Multivariate Statistical Analysis Applied to an IL6 Signal Transduction Model in Hepatocytes

This paper introduces the application of linear multivariate statistical techniques, including partial least squares (PLS), canonical correlation analysis (CCA) and reduced rank regression (RRR), into the area of Systems Biology. This new approach aims to extract the important proteins embedded in complex signal transduction pathway models.The analysis is performed on a model of intracellular signalling along the janus-associated kinases/signal transducers and transcription factors (JAK/STAT) and mitogen activated protein kinases (MAPK) signal transduction pathways in interleukin-6 (IL6) stimulated hepatocytes, which produce signal transducer and activator of transcription factor 3 (STAT3).A region of redundancy within the MAPK pathway that does not affect the STAT3 transcription was identified using CCA. This is the core finding of this analysis and cannot be obtained by inspecting the model by eye. In addition, RRR was found to isolate terms that do not significantly contribute to changes in protein concentrations, while the application of PLS does not provide such a detailed picture by virtue of its construction.This analysis has a similar objective to conventional model reduction techniques with the advantage of maintaining the meaning of the states prior to and after the reduction process. A significant model reduction is performed, with a marginal loss in accuracy, offering a more concise model while maintaining the main influencing factors on the STAT3 transcription.The findings offer a deeper understanding of the reaction terms involved, confirm the relevance of several proteins to the production of Acute Phase Proteins and complement existing findings regarding cross-talk between the two signalling pathways.

A new social-family model for eating disorders: A European multicentre project using a case-control design

OBJECTIVE: To examine a new socio-family risk model of Eating Disorders (EDs) using path-analyses. METHOD: The sample comprised 1264 (ED patients = 653; Healthy Controls = 611) participants, recruited into a multicentre European project. Socio-family factors assessed included: perceived maternal and parental parenting styles, family, peer and media influences, and body dissatisfaction. Two types of path-analyses were run to assess the socio-family model: 1.) a multinomial logistic path-model including ED sub-types [Anorexia Nervosa-Restrictive (AN-R), AN-Binge-Purging (AN-BP), Bulimia Nervosa (BN) and EDNOS)] as the key polychotomous categorical outcome and 2.) a path-model assessing whether the socio-family model differed across ED sub-types and healthy controls using body dissatisfaction as the outcome variable. RESULTS: The first path-analyses suggested that family and media (but not peers) were directly and indirectly associated (through body dissatisfaction) with all ED sub-types. There was a weak effect of perceived parenting directly on ED sub-types and indirectly through family influences and body dissatisfaction. For the second path-analyses, the socio-family model varied substantially across ED sub-types. Family and media influences were related to body dissatisfaction in the EDNOS and control sample, whereas perceived abusive parenting was related to AN-BP and BN. DISCUSSION: This is the first study providing support for this new socio-family model, which differed across ED sub-types. This suggests that prevention and early intervention might need to be tailored to diagnosis-specific ED profiles.

DNA incisions stimulate genetic instability of triplet repeat sequences related to human hereditary neurological diseases

The molecular mechanisms responsible for the expansion and deletion of trinucleotide repeat sequences (TRS) are the focus of our studies. Several hereditary neurological diseases including Huntington's disease, myotonic dystrophy, and fragile X syndrome are associated with the instability of TRS. Using the well defined and controllable model system of Escherichia coli, the influences of three types of DNA incisions on genetic instability of CTG•CAG repeats were studied: DNA double-strand breaks (DSB), single-strand nicks, and single-strand gaps. The DNA incisions were generated in pUC19 derivatives by in vitro cleavage with restriction endonucleases. The cleaved DNA was then transformed into E. coli parental and mutant strains. Double-strand breaks induced deletions throughout the TRS region in an orientation dependent manner relative to the origin of replication. The extent of instability was enhanced by the repeat length and sequence (CTG•CAG vs. CGG•CCG). Mutations in recA and recBC increased deletions, mutations in recF stabilized the TRS, whereas mutations in ruvA had no effect. DSB were repaired by intramolecular recombination, versus an intermolecular gene conversion or crossover mechanism. 30 nt gaps formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nts did not induce expansions or deletions. Formation of this deletion product required the CTG•CAG repeats to be present in the single-stranded region and was stimulated by E. coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the DSB induced instabilities and formation of the 30 nucleotide deletion product. In addition to the in vitro creation of DSBs, several attempts to generate this incision in vivo with the use of EcoR I restriction modification systems were conducted. ^

Activation of the Wnt pathway plays a pathogenic role in diabetic retinopathy in humans and animal models

Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of beta-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of beta-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target.

Molecular models for shikimate pathway enzymes of Xylella fastidiosa

The Xylella fastidiosa is a bacterium that is the cause of citrus variegated chlorosis (CVC). The shikimate pathway is of pivotal importance for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Putative structural differences in the enzymes from the shikimate pathway, between the proteins of bacterial origin and those of plants, could be used for the development of a drug for the control of CVC. However, inhibitors for shikimate pathway enzymes should have high specificity for X. fastidiosa enzymes, since they are also present in plants. In order to pave the way for structural and functional efforts towards antimicrobial agent development, here we describe the molecular modeling of seven enzymes of the shikimate pathway of X. fastidiosa. The structural models of shikimate pathway enzymes, complexed with inhibitors, strongly indicate that the previously identified inhibitors may also inhibit the X. fastidiosa enzymes. (C) 2004 Elsevier B.V. All rights reserved.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Inhibition of p38 pathway leads to OA-like changes in a rat animal model

Objectives The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is involved in a variety of inflammatory responses, including cytokine generation, cell differentiation proliferation and apoptosis. Here, we examined the effects of systemic p38 MAPK inhibition on cartilage cells and osteoarthritis (OA) disease progression by both in vitro and in vivo approaches. Methods p38 kinase activity was evaluated in normal and OA cartilage cells by measuring the amount of phosphorylated protein. To examine the function of p38 signaling pathway in vitro, normal chondrocytes were isolated and differentiated in the presence or absence of p38 inhibitor; SB203580 and analysed for chondrogenic phenotype. Effect of systemic p38 MAPK inhibition in normal and OA (induced by menisectomy) rats were analysed by treating animals with vehicle alone (DMS0) or p38 inhibitor (SB203580). Damage to the femur and tibial plateau was evaluated by modified Mankin score, histology and immunohistochemistry. Results Our in vitro studies have revealed that a down-regulation of chondrogenic and increase of hypertrophic gene expression occurs in the normal chondrocytes, when p38 is neutralized by a pharmacological inhibitor. We further observed that the basal levels of p38 phosphorylation were decreased in OA chondrocytes compared with normal chondrocytes. These findings together indicate the importance of this pathway in the regulation of cartilage physiology and its relevance to OA pathogenesis. At in vivo level, systematic administration of a specific p38 MAPK inhibitor, SB203580, continuously for over a month led to a significant loss of proteoglycan; aggrecan and cartilage thickness. On the other hand, SB203580 treated normal rats showed a significant increase in TUNEL positive cells, cartilage hypertrophy markers such as Type 10 collagen, Runt-related transcription factor and Matrix metalloproteinase-13 and substantially induced OA like phenotypic changes in the normal rats. In addition, menisectomy induced OA rat models that were treated with p38 inhibitor showed aggravation of cartilage damage. Conclusions In summary, this study has provided evidence that the component of the p38 MAPK pathway is important to maintain the cartilage health and its inhibition can lead to severe cartilage degenerative changes. The observations in this study highlight the possibility of using activators of the p38 pathway as an alternative approach in the treatment of OA.

Stochastic models for regulatory networks of the genetic toggle switch

Bistability arises within a wide range of biological systems from the λ phage switch in bacteria to cellular signal transduction pathways in mammalian cells. Changes in regulatory mechanisms may result in genetic switching in a bistable system. Recently, more and more experimental evidence in the form of bimodal population distributions indicates that noise plays a very important role in the switching of bistable systems. Although deterministic models have been used for studying the existence of bistability properties under various system conditions, these models cannot realize cell-to-cell fluctuations in genetic switching. However, there is a lag in the development of stochastic models for studying the impact of noise in bistable systems because of the lack of detailed knowledge of biochemical reactions, kinetic rates, and molecular numbers. In this work, we develop a previously undescribed general technique for developing quantitative stochastic models for large-scale genetic regulatory networks by introducing Poisson random variables into deterministic models described by ordinary differential equations. Two stochastic models have been proposed for the genetic toggle switch interfaced with either the SOS signaling pathway or a quorum-sensing signaling pathway, and we have successfully realized experimental results showing bimodal population distributions. Because the introduced stochastic models are based on widely used ordinary differential equation models, the success of this work suggests that this approach is a very promising one for studying noise in large-scale genetic regulatory networks.

Enhancing teacher education : the School-Community Intergrated Learning (SCIL) pathway

National Australian reviews advocate exploring new models for preservice teacher education. This study investigates the outcomes of the School-Community Integrated Learning (SCIL) pathway as a model for advancing preservice teachers’ understandings of teaching. Thirty-two final-year preservice teachers were surveyed with extended written responses on how the SCIL pathway advanced their understandings of teaching. Results indicated 100% agreement on 6 of the 27 survey items. Indeed, 78% or more preservice teachers agreed that they had a range of experiences across the five categories (i.e., personal-professional skill development, understandings of system requirements, teaching practices, student behaviour and reflective practices). Extended responses suggested they had developed understandings around setting up classrooms, whole school planning processes with professional development, the allocation of teacher responsibilities (e.g., playground duties), parent-teacher interviews, diagnostic testing for literacy and numeracy, commencing running records of students’ assessment results, and the development of relationships (students, teachers and parents). Although a longitudinal study is required to determine long-term effects, the SCIL pathway may be viewed as a positive step towards preparing final-year preservice teachers for their first year as fully-fledged teachers.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244)

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

Signaling pathway genes for blood pressure, folate and cholesterol levels among hypertensives : an epistasis analysis

Irregular atrial pressure, defective folate and cholesterol metabolism contribute to the pathogenesis of hypertension. However, little is known about the combined roles of the methylenetetrahydrofolate reductase (MTHFR), apolipoprotein-E (ApoE) and angiotensin-converting enzyme (ACE) genes, which are involved in metabolism and homeostasis. The objective of this study is to investigate the association of the MTHFR 677 C>T and 1298A>C, ACE insertion–deletion (I/D) and ApoE genetic polymorphisms with hypertension and to further explore the epistasis interactions that are involved in these mechanisms. A total of 594 subjects, including 348 normotensive and 246 hypertensive ischemic stroke subjects were recruited. The MTHFR 677 C>T and 1298A>C, ACE I/D and ApoEpolymorphisms were genotyped and the epistasis interaction were analyzed. The MTHFR 677 C>T and ApoE polymorphisms demonstrated significant associations with susceptibility to hypertension in multiple logistic regression models, multifactor dimensionality reduction and a classification and regression tree. In addition, the logistic regression model demonstrated that significant interactions between the ApoE E3E3, E2E4, E2E2 and MTHFR 677 C>T polymorphisms existed. In conclusion, the results of this epistasis study indicated significant association between the ApoE and MTHFR polymorphisms and hypertension.

N-ethyl-N-nitrosourea (ENU) induced mutations within the Klotho gene lead to ectopic calcification and reduced lifespan in mouse models

Ectopic calcification (EC), which is the pathological deposition of calcium and phosphate in extra-skeletal tissues, may be associated with hypercalcaemic and hyperphosphataemic disorders, or it may occur in the absence of metabolic abnormalities. In addition, EC may be inherited as part of several monogenic disorders and studies of these have provided valuable insights into the metabolic pathways regulating mineral metabolism. For example, studies of tumoural calcinosis, a disorder characterised by hyperphosphataemia and progressive EC, have revealed mutations of fibroblast growth factor 23 (FGF23), polypeptide N-acetyl galactosaminyltransferase 3 (GALNT3) and klotho (KL), which are all part of a phosphate-regulating pathway. However, such studies in humans are limited by the lack of available large families with EC, and to facilitate such studies we assessed the progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for EC. This identified two mutants with autosomal recessive forms of EC, and reduced lifespan, designated Ecalc1 and Ecalc2. Genetic mapping localized the Ecalc1 and Ecalc2 loci to a 11.0 Mb region on chromosome 5 that contained the klotho gene (Kl), and DNA sequence analysis identified nonsense (Gln203Stop) and missense (Ile604Asn) Kl mutations in Ecalc1 and Ecalc2 mice, respectively. The Gln203Stop mutation, located in KL1 domain, was severely hypomorphic and led to a 17-fold reduction of renal Kl expression. The Ile604Asn mutation, located in KL2 domain, was predicted to impair klotho protein stability and in vitro expression studies in COS-7 cells revealed endoplasmic reticulum retention of the Ile604Asn mutant. Further phenotype studies undertaken in Ecalc1 (kl203X/203X) mice demonstrated elevations in plasma concentrations of phosphate, FGF23 and 1,25-dihydroxyvitamin D. Thus, two allelic variants of Kl that develop EC and represent mouse models for tumoural calcinosis have been established. © 2015 Esapa et al.