral biopsy cases previously assessed as epithelial dysplasia of various grades. MATERIALS AND METHODS: Using Visiopharm image analysis system, we analyzed an independent retrospective cohort of 51 oral biopsy samples with known outcomes and a follow-up history of up to 12years, to verify Straticyte, an individualized 5-year risk assessment for progression of oral potentially malignant lesions to invasive squamous cell carcinoma. RESULTS: Straticyte classified the lesions more accurately than histopathological oral epithelial dysplasia grading for risk for progression to cancer over five years. The sensitivity of low-risk vs. non-low-risk Straticyte groups was 100% compared to 68% for mild vs. non-mild dysplasia. The sensitivity of high-risk vs. non-high-risk Straticyte was 71% compared to 3% for severe vs. non-severe dysplasia. Furthermore, the Negative Predictive Value (NPV) for Straticyte was 100% for low-risk vs. non-low-risk, whereas the NPV for mild vs. non-mild dysplasia was 38%. CONCLUSION: In this cohort, Straticyte ascertains as a more useful assessment for risk of cancer progression in oral potentially malignant lesions than oral epithelial dysplasia grade.

Efficacy of single-incision needle-knife biopsy for sampling subepithelial lesions.

Background and study aims Single-incision needle-knife (SINK) biopsy is a diagnostic method for acquiring tissue samples for subepithelial lesions (SELs). A single linear incision is made in the overlying mucosa and tissue samples are obtained by passing conventional biopsy forceps through the opening and deep into the lesion. The aim of this study was to describe the efficacy and safety of this technique. Patients and methods Consecutive patients who underwent SINK biopsy for an upper gastrointestinal SEL between October 2013 and September 2015 were retrospectively reviewed. Results Forty-nine patients underwent 50 SINK biopsies. Sufficient sampling for a definite pathologic diagnosis was obtained in 42 (86 %) cases, with 91 % (40/44) having sufficient sample to perform immunohistochemistry when deemed clinically relevant. Of the 26 patients with prior non-diagnostic biopsies or FNA, a specific diagnosis was obtained in 85 % (22/26). There were no significant adverse events. Conclusions SINK biopsy is a safe and feasible strategy for obtaining a definitive tissue diagnosis with immunohistochemistry for SELs.

Individualized five-year risk assessment for oral premalignant lesion progression to cancer.

OBJECTIVE: The standard of care for premalignant lesion risk assessment is dysplasia grading by histopathology. With significant overlap between dysplasia grades and high inter- and intraobserver variations, histopathology dysplasia grading alone is not a useful prognostic tool. Our aim is to investigate whether a method for quantitatively assessing S100A7, a prognostic biomarker, using image analysis can better predict clinical outcome in cases with oral dysplasia. STUDY DESIGN: Using the Visiopharm image analysis system, we analyzed a cohort of 150 oral biopsy samples to build and test Straticyte, a model for individualized assessment of the 5-year risk of progression of oral precancerous lesions to invasive squamous cell carcinomas. RESULTS: Straticyte classified lesions more accurately than histopathological dysplasia grading for risk to progression to cancer over the following 5 years. The sensitivity of low-risk versus intermediate- and high-risk Straticyte groups was 95% compared to 75% for mild versus moderate and severe dysplasia. Furthermore, the negative predictive value for low-risk versus intermediate- and high-risk Straticyte groups was 78% compared to 59% for mild versus moderate and severe dysplasia. CONCLUSION: By quantitatively assessing S100A7, Straticyte better defines the risk for developing oral squamous cell carcinoma than histopathological dysplasia grading alone.

Obligatory Role of EP1 Receptors in the Increase in Cerebral Blood Flow Produced by Hypercapnia in the Mice.

Hypercapnia induces potent vasodilation in the cerebral circulation. Although it has long been known that prostanoids participate in the cerebrovascular effects of hypercapnia, the role of prostaglandin E2 (PGE2) and PGE2 receptors have not been fully investigated. In this study, we sought to determine whether cyclooxygenase-1 (COX-1)-derived PGE2 and EP1 receptors are involved in the cerebrovascular response induced by hypercapnia. Cerebral blood flow (CBF) was recorded by laser-Doppler flowmetry in the somatosenasory cortex of anesthetized male EP1-/- mice and wild type (WT) littermates. In WT mice, neocortical application of the EP1 receptor antagonist SC-51089 attenuated the increase in CBF elicited by systemic hypercapnia (pCO2 = 50-60 mmHg). SC-51089 also attenuated the increase in CBF produced by neocortical treatment of arachidonic acid or PGE2. These CBF responses were also attenuated in EP1-/- mice. In WT mice, the COX-1 inhibitor SC-560, but not the COX-2 inhibitor NS-398, attenuated the hypercapnic CBF increase. Neocortical application of exogenous PGE2 restored the attenuation in resting CBF and the hypercapnic response induced by SC-560. In contrast, exogenous PGE2 failed to rescue the attenuation both in WT mice induced by SC-51089 and EP1-/- mice, attesting to the obligatory role of EP1 receptors in the response. These findings indicate that the hypercapnic vasodilatation depends on COX-1-derived PGE2 acting on EP1 receptors and highlight the critical role that COX-1-derived PGE2 and EP1 receptors play in the hypercapnic regulation of the cerebral circulation.

Seeing better--Evidence based recommendations on optimizing colonoscopy adenoma detection rate.

Colorectal cancer is one of the three most frequent causes of cancer deaths in men and women in Europe and North America. Diagnosis and resection of adenomas has convincingly demonstrated its utility in diminishing colorectal cancer incidence. Therefore, colonoscopy is now the gold standard for colorectal cancer screening. But it is also known that colonoscopy effectiveness varies among endoscopists. Among different quality indicators, the most used is the adenoma detection rate (ADR) which is the percentage of average-risk patients for colorectal cancer who are found to have at least one adenoma or adenocarcinoma during a screening colonoscopy. There is compelling evidence supporting an inverse correlation between ADR and interval colorectal cancer (cancer found after a screening colonoscopy). Many factors such as quality of precolonoscopy preparation, additional observers, manoeuvres with the endoscope (second view, retroflexion, water inflation rather than air), time spent during withdrawal, changes in patient position, fold-flattener devices, new imaging or endoscopic modalities and use of intravenous or through the scope sprayed drugs, have been studied and developed with the aim of increasing the ADR. This reviews discusses these factors, and the current evidence, to "see better" in the colon and optimize ADR.

Redox regulation of canonical Wnt signaling affects extraembryonic endoderm formation.

Retinoic acid (RA) induces mouse F9 cells to form primitive endoderm (PrE) and increased levels of reactive oxygen species (ROS) accompany differentiation. ROS are obligatory for differentiation and while H2O2 alone induces PrE, antioxidants attenuate the response to RA. Evidence shows that ROS can modulate the Wnt/ß-catenin pathway and in this study, we show that extraembryonic endoderm formation is dependent on the redox state of nucleoredoxin (NRX). In undifferentiated F9 cells, NRX interacted with dishevelled 2 (Dvl2) and while this association was enhanced under reduced conditions, it decreased following H2O2 treatment. Depleting NRX levels caused morphological changes like those induced by RA, while increasing protein kinase A activity further induced these PrE cells to parietal endoderm. Reduced NRX levels also correlated to an increase in T-cell-factors-lymphoid enhancer factors-mediated transcription, indicative of canonical Wnt signaling. Together these results indicate that a mechanism exists whereby NRX maintains canonical Wnt signaling in the off state in F9 cells, while increased ROS levels lift these constraints. Dvl2 no longer bound to NRX is now positioned to prime the Wnt pathway(s) required for PrE formation.

Reactive oxygen species and Wnt signalling crosstalk patterns mouse extraembryonic endoderm.

Primitive endoderm formation from the inner cell mass is one of the earliest known cell fate decisions made in the mouse embryo. The mechanisms involved in orchestrating this process are not fully understood and are difficult to study in vivo. The F9 teratocarcinoma cell line is an in vitro model used to circumvent many technical problems surrounding the study of extraembryonic endoderm differentiation. F9 cells treated with retinoic acid differentiate to primitive endoderm and this is accompanied by the activation of canonical Wnt-ß-catenin signalling. Reactive oxygen species can modulate this signalling pathway, but whether they are sufficient to induce extraembryonic endoderm in vitro is not known. In the present study, a sustained increase in ROS levels was found in retinoic acid-treated F9 cells. An increase in Tcf-Lef transcriptional activity, a read out of Wnt-ß-catenin signalling, was also seen in response to exogenous H(2)O(2). Analysis from immunoblots, immunocytochemistry and real time PCR revealed the presence of markers of differentiation and a reduction in the expression of a marker of proliferation, confirming that H(2)O(2)-treated F9 cells developed into primitive endoderm. In contrast, exposing retinoic acid-treated cells to antioxidants impeded differentiation. Real time PCR was also used to identify candidates responsible for the observed elevation in ROS production. Results indicated that the NADPH oxidase 1, 2, 3 and 4 and Duox2 genes were RA responsive. Furthermore, the NADPH oxidase inhibitor, diphenyleneiodonium chloride was shown to attenuate primitive endoderm formation. Together, these results shed new light on how early mouse embryogenesis might be influenced by the crosstalk involving ROS and the Wnt-ß-catenin signalling pathway.

GATA6 and FOXA2 regulate Wnt6 expression during extraembryonic endoderm formation.

One of the earliest epithelial-to-mesenchymal transitions in mouse embryogenesis involves the differentiation of inner cell mass cells into primitive and then into parietal endoderm. These processes can be recapitulated in vitro using F9 teratocarcinoma cells, which differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm with subsequent treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP). Our previous work on how primitive endoderm develops revealed that the Wnt6 gene is upregulated by RA, leading to the activation of the canonical WNT-ß-catenin pathway. The mechanism by which Wnt6 is regulated was not determined, but in silico analysis of the human WNT6 promoter region had suggested that the GATA6 and FOXA2 transcription factors might be involved [1]. Subsequent analysis determined that both Gata6 and Foxa2 mRNA are upregulated in F9 cells treated with RA or RA and db-cAMP. More specifically, overexpression of Gata6 or Foxa2 alone induced molecular and morphological markers of primitive endoderm, which occurred concomitantly with the upregulation of the Wnt6 gene. Gata6- or Foxa2-overexpressing cells were also found to have increased levels in T-cell factor (TCF)-dependent transcription, and when these cells were treated with db-cAMP, they developed into parietal endoderm. Chromatin immunoprecipitation analysis revealed that GATA6 and FOXA2 were bound to the Wnt6 promoter, and overexpression studies showed that these transcription factors were sufficient to switch on the gene expression of a Wnt6 reporter construct. Together, these results provide evidence for the direct regulation of Wnt6 that leads to the activation of the canonical WNT-ß-catenin pathway and subsequent induction of primitive extraembryonic endoderm.