The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy

The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic β cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (ΔF1388) causes PHHI. Previous studies have shown that coexpression of ΔF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of KATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ΔF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ΔF1388 SUR1 by mutation to AAA (ΔF1388 SUR1AAA). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ΔF508. We found that coexpression of ΔF1388 SUR1AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR → AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of KATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 → Trp and Pro-253 → Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2) . These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2–FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 → Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 → Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the “ear” domain of the clathrin adaptor AP-1 γ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Δ), the major Gga protein, accentuates growth and α-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Δ or a deletion of the AP-1 β subunit gene (apl2Δ) alone are phenotypically normal, but cells carrying both gga2Δ and apl2Δ are defective in growth, α-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.

Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Function of the oxidative burst in hypersensitive disease resistance.

Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. Recent findings indicate that H2O2 from this oxidative burst plays a central role in the orchestration of the hypersensitive response: (i) as the substrate driving the cross-linking of cell wall structural proteins to slow microbial ingress prior to the deployment of transcription-dependent defenses and to trap pathogens in cells destined to undergo hypersensitive cell death, (ii) as a local threshold trigger of this programmed death in challenged cells, and (iii) as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. These findings provide the basis for an integrated model for the orchestration of the localized hypersensitive resistance response to attack by an avirulent pathogen.

Modulation of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) function by acetylation following the treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.

Orchestration of energy efficiency capabilities for a sustainable network management

The energy demand for operating Information and Communication Technology (ICT) systems has been growing, implying in high operational costs and consequent increase of carbon emissions. Both in datacenters and telecom infrastructures, the networks represent a significant amount of energy spending. Given that, there is an increased demand for energy eficiency solutions, and several capabilities to save energy have been proposed. However, it is very dificult to orchestrate such energy eficiency capabilities, i.e., coordinate or combine them in the same network, ensuring a conflict-free operation and choosing the best one for a given scenario, ensuring that a capability not suited to the current bandwidth utilization will not be applied and lead to congestion or packet loss. Also, there is no way in the literature to do this taking business directives into account. In this regard, a method able to orchestrate diferent energy eficiency capabilities is proposed considering the possible combinations and conflicts among them, as well as the best option for a given bandwidth utilization and network characteristics. In the proposed method, the business policies specified in a high-level interface are refined down to the network level in order to bring highlevel directives into the operation, and a Utility Function is used to combine energy eficiency and performance requirements. A Decision Tree able to determine what to do in each scenario is deployed in a Software Defined Network environment. The proposed method was validated with diferent experiments, testing the Utility Function, checking the extra savings when combining several capabilities, the decision tree interpolation and dynamicity aspects. The orchestration proved to be valid to solve the problem of finding the best combination for a given scenario, achieving additional savings due to the combination, besides ensuring a conflict-free operation.

Schleiermacher's Doctrine of Biblical Authority: An Alternative to Content-Based/Supernaturalist and Function-Based Rationalist Models

Author: Kerry W. Holton Title: SCHLEIERMACHER’S DOCTRINE OF BIBLICAL AUTHORITY: AN ALTERNATIVE TO CONTENT-BASED/SUPERNATURALIST AND FUNCTION- BASED/RATIONALIST MODELS Advisor: Theodore M. Vial, Jr. Degree Date: August 2015 This dissertation examines Friedrich Schleiermacher’s understanding of biblical authority and argues that, as an alternative to strictly supernaturalistic and rationalistic models, his understanding allows the New Testament to speak authoritatively in Christian religion in an age of critical, historical awareness. After classifying Schleiermacher’s position in a typology of the doctrine of biblical authority, this dissertation explores his conception of divine revelation and inspiration vis-à-vis scripture. It demonstrates that although he did not believe there is warrant for the claim of a direct connection between divine revelation and scripture, or that scripture is the foundation of faith, he nonetheless asserted that the New Testament is authoritative. He asserted the normative authority of the New Testament on the basis that it is the first presentation of Christian faith. This dissertation examines Schleiermacher’s “canon within the canon,” as well as his denial that the Old Testament shares the same normative worth and inspiration of the New. Although this dissertation finds difficulty with some of Schleiermacher’s views regarding the Old Testament, it names two significant strengths of what is identified as his evangelical, content-based, and rationalist approach to biblical authority. First, it recognizes and values the co-presence and co-activity of the supernatural and the natural !ii in the production of the New Testament canon. This allows both scripture and the church to share religious authority. Second, it allows Christian faith and the historical-method to coexist, as it does not require people to contradict what they know to be the case about science, history, and philosophy. Thus, this dissertation asserts that Schleiermacher’s understanding of biblical authority is a robust one, since, for him, the authority of scripture does not lie in some property of the texts themselves that historians or unbelievers can take away.

Representation of 2D objects with a topology preserving network

Comunicación presentada en el 2nd International Workshop on Pattern Recognition in Information Systems, Alicante, April, 2002.

Innovation in the teaching of sustainable development in Europe: the case of ISLE Erasmus Network

Sustainable Development (SD) is one of the most widely used terms during the last years. It is a multidisciplinary concept, which applies mostly to life sciences but is not limited to them. Even though the short survey conducted by the authors revealed that there are only a few cases of Higher Educational Institutes (HEIs) around Europe that provide programs dedicated to SD, it is obvious that there is a constant raise in the need for implementing courses related to SD in existing programs. This paper discusses the case study of I.S.L.E., an Erasmus Academic Network, which aims to use the existing knowledge and tools in the context of teaching sustainable development topics in Universities and HEIs around Europe as a basis, and elaborate further by introducing an innovative approach towards the improvement of teaching SD in HEIs, based on the current needs as they are identified by the actions of the Network.

A Model to Visualize Information in a Complex Streets’ Network

his paper discusses a process to graphically view and analyze information obtained from a network of urban streets, using an algorithm that establishes a ranking of importance of the nodes of the network itself. The basis of this process is to quantify the network information obtained by assigning numerical values to each node, representing numerically the information. These values are used to construct a data matrix that allows us to apply a classification algorithm of nodes in a network in order of importance. From this numerical ranking of the nodes, the process finish with the graphical visualization of the network. An example is shown to illustrate the whole process.

On Modeling an Innovative Monitoring Network for Protecting and Managing Cultural Heritage from Risk Events

In this paper the model of an Innovative Monitoring Network involving properly connected nodes to develop an Information and Communication Technology (ICT) solution for preventive maintenance of historical centres from early warnings is proposed. It is well known that the protection of historical centres generally goes from a large-scale monitoring to a local one and it could be supported by a unique ICT solution. More in detail, the models of a virtually organized monitoring system could enable the implementation of automated analyses by presenting various alert levels. An adequate ICT solution tool would allow to define a monitoring network for a shared processing of data and results. Thus, a possible retrofit solution could be planned for pilot cases shared among the nodes of the network on the basis of a suitable procedure utilizing a retrofit catalogue. The final objective would consist in providing a model of an innovative tool to identify hazards, damages and possible retrofit solutions for historical centres, assuring an easy early warning support for stakeholders. The action could proactively target the needs and requirements of users, such as decision makers responsible for damage mitigation and safeguarding of cultural heritage assets.

Analyzing the commercial activities of a street network by ranking their nodes: a case study in Murcia, Spain

Urban researchers and planners are often interested in understanding how economic activities are distributed in urban regions, what forces influence their special pattern and how urban structure and functions are mutually dependent. In this paper, we want to show how an algorithm for ranking the nodes in a network can be used to understand and visualize certain commercial activities of a city. The first part of the method consists of collecting real information about different types of commercial activities at each location in the urban network of the city of Murcia, Spain. Four clearly differentiated commercial activities are studied, such as restaurants and bars, shops, banks and supermarkets or department stores, but obviously we can study other. The information collected is then quantified by means of a data matrix, which is used as the basis for the implementation of a PageRank algorithm which produces a ranking of all the nodes in the network, according to their significance within it. Finally, we visualize the resulting classification using a colour scale that helps us to represent the business network.