Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.     

Achillea ligustica: composition and antimicrobial activity of essential oils from the leaves, flowers and some pure constituents

The composition of the essential oils obtained from the leaves and the flowers of Achillea ligustica (Asteraceae) growing in Sicily has been studied. The main constituents of the leaves were 4-terpineol (19.3%), carvone (8.9%), γ-terpinene (7.2%) and β-phellandrene (6.8%). 4-terpineol (12.0%), carvone (10.0%), and β-phellandrene (5.4%), along with linalool (20.4%) and cedrol (4.3%) were detected in the flower’s oil. Furthermore, the antimicrobial activity of the essential oils and of some of the main constituents were assayed on bacteria and fungi. In memory of Prof. Ivano Morelli (1940–2005)     

Role of ischemia and deformation in the onset of compression-induced deep tissue injury: MRI-based studies in a rat model.

A rat model was used to distinguish between the different factors that contribute to muscle tissue damage related to deep pressure ulcers that develop after compressive loading. The separate and combined effects of ischemia and deformation were studied. Loading was applied to the hindlimb of rats for 2 h. Muscle tissue was examined using MR imaging (MRI) and histology. An MR-compatible loading device allowed simultaneous loading and measurement of tissue status. Two separate loading protocols incorporated uniaxial loading, resulting in tissue compression and ischemic loading. Uniaxial loading was applied to the tibialis anterior by means of an indenter, and ischemic loading was accomplished with an inflatable tourniquet. Deformation of the muscle tissue during uniaxial loading was measured using MR tagging. Compression of the tissues for 2 h led to increased T2 values, which were correlated to necrotic regions in the tibialis anterior. Perfusion measurements, by means of contrast-enhanced MRI, indicated a large ischemic region during indentation. Pure ischemic loading for 2 h led to reversible tissue changes. From the MR-tagging experiments, local strain fields were calculated. A 4.5-mm deformation, corresponding to a surface pressure of 150 kPa, resulted in maximum shear strain up to 1.0. There was a good correlation between the location of damage and the location of high shear strain. It was concluded that the large deformations, in conjunction with ischemia, provided the main trigger for irreversible muscle damage.     

Sheoak woodlands: a newly identified habitat for western ringtail possums

Although critically endangered western ringtail possums (Pseudocheirus occidentalis) have been recorded from a variety of vegetation types in southwestern Australia, the extent to which many of these vegetation types are occupied by the species remains unknown. We conducted spotlight surveys for western ringtail possums between March and April 2018 in Albany, southwestern Australia, to determine the species’ occupancy in 2 vegetation types. Using occupancy models, we demonstrated that sheoak (Allocasuarina fraseriana) woodlands, previously unrecognized as providing western ringtail possum habitat, support the species, although the median probability of their occupancy was lower than in marri (Corymbia calophylla) and eucalypt (Eucalyptus marginata and E. staeri) woodlands. Use of trees and other habitat components by western ringtail possums varied within and between vegetation types. Sheoak woodlands are likely critical for western ringtail possums and given the apparent flexibility in vegetation types used by the species, investigation of the potential value of other vegetation types for this species has conservation merit. © 2019 The Wildlife Society.     

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type–specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP

Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins.     

Bacterial toxins: useful for studying G-proteins implicated in the mechanism of exocytosis in neuroendocrine cells

In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitment and priming of secretory granules, their docking to the exocytotic sites, and the subsequent fusion of granules with the plasma membrane leading to the release of secretory products into the extracellular space. Using bacterial toxins which specially inactivate subsets of G proteins, we were able to demonstrate that both trimeric and monomeric G proteins directly control the late stages of exocytosis in chromaffin cells. Indeed, in secretagogue-stimulated chromaffin cells, the subplasmalemmal actin cytoskeleton undergoes a specific reorganization that is a prerequisite for exocytosis. Our results suggest that a granule-bound trimeric Go protein controls the actin network surrounding secretory granules through a pathway involving the GTPase RhoA and a downstream phosphatidylinositol 4-kinase. Furthermore, the GTPase Cdc42 plays a active role in exocytosis, most likely by providing specific actin structures to the late docking and/or fusion steps. We propose that G proteins tightly control secretion in neuroendocrine cells by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis. Our data highlight the use of bacterial toxins, which proved to be powerful tools to dissect the exocytotic machinery at the molecular level.