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.

Bioassay for detection of transgenic soybean seeds tolerant to glyphosate

Glyphosate is a systemic, nonselective, postemergence herbicide that inhibits growth of both weeds and crop plants. Once inside the plant, glyphosate interferes with biosynthesis of aromatic amino acids phenylalanine, tyrosine, and tryptophan, by inhibiting the activity of 5enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme of the shikimate pathway. The objective of this work was to develop a simple, effective and inexpensible method for identification of transgenic soybean tolerant to glyphosate. This technique consisted in germinating soybean seeds in filter paper moistened with 100 to 200 muM of glyphosate. Transgenic soybean seeds tolerant to glyphosate germinated normally in this solution and, between 7 and 10 days, started to develop a primary root system. However non-transgenic seeds stopped primary root growth and emission of secondary roots.

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.

Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background: Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria. Methodology/Principal Findings: We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells. Conclusion/Significance: Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.

Detecting the oxidative reactivity of nanoparticles: a new protocol for reducing artifacts

Understanding the oxidative reactivity of nanoparticles (NPs; <100 nm) could substantially contribute to explaining their toxicity. We attempted to refine the use of 2′7-dichlorodihydrofluorescein (DCFH) to characterize NP generation of reactive oxygen species (ROS). Several fluorescent probes have been applied to testing oxidative reactivity, but despite DCFH being one of the most popular for the detection of ROS, when it has been applied to NPs there have been an unexplainably wide variability in results. Without a uniform methodology, validating even robust results is impossible. This study, therefore, identified sources of conflicting results and investigated ways of reducing occurrence of artificial results. Existing techniques were tested and combined (using their most desirable features) to form a more reliable method for the measurement of NP reactivity in aqueous dispersions. We also investigated suitable sample ranges necessary to determine generation of ROS. Specifically, ultrafiltration and time-resolved scan absorbance spectra were used to study possible optical interference when using high sample concentrations. Robust results were achieved at a 5 µM DCFH working solution with 0.5 unit/mL horseradish peroxidase (HRP) dissolved in ethanol. Sonication in DCFH-HRP working solution provided more stable data with a relatively clean background. Optimal particle concentration depends on the type of NP and in general was in the µg/mL range. Major reasons for previously reported conflicting results due to interference were different experimental approaches and NP sample concentrations. The protocol presented here could form the basis of a standardized method for applying DCFH to detect generation of ROS by NPs.

Ubiquitin Ligases of the N-End Rule Pathway: Assessment of Mutations in UBR1 That Cause the Johanson-Blizzard Syndrome.

Background: Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.Methodology/Principal Findings: Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.Conclusions/Significance: These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.

Pathogenic role of basic calcium phosphate crystals in destructive arthropathies.

BACKGROUND: basic calcium phosphate (BCP) crystals are commonly found in osteoarthritis (OA) and are associated with cartilage destruction. BCP crystals induce in vitro catabolic responses with the production of metalloproteases and inflammatory cytokines such as interleukin-1 (IL-1). In vivo, IL-1 production induced by BCP crystals is both dependant and independent of NLRP3 inflammasome. We aimed to clarify 1/ the role of BCP crystals in cartilage destruction and 2/ the role of IL-1 and NLRP3 inflammasome in cartilage degradation related to BCP crystals. METHODOLOGY PRINCIPAL FINDINGS: synovial membranes isolated from OA knees were analysed by alizarin Red and FTIR. Pyrogen free BCP crystals were injected into right knees of WT, NLRP3 -/-, ASC -/-, IL-1α -/- and IL-1β-/- mice and PBS was injected into left knees. To assess the role of IL-1, WT mice were treated by intra-peritoneal injections of anakinra, the IL-1Ra recombinant protein, or PBS. Articular destruction was studied at d4, d17 and d30 assessing synovial inflammation, proteoglycan loss and chondrocyte apoptosis. BCP crystals were frequently found in OA synovial membranes including low grade OA. BCP crystals injected into murine knee joints provoked synovial inflammation characterized by synovial macrophage infiltration that persisted at day 30, cartilage degradation as evidenced by loss of proteoglycan staining by Safranin-O and concomitant expression of VDIPEN epitopes, and increased chondrocyte apoptosis. BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β. Similarly, treatment with anakinra did not prevent BCP crystal effects. In vitro, BCP crystals elicited enhanced transcription of matrix degrading and pro-inflammatory genes in macrophages. CONCLUSIONS SIGNIFICANCE: intra-articular BCP crystals can elicit synovial inflammation and cartilage degradation suggesting that BCP crystals have a direct pathogenic role in OA. The effects are independent of IL-1 and NLRP3 inflammasome.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Basic calcium phosphate crystals induce IL-1B secretion in vitro through activation of the Nalp3 inflammasome

Objectives: We tested the effects of the three forms of basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbonate-substituted apatite (CA) and hydroxyapatite (HA)) on monocytes and macrophages on IL-1β secretion. The requirement for the NALP3 inflammasome and TLR2 and TLR4 receptors in this acute response was analyzed.

Twofold cost of reproduction: an increase in parental effort leads to higher malarial parasitaemia and to a decrease in resistance to oxidative stress.

Parental effort is usually associated with high metabolism that could lead to an increase in the production of reactive oxidative species giving rise to oxidative stress. Since many antioxidants involved in the resistance to oxidative stress can also enhance immune function, an increase in parental effort may diminish the level of antioxidants otherwise involved in parasite resistance. In the present study, we performed brood size manipulation in a population of great tits (Parus major) to create different levels of parental effort. We measured resistance to oxidative stress and used a newly developed quantitative PCR assay to quantify malarial parasitaemia. We found that males with an enlarged brood had significantly higher level of malarial parasites and lower red blood cell resistance to free radicals than males rearing control and reduced broods. Brood size manipulation did not affect female parasitaemia, although females with an enlarged brood had lower red blood cell resistance than females with control and reduced broods. However, for both sexes, there was no relationship between the level of parasitaemia and resistance to oxidative stress, suggesting a twofold cost of reproduction. Our results thus suggest the presence of two proximate and independent mechanisms for the well-documented trade-off between current reproductive effort and parental survival.

Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

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]

Phosphate solubilization and synergism between P-solubilizing and arbuscular mycorrhizal fungi

The objective of this work was to evaluate the ability of several P-solubilizing fungi to solubilize aluminum phosphate and Araxá apatite as well as the synergism between the P-solubilizing fungus, PSF 7, and arbuscular mycorrhizal fungi to promote clover growth amended with aluminum phosphate. Two experiments were carried out, the first under laboratory conditions and the second in a controlled environmental chamber. In the first experiment, PSF 7, PSF 9, PSF 21 and PSF 22 isolates plus control were incubated in liquid medium at 28ºC for eight days. On the 2nd, 4th and 8th day of incubation, pH and soluble P were determined. In the second experiment, clover was sowed in plastic pots containing 300 g of sterilized substrate amended with aluminum phosphate, 3 g L-1, in presence and absence of PSF 7 isolate and arbuscular mycorrhizal fungi. A completely randomized design, in factorial outline 2x2 (presence and absence of PSF 7 and arbuscular mycorrhizal fungi) and five replicates were used. In the first experiment, higher P content was detected in the medium containing aluminum phosphate. PSF 7 is the best fungi isolate which increases aluminum solubilization with major tolerance to Al3+. Clover growth was stimulated by presence of PSF 7 and arbuscular mycorrhizal fungi. There is synergism between microorganisms utilized to improve plant nutrition.