Identification of rat ventral tegmental area GABAergic neurons

The canonical two neuron model of opioid reward posits that mu opioid receptor (MOR) activation produces reward by disinhibiting midbrain ventral tegmental area (VTA) dopamine neurons through inhibition of local GABAergic interneurons. Although indirect evidence supports the neural circuit postulated by this model, its validity has been called into question by growing evidence for VTA neuronal heterogeneity and the recent demonstration that MOR agonists inhibit GABAergic terminals in the VTA arising from extrinsic neurons. In addition, VTA MOR reward can be dopamine-independent. To directly test the assumption that MOR activation directly inhibits local GABAergic neurons, we investigated the properties of rat VTA GABA neurons directly identified with either immunocytochemistry for GABA or GAD65/67, or in situ hybridization for GAD65/67 mRNA. Utilizing co-labeling with an antibody for the neural marker NeuN and in situ hybridization against GAD65/67, we found that 23±3% of VTA neurons are GAD65/67(+). In contrast to the assumptions of the two neuron model, VTA GABAergic neurons are heterogeneous, both physiologically and pharmacologically. Importantly, only 7/13 confirmed VTA GABA neurons were inhibited by the MOR selective agonist DAMGO. Interestingly, all confirmed VTA GABA neurons were insensitive to the GABA(B) receptor agonist baclofen (0/6 inhibited), while all confirmed dopamine neurons were inhibited (19/19). The heterogeneity of opioid responses we found in VTA GABAergic neurons, and the fact that GABA terminals arising from neurons outside the VTA are inhibited by MOR agonists, make further studies essential to determine the local circuit mechanisms underlying VTA MOR reward.     

Screening antimicrobial activity of various extracts of Artemisia dracunculus L

The antimicrobial activities of chloroform, acetone and two different concentrations of methanol extracts of Artemisia dracunculus L. were studied. These extracts were tested against nine bacteria and four yeasts strains by the disc diffusion method. The results indicated that the methanol extract of A. dracunculus is more effective against tested microorganisms than chloroform or acetone extracts. The chloroform and acetone extracts were inhibitory only towards Pseudomonas aeruginosa (ATCC 27853). While the methanol extract that was diluted with 10 ml distilled water showed inhibition zones against Shigella (RSHI), Listeria monocytogenes ATCC 7644, P. aeruginosa (ATCC 27853), the methanol extract that was diluted with 5 ml distilled water showed inhibition zones against two different strains of Escherichia coli (RSHI, ATCC 25922), Shigella (RSHI), L. monocytogenes (ATCC 7644), and P. aeruginosa ATCC 27853. The cells of microorganisms treated with plant extracts and normal microorganism cells were observed by scanning electron microscope. It was apparent that cells are damaged after treatment with A. dracunculus.     

Ecological change in dynamic environments: Accounting for temporal environmental variability in studies of ocean change biology

The environmental conditions in the ocean have long been considered relatively more stable through time compared to the conditions on land. Advances in sensing technologies, however, are increasingly revealing substantial fluctuations in abiotic factors over ecologically and evolutionarily relevant timescales in the ocean, leading to a growing recognition of the dynamism of the marine environment as well as new questions about how this dynamism may influence species' vulnerability to global environmental change. In some instances, the diurnal or seasonal variability in major environmental change drivers, such as temperature, pH and seawater carbonate chemistry, and dissolved oxygen, can exceed the changes expected with continued anthropogenic global change. While ocean global change biologists have begun to experimentally test how variability in environmental conditions mediates species' responses to changes in the mean, the extensive literature on species' adaptations to temporal variability in their environment and the implications of this variability for their evolutionary responses has not been well integrated into the field. Here, we review the physiological mechanisms underlying species' responses to changes in temperature, pCO₂/pH (and other carbonate parameters), and dissolved oxygen, and discuss what is known about behavioral, plastic, and evolutionary strategies for dealing with variable environments. In addition, we discuss how exposure to variability may influence species' responses to changes in the mean conditions and highlight key research needs for ocean global change biology.     

Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells

With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.     

RhoC Impacts the Metastatic Potential and Abundance of Breast Cancer Stem Cells

Cancer stem cells (CSCs) have been shown to promote tumorigenesis of many tumor types, including breast, although their relevance to cancer metastasis remains unclear. While subpopulations of CSCs required for metastasis have been identified, to date there are no known molecular regulators of breast CSC (BCSC) metastasis. Here we identify RhoC GTPase as an important regulator of BCSC metastasis, and present evidence suggesting that RhoC also modulates the frequency of BCSCs within a population. Using an orthotopic xenograft model of spontaneous metastasis we discover that RhoC is both necessary and sufficient to promote SUM149 and MCF-10A BCSC metastasis-often independent from primary tumor formation-and can even induce metastasis of non-BCSCs within these cell lines. The relationship between RhoC and BCSCs persists in breast cancer patients, as expression of RhoC and the BCSC marker ALDH1 are highly correlated in clinical specimens. These results suggest new avenues to combating the deadliest cells driving the most lethal stage of breast cancer progression.     

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA⁻ and three RNA⁺ strains [temperature-sensitive mutants unable (RNA⁻) and able (RNA⁺) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.     

In vitro depression of cellular immunity by Friend virus leukemic spleen cells.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.     

Cost-Effectiveness and Cost Thresholds of Generic and Brand Drugs in a National Chronic Hepatitis B Treatment Program in China

Chronic liver disease and liver cancer associated with chronic hepatitis B (CHB) are leading causes of death among adults in China. Although newborn hepatitis B immunization has successfully reduced the prevalence of CHB in children, about 100 million Chinese adults remain chronically infected. If left unmanaged, 15–25% will die from liver cancer or liver cirrhosis. Antiviral treatment is not necessary for all patients with CHB, but when it is indicated, good response to treatment would prevent disease progression and reduce disease mortality and morbidity, and costly complications. The aim of this study is to analyze the cost-effectiveness of generic and brand antiviral drugs for CHB treatment in China, and assessing various thresholds at which a highly potent, low resistance antiviral drug would be cost-saving and/or cost-effective to introduce in a national treatment program. We developed a Markov simulation model of disease progression using effectiveness and cost data from the medical literature. We measured life-time costs, quality adjusted life years (QALYs), incremental cost-effectiveness ratios (ICERs), and clinical outcomes. The no treatment strategy incurred the highest health care costs ($12,932-$25,293) per patient, and the worst health outcomes, compared to the antiviral treatment strategies. Monotherapy with either entecavir or tenofovir yielded the most QALYs (14.10–19.02) for both HBeAg-positive and negative patients, with or without cirrhosis. Threshold analysis showed entercavir or tenofovir treatment would be cost saving if the drug price is $32–75 (195–460 RMB) per month, highly cost-effective at $62–110 (379–670 RMB) per month and cost-effective at $63–120 (384–734 RMB) per month. This study can support policy decisions regarding the implementation of a national health program for chronic hepatitis B treatment in China at the population level.     

Cyclic hexapeptides related to somatostatin. Conformational analysis employing 1H-NMR and molecular dynamics

We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure-activity relationship of somatostatin. For most of the molecules examined, a beta II' turn about the D-tryptophan-lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and D-tryptophan extended away from the 18-membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the beta II' turn at the D-tryptophan-lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe-Pro dipeptide, is to stabilize the beta II' turn and help maintain the proper orientation of the biologically important side chains.