bluuebunny/crossref_metadata_2025 huggingface dataset:
A statistical approach to central monitoring of data quality in clinical trials
Authors: Venet David , Doffagne Erik , Burzykowski Tomasz , Beckers François , Tellier Yves , Genevois-Marlin Eric , Becker Ursula , Bee Valerie , Wilson Veronique , Legrand Catherine , Buyse Marc
Published: June 2012

Background

Classical monitoring approaches rely on extensive on-site visits and source data verification. These activities are associated with high cost and a limited contribution to data quality. Central statistical monitoring is of particular interest to address these shortcomings.

Purpose

This article outlines the principles of central statistical monitoring and the challenges of implementing it in actual trials.

Methods

A statistical approach to central monitoring is based on a large number of statistical tests performed on all variables collected in the database, in order to identify centers that differ from the others. The tests generate a high-dimensional matrix of p-values, which can be analyzed by statistical methods and bioinformatic tools to identify extreme centers.

Results

Results from actual trials are provided to illustrate typical findings that can be expected from a central statistical monitoring approach, which detects abnormal patterns that were not (or could not have been) detected by on-site monitoring.

Limitations

Central statistical monitoring can only address problems present in the data. Important aspects of trial conduct such as a lack of informed consent documentation, for instance, require other approaches. The results provided here are empirical examples from a limited number of studies.

Conclusion

Central statistical monitoring can both optimize on-site monitoring and improve data quality and as such provides a cost-effective way of meeting regulatory requirements for clinical trials.
Therapeutic targeting of the phosphatidylinositol 3-kinase signaling pathway: novel targeted therapies and advances in the treatment of colorectal cancer
Authors: Yu Ming , Grady William M.
Published: June 2012

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the USA, and more effective treatment of CRC is therefore needed. Advances in our understanding of the molecular pathogenesis of this malignancy have led to the development of novel molecule-targeted therapies. Among the most recent classes of targeted therapies being developed are inhibitors targeting the phosphatidylinositol 3-kinase (PI3K) signaling pathway. As one of the most frequently deregulated pathways in several human cancers, including CRC, aberrant PI3K signaling plays an important role in the growth, survival, motility and metabolism of cancer cells. Targeting this pathway therefore has considerable potential to lead to novel and more effective treatments for CRC. Preclinical and early clinical studies have revealed the potential efficacy of drugs that target PI3K signaling for the treatment of CRC. However, a major challenge that remains is to study these agents in phase III clinical trials to see whether these early successes translate into better patient outcomes. In this review we focus on providing an up-to-date assessment of our current understanding of PI3K signaling biology and its deregulation in the molecular pathogenesis of CRC. Advances in available agents and challenges in targeting the PI3K signaling pathway in CRC treatment will be discussed and placed in the context of the currently available therapies for CRC.
Extended-release dalfampridine in the management of multiple-sclerosis-related walking impairment
Authors: Hersh Carrie , Rae-Grant Alex
Published: June 2012

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system that causes neurological impairment in young adults. As part of the disease, ambulation remains one of the most disabling features of multiple sclerosis. Extended-release dalfampridine is a long-acting form of 4-aminopyridine that has been shown in two phase III trials to increase ambulation speed in a subset of patients with multiple sclerosis (timed walk responders). The primary endpoint of these studies was ‘responder status’, analyzing difference in the proportion of timed walk responders between extended-release dalfampridine and placebo groups. Extended-release dalfampridine exerts its effects by inhibiting voltage-activated K+ channels and has been previously demonstrated to improve action potential propagation in demyelinated nerve fibers in vitro. Side effects of extended-release dalfampridine include increased urinary tract infections, insomnia, headache, asthenia, dizziness, back pain, and paresthesias. Rare seizure events are also reported on the approved dose of 10 mg every 12 h. In this review we will summarize the results of key phase II and phase III trials of extended-release dalfampridine, its safety, and potential use in patients with multiple sclerosis.
Aberrant STAT5 and PI3K/mTOR pathway signaling occurs in human CRLF2-rearranged B-precursor acute lymphoblastic leukemia
Authors: Tasian Sarah K. , Doral Michelle Y. , Borowitz Michael J. , Wood Brent L. , Chen I-Ming , Harvey Richard C. , Gastier-Foster Julie M. , Willman Cheryl L. , Hunger Stephen P. , Mullighan Charles G. , Loh Mignon L.
Published: June 2012

Adults and children with high-risk CRLF2-rearranged acute lymphoblastic leukemia (ALL) respond poorly to current cytotoxic chemotherapy and suffer unacceptably high rates of relapse, supporting the need to use alternative therapies. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor, which activates cell signaling in normal lymphocytes on binding its ligand, TSLP. We hypothesized that aberrant cell signaling occurs in CRLF2-rearranged ALL and can be targeted by signal transduction inhibitors of this pathway. In a large number of primary CRLF2-rearranged ALL samples, we observed increased basal levels of pJAK2, pSTAT5, and pS6. We thus characterized the biochemical sequelae of CRLF2 and JAK alterations in CRLF2-rearranged ALL primary patient samples via analysis of TSLP-mediated signal transduction. TSLP stimulation of these leukemias further induced robust JAK/STAT and PI3K/mTOR pathway signaling. JAK inhibition abrogated phosphorylation of JAK/STAT and, surprisingly, of PI3K/mTOR pathway members, suggesting an interconnection between these signaling networks and providing a rationale for testing JAK inhibitors in clinical trials. The PI3K/mTOR pathway inhibitors rapamycin, PI103, and PP242 also inhibited activated signal transduction and translational machinery proteins of the PI3K/mTOR pathway, suggesting that signal transduction inhibitors targeting this pathway also may have therapeutic relevance for patients with CRLF2-rearranged ALL and merit further preclinical testing.
Pivotal Advance: Protein synthesis modulates responsiveness of differentiating and malignant plasma cells to proteasome inhibitors
Authors: Cenci Simone , Oliva Laura , Cerruti Fulvia , Milan Enrico , Bianchi Giada , Raule Mary , Mezghrani Alexandre , Pasqualetto Elena , Sitia Roberto , Cascio Paolo
Published: June 2012

Protein synthesis determines sensitivity of differentiating and malignant plasma cells to proteasome inhibitors by saturating cellular proteolytic capacity.

A previously unsuspected, considerable proportion of newly synthesized polypeptides are hydrolyzed rapidly by proteasomes, possibly competing with endogenous substrates and altering proteostasis. In view of the anti-cancer effects of PIs, we set out to achieve a quantitative assessment of proteasome workload in cells hallmarked by different PI sensitivity, namely, a panel of MM cells, and in a dynamic model of plasma cell differentiation, a process that confers exquisite PI sensitivity. Our results suggest that protein synthesis is a key determinant of proteasomal proteolytic burden and PI sensitivity. In different MM cells and in differentiating plasma cells, the average proteolytic work accomplished per proteasome ranges over different orders of magnitude, an unexpected degree of variability, with increased workload invariably associated to increased PI sensitivity. The unfavorable load-versus-capacity balance found in highly PI-sensitive MM lines is accounted for by a decreased total number of immunoproteasomes/cell coupled to enhanced generation of RDPs. Moreover, indicative of cause-effect relationships, attenuating general protein synthesis by the otherwise toxic agent CHX reduces PI sensitivity in activated B and in MM cells. Our data support the view that in plasma cells protein synthesis contributes to determine PI sensitivity by saturating the proteasomal degradative capacity. Quantitating protein synthesis and proteasome workload may thus prove crucial to design novel negative proteostasis regulators against cancer.
Deletion of TGF-β signaling in myeloid cells enhances their anti-tumorigenic properties
Authors: Novitskiy Sergey V , Pickup Michael W , Chytil Anna , Polosukhina Dina , Owens Philip , Moses Harold L
Published: June 2012

Specific deletion of TGF-β receptor II in macrophages, CD11b+Gr1+, and dendritic cells inhibit tumor growth by increasing efficiency of the immune system.

By crossing LysM-Cre and TGF-β type II receptor (Tgfbr2) floxed mice we achieved specific deletion of Tgfbr2 in myeloid cells (Tgfbr2MyeKO mice). S.c.-injected (LLC, EL4-OVA) and implanted (MMTV-PyMT) carcinoma cells grow slower in Tgfbr2MyeKO mice. The number of CD45+ cells in the tumor tissue was the same in both genotypes of mice, but upon analysis, the percentage of T cells (CD45+CD3+) in the KO mice was increased. By flow cytometry analysis, we did not detect any differences in the number and phenotype of TAMs, CD11b+Gr1+, and DCs in Tgfbr2MyeKO compared with Tgfbr2MyeWT mice. ELISA and qRT-PCR data showed differences in myeloid cell functions. In Tgfbr2MyeKO TAMs, TNF-α secretion was increased, basal IL-6 secretion was down-regulated, TGF-β did not induce any VEGF response, and there was decreased MMP9 and increased MMP2 and iNOS expression. TGF-β did not have any effect on CD11b+Gr1+ cells isolated from Tgfbr2MyeKO mice in the regulation of Arg, iNOS, VEGF, and CXCR4, and moreover, these cells have decreased suppressive activity relative to T cell proliferation. Also, we found that DCs from tumor tissue of Tgfbr2MyeKO mice have increased antigen-presented properties and an enhanced ability to stimulate antigen-specific T cell proliferation. We conclude that Tgfbr2 in myeloid cells has a negative role in the regulation of anti-tumorigenic functions of these cells, and deletion of this receptor decreases the suppressive function of CD11b+Gr1+ cells and increases antigen-presenting properties of DCs and anti-tumorigenic properties of TAMs.
Overlap of Endocrine Hormone Expression in the Mouse Intestine Revealed by Transcriptional Profiling and Flow Cytometry
Authors: Habib Abdella M. , Richards Paul , Cairns Lynne S. , Rogers Gareth J. , Bannon Christopher A. M. , Parker Helen E. , Morley Tom C. E. , Yeo Giles S. H. , Reimann Frank , Gribble Fiona M.
Published: June 2012

The intestine secretes a range of hormones with important local and distant actions, including the control of insulin secretion and appetite. A number of enteroendocrine cell types have been described, each characterized by a distinct hormonal signature, such as K-cells producing glucose-dependent insulinotropic polypeptide (GIP), L-cells producing glucagon-like peptide-1 (GLP-1), and I-cells producing cholecystokinin (CCK). To evaluate similarities between L-, K-, and other enteroendocrine cells, primary murine L- and K-cells, and pancreatic α- and β-cells, were purified and analyzed by flow cytometry and microarray-based transcriptomics. By microarray expression profiling, L cells from the upper small intestinal (SI) more closely resembled upper SI K-cells than colonic L-cells. Upper SI L-cell populations expressed message for hormones classically localized to different enteroendocrine cell types, including GIP, CCK, secretin, and neurotensin. By immunostaining and fluorescence-activated cell sorting analysis, most colonic L-cells contained GLP-1 and PeptideYY In the upper SI, most L-cells contained CCK, approximately 10% were GIP positive, and about 20% were PeptideYY positive. Upper SI K-cells exhibited approximately 10% overlap with GLP-1 and 6% overlap with somatostatin. Enteroendocrine-specific transcription factors were identified from the microarrays, of which very few differed between the enteroendocrine cell populations. Etv1, Prox1, and Pax4 were significantly enriched in L-cells vs. K cells by quantitative RT-PCR. In summary, our data indicate a strong overlap between upper SI L-, K-, and I-cells and suggest they may rather comprise a single cell type, within which individual cells exhibit a hormonal spectrum that may reflect factors such as location along the intestine and exposure to dietary nutrients.
TNFR1-Activated Reactive Oxidative Species Signals Up-Regulate Osteogenic Msx2 Programs in Aortic Myofibroblasts
Authors: Lai Chung-Fang , Shao Jian-Su , Behrmann Abraham , Krchma Karen , Cheng Su-Li , Towler Dwight A.
Published: June 2012

In LDLR−/− mice fed high-fat diabetogenic diets, osteogenic gene-regulatory programs are ectopically activated in vascular myofibroblasts and smooth muscle cells that promote arteriosclerotic calcium deposition. Msx2-Wnt signaling pathways previously identified as important for craniofacial skeletal development are induced in the vasculature by TNF, a prototypic cytokine mediator of the low-grade systemic inflammation of diabesity. To better understand this biology, we studied TNF actions on Msx2 in aortic myofibroblasts. TNF up-regulated Msx2 mRNA 4-fold within 3 h but did not regulate Msx1. Although IL-1β could also induce Msx2 expression, TNF-related apoptosis inducing ligand, receptor activator of nuclear factor-κB ligand, and IL-6 were inactive. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (Nox) activity and genetically induced Nox deficiency (p47phox−/−) reduced Msx2 induction, indicating contributions of reactive oxygen species (ROS) and redox signaling. Consistent with this, rotenone, an antagonist of mitochondrial complex I, inhibited TNF induction of Msx2 and Nox2, whereas pyruvate, an anapleurotic mitochondrial metabolic substrate, enhanced induction. Moreover, the glutathione peroxidase-mimetic ebselen abrogated this TNF response. Treatment of aortic myofibroblasts with hydrogen peroxide up-regulated Msx2 mRNA, promoter activity, and DNA-protein interactions. In vivo, SM22-TNF transgenic mice exhibit increased aortic Msx2 with no change in Msx1. Dosing SM22-TNF mice with either 20 ng/g Nox1 + 20 ng/g Nox2 antisense oligonucleotides or low-dose rotenone reduced arterial Msx2 expression. Aortic myofibroblasts from TNFR1−/− mice expressed levels of Msx2 that were 5% that of wild-type and were not inducible by TNF. Wnt7b and active β-catenin levels were also reduced. By contrast, TNF-inducible Msx2 expression was not reduced in TNFR2−/− cells. Finally, when cultured under mineralizing conditions, TNFR1−/− aortic myofibroblasts exhibited reduced calcification compared with wild-type and TNFR2−/− cells. Thus, ROS metabolism contributes to TNF induction of Msx2 and procalcific responses in myofibroblasts via TNFR1. Strategies that reduce vascular Nox- or mitochondrially activated ROS signals may prove useful in mitigating arteriosclerotic calcification.
Different Signaling Mechanisms Are Involved in the Norepinephrine-Stimulated TORC1 and TORC2 Nuclear Translocation in Rat Pinealocytes
Authors: McTague J. , Amyotte N. , Kanyo R. , Ferguson M. , Chik C. L. , Ho A. K.
Published: June 2012

The distribution of transducers of regulated cAMP-response element-binding protein activity (TORC) between the cytoplasm and the nucleus is tightly regulated and represents one of the main mechanisms whereby the cAMP response element activation activities of TORC are controlled. Whereas both cAMP and Ca2+ pathways can cause translocation of TORC, the relative importance of these two pathways in regulating different TORC within the same cell is unclear. In this study, we determined the mechanism that regulated TORC1 translocation and compared it with that of TORC2 in rat pinealocytes. Stimulation of pinealocytes with norepinephrine (NE), although having no effect on Torc1 transcription, caused rapid dephosphorylation of TORC1. Although NE also caused rapid dephosphorylation of TORC2, pharmacological studies revealed that TORC1 dephosphorylation could be induced by both β-adrenoceptor/cAMP and α-adrenoceptor/intracellular Ca2+ pathways contrasting with TORC2 dephosphorylation being induced mainly through the β-adrenoceptor/cAMP pathway. PhosTag gel indicated a different pattern of TORC1 desphosphorylation resulting from the selective activation of α- or β-adrenoceptors. Interestingly, only the α-adrenoceptor/intracellular Ca2+-mediated dephosphorylation could translocate TORC1 to the nucleus, whereas the β-adrenoceptor/cAMP-mediated dephosphorylation of TORC1 was ineffective. In comparison, translocation of TORC2 was induced predominantly by the β-adrenoceptor/cAMP pathway. Studies with different protein phosphatase (PP) inhibitors indicated that the NE-mediated translocation of TORC1 was blocked by cyclosporine A, a PP2B inhibitor, but that of TORC2 was blocked by okadaic acid, a PP2A inhibitor. Together these results highlight different intracellular signaling pathways that are involved in the NE-stimulated dephosphorylation and translocation of TORC1 and TORC2 in rat pinealocytes.
Mechanisms for the Antihyperglycemic Effect of Sitagliptin in Patients with Type 2 Diabetes
Authors: Muscelli Elza , Casolaro Arturo , Gastaldelli Amalia , Mari Andrea , Seghieri Giuseppe , Astiarraga Brenno , Chen Yu , Alba Maria , Holst Jens , Ferrannini Ele
Published: June 2012

Context:

Dipeptidyl peptidase IV (DPP-4) inhibitors improve glycemic control in patients with type 2 diabetes. The underlying mechanisms (incretin effect, β-cell function, endogenous glucose production) are not well known.

Objective:

The aim of the study was to examine mechanisms of the antihyperglycemic effect of DPP-4 inhibitors.

Design, Setting, and Patients:

We administered a mixed meal with glucose tracers ([6,6-2H2]-glucose infused, [1-2H]-glucose ingested), and on a separate day, a glucose infusion matched the glucose responses to the meal (isoglycemic test) in 50 type 2 diabetes patients (hemoglobin A1c = 7.4 ± 0.8%) and seven controls; 47 diabetic completers were restudied after 6 wk. Glucose fluxes were calculated, and β-cell function was assessed by mathematical modeling. The incretin effect was calculated as the ratio of oral to iv insulin secretion.

Intervention:

We conducted a 6-wk, double-blind, randomized treatment with sitagliptin (100 mg/d; n = 25) or placebo (n = 22).

Results:

Relative to placebo, meal-induced changes in fasting glucose and glucose area under the curve (AUC) were greater with sitagliptin, in parallel with a lower appearance of oral glucose [difference (post-pre) AUC = −353 ± 915 vs. +146 ± 601 μmol · kg−1 · 5 h] and greater suppression of endogenous glucose production. Insulin sensitivity improved 10%, whereas total insulin secretion was unchanged. During the meal, β-cell glucose sensitivity improved (+19[29] vs. 5[21] pmol · min−1 · m−2 · mm−1; median [interquartile range]) and glucagon AUC decreased (19.6 ± 7.5 to 17.3 ± 7.1 ng · ml−1 · 5 h), whereas intact glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 AUC increased with sitagliptin vs. placebo. The incretin effect was unchanged because sitagliptin increased β-cell glucose sensitivity also during the isoglycemic test.

Conclusions:

Chronic sitagliptin treatment improves glycemic control by lowering the appearance of oral glucose, postprandial endogenous glucose release, and glucagon response, and by improving insulin sensitivity and β-cell glucose sensing in response to both oral and iv glucose.
The loss ofHoxa5function promotes Notch-dependent goblet cell metaplasia in lung airways
Authors: Boucherat Olivier , Chakir Jamila , Jeannotte Lucie
Published: June 2012

Hox genes encode transcription factors controlling complex developmental processes in various organs. Little is known, however, about how HOX proteins control cell fate. Herein, we demonstrate that the goblet cell metaplasia observed in lung airways from Hoxa5−/− mice originates from the transdifferentiation of Clara cells. Reduced CC10 expression in Hoxa5−/− embryos indicates that altered cell specification occurs prior to birth. The loss of Hoxa5 function does not preclude airway repair after naphthalene exposure, but the regenerated epithelium presents goblet cell metaplasia and less CC10-positive cells, demonstrating the essential role of Hoxa5 for correct differentiation. Goblet cell metaplasia in Hoxa5−/− mice is a FOXA2-independent process. However, it is associated with increased Notch signaling activity. Consistent with these findings, expression levels of activated NOTCH1 and the effector gene HEY2 are enhanced in patients with chronic obstructive pulmonary disease. In vivo administration of a γ-secretase inhibitor attenuates goblet cell metaplasia in Hoxa5−/− mice, highlighting the contribution of Notch signaling to the phenotype and suggesting a potential therapeutic strategy to inhibit goblet cell differentiation and mucus overproduction in airway diseases. In summary, the loss of Hoxa5 function in lung mesenchyme impacts on epithelial cell fate by modulating Notch signaling.
Advances in high-resolution imaging – techniques for three-dimensional imaging of cellular structures
Authors: Lidke Diane S. , Lidke Keith A.
Published: June 2012

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.
Inhibiting myostatin reverses muscle fibrosis through apoptosis
Authors: Li Zhao Bo , Zhang Jiangyang , Wagner Kathryn R.
Published: June 2012

Skeletal muscle fibrosis is a defining feature of the muscular dystrophies in which contractile myofibers are replaced by fibroblasts, adipocytes and extracellular matrix. This maladaptive response of muscle to repetitive injury is progressive, self-perpetuating and thus far, has been considered irreversible. We have previously shown that myostatin, a known endogenous modulator of muscle growth, stimulates normal muscle fibroblasts to proliferate. Here, we demonstrate that myostatin also regulates the proliferation of dystrophic muscle fibroblasts, and increases resistance of fibroblasts to apoptosis through Smad and MAPK signaling. Inhibiting myostatin signaling pathways with a soluble activin IIB receptor (ActRIIB.Fc), reduces resistance of muscle fibroblasts to apoptosis in vitro. Systemic administration of ActRIIB.Fc in senescent mdx mice, a model of muscular dystrophy, significantly increases the number of muscle fibroblasts undergoing apoptosis. This leads to the reversal of pre-existed muscle fibrosis as determined by histological, biochemical and radiographical criteria. These results demonstrate that skeletal muscle fibrosis can be pharmacologically reversed through induction of fibroblast apoptosis.
ATF2 – at the crossroad of nuclear and cytosolic functions
Authors: Lau Eric , Ronai Ze'ev A.
Published: June 2012

An increasing number of transcription factors have been shown to elicit oncogenic and tumor suppressor activities, depending on the tissue and cell context. Activating transcription factor 2 (ATF2; also known as cAMP-dependent transcription factor ATF-2) has oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin tumors and breast cancer. Recent work has shown that the opposing functions of ATF2 are associated with its subcellular localization. In the nucleus, ATF2 contributes to global transcription and the DNA damage response, in addition to specific transcriptional activities that are related to cell development, proliferation and death. ATF2 can also translocate to the cytosol, primarily following exposure to severe genotoxic stress, where it impairs mitochondrial membrane potential and promotes mitochondrial-based cell death. Notably, phosphorylation of ATF2 by the epsilon isoform of protein kinase C (PKCε) is the master switch that controls its subcellular localization and function. Here, we summarize our current understanding of the regulation and function of ATF2 in both subcellular compartments. This mechanism of control of a non-genetically modified transcription factor represents a novel paradigm for ‘oncogene addiction’.
The translational repressor 4E-BP mediates the hypoxia-induced defects in myotome cells
Authors: Hidalgo Magdalena , Le Bouffant Ronan , Bello Valérie , Buisson Nicolas , Cormier Patrick , Beaudry Michèle , Darribère Thierry
Published: June 2012

Cell growth, proliferation, differentiation, and survival are influenced by the availability of oxygen. The effect of hypoxia on embryonic cells and the underlying molecular mechanisms to maintain cellular viability are still poorly understood. In this study, we show that hypoxia during Xenopus embryogenesis rapidly leads to a significant developmental delay and to cell apoptosis after prolonged exposure. We provide strong evidence that hypoxia does not affect somitogenesis but affects the number of mitotic cells and muscle-specific protein accumulation in somites, without interfering with the expression of MyoD and MRF4 transcription factors. We also demonstrate that hypoxia reversibly decreases Akt phosphorylation and increases the total amount of the translational repressor 4E-BP, in combination with an increase of the 4E-BP associated with eIF4E. Interestingly, the inhibition of PI3-Kinase or mTOR, with LY29002 or rapamycin respectively, triggers the 4E-BP accumulation in Xenopus embryos. Finally, the overexpression of the non-phosphorylatable 4E-BP protein induces, similar to hypoxia, a decrease in mitotic cells and a decrease in muscle-specific protein accumulation in somites. Taken together, our studies suggest that 4E-BP plays a central role under hypoxia in promoting the cap-independent translation at the expense of cap-dependent translation and triggers specific defects in muscle development.
Eya1 protein phosphatase regulates tight junction formation in lung distal epithelium
Authors: El-Hashash Ahmed H. K. , Turcatel Gianluca , Varma Saaket , Al Alam Denise , Warburton David
Published: June 2012

Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity such as aPKCζ. We recently reported that Eya1 phosphatase functions through aPKCζ-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKCζ phosphorylation levels, aPKCζ-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wildtype Eya1 or partially inhibiting aPKCζ in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1−/− lung epithelial TJ defects. These findings uncover novel functions for Eya1-aPKCζ-Notch1-Cdc42 as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.
Rab11 regulates exocytosis of recycling vesicles at the plasma membrane
Authors: Takahashi Senye , Kubo Keiji , Waguri Satoshi , Yabashi Atsuko , Shin Hye-Won , Katoh Yohei , Nakayama Kazuhisa
Published: June 2012

Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We here show that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of a component of the exocyst tethering complex, Sec15 or Exo70, the former which interacts with Rab11, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.
Perilipin family members preferentially sequester to either triacylglycerol- or cholesteryl ester-specific intracellular lipid storage droplets
Authors: Hsieh Kai , Lee Yun Kyung , Londos Constantine , Raaka Bruce M. , Dalen Knut Tomas , Kimmel Alan R.
Published: June 2012

Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes encode 5 distinct Plin gene members and additional protein forms derive from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging, and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to lipid droplets, comprised distinctly of either triacylcerides or of cholesterol esters. Further, Plins with strong association preferences to TAG (or CE) droplets can re-direct the relative intracellular TAG/CE balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.
Semaphorin 3A elevates endothelial cell permeability through PP2A inactivation
Authors: Le Guelte Armelle , Galan-Moya Eva-Maria , Dwyer Julie , Treps Lucas , Kettler Garance , Hebda Jagoda K. , Dubois Sonia , Auffray Cedric , Chneiweiss Herve , Bidere Nicolas , Gavard Julie
Published: June 2012

VE-cadherin-mediated cell-cell junction weakening increases paracellular permeability in response to both angiogenic and inflammatory stimuli. Although Semaphorin 3A has emerged as one of the few known anti-angiogenic factors to exhibit pro-permeability activity, little is known about how it triggers vascular leakage. Here we report that Semaphorin 3A induced VE-cadherin serine phosphorylation and internalization, cell-cell junction destabilization, and loss of barrier integrity in brain endothelial cells. In addition, high-grade glioma-isolated tumour initiating cells were found to secrete Semaphorin 3A, which promoted brain endothelial monolayer permeability. From a mechanistic standpoint, Semaphorin 3A impinged upon the basal activity of the serine phosphatase PP2A and disrupted PP2A interaction with VE-cadherin, leading to cell-cell junction disorganization and increased permeability. Accordingly, both pharmacological inhibition and siRNA-based knockdown of PP2A mimicked Semaphorin 3A effects on VE-cadherin. Hence, local Semaphorin 3A production impacts on the PP2A/VE-cadherin equilibrium and contributes to elevated vascular permeability.
In vivo studies on non-viral transdifferentiation of liver cells towards pancreatic β cells
Authors: Cim Abdullah , Sawyer Greta J , Zhang Xiaohong , Su Haibin , Collins Louise , Jones Peter , Antoniou Michael , Reynes Jean-Paul , Lipps Hans-Joachim , Fabre John W
Published: June 2012

Transdifferentiationin vivois an attractive option for autologous replacement of pancreatic β cells in patients with type 1 diabetes. It has been achieved by adenoviral delivery of genes for transcription factors in the liver and pancreas of hyperglycaemic mice. However, these viral approaches are not clinically applicable. We used the hydrodynamic approach to deliver genesPdx1,Ngn3(Neurog3) andMafAsingly and in combination to livers of normoglycaemic rats. Five expression plasmids were evaluated. Livers were removed 1, 3, 7, 14 and 28 days after gene delivery and assayed by quantitative PCR, semi-quantitative PCR and immunohistology. Functional studies on hyperglycaemic rats were performed. The highest and most sustained expression was from a CpG-depleted plasmid (pCpG) and a plasmid with an in-frame scaffold/matrix attachment region ((pEPI(CMV)). WhenPdx1,Ngn3andMafAwere delivered together to normoglycaemic rats with these plasmids, insulin mRNA was detected at all time points and was ∼50-fold higher with pCpG. Insulin mRNA content of livers at days 3 and 7 was equivalent to that of a pancreas, with scattered insulin-positive cells detected by immunohistology, but levels declined thereafter. Prohormone convertase 1/3 was elevated at days 3 and 7. In hyperglycaemic rats, fasting blood glucose was lower at days 1, 3 and 7 but not thereafter, and body weight was maintained to day 28. We conclude that hydrodynamic gene delivery of multiple transcription factors to rat liver can initiate transdifferentiation to pancreatic β cells, but the process is reversible and probably requires more sustained transcription factor expression.
Extracellular ATP signaling via P2X4 receptor and cAMP/PKA signaling mediate ATP oscillations essential for prechondrogenic condensation
Authors: Kwon Hyuck Joon
Published: June 2012

Prechondrogenic condensation is the most critical process in skeletal patterning. A previous study demonstrated that ATP oscillations driven by Ca2+oscillations play a critical role in prechondrogenic condensation by inducing oscillatory secretion. However, it remains unknown what mechanisms initiate the Ca2+-driven ATP oscillations, mediate the link between Ca2+and ATP oscillations, and then result in oscillatory secretion in chondrogenesis. This study has shown that extracellular ATP signaling was required for both ATP oscillations and prechondrogenic condensation. Among P2 receptors, the P2X4receptor revealed the strongest expression level and mediated ATP oscillations in chondrogenesis. Moreover, blockage of P2X4activity abrogated not only chondrogenic differentiation but also prechondrogenic condensation. In addition, both ATP oscillations and secretion activity depended on cAMP/PKA signaling but not on KATPchannel activity and PKC or PKG signaling. This study proposes that Ca2+-driven ATP oscillations essential for prechondrogenic condensation is initiated by extracellular ATP signaling via P2X4receptor and is mediated by cAMP/PKA signaling and that cAMP/PKA signaling induces oscillatory secretion to underlie prechondrogenic condensation, in cooperation with Ca2+and ATP oscillations.
Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility
Authors: Guo-Parke Hong , McCluskey Jane T , Kelly Catriona , Hamid Muhajir , McClenaghan Neville H , Flatt Peter R
Published: June 2012

Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human β-cell line. Structural pseudoislets formed readily over 3–7 days in culture using ultra-low-attachment plastic, attaining a static size of 100–200 μm in diameter, corresponding to ∼6000 β cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1.7- to 12.5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human β cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.
Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner
Authors: Xu Hongbin , Liu Fang , Srakaew Nopparat , Koppisetty Chaitanya , Nyholm Per-Georg , Carmona Euridice , Tanphaichitr Nongnuj
Published: June 2012

We have shown previously that sperm surface arylsulfatase A (ASA) of mouse, pig, and human is involved in sperm–egg zona pellucida (ZP) binding. By treating capacitated mouse sperm with A23187 to induce the acrosome reaction, we demonstrated by immunoblotting that ASA also existed in the acrosomal content and on the inner acrosomal membrane. Since mZP2 and mZP3 are known as sperm receptors, whereas mZP1 as a cross-linker of mZP2/mZP3, we determined whether purified ASA bound to mZP2 and mZP3 selectively. The three mZP glycoproteins were purified from solubilized ovarian ZP by size exclusion column chromatography. Immuno-dot blot analyses revealed that purified sperm ASA bound to mZP2 at the highest level followed by mZP3, whereas the binding of ASA to mZP1 was minimal. The results confirmed the physiological significance of sperm ASA in the ZP binding process. The binding of ASA to mZP2 and mZP3 was, however, not dependent on the active site pocket amino acids, Cys69, Lys123, and Lys302, which are pertinent to the capturing of an arylsulfate substrate, since ASA mutant with Ala substitution at these three residues still bound to mZP2 and mZP3. The availability of the active site pocket of ASA bound to the ZP suggested that ASA would still retain enzymatic activity, which might be important for subsequent sperm penetration through the ZP.
Investigations into the mechanisms controlling parturition in cattle
Authors: Shenavai Sima , Preissing Susanne , Hoffmann Bernd , Dilly Marc , Pfarrer Christiane , Özalp Gözde R , Caliskan Caglar , Seyrek-Intas Kamil , Schuler Gerhard
Published: June 2012

A pronounced increase in fetal cortisol concentrations stimulating an increase in estrogen production at the expense of progesterone precursors in the placenta, luteolysis, and progesterone withdrawal is considered as a key event during the complex signal cascade leading to the initiation of parturition in cattle. However, there are many questions concerning the exact functional and/or temporal relationships between these individual processes which finally result in the expulsion of the calf and the timely release of the placenta. Thus, parturition was induced in 270-day pregnant cows using the progesterone receptor blocker aglepristone (group AG,n=3), the prostaglandin F2αanalog cloprostenol (group PG,n=4), and the glucocorticoid dexamethasone (group GC,n=4) to characterize the effect on maternal steroid and prostaglandin levels and to identify immediate subsequent changes in placental morphology and gene expression as compared with untreated controls sampled on day 272 (group D272,n=3) and cows during normal parturition (group NT,n=4). All calves of the treatment groups were born on days 271–272, whereas gestational length in NT cows was 280.5±1.3 days. However, none of the treatments significantly induced the prepartal remodeling of placentomes characterized by a decline in trophoblast giant cells and reduction of the caruncular epithelium. Data on placental CYP17 and COX2 expression confirm that these key enzymes are upregulated by GC, whereas placental aromatase expression was not affected by any treatment. Maternal progesterone and prostaglandin profiles suggest differential effects of the treatments on luteal function and placental or uterine prostaglandin production. The results provide new information on the initiation of parturition in cattle but raise many new questions.
Effect of aging on follicular function may be relieved by exogenous gonadotropin treatment in a sheep model
Authors: Berlinguer Fiammetta , Gonzalez-Bulnes Antonio , Spezzigu Antonio , Contreras-Solis Ignacio , Succu Sara , McNeilly Alan S , Naitana Salvatore , Leoni Giovanni G
Published: June 2012

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12–14 years) and young adult ewes (4–5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E2) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E2 and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen.
Income convergence in Europe: Catching up or falling behind?
Authors: Czasonis Megan , Quinn Michael
Published: June 2012

One of the motivations for a country to join the European Union is the belief that this will boost short- and long-run incomes. Researchers have tested the hypothesis of income convergence in different settings using either regression or unit root analysis, with mixed results. In this paper, we use both methods on the same samples over a significant time period. This allows us to judge differences in results across varied time-frames and methodologies. The focus of these tests is on convergence to German and EMU average incomes by Eastern European countries and those within the Euro-zone from 1971–2007. The evidence for convergence is mixed. Among the Euro-zone countries, there is more evidence of convergence in the 1970s and 1980s than recently. There is significant evidence that Eastern Europe experienced convergence and that capital formation was one of the root causes. While the results do not support the hypothesis that joining the EU increases convergence, reforms undertaken in the 1990s by Eastern European countries in preparation for joining may have helped them to “catch up”, even if the act of joining the EU did not directly impact convergence.
Data reduction and univariate splitting — Do they together provide better corporate bankruptcy prediction?
Authors: Kristóf Tamás , Virág Miklós
Published: June 2012

Discussion on methodological problems of corporate survival and solvency prediction is enjoying a renaissance in the era of financial and economic crisis. Within the framework of this article, the most frequently applied bankruptcy prediction methods are competed on a Hungarian corporate database. Model reliability is evaluated by Receiver Operating Characteristic (ROC) curve analysis. The article attempts to answer the question of whether the simultaneous application of data reduction and univariate splitting (or just one of them) improves model performance, and for which methods it is worth applying such transformations.
Capital structure and its choice in Central and Eastern Europe
Authors: Hernádi Péter , Ormos Mihály
Published: June 2012

We analyze the determinants of capital structure and its choice by small and medium-sized enterprises in Central and Eastern Europe from 2002 to 2007. We test the relevance of the three main theories: the Static Trade-off Theory, the Pecking Order Theory, and the Agency Theory, which have been derived primarily for developed markets, because our knowledge on their validity for emerging European countries is limited. We confirm the positive impact of size and asset tangibility on the leverage, while rejecting both the positive impact of profitability and tax, as well as the negative impact of business risk and non-debt tax shields. We report that SMEs behave homogeneously, and the relevant capital structure determinants show remarkable steadiness. Our results show a special time varying behaviour, in which the relevant determinants become stronger, while most of the country-specific factors present weakening effects. We argue that firms of the CEE countries remarkably converged their financial decision-making procedure to that of developed countries through the investigated period. The relevance of the Trade-off Theory is weak, as firms respect a one-sided upper threshold rather than converging to a fixed target on both sides, while they are not indifferent to the hierarchy of financing alternatives.