Quantification of Neurite Degeneration with Enhanced Accuracy and Efficiency in an In Vitro Model of Parkinson's Disease.

Neurite degeneration is associated with early stages of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease (PD), and amyotrophic lateral sclerosis. One method that is commonly used to analyze neurite degeneration involves calculation of a Degeneration Index (DI) following utilization of the Analyze Particles tool of ImageJ to detect neurite fragments in micrographs of cultured cells. However, DI analyses are prone to several types of measurement error, can be time consuming to perform, and are limited in application. Here, we describe an improved method for performing DI analyses. Accuracy of measurements was enhanced through modification of selection criteria for detecting neurite fragments, removal of image artifacts and non-neurite materials from images, and optimization of image contrast. Such enhancements were implemented into an ImageJ macro that enables rapid and fully automated DI analysis of multiple images. The macro features operations for automated removal of cell bodies from micrographs, thus expanding the application of DI analyses to use in experiments involving dissociated cultures. We present experimental findings supporting that, compared with the conventional method, the enhanced analysis method yields measurements with increased accuracy and requires significantly less time to perform. Furthermore, we demonstrate the utility of the method to investigate neurite degeneration in a cell culture model of PD by conducting an experiment revealing the effects of c-Jun N-terminal kinase (JNK) on neurite degeneration induced by oxidative stress in human mesencephalic cells. This improved analysis method may be used to gain novel insight into factors underlying neurite degeneration and the progression of neurodegenerative disorders.

Circulating adrenocorticotropic hormone levels, lactate levels, hematocrit and osmolality in relation to capture stress in Atlantic sharpnose sharks, Rhizoprionodon terraenovae.

Incidental capture of sharks during commercial and recreational fishing is of major conservation concern because of the potential effects it can have on physiological stress responses and survival. Endocrine aspects of the stress response are, however, poorly understood in elasmobranchs because of difficulties in measuring the primary glucocorticoid (1α-hydroxycorticosterone). Here, we combined measures of plasma adrenocorticotropic hormone (ACTH), the highly conserved pituitary hormone responsible for stimulating the release of adrenal/interrenal glucocorticoids, with measures of plasma lactate, osmolality, hematocrit, and behavior to gain a greater understanding of the capture stress response in Atlantic Sharpnose sharks, Rhizoprionodon terraenovae. Individuals were subject to a non-repeated measures blood sampling protocol in which blood samples were obtained following exposure to capture stress for <3 min (designated baseline), and 15, 30, 45 and 60 min, after which behavior was categorized during release. Results revealed that ACTH was significantly higher at 15, 30, 45, and 60 min than at baseline. Lactate levels were highest at 45 and 60 min whereas osmolality and hematocrit did not differ significantly among the sampling periods. Lactate was the only variable to significantly predict the shark's behavior upon release with higher lactate levels correlating with sluggish behavior. Measurements of stress indicators are important in understanding the effects of capture on shark populations, which has been implicated in population declines.

Barriers to management of tree diseases: Framing perspectives of pinewood managers around Dothistroma Needle Blight.

Forest Managers in the UK and elsewhere are facing new threats such as climate change and novel pests and diseases. Strategies seek to coordinate and steer appropriate responses through raising awareness and encouraging action but little is known about how individual managers respond to disease threats. We studied how managers have responded to the threat of Dothistroma Needle Blight (DNB), a disease which can affect growth and cause mortality of many pine species, and the key frames influencing their responses. Frames involve values and beliefs and allow people to make sense of, and interpret, events, experiences or issues. Interviews revealed broad awareness of the disease and threat it poses, but also high levels of inaction. Lack of action was associated with several framings grouped around causes and perceived severity of DNB, locating responsibility for prevention and management, mistrust/scepticism of advice and uncertainties over the future impact. These framings need to be considered as strategies are refined and new knowledge of disease behaviour is communicated to those from whom action is required.

Interactions between PTEN and the c-Met pathway in glioblastoma and implications for therapy.

The tyrosine kinase receptor c-Met and its ligand hepatocyte growth factor (HGF) are frequently overexpressed and the tumor suppressor PTEN is often mutated in glioblastoma. Because PTEN can interact with c-Met-dependent signaling, we studied the effects of PTEN on c-Met-induced malignancy and associated molecular events and assessed the potential therapeutic value of combining PTEN restoration approaches with HGF/c-Met inhibition. We studied the effects of c-Met activation on cell proliferation, cell cycle progression, cell migration, cell invasion, and associated molecular events in the settings of restored or inhibited PTEN expression in glioblastoma cells. We also assessed the experimental therapeutic effects of combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition. PTEN significantly inhibited HGF-induced proliferation, cell cycle progression, migration, and invasion of glioblastoma cells. PTEN attenuated HGF-induced changes of signal transduction proteins Akt, GSK-3, JNK, and mTOR as well as cell cycle regulatory proteins p27, cyclin E, and E2F-1. Combining PTEN restoration to PTEN-null glioblastoma cells with c-Met and HGF inhibition additively inhibited tumor cell proliferation and cell cycle progression. Similarly, combining a monoclonal anti-HGF antibody (L2G7) with the mTOR inhibitor rapamycin had additive inhibitory effects on glioblastoma cell proliferation. Systemic in vivo delivery of L2G7 and PTEN restoration as well as systemic in vivo deliveries of L2G7 and rapamycin additively inhibited intracranial glioma xenograft growth. These preclinical studies show for the first time that PTEN loss amplifies c-Met-induced glioblastoma malignancy and suggest that combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition is worth testing in a clinical setting.

PTEN has tumor-promoting properties in the setting of gain-of-function p53 mutations.

We show, for the first time, that the tumor suppressor PTEN can have tumor-promoting properties. We show that PTEN acquires these unexpected properties by enhancing gain-of-function mutant p53 (mut-p53) protein levels. We find that PTEN restoration to cells harboring mut-p53 leads to induction of G(1)-S cell cycle progression and cell proliferation and to inhibition of cell death. Conversely, PTEN inhibition in cells expressing wild-type PTEN and mut-p53 leads to inhibition of cell proliferation and inhibition of in vivo tumor growth. We show the dependency of the tumor-promoting effects of PTEN on mut-p53 by showing that knockdown of mut-p53 expression inhibits or reverses the tumor-promoting effects of PTEN. Mechanistically, we show that PTEN expression enhances mut-p53 protein levels via inhibition of mut-p53 degradation by Mdm2 and possibly also via direct protein binding. These findings describe a novel function of PTEN and have important implications for experimental and therapeutic strategies that aim at manipulating PTEN or p53 in human tumors. They suggest that the mutational status of PTEN and p53 should be considered to achieve favorable therapeutic outcomes. The findings also provide an explanation for the low frequency of simultaneous mutations of PTEN and p53 in human cancer.

12/15 lipoxygenase mediates monocyte adhesion to aortic endothelium in apolipoprotein E-deficient mice through activation of RhoA and NF-kappaB.

OBJECTIVE: 12/15 lipoxygenase (12/15LO) has been implicated as a mediator of inflammation and atherosclerosis. In the current study, we identified mechanisms through which 12/15LO mediates monocyte:endothelial interactions in vivo in apolipoprotein E-deficient mice (apoEKO), a well-characterized mouse model of atherosclerosis. METHODS AND RESULTS: In apoEKO mice that are also deficient in 12/15LO (doubleKO), monocyte adhesion to aorta in vivo was reduced by 95% in doubleKO mice compared with apoEKO mice. Inhibition of 12/15LO in apoEKO mice in vivo using CDC (Cinnamyl-3,4-Dihydroxy-a-Cyanocinnamate) prevented monocyte adhesion to aortic endothelium in apoEKO mice. Aortic endothelium of apoEKO mice had significant activation of rhoA compared with doubleKO aortic endothelium. Further, apoEKO aorta displayed significant activation of NF-kappaB. DoubleKO aorta displayed little nuclear localization of NF-kappaB. Finally, we found significant upregulation of intercellular adhesion molecule-1 (ICAM-1) on apoEKO aortic endothelium compared with doubleKO endothelium. Inhibition of rhoA and PKCalpha significantly reduced NF-kappaB activation, ICAM-1 induction, and monocyte adhesion to aorta. CONCLUSIONS: We conclude that 12/15LO products activate endothelial rhoA and PKCalpha. Activation of rhoA and PKCalpha cause activation and translocation of NF-kappaB to the nucleus, which, in turn, results in induction of ICAM-1. Induction of ICAM-1 on aortic endothelium stimulates monocyte:endothelial adhesion in vivo in apoEKO mice.