Identification of all steady states in large networks by logical analysis

The goal of generalized logical analysis is to model complex biological systems, especially so-called regulatory systems, such as genetic networks. This theory is mainly characterized by its capacity to find all the steady states of a given system and the functional positive and negative circuits, which generate multistationarity and a cycle in the state sequence graph, respectively. So far, this has been achieved by exhaustive enumeration, which severely limits the size of the systems that can be analysed. In this paper, we introduce a mathematical function, called image function, which allows the calculation of the value of the logical parameter associated with a logical variable depending on the state of the system. Thus the state table of the system is represented analytically. We then show how all steady states can be derived as solutions to a system of steady-state equations. Constraint programming, a recent method for solving constraint satisfaction problems, is applied for that purpose. To illustrate the potential of our approach, we present results from computer experiments carried out on very large randomly-generated systems (graphs) with hundreds, or even thousands, of interacting components, and show that these systems can be solved using moderate computing time. Moreover, we illustrate the approach through two published applications, one of which concerns the computation times of all steady states for a large genetic network.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis

Introduction

The microenvironment surrounding inflamed synovium leads to the activation of fibroblast-like synoviocytes (FLSs), which are important contributors to cartilage destruction in rheumatoid arthritic (RA) joints. Transglutaminase 2 (TG2), an enzyme involved in extracellular matrix (ECM) cross-linking and remodeling, is activated by inflammatory signals. This study was undertaken to assess the potential contribution of TG2 to FLS-induced cartilage degradation.

Methods

Transglutaminase (TGase) activity and collagen degradation were assessed with the immunohistochemistry of control, collagen-induced arthritic (CIA) or TG2 knockdown (shRNA)-treated joint tissues. TGase activity in control (C-FLS) and arthritic (A-FLS) rat FLSs was measured by in situ 5-(biotinamido)-pentylamine incorporation. Invadopodia formation and functions were measured in rat FLSs and cells from normal (control; C-FLS) and RA patients (RA-FLS) by in situ ECM degradation. Immunoblotting, enzyme-linked immunosorbent assay (ELISA), and p3TP-Lux reporter assays were used to assess transforming growth factor-β (TGF-β) production and activation.

Results

TG2 and TGase activity were associated with cartilage degradation in CIA joints. In contrast, TGase activity and cartilage degradation were reduced in joints by TG2 knockdown. A-FLSs displayed higher TGase activity and TG2 expression in ECM than did C-FLSs. TG2 knockdown or TGase inhibition resulted in reduced invadopodia formation in rat and human arthritic FLSs. In contrast, increased invadopodia formation was noted in response to TGase activity induced by TGF-β, dithiothreitol (DTT), or TG2 overexpression. TG2-induced increases in invadopodia formation were blocked by TGF-β neutralization or inhibition of TGF-βR1.

Conclusions

TG2, through its TGase activity, is required for ECM degradation in arthritic FLS and CIA joints. Our findings provide a potential target to prevent cartilage degradation in RA.     

Biosynthesis and metabolism of testosterone by sertoli cell-enriched seminiferous tubules

Sertoli cell-enriched tubules isolated from rats which had been treated with 1,4-dimethyl sulfonyloxybutane were incubated with either [¹⁴C] progesterone or [¹⁴C] testosterone for 2 hours. Tubules of normal rats and fragments of Sertoli cell-enriched testes were incubated under the same conditions. Sertoli cell-enriched tubules converted progesterone to 20α-dihydroprogesterone, 17α-hydroxyprogesterone, androstenedione and testosterone. The major metabolite was 20α-dihydroprogesterone. The percentage conversion of progesterone into testosterone corresponded to a production of 10–20 ng testosterone. Sertoli cell-enriched tubules converted testosterone to dihydrotestosterone, androstenedione, 3α-androstanediol and 3β-androstanediol. Under our experimental conditions, dihydrotestosterone was the major 5α-reduced metabolite. Normal tubules converted progesterone and testosterone to the same metabolites as Sertoli cell-enriched tubules. Fragments of Sertoli cell-enriched testes were much more active than isolated tubules in metabolizing progesterone. They produced the same amounts of 5α-reduced metabolites of testosterone.     

An internal standard improves the reliability of transient expression studies in plant protoplasts

Transient expression of foreign genes introduced on a plasmid into isolated plant protoplasts is widely used to study the control of gene expression. Unfortunately, many experimental variables implicated in this technique are difficult or impossible to control, resulting in a disturbing degree of variability between otherwise identical experiments. We have studied the co-expression of two constitutively expressed genes located on the same plasmid. This has allowed us to identify the lot of plasmid DNA as an important source of variation, along with the protoplast lot. Plasmid DNA concentration was found to be of minor importance. Since the variation of expression level of the two genes was identical for the two genes in all experiments, we propose the use of an internal standard in all comparative transient expression studies, which allows the reduction of the variation between experiments by one order of magnitude.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase synthase - MS medium after Murashige and Skoog (1962) - MU methyl umbelliferone - NOS nopaline synthase - NPT II-neomycin phospho transferase - PEG polyethylene glycol - Tris tris-hydroxymethyl aminoethane     

Linguistic feature analysis for protein interaction extraction

Background  

The rapid growth of the amount of publicly available reports on biomedical experimental results has recently caused a boost of text mining approaches for protein interaction extraction. Most approaches rely implicitly or explicitly on linguistic, i.e., lexical and syntactic, data extracted from text. However, only few attempts have been made to evaluate the contribution of the different feature types. In this work, we contribute to this evaluation by studying the relative importance of deep syntactic features, i.e., grammatical relations, shallow syntactic features (part-of-speech information) and lexical features. For this purpose, we use a recently proposed approach that uses support vector machines with structured kernels.     

Early chloroplastic alterations analysed by optical coherence tomography during a harpin-induced hypersensitive response

The hypersensitive response has been mostly studied by molecular and biochemical methods after sample destruction. The development of imaging techniques allows the monitoring of physiological changes before any signs of cell death. Here, we follow the early steps of a hypersensitive-like response induced by the bacterial elicitor harpin in Nicotiana sp. We describe cytological modifications after inoculation of the harpin protein, using confocal fluorescence microscopy (CFM) and optical coherence tomography (OCT), an interferometric-based microscopy. The changes detected by CFM occurred 5 h after harpin infiltration and corresponded to a redistribution of the chloroplasts from the upper to the inner regions of the palisade mesophyll cells which could be related to a perturbation in the microtubule network. Using OCT, we were able to detect a decrease in chloroplast backscattered signal as early as 30 min after harpin infiltration. A simple physical model, which accounted for the structure and distribution of thylakoid membranes, suggested that this loss of scattering could be associated with a modification in the refractive index of the thylakoid membranes. Our OCT observations were correlated with a decrease in photosynthesis, emphasizing changes in chloroplast structure as one of the earliest hallmarks of plant hypersensitive cell death.     

Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection

Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

What is the role of autophagy in HIV-1 infection?

HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.