Pathway databases and tools for their exploitation: benefits, current limitations and challenges

In past years, comprehensive representations of cell signalling pathways have been developed by manual curation from literature, which requires huge effort and would benefit from information stored in databases and from automatic retrieval and integration methods. Once a reconstruction of the network of interactions is achieved, analysis of its structural features and its dynamic behaviour can take place. Mathematical modelling techniques are used to simulate the complex behaviour of cell signalling networks, which ultimately sheds light on the mechanisms leading to complex diseases or helps in the identification of drug targets. A variety of databases containing information on cell signalling pathways have been developed in conjunction with methodologies to access and analyse the data. In principle, the scenario is prepared to make the most of this information for the analysis of the dynamics of signalling pathways. However, are the knowledge repositories of signalling pathways ready to realize the systems biology promise? In this article we aim to initiate this discussion and to provide some insights on this issue.

Role of homer 1 proteins in the calcium signaling pathway(s) underlying exo-endocytosis processes of glutamatergic slmvs in astrocytes

The discovery that astrocytes possess a nonelectrical form of excitability (calcium excitability) that leads to the release of chemical transmitters, an activity called gliotransmission, indicates that these cells may have additional important roles in brain function. Elucidating the stimulussecretion coupling leading to the exocytic release of chemical transmitters (such as glutamate, Bezzi et al., Nature Neurosci, 2004) may therefore clarify i) whether astrocytes represent in full a new class of secretory cells in the brain and ii) whether they can participate to the fast brain signaling in the brain. We have recently discovered the existence in astrocytes of functional sub-membrane microdomains of calcium release from the internal stores in response to mGluR5 activation (Marchaland et al., J of Neurosci., 2008). Such sub-plasma membrane calcium microdomains control exocytosis of astrocytic glutamate signaling to neurons. Homer proteins are scaffold proteins controlling calcium signaling in different cellular microdomains, including dendritic spines in neurons (Sala et al., J of Neurosci., 2005). Thus, similarly to dendritic pines, Homer1 could be implicated in the coupling between astrocytic mGluR5 and IP3Rs on the ER. Here, by using a recently developed approach for studying vesicle recycling dynamics at synapses (Voglmaier et al., Neuron, 2006; Balaji and Ryan, PNAS, 2007) combined with epifluorescence and total internal reflection fluorescence (TIRF) imaging, we investigated the involvement of Homer1 proteins in the calcium dependent stimulus-secretion coupling leading glutamate exocytosis of synaptic-like microvesicles (SLMVs) in astrocytes.

Enhanced recovery pathway for urgent colectomy.

BACKGROUND: Enhanced recovery protocols have been proven to decrease complications and hospital stay following elective colorectal surgery. However, these principles have not yet been reported for urgent surgery procedures. We aimed to assess our initial experience with urgent colectomies performed within an established enhanced recovery pathway. METHODS: In a prospective cohort study, all patients undergoing colonic resection between April 2012 and March 2013 were treated according to a standardized enhanced recovery protocol. Urgent surgeries were compared with the elective procedures with regards to baseline characteristics, compliance with enhanced recovery items, and clinical outcome. RESULTS: Patients (N = 28) requiring urgent colonic resection were included and compared with patients undergoing elective colectomy (N = 63). Overall compliance with the protocol was 57% for the urgent compared with 77% for the elective procedures (p = 0.006). The pre-operative compliance was 64 versus 96% (p < 0.001), the intra-operative compliance was 77 versus 86% (p = 0.145), and the post-operative compliance was 49 versus 67% (p = 0.015), for the urgent and elective resections, respectively. Overall, 18 urgent patients (64%) and 32 elective patients (51%) developed postoperative complications (p = 0.261). Median postoperative length of stay was 8 days in the urgent setting compared with 5 days in the elective setting (p = 0.006). CONCLUSIONS: Many of the intra-operative and post-operative enhanced recovery items can also be applied to urgent colectomy, entailing outcomes that approach the results achieved in the elective setting.

Neutralizing antibodies in Multiple Sclerosis a model-based analysis of Interferon beta signaling pathway in macrophages

Multiple Sclerosis is the most common non-traumatic cause of neurologicaldisability in young people. There is no cure yet, and until recently, few long-termtherapies existed. Interferon beta (IFNβ) was the first treatment, and remains the mostcommonly prescribed. One of the most significant problems of IFNβ therapy is theproduction of drug specific antibodies. Up to 45% of patients develop neutralizingantibodies (NAbs) to IFNβ products. The neutralizing antibody binds to the biologicalagent preventing its interaction with its receptor, inhibiting the biological action of theprotein, which abrogates the clinical efficacy of IFNβ treatment. Interferon-betamediates its response by binding to its high affinity cell surface receptor and initiatingthe JAK/STAT signalling cascade. In this project we have analyzed the IFNβ signalingpathway in macrophages when neutralizing antibodies are present. The response tothis pathway after IFNβ stimulation shows a transient oscillatory rhythm of STAT1phosphorylation, which varies as NAbs concentration increases. To improve ourunderstanding of that behavior, we extended an existing mathematical model based onnonlinear ordinary differential equations of JAK/STAT pathway by including IFN-NAbassociation and IFN-activation receptor. Combining our theoretical model withexperimental data we could study the role of neutralizing antibodies on the molecularresponse and determine its lifetime after cytokine stimulation.

Beta-oxidation in fatty acid degradation and beyond.

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the beta-oxidation cycle. Enzymes that are involved in various aspects of beta-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for beta-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. Beta-oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid.

The clc element of Pseudomonas sp. strain B13, a genomic island with various catabolic properties.

Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3- and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.

Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis.

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.

The jasmonate biochemical pathway.

Plants possess an interrelated and interacting family of potent fatty acid-derived regulators--the jasmonates. These compounds, which play roles in both defense and development, are derived from tri-unsaturated fatty acids [alpha-linolenic acid (18:3) or 7Z,10Z,13Z-hexadecatrienoic acid (16:3)]. The lipoxygenase-catalyzed addition of molecular oxygen to alpha-linolenic acid initiates jasmonate synthesis by providing a 13-hydroperoxide substrate for the formation of an unstable allene oxide that is then subject to enzyme-guided cyclization to produce 12-oxo-phytodienoic acid (OPDA). OPDA, a key regulatory lipid in the plant immune system, has several fates, including esterification into plastid lipids or transformation into the 12-carbon co-regulator jasmonic acid (JA). JA, the best-characterized member of the family, regulates both male and female fertility (depending on the plant species), and is an important mediator of defense gene expresssion. JA is itself a substrate for further diverse modifications. Genetic dissection of the pathway is revealing how the different jasmonates modulate different physiological processes. Each new family member that is discovered provides another key to understanding the fine control of gene expression in immune responses, in the initiation and maintenance of long-distance signal transfer in response to wounding, and in the regulation of fertility, among other processes. The Jasmonate Biochemical Pathway provides an overview of the growing jasmonate family, and new members will be included in future versions of the Connections Map. Science Viewpoint R. Liechti, E. E. Farmer, The jasmonate pathway. Science 296, 1649-1650 (2002). [Abstract] [Full Text]

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.

Energy relaxation in nonlinear one-dimensional lattices

We study energy relaxation in thermalized one-dimensional nonlinear arrays of the Fermi-Pasta-Ulam type. The ends of the thermalized systems are placed in contact with a zero-temperature reservoir via damping forces. Harmonic arrays relax by sequential phonon decay into the cold reservoir, the lower-frequency modes relaxing first. The relaxation pathway for purely anharmonic arrays involves the degradation of higher-energy nonlinear modes into lower-energy ones. The lowest-energy modes are absorbed by the cold reservoir, but a small amount of energy is persistently left behind in the array in the form of almost stationary low-frequency localized modes. Arrays with interactions that contain both a harmonic and an anharmonic contribution exhibit behavior that involves the interplay of phonon modes and breather modes. At long times relaxation is extremely slow due to the spontaneous appearance and persistence of energetic high-frequency stationary breathers. Breather behavior is further ascertained by explicitly injecting a localized excitation into the thermalized arrays and observing the relaxation behavior.

Immunohistochemical analysis of bone morphological protein signaling pathway in human myometrium.

We assessed by immunohistochemistry the expression of the phosphorylated (activated) form of Smad1 and 5 (P-SMAD1/5), of Noggin and of two smooth muscle cell markers (α-SMA and SM22) in a series of human myometrium samples and in a smooth muscle cell line derived from human myometrium (HUt-SMC, PromoCell, USA). Myometrium samples were removed from two cadavers (a fetus at 26weeks of gestation and a neonate) and from ten non-menopausal women who underwent hysterectomy for adenomyosis and leiomyoma. P-SMAD1/5 expression was never detected in myometrium (both normal and pathological specimens), but only as a nuclear positive staining in glandular and luminal epithelial cells in sections in which also the endometrial mucosa was present. Noggin was strongly expressed especially in myometrium and adenomyosis samples from non-menopausal patients in comparison to the neonatal and fetal myometrium specimens in which muscle cells were less positive. In more than 95% of HUt-SMCs, α-SMA and Desmin were co-expressed, indicating a pure smooth muscle phenotype. When progesterone was added to the culture medium, no P-SMAD1/5 expression was detected, whereas the expression Noggin and SM22, a marker of differentiated smooth muscle cells, increased by 3 fold (p=0.002) and 4.3 fold (p=0.001), respectively (p=0.002). Our results suggest that, in non-menopausal normal human myometrium, the BMP pathway might be inhibited and that this inhibition might be enhanced by progesterone, which increases the differentiation of smooth muscle cells (SM22 levels). These findings could help in the identification of new mechanisms that regulate uterine motility.

Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway.

OBJECTIVE: The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) generates pancreatic beta-cells apoptosis mainly through activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced beta-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS: Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1 beta. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Ex-4 inhibited induction of the JNK pathway elicited by IL-1 beta. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1 beta. CONCLUSIONS: The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1 beta and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

The MyD88 protein 88 pathway is differently involved in immune responses induced by distinct substrains of Leishmania major.

Host resistance to Leishmania major is highly dependent on the development of a Th1 immune response. The TLR adaptator myeloid differentiation protein 88 (MyD88) has been implicated in the Th1 immune response associated with the resistant phenotype observed in C57BL/6 mice after infection with L. major. To investigate whether the MyD88 pathway is differentially used by distinct substrains of parasites, MyD88(-/-) C57BL/6 mice were infected with two substrains of L. major, namely L. major LV39 and L. major IR75. MyD88(-/-) mice were susceptible to both substrains of L. major, although with different kinetics of infection. The mechanisms involved during the immune response associated with susceptibility of MyD88(-/-) mice to L. major is however, parasite substrain-dependent. Susceptibility of MyD88(-/-) mice infected with L. major IR75 is a consequence of Th2 immune-deviation, whereas susceptibility of MyD88(-/-) mice to infection with L. major LV39 resulted from an impaired Th1 response. Depletion of regulatory T cells (Treg) partially restored IFN-gamma secretion and the Th1 immune response in MyD88(-/-) mice infected with L. major LV39, demonstrating a role of Treg activity in the development of an impaired Th1 response in these mice.

Impact of the phosphatidylinositide 3-kinase signaling pathway on the cardioprotection induced by intermittent hypoxia.

BACKGROUND: Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels. METHODS: Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 µg/kg ip) 15 min before LAD. RESULTS: We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size. CONCLUSION: We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.