Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

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.     

Cannabis, pain, and sleep: lessons from therapeutic clinical trials of Sativex, a cannabis-based medicine

Cannabis sativa L. has been utilized for treatment of pain and sleep disorders since ancient times. This review examines modern studies on effects of Delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on sleep. It goes on to report new information on the effects on sleep in the context of medical treatment of neuropathic pain and symptoms of multiple sclerosis, employing standardized oromucosal cannabis-based medicines containing primarily THC, CBD, or a 1 : 1 combination of the two (Sativex). Sleep-laboratory results indicate a mild activating effect of CBD, and slight residual sedation with THC-predominant extracts. Experience to date with Sativex in numerous Phase I-III studies in 2000 subjects with 1000 patient years of exposure demonstrate marked improvement in subjective sleep parameters in patients with a wide variety of pain conditions including multiple sclerosis, peripheral neuropathic pain, intractable cancer pain, and rheumatoid arthritis, with an acceptable adverse event profile. No tolerance to the benefit of Sativex on pain or sleep, nor need for dosage increases have been noted in safety extension studies of up to four years, wherein 40-50% of subjects attained good or very good sleep quality, a key source of disability in chronic pain syndromes that may contribute to patients' quality of life.     

Greater sage‐grouse respond positively to intensive post‐fire restoration treatments

Habitat loss is the most prevalent threat to biodiversity in North America. One of the most threatened landscapes in the United States is the sagebrush (Artemisia spp.) ecosystem, much of which has been fragmented or converted to non‐native grasslands via the cheatgrass‐fire cycle. Like many sagebrush obligates, greater sage‐grouse (Centrocercus urophasianus) depend upon sagebrush for food and cover and are affected by changes to this ecosystem. We investigated habitat selection by 28 male greater sage‐grouse during each of 3 years after a 113,000‐ha wildfire in a sagebrush steppe ecosystem in Idaho and Oregon. During the study period, seeding and herbicide treatments were applied for habitat restoration. We evaluated sage‐grouse responses to vegetation and post‐fire restoration treatments. Throughout the 3 years post‐fire, sage‐grouse avoided areas with high exotic annual grass cover but selected strongly for recovering sagebrush and moderately strongly for perennial grasses. By the third year post‐fire, they preferred high‐density sagebrush, especially in winter when sagebrush is the primary component of the sage‐grouse diet. Sage‐grouse preferred forb habitat immediately post‐fire, especially in summer, but this selection preference was less strong in later years. They also selected areas that were intensively treated with herbicide and seeded with sagebrush, grasses, and forbs, although these responses varied with time since treatment. Wildfire can have severe consequences for sagebrush‐obligate species due to loss of large sagebrush plants used for food and for protection from predators and thermal extremes. Our results show that management efforts, including herbicide application and seeding of plants, directed at controlling exotic annual grasses after a wildfire can positively affect habitat selection by sage‐grouse.     

Glucose-6-phosphatase catalytic subunit 2 negatively regulates glucose oxidation and insulin secretion in pancreatic β-cells

Elevated fasting blood glucose (FBG) is associated with increased risks of developing type 2 diabetes (T2D) and cardiovascular-associated mortality. G6PC2 is predominantly expressed in islets, encodes a glucose-6-phosphatase catalytic subunit that converts glucose-6-phosphate (G6P) to glucose, and has been linked with variations in FBG in genome-wide association studies. Deletion of G6pc2 in mice has been shown to lower FBG without affecting fasting plasma insulin levels in vivo. At 5 mM glucose, pancreatic islets from G6pc2 knockout (KO) mice exhibit no glucose cycling, increased glycolytic flux, and enhanced glucose-stimulated insulin secretion (GSIS). However, the broader effects of G6pc2 KO on β-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in βTC3 cells, a murine pancreatic β-cell line, to examine the role of G6pc2 in regulating glycolytic and mitochondrial fluxes. We found that deletion of G6pc2 led to ∼60% increases in glycolytic and citric acid cycle (CAC) fluxes at both 5 and 11 mM glucose concentrations. Furthermore, intracellular insulin content and GSIS were enhanced by approximately two-fold, along with increased cytosolic redox potential and reductive carboxylation flux. Normalization of fluxes relative to net glucose uptake revealed upregulation in two NADPH-producing pathways in the CAC. These results demonstrate that G6pc2 regulates GSIS by modulating not only glycolysis but also, independently, citric acid cycle activity in β-cells. Overall, our findings implicate G6PC2 as a potential therapeutic target for enhancing insulin secretion and lowering FBG, which could benefit individuals with prediabetes, T2D, and obesity.     

A new principle for rapid immunoassay of proteins based on in situ precipitate-enhanced ellipsometry

A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10-20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.     

Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay

The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2.     

Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins

Formation of synaptic connections requires alignment of neurotransmitter receptors on postsynaptic dendrites opposite matching transmitter release sites on presynaptic axons. beta-neurexins and neuroligins form a trans-synaptic link at glutamate synapses. We show here that neurexin alone is sufficient to induce glutamate postsynaptic differentiation in contacting dendrites. Surprisingly, neurexin also induces GABA postsynaptic differentiation. Conversely, neuroligins induce presynaptic differentiation in both glutamate and GABA axons. Whereas neuroligins-1, -3, and -4 localize to glutamate postsynaptic sites, neuroligin-2 localizes primarily to GABA synapses. Direct aggregation of neuroligins reveals a linkage of neuroligin-2 to GABA and glutamate postsynaptic proteins, but the other neuroligins only to glutamate postsynaptic proteins. Furthermore, mislocalized expression of neuroligin-2 disperses postsynaptic proteins and disrupts synaptic transmission. Our findings indicate that the neurexin-neuroligin link is a core component mediating both GABAergic and glutamatergic synaptogenesis, and differences in isoform localization and binding affinities may contribute to appropriate differentiation and specificity.