Functional antagonism between ΔNp63α and GCM1 regulates human trophoblast stemness and differentiation.

The combination of EGF, CHIR99021, A83-01, SB431542, VPA, and Y27632 (EGF/CASVY) facilitates the derivation of trophoblast stem (TS) cells from human blastocysts and first-trimester, but not term, cytotrophoblasts. The mechanism underlying this chemical induction of TS cells remains elusive. Here we demonstrate that the induction efficiency of cytotrophoblast is determined by functional antagonism of the placental transcription factor GCM1 and the stemness regulator ΔNp63α. ΔNp63α reduces GCM1 transcriptional activity, whereas GCM1 inhibits ΔNp63α oligomerization and autoregulation. EGF/CASVY cocktail activates ΔNp63α, thereby partially inhibiting GCM1 activity and reverting term cytotrophoblasts into stem cells. By applying hypoxia condition, we can further reduce GCM1 activity and successfully induce term cytotrophoblasts into TS cells. Consequently, we identify mitochondrial creatine kinase 1 (CKMT1) as a key GCM1 target crucial for syncytiotrophoblast differentiation and reveal decreased CKMT1 expression in preeclampsia. Our study delineates the molecular underpinnings of trophoblast stemness and differentiation and an efficient method to establish TS cells from term placentas.

Large papillary fibroelastoma of right atrium, an unusual case of respiratory distress in a young man.

BACKGROUND: Papillary fibroelastomas are rare but benign cardiac tumour that are often found on cardiac valvular surfaces. Their clinical manifestations ranging from clinically asymptomatic to substantial complications that are usually secondary to systemic embolism. Multiple theories have been proposed to explain the pathophysiology of its formation. CASE PRESENTATION: We reported a rare case of large papillary fibroelastoma in the right atrium of a young gentleman which was complicated with pulmonary embolism. Transthoracic echocardiography identified a large pedunculated mass measuring 3.4cmX3.4cmX2cm in right atrium with stalk attached to interatrial septum. The intracardiac mass was resected surgically, which revealed papillary fibroelastoma in histology examination. CONCLUSION: Differential diagnosis of intracardiac masses requires clinical information, laboratory tests and imaging modalities including echocardiography. Incidentally discovered papillary fibroelastomas are treated on the basis of their sizes, site, mobility and potential embolic complications. Due to the embolic risk inherent to intraacardiac masses, surgical resection represents an effective curative protocol in treating both symptomatic and asymptomatic right sided and left sided papillary fibroelastomas, with excellent long term postoperative prognosis.

HDAC11 deficiency disrupts oncogene-induced hematopoiesis in myeloproliferative neoplasms.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

SFRP3 negatively regulates placental extravillous trophoblast cell migration mediated by the GCM1-WNT10B-FZD7 axis.

Migration of placental extravillous trophoblast (EVT) cells into uterine decidua facilitates the establishment of blood circulation between mother and fetus and is modulated by EVT-decidual cell interaction. Poor or excessive EVT migration is associated with pregnancy complications such as preeclampsia or placenta accreta. Glial cells missing 1 (GCM1) transcription factor is essential for placental development, and decreased GCM1 activity is detected in preeclampsia. To study whether GCM1 regulates trophoblast cell migration, here we showed that GCM1 promotes BeWo and JAR trophoblast cell migration through a novel target gene, WNT10B. Moreover, WNT10B signaling stimulated cytoskeletal remodeling via Rac1 and frizzled 7 (FZD7) was identified as the cognate receptor for WNT10B to up-regulate cell migration. We further showed that secreted frizzled-related protein 3 (SFRP3) is expressed in uterine decidual cells by immunohistochemistry and that SFRP3 expression in telomerase-transformed human endometrial stromal cells (T-HESCs) is elevated under decidualization stimuli and further enhanced by bone morphogenetic protein 2 via SMAD1. SFRP3 blocked the interaction between FZD7 and WNT10B to decrease BeWo cell migration, which corroborated the elevated BeWo cell migration when cocultured with decidualized and SFRP3-knockdown T-HESC monolayer. Our results suggest that GCM1 up-regulates EVT cell migration through WNT10B and FZD7, which is negatively modulated by decidual SFRP3.-Wang, L.-J., Lo, H.-F., Lin, C.-F., Ng, P.-S., Wu, Y.-H., Lee, Y.-S., Cheong, M.-L., Chen, H. SFRP3 negatively regulates placental extravillous trophoblast cell migration mediated by the GCM1-WNT10B-FZD7 axis.

Discovery of novel N-hydroxy-2-arylisoindoline-4-carboxamides as potent and selective inhibitors of HDAC11.

N-Hydroxy-2-arylisoindoline-4-carboxamides are potent and selective inhibitors of HDAC11. The discovery, synthesis, and structure activity relationships of this novel series of inhibitors are reported. An advanced analog (FT895) displays promising cellular activity and pharmacokinetic properties that make it a useful tool to study the biology of HDAC11 and its potential use as a therapeutic target for oncology and inflammation indications.

Mechanical Behavior of Steel Fiber-Reinforced Concrete Beams Bonded with External Carbon Fiber Sheets.

This study investigates the mechanical behavior of steel fiber-reinforced concrete (SFRC) beams internally reinforced with steel bars and externally bonded with carbon fiber-reinforced polymer (CFRP) sheets fixed by adhesive and hybrid jointing techniques. In particular, attention is paid to the load resistance and failure modes of composite beams. The steel fibers were used to avoiding the rip-off failure of the concrete cover. The CFRP sheets were fixed to the concrete surface by epoxy adhesive as well as combined with various configurations of small-diameter steel pins for mechanical fastening to form a hybrid connection. Such hybrid jointing techniques were found to be particularly advantageous in avoiding brittle debonding failure, by promoting progressive failure within the hybrid joints. The use of CFRP sheets was also effective in suppressing the localization of the discrete cracks. The development of the crack pattern was monitored using the digital image correlation method. As revealed from the image analyses, with an appropriate layout of the steel pins, brittle failure of the concrete-carbon fiber interface could be effectively prevented. Inverse analysis of the moment-curvature diagrams was conducted, and it was found that a simplified tension-stiffening model with a constant residual stress level at 90% of the strength of the SFRC is adequate for numerically simulating the deformation behavior of beams up to the debonding of the CFRP sheets.

Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma.

UNLABELLED: Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I-mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K-AKT-mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eµ-Myc lymphoma-bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies. SIGNIFICANCE: Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eµ-Myc lymphoma-bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer.

Synergistic inhibition of ovarian cancer cell growth by combining selective PI3K/mTOR and RAS/ERK pathway inhibitors.

BACKGROUND: Ovarian cancer is the major cause of death from gynaecological malignancy with a 5year survival of only ∼30% due to resistance to platinum and paclitaxel-based first line therapy. Dysregulation of the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) and RAS/extracellular signal-regulated kinase (ERK) pathways is common in ovarian cancer, providing potential new targets for 2nd line therapy. METHODS: We determined the inhibition of proliferation of an extensive panel of ovarian cancer cell lines, encompassing all the major histotypes, by the dual PI3K/mTOR inhibitor PF-04691502 and a MEK inhibitor, PD-0325901. In addition, we analysed global gene expression, mutation status of key PI3K/mTOR and RAS/ERK pathway members and pathway activation to identify predictors of drug response. RESULTS: PF-04691502 inhibits proliferation of the majority of cell lines with potencies that correlate with the extent of pathway inhibition. Resistant cell lines were characterised by activation of the RAS/ERK pathway as indicated by differential gene expression profiles and pathway activity analysis. PD-0325901 suppressed growth of a subset of cell lines that were characterised by high basal RAS/ERK signalling. Strikingly, using PF-04691502 and PD-0325901 in combination resulted in synergistic growth inhibition in 5/6 of PF-04691502 resistant cell lines and two cell lines resistant to both single agents showed robust synergistic growth arrest. Xenograft studies confirm the utility of combination therapy to synergistically inhibit tumour growth of PF-04691502-resistant tumours in vivo. CONCLUSIONS: These studies identify dual targeted inhibitors of PI3K/mTOR in combination with inhibitors of RAS/ERK signalling as a potentially effective new approach to treating ovarian cancer.

Evidence for a common mechanism of SIRT1 regulation by allosteric activators.

A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

AKT signalling is required for ribosomal RNA synthesis and progression of Eµ-Myc B-cell lymphoma in vivo.

The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a 'super signalling' network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eµ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eµ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.

Endoscopic Ultrasound-guided Drainage of Pancreatic Pseudocysts: Medium-Term Assessment of Outcomes and Complications.

OBJECTIVE: Endoscopic ultrasound (EUS)-guided drainage is a widely used treatment modality for pancreatic pseudocysts (PPC). However, data on the clinical outcome and complication rates are conflicting. Our study aims to evaluate the rates of technical success, treatment success and complications of EUS-guided PPC drainage in a medium-term follow-up of 45 weeks. MATERIALS AND METHODS: A retrospective review was conducted for 55 patients with symptomatic PPC from December 2005 to August 2010 drained by EUS. Medium-term follow-up data were obtained by searching their medical history or by telephonic interview. RESULTS: A total of 61 procedures were performed. The symptoms that indicated drainage were abdominal pain (n = 43), vomiting (n = 7) and jaundice (n = 5). The procedure was technically successful in 57 of the 61 procedures (93%). The immediate complication rate was 5%. At a mean follow-up of 45 weeks, the treatment success was 75%. The medium term complications appeared in 25% of cases, which included three cases each of stent clogging, stent migration, infection and six cases of recurrence. There was no mortality. CONCLUSION: EUS-guided drainage is an effective treatment for PPC with a successful outcome in most of patients. Most of the complications require minimal invasive surgical treatment or repeated EUS-guided drainage procedures.

AKT promotes rRNA synthesis and cooperates with c-MYC to stimulate ribosome biogenesis in cancer.

Precise regulation of ribosome biogenesis is fundamental to maintain normal cell growth and proliferation, and accelerated ribosome biogenesis is associated with malignant transformation. Here, we show that the kinase AKT regulates ribosome biogenesis at multiple levels to promote ribosomal RNA (rRNA) synthesis. Transcription elongation by RNA polymerase I, which synthesizes rRNA, required continuous AKT-dependent signaling, an effect independent of AKT's role in activating the translation-promoting complex mTORC1 (mammalian target of rapamycin complex 1). Sustained inhibition of AKT and mTORC1 cooperated to reduce rRNA synthesis and ribosome biogenesis by additionally limiting RNA polymerase I loading and pre-rRNA processing. In the absence of growth factors, constitutively active AKT increased synthesis of rRNA, ribosome biogenesis, and cell growth. Furthermore, AKT cooperated with the transcription factor c-MYC to synergistically activate rRNA synthesis and ribosome biogenesis, defining a network involving AKT, mTORC1, and c-MYC as a master controller of cell growth. Maximal activation of c-MYC-dependent rRNA synthesis in lymphoma cells required AKT activity. Moreover, inhibition of AKT-dependent rRNA transcription was associated with increased lymphoma cell death by apoptosis. These data indicate that decreased ribosome biogenesis is likely to be a fundamental component of the therapeutic response to AKT inhibitors in cancer.

Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes.

Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme-peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

Life-table analysis of the success of thermal balloon endometrial ablation in the treatment of menorrhagia.

OBJECTIVE: To evaluate the change in intrauterine pressure during thermal balloon endometrial ablation and to identify risk factors associated with treatment failure. DESIGN: Prospective observational study. SETTING: University-affiliated teaching hospital. PATIENT(S): Seventy two consecutive patients with idiopathic menorrhagia refractory to medical treatment. INTERVENTION(S): Thermal balloon endometrial ablation under patient-controlled sedation. MAIN OUTCOME MEASURE(S): Change in intrauterine pressure during the treatment cycle and risk factors associated with treatment failure. RESULT(S): A spontaneous decrease in intrauterine pressure occurred in most patients (93%). The mean (+/-SD) decrease was 34.1 +/- 14.9 mm Hg, or 19.5% +/- 9.1%. The treatment failed in 10 patients (13.9%), and the mean end pressure was significantly lower in this group (131.1 +/- 14.1 mm Hg vs. 145.1 +/- 18.0 mm Hg; P=.02). The chance of success of treatment was significantly lower when the end pressure was <140 mm Hg (odds ratio, 0.42 [95% CI, 0.27 to 0.68]; P=.01), the intrauterine volume was >10 mL (odds ratio, 0.43 [95% CI, 0.22 to 0.83]; P=.058) and the uterus was retroverted (odds ratio, 0.36 [95% CI, 0.20 to 0.65]; P=.008). CONCLUSION(S): Maintaining high intrauterine pressure during the treatment cycle and correction of the retroversion may help to improve treatment success in thermal balloon endometrial ablation.

Comparison of peritoneal oxidative stress during laparoscopy and laparotomy.

STUDY OBJECTIVE: To identify oxidative stress in peritoneum during laparoscopic and open surgery by measuring products of lipid peroxidation, and to determine whether surgical approach influences the type of oxidative metabolite synthesized. DESIGN: Retrospective analysis (Canadian Task Force classification II-2). SETTING: University-affiliated hospital. PATIENTS: Twenty-eight consecutive women with uterine myomas or ovarian cysts. INTERVENTION: Laparoscopic or open surgery (14 patients each). MEASUREMENTS AND MAIN RESULTS: We obtained 1 x 1-cm squares of peritoneum at the beginning and end of surgical procedures away from sites of surgery. 8-Isoprostaglandin F(2alpha), hydroxyeicosatetranoic acids (HETEs), and malondyaldehyde (MDA) were measured by enzyme-immunoassay, high-performance liquid chromatography, and thiobarbituric acid adduction method, respectively. Comparisons showed significant increases in 5-HETE and 8-prostane in the laparoscopy group, which were correlated with duration of pneumoperitoneum and volume of carbon dioxide (CO(2)) insufflated, respectively. In the laparotomy group only MDA rose significantly related to duration of surgery. CONCLUSIONS: Lipid peroxidation was observed in peripheral peritoneum during laparoscopic surgery, mediated through noncyclooxygenase and lipoxygenase pathways, and appears to be due to effects of CO(2) pneumoperitoneum. Biochemical reactions were also observed in the laparotomy group, but are thought to be related to mechanisms other than lipid peroxidation.

Measurement of trocar insertion force using a piezoelectric transducer.

We attempted to establish a model to measure the force required for trocar insertion at laparoscopy. A 3-cm, circular transducer was constructed from piezoresistive material that changes its impedance as force is exerted on its surface. The transducer is connected by an interface box to a personal computer to record surface contact pressure digitally (pressure = force/area) profile continuously during trocar insertion. Each subject had three trocars inserted: a 10-mm trocar at the umbilicus after creation of pneumoperitoneum, and 5-mm trocars at corresponding sites on the left and right sides of the lower abdomen. All insertions were performed by the same operator using reusable trocar with a conical tip. Each subject acted as her own control. Recordings were successfully obtained from eight women. There was no instance of transducer failure. The mean (SE) peak contact surface pressure for the 10-mm and 5-mm left and right trocars were 5.3 (0.32), 6.4 (0.51), and 6.81 (0.27) pounds/square inch, respectively. Placement of the 10-mm trocar required less insertion force than placement of the 5-mm trocars. There was a strong negative correlation (r = -0.97, p < 0.001) between body weight and peak insertion force for the 10-mm trocar.