Transcriptional profiling reveals functional links between RasGrf1 and Pttg1 in pancreatic beta cells

Background: Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those"pancreatic" phenotypes. Results: Here, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways,suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation,together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins. Analysis of the mPttg1 promoter region identified specific recognition sites for numerous transcription factors which were also found to be differentially expressed in RasGrf1 KO pancreatic islets and are known to be relevant for Ras-ERK signaling as well as beta cell function. Reporter luciferase assays in BT3 insulinoma cells demonstrated the ability of RasGrf1 to modulate mPttg1 promoter activity through ERK-mediated signals. Analysis of the phenotypic interplay between RasGrf1 and Pttg1 in double knockout RasGrf1/Pttg1 mice showed that combined elimination of the two loci resulted in dramatically reduced values of islet and beta cell count and glucose homeostasis function which neared those measured in single Pttg1 KO mice and were significantly lower than those observed in individual RasGrf1 KO mice. Conclusions: The specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological response

Transcriptional profiling reveals functional links between RasGrf1 and Pttg1 in pancreatic beta cells

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License .[Background]: Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those >pancreatic> phenotypes. [Results]: Here, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins. [Conclusions]: The specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses.Work supported by grants FIS PI13/02846 and RTICC RD12/0036/0001 from Instituto de Salud Carlos III (ISCIII), and grant SA181U13 from JCyL, Spain. We are grateful to Dr. Douglas Lowy (National Cancer Institute, Bethesda, MD) for providing plasmid pBK-CMV RasGrf1 and to Dr. Shlomo Melmed (Cedars-Sinai Medical Center, Los Angeles, CA) for providing reporter plasmid pGL3-Pttg1 and the single PTTG1 KO mouse strain used in these studies.Peer Reviewe

Nanomechanics: A new approach for studying the mechanical properties of materials

Mitjançant l'espectroscòpia de forces atòmiques s'ha estudiat la resposta nanomecànica a la nanoindentació de la superfície més estable d'un material trencadís FCC, com és ara el MgO (100). L'expulsió del material en forma de capes demostra que la fallida trencadissa implica, de fet, l'inici de la deformació plàstica o estrès crític, i que la deformació plàstica posterior consisteix en una sèrie d'esdeveniments discrets. Es pot determinar amb precisió el mòdul de Young, E, a partir de la regió de deformació elàstica mitjançant una mecànica senzilla, atesa l'absència de dislocacions induïdes per la nanoindentació. Amb aquesta finalitat s'ha desenvolupat un nou model fisicomatemàtic, que té en compte les interaccions laterals. El valor de l'estrès crític de fricció també s'ha calculat i comentat. Com a conseqüència d'aquesta expulsió en capes, també s'ha estudiat la resposta nanomecànica de superfícies de capes primes (gruix & 1 µm) de molècules orgàniques altament orientades, ja que es tracta de materials en capes amb interaccions de tipus Van der Waals. També en aquests materials la superfície es deforma plàsticament i presenta discontinuïtats discretes en les corbes d'indentació, associades ara a les capes moleculars expulsades per l'indentador. En el cas del metall quasiunidimensional tetratiofulvalè tetracianoquinodimetà (TTFTCNQ), el valor del mòdul de Young, E & 20 GPa, coincideix amb l'obtingut per altres mètodes. En el cas de la fase ! del radical p-nitrofenil nitronil nitròxid (p-NPNN) no es disposa d'informació per a monocristalls, i el valor obtingut per a les capes primes és de E & 2 GPa.Atomic force spectroscopy was used to study the nanomechanical response to nanoindentations on the most stable face (100) of FCC brittle materials such as MgO and alkali halides. The layered expulsion of material demonstrates that brittle failure results from the critical stress brought on by plastic deformation and that plastic deformation consists of a series of discrete events. Due to the absence of indentation- induced dislocations, Young?s modulus E can be correctly estimated from the elastic deformation region using simple mechanics. A new model is developed taking into account lateral interactions. Critical shear stress is also evaluated and discussed. As a result of the layered expulsion we also studied the nanomechanical response of surfaces of highly-oriented molecular organic thin films (ca. 1 µm thickness) because these are Van der Waals layered materials. The surfaces were again found to deform plastically and there were discrete discontinuities in the indentation curves, representing the molecular layers being expelled by the penetrating tip. Here, the Hertz model is quite good at revealing the role of lateral interactions in the indentation process. For the quasi-one-dimensional metal tetrathiafulvalene tetracyanoquinodimethane (TTF-TCNQ) the value of Young?s modulus, E & 20 GPa, coincides with that obtained by other bulk methods. For the !-phase of the p-nitrophenyl nitronyl nitroxide (p-NPNN) radical, no information is available for single crystals and the estimated value obtained for the film is E & 2 GPa

Mechanically activated rupture of single covalent bonds: evidence of force induced bond hydrolysis.

We have used temperature-dependent single molecule force spectroscopy to stretch covalently anchored carboxymethylated amylose (CMA) polymers attached to an amino-functionalized AFM cantilever. Using an Arrhenius kinetics model based on a Morse potential as a one-dimensional representation of covalent bonds, we have extracted kinetic and structural parameters of the bond rupture process. With 35.5 kJ mol−1, we found a significantly smaller dissociation energy and with 9.0 × 102 s−1 to 3.6 × 103 s−1 also smaller Arrhenius pre-factors than expected for homolytic bond scission. One possible explanation for the severely reduced dissociation energy and Arrhenius pre-factors is the mechanically activated hydrolysis of covalent bonds. Both the carboxylic acid amide and the siloxane bond in the amino-silane surface linker are in principle prone to bond hydrolysis. Scattering, slope and curvature of the scattered data plots indicate that in fact two competing rupture mechanisms are observed

Ions modulate stress-induced nano-texture in supported fluid lipid bilayers.

Most plasma membranes comprise a large number of different molecules including lipids and proteins. In the standard fluid mosaic model, the membrane function is effected by proteins whereas lipids are largely passive and serve solely in the membrane cohesion. Here we show, using supported 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers in different saline solutions, that ions can locally induce ordering of the lipid molecules within the otherwise fluid bilayer when the latter is supported. This nanoordering exhibits a characteristic length scale of ∼20 nm, and manifests itself clearly when mechanical stress is applied to the membrane. Atomic force microscopy (AFM) measurements in aqueous solutions containing NaCl, KCl, CaCl2, and Tris buffer show that the magnitude of the effect is strongly ion-specific, with Ca2+ and Tris, respectively, promoting and reducing stress-induced nanotexturing of the membrane. The AFM results are complemented by fluorescence recovery after photobleaching experiments, which reveal an inverse correlation between the tendency for molecular nanoordering and the diffusion coefficient within the bilayer. Control AFM experiments on other lipids and at different temperatures support the hypothesis that the nanotexturing is induced by reversible, localized gel-like solidification of the membrane. These results suggest that supported fluid phospholipid bilayers are not homogenous at the nanoscale, but specific ions are able to locally alter molecular organization and mobility, and spatially modulate the membrane’s properties on a length scale of ∼20 nm. To illustrate this point, AFM was used to follow the adsorption of the membrane-penetrating antimicrobial peptide Temporin L in different solutions. The results confirm that the peptides do not absorb randomly, but follow the ion-induced spatial modulation of the membrane. Our results suggest that ionic effects have a significant impact for passively modulating the local properties of biological membranes, when in contact with a support such as the cytoskeleton

The Nanomechanics of Lipid Multibilayer Stacks Exhibits Complex Dynamics

The nanomechanics of lipid membranes regulates a large number of cellular functions. However, the molecular mechanisms underlying the plastic rupture of individual bilayers remain elusive. This study uses force clamp spectroscopy to capture the force-dependent dynamics of membrane failure on a model diphytanoylphosphatidylcholine multilayer stack, which is devoid of surface effects. The obtained kinetic measurements demonstrate that the rupture of an individual lipid bilayer, occurring in the bilayer parallel plane, is a stochastic process that follows a log-normal distribution, compatible with a pore formation mechanism. Furthermore, the vertical individual force-clamp trajectories, occurring in the bilayer orthogonal bilayer plane, reveal that rupturing process occurs through distinct intermediate mechanical transition states that can be ascribed to the fine chemical composition of the hydrated phospholipid moiety. Altogether, these results provide a first description of unanticipated complexity in the energy landscape governing the mechanically induced bilayer rupture process.</p

Mitigating AFB1 and OTA hepatic residues using pumpkin and milk fermented whey as functional ingredients

The consumption of antioxidant-rich commodities acts as a preventive measure to regulate mycotoxin levels, which pose a significant threat to human and animal health worldwide. This study aimed to evaluate the potential reduction of mycotoxin liver bioaccumulation through an in vivo experimental study on 120 rats fed with pumpkin, milk fermented whey, or combinations. The levels of Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) in liver were monitored through Ultra High-Resolution Liquid Chromatography-Orbitrap Mass Spectrometry (UHPLC-Q-Orbitrap HRMS). The methodology was appropriately optimized according to current regulations. Limit of quantification (LOQ) for AFB1 and OTA were 0.78 and 6.25 ng/g respectively, whereas recovery rates were up to 70% for both mycotoxins. Mycotoxin residues in the liver of rats fed with functional ingredients showed up to a 100 % reduction for AFB1 and 80% for OTA. The combination of pumpkin and fermented whey in feed formulations has proven to be an effective approach easily implemented by the food industry to safeguard consumer health

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

In vitro and in vivo evaluation of AFB1 and OTA-toxicity through immunofluorescence and flow cytometry techniques : A systematic review

Due to the globalization, mycotoxins have been considered a major risk to human health being the main con- taminants of foodstuffs. Among them, AFB1 and OTA are the most toxic and studied. Therefore, the goal of this review is to deepen the knowledge about the toxicological effects that AFB1 and OTA can induce on human health by using flow cytometry and immunofluorescence techniques in vitro and in vivo models. The examination of the selected reports shows that the majority of them are focused on immunotoxicity while the rest are con- cerned about nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, neurotoxicity, embryotoxicity, reproduc- tive system, breast, esophageal and lung toxicity. In relation to immunofluorescence analysis, biological processes related to AFB1- and OTA-toxicity were evaluated such as inflammation, neuronal differentiation, DNA damage, oxidative stress and cell death. In flow cytometry analysis, a wide range of assays have been performed across the reviewed studies being apoptosis assay, cell cycle analysis and intracellular ROS measurement the most employed. Although, the toxic effects of AFB1 and OTA have been reported, further research is needed to clarify AFB1 and OTA-mechanism of action on human health