Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1.

BACKGROUND: Oncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response. METHODS: To sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R-). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R- mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor nude mouse xenograft models and systemic anti-tumor effects tested using dual flank syngeneic 4434 (melanoma), A20 (lymphoma) mouse tumor models alone and in combination with a murine anti-PD1 antibody, and 9 L (gliosarcoma) tumors in rats. RESULTS: The twenty nine clinical strains of HSV-1 isolated and tested demonstrated a broad range of tumor cell killing abilities allowing the most potent strain to be identified which was then used for further development. Oncolytic ability was demonstrated to be further augmented by the expression of GALV-GP-R- in a range of tumor cell lines in vitro and in mouse xenograft models in nude mice. The expression of GALV-GP-R- was also demonstrated to lead to enhanced immunogenic cell death in vitro as confirmed by the increased release of HMGB1 and ATP and increased levels of calreticulin on the cell surface. Experiments using the rat 9 L syngeneic tumor model demonstrated that GALV-GP-R- expression increased abscopal uninjected (anenestic) tumor responses and data using mouse 4434 tumors demonstrated that virus treatment increased CD8+ T cell levels both in the injected and uninjected tumor, and also led to increased expression of PD-L1. A combination study using varying doses of a virus expressing GALV-GP-R- and mGM-CSF and an anti-murine PD1 antibody showed enhanced anti-tumor effects with the combination which was most evident at low virus doses, and also lead to immunological memory. Finally, treatment of mice with derivatives of this virus which additionally expressed anti-mCTLA-4, mCD40L, m4-1BBL, or mOX40L demonstrated enhanced activity, particularly in uninjected tumors. CONCLUSION: The new HSV-1 based platform described provides a potent and versatile approach to developing new oncolytic immunotherapies for clinical use. Each of the modifications employed was demonstrated to aid in optimizing the potential of the virus to both directly kill tumors and to lead to systemic therapeutic benefit. For clinical use, these viruses are expected to be most effective in combination with other anti-cancer agents, in particular PD1/L1-targeted immune checkpoint blockade. The first virus from this program (expressing GALV-GP-R- and hGM-CSF) has entered clinical development alone and in combination with anti-PD1 therapy in a number of tumor types (NCT03767348).

Substrate Specificity and Engineering of Mevalonate 5-Phosphate Decarboxylase.

A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)-mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)-MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomic-resolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)-MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically, plants, which universally encode a structural and functional homologue of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway.

Contribution of isopentenyl phosphate to plant terpenoid metabolism.

Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases. Increasing metabolically available IP by overexpression of a bacterial phosphomevalonate decarboxylase (MPD) in Nicotiana tabacum resulted in significant enhancement in both monoterpene and sesquiterpene production. These results indicate that perturbing IP metabolism results in measurable changes in terpene products derived from both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Moreover, the unpredicted peroxisomal localization of bacterial MPD led us to discover that the step catalysed by phosphomevalonate kinase (PMK) imposes a hidden constraint on flux through the classical MVA pathway. These complementary findings fundamentally alter conventional views of metabolic regulation of terpenoid metabolism in plants and provide new metabolic engineering targets for the production of high-value terpenes in plants.

Sleep Disturbances in OEF/OIF/OND Veterans: Associations with PTSD, Personality, and Coping.

STUDY OBJECTIVES: Sleep disturbances are well documented in relation to trauma exposure and posttraumatic stress disorder (PTSD), but correlates of such disturbances remain understudied in veteran populations. We conducted a preliminary study of sleep disturbances in Operation Enduring Freedom, Operation Iraqi Freedom, and Operation New Dawn veterans (n = 133; mean [standard deviation] age = 29.8 [4.7] y). METHODS: Veterans were assigned to one of three groups based on responses to the Clinician Administered PTSD Scale: control (no trauma-exposure [TE] or PTSD), TE, and PTSD. Sleep disturbance was assessed using the Pittsburgh Sleep Quality Index (PSQI). Measures of resilience, trauma load, personality, coping, alcohol use, and mild traumatic brain injury were also assessed via self-report. RESULTS: The PTSD group had significantly more disturbed sleep (PSQI global score mean = 8.94, standard deviation = 3.12) than control (mean = 5.27, standard deviation = 3.23) and TE (mean = 5.34, standard deviation = 3.17) groups, but there were no differences between TE and control. The same pattern emerged across most PSQI subscales. Results of linear regression analyses indicated that current smoking, Army (versus other military branches), neuroticism, and using substances to cope were all significant correlates of higher sleep disturbance, whereas post-deployment social support was associated with less sleep disturbance. However, when combined together into a model with PTSD status, only neuroticism and substance use coping remained significant as predictors of more disturbed sleep. CONCLUSIONS: These initial findings suggest that TE itself may not be an independent risk factor for disturbed sleep in veterans, and that neurotic personality and a tendency to cope by using substances may partially explain sleep disturbance, above and beyond a diagnosis of PTSD.

Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants.

Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.

An online alcohol and oral health curriculum for dental students.

Regular heavy alcohol use can cause or worsen several oral health disorders and is associated with complications during and after dental procedures. Dental student education should provide detailed knowledge of these issues together with skills needed to detect and counsel patients with unhealthy drinking patterns. This project was designed to develop and evaluate a five-module, online program to teach dental students about alcohol and oral health, systemic and oral biological effects of heavy drinking, required changes to treatment protocols for heavy drinkers, reliable methods of alcohol screening, and ways to provide heavy drinkers with brief interventions. Results indicated that the online program resulted in significant changes in knowledge, attitudes, and behavior. This online format could easily be incorporated into an already crowded dental school curriculum, with students learning the material at their own pace and in their own available time.

Discovery of a metabolic alternative to the classical mevalonate pathway.

Eukarya, Archaea, and some Bacteria encode all or part of the essential mevalonate (MVA) metabolic pathway clinically modulated using statins. Curiously, two components of the MVA pathway are often absent from archaeal genomes. The search for these missing elements led to the discovery of isopentenyl phosphate kinase (IPK), one of two activities necessary to furnish the universal five-carbon isoprenoid building block, isopentenyl diphosphate (IPP). Unexpectedly, we now report functional IPKs also exist in Bacteria and Eukarya. Furthermore, amongst a subset of species within the bacterial phylum Chloroflexi, we identified a new enzyme catalyzing the missing decarboxylative step of the putative alternative MVA pathway. These results demonstrate, for the first time, a functioning alternative MVA pathway. Key to this pathway is the catalytic actions of a newly uncovered enzyme, mevalonate phosphate decarboxylase (MPD) and IPK. Together, these two discoveries suggest that unforeseen variation in isoprenoid metabolism may be widespread in nature. DOI: http://dx.doi.org/10.7554/eLife.00672.001.

GnRH receptor antagonists for prostate cancer.

GnRH receptor antagonists can reduce testosterone levels without the adverse reactions caused by other drugs used to treat prostate cancer. These drugs also offer hope for prolonged control of metastasis.

Client attitudes toward alcohol use self-report.

Because psychiatric illnesses and problematic alcohol use frequently co-occur and heavy alcohol use can exacerbate depression and anxiety, mental health clinicians should perform alcohol-use screenings. The aim of this study was to determine if psychiatric patients would be accepting of their mental health clinician screening them for heavy alcohol use. Using a written survey, patients rated their levels of agreement with 9 statements regarding opinions about alcohol screening by their mental-health providers. They also completed the Alcohol Use Disorders Identification Test-C (AUDIT-C), a screening instrument for heavy alcohol use. One hundred fifty-four patients were surveyed in 2 psychiatric outpatient clinics. Nearly 40% screened positively for heavy alcohol use on the AUDIT-C. Nearly 8 out of 10 psychiatric patients were in favor of being screened for alcohol use by either self-report or biomarkers, independent of AUDIT-C status and gender. Thus, mental health clinicians should not be deterred from alcohol screening by perceived negative attitudes from patients.

Pathway profiling in Mycobacterium tuberculosis: elucidation of cholesterol-derived catabolite and enzymes that catalyze its metabolism.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-(14)C]cholesterol or [1,7,15,22,26-(13)C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1ß-(2'-propanoate)-3aα-H-4α-(3'-propanoic acid)-7aß-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2'-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2'-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites.

Patients who endorse more binge eating triggers before gastric bypass lose less weight at 6 months.

BACKGROUND: Patients presenting for gastric bypass surgery often demonstrate binge eating behaviors. The present study sought to determine whether binge eating triggers are predictive of weight loss outcomes in bariatric surgery patients at 6 months postoperatively in the setting of a university hospital. METHODS: A total of 48 patients presenting for gastric bypass surgery at an academic medical center in the Southeastern United States and who had returned for the 6-month follow-up visit were included in the present study. The patients were mostly women (85%), white (71%), and middle-aged (mean age 47 years), with an average weight of 100.9 kg. The patients completed the Inventory of Binge Eating Situations at baseline, and weight loss outcomes were assessed at 6 months. Weight loss success was indexed using 2 methods: the percentage of excess weight lost (continuous variable) and whether the patient was on track with their weight loss as defined by a ≥ 50% excess weight loss (dichotomous variable). RESULTS: A significant negative correlation (r = -.31, P = .03) was found between the preoperative Inventory of Binge Eating Situations scores and the percentage of excess weight loss at 6 months after gastric bypass surgery. Logistic regression analysis showed that "on track" status at 6 months was predicted by the Inventory of Binge Eating Situations score at baseline (Wald chi-square = 3.97, df = 1, P = .046). CONCLUSION: Careful assessment of binge eating situations could serve as a potential predictor of poor weight loss outcomes in patients seeking gastric bypass surgery. These findings support the baseline assessment of binge eating triggers and future research to examine the effectiveness of interventions for coping with binge eating triggers for gastric bypass surgery patients.

Alcohol screening in dental patients: the prevalence of hazardous drinking and patients' attitudes about screening and advice.

BACKGROUND: Because heavy drinking is a risk factor for oral cancer, dentists should screen patients for alcohol use. The authors investigated heavy drinking in dental patients and patients' attitudes about alcohol screening. METHODS: A convenience sample of 408 patients attending an emergency walk-in dental clinic served as subjects. Patients completed the Alcohol Use Disorders Identification Test-C (AUDIT-C), a three-item alcohol screening test, and an opinion survey regarding attitudes about the acceptability of alcohol screening and counseling by dentists. RESULTS: One in four patients had positive screening results for heavy alcohol use. The majority of subjects (> 75 percent) were in support of dentists' inquiries and advice about alcohol use. Age, sex and drinking status were not predictive of patients' opinions about alcohol screening. CONCLUSIONS: One hundred three of the dental patients exhibited evidence of hazardous alcohol consumption, a risk factor for oropharyngeal cancer. The majority of patients reported that they would readily accept alcohol screening and alcohol counseling by dentists. CLINICAL IMPLICATIONS: Because studies have shown that some dentists hesitate to screen for alcohol use because of a belief that screening is unacceptable to patients, these results may encourage a change in practice.

Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression.

The antioxidant response element (ARE) is a transcriptional control element that mediates expression of a set of antioxidant proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that activates ARE-containing genes. In endothelial cells, the ARE-mediated genes are upregulated by atheroprotective laminar flow through a Nrf2-dependent mechanism. We tested the hypothesis that activation of ARE-regulated genes via adenovirus-mediated expression of Nrf2 may suppress redox-sensitive inflammatory gene expression. Expression of Nrf2 in human aortic endothelial cells (HAECs) resulted in a marked increase in ARE-driven transcriptional activity and protected HAECs from H2O2-mediated cytotoxicity. Nrf2 suppressed TNF-alpha-induced monocyte chemoattractant protein (MCP)-1 and VCAM-1 mRNA and protein expression in a dose-dependent manner and inhibited TNF-alpha-induced monocytic U937 cell adhesion to HAECs. Nrf2 also inhibited IL-1beta-induced MCP-1 gene expression in human mesangial cells. Expression of Nrf2 inhibited TNF-alpha-induced activation of p38 MAP kinase. Furthermore, expression of a constitutively active form of MKK6 (an upstream kinase for p38 MAP kinase) partially reversed Nrf2-mediated inhibition of VCAM-1 expression, suggesting that p38 MAP kinase, at least in part, mediates Nrf2's anti-inflammatory action. In contrast, Nrf2 did not inhibit TNF-alpha-induced NF-kappaB activation. These data identify the Nrf2/ARE pathway as an endogenous atheroprotective system for antioxidant protection and suppression of redox-sensitive inflammatory genes, suggesting that targeting the Nrf2/ARE pathway may represent a novel therapeutic approach for the treatment of inflammatory diseases such as atherosclerosis.

Efeitos das mudanças do uso da terra na biogeoquímica dos corpos d'água da bacia do rio Ji-Paraná, Rondônia / Effects of land use changes in the biogeochemistry of fluvial systems of the Ji-Paraná river basin, Rondônia

Este trabalho discute os efeitos das mudanças do uso do solo na biogequímica dos rios da bacia de drenagem do rio Ji-Paraná (Rondônia). Nesta região, a distribuição espacial do desmatamento e das propriedades do solo resultam em sinais diferentes, possibilitando a divisão dos sistemas fluviais em três grupos: rios com águas pobres em íons e baixo impacto; rios com conteúdo iônico intermediário e impacto médio e rios com elevados conteúdo iônico e impacto antropogênico. As características biogeoquímicas dos rios têm relação significativa com a área de pasto, melhor parâmetro para prever a condutividade elétrica (r² = 0,87) e as concentrações de sódio (r² = 0,75), cloreto (r² = 0,69), potássio (r² = 0,63), fosfato (r² = 0.78), nitrogênio inorgânico (r² = 0.52), carbono inorgânico (r² = 0.81) e carbono orgânico (rain ² = 0.51) dissolvidos. Cálcio e magnésio tiveram sua variância explicada pelas características do solo e pastagem. Nossos resultados indicam que as mudanças observadas na micro-escala constituem "sinais biogeoquímicos" gerados pelo processamento do material nas margens dos rios. A medida em que os rios evoluem para ordens superiores, os sinais persistentes nos canais fluviais estão mais associdados às características da bacia de drenagem (solos e uso da terra). Apesar dos efeitos das mudanças observadas no uso do solo não serem ainda detectáveis na macro-escala (bacia amazônica), a disrupção da estrutura e funcionamento dos ecossistemas é detectável nas micro e meso escalas, com alterações significativas na ciclagem de nutrientes nos ecossistemas fluviais.

AGIX-4207 [2-[4-[[1-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio]-1-methylethyl]thio]-2,6-bis(1,1-dimethylethyl)phenoxy]acetic acid], a novel antioxidant and anti-inflammatory compound: cellular and biochemical characterization of antioxidant activity and inhibition of redox-sensitive inflammatory gene expression.

The pathogenesis of chronic inflammatory diseases, including rheumatoid arthritis, is regulated, at least in part, by modulation of oxidation-reduction (redox) homeostasis and the expression of redox-sensitive inflammatory genes including adhesion molecules, chemokines, and cytokines. AGIX-4207 [2-[4-[[1-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]thio]-1-methylethyl]thio]-2,6-bis(1,1-dimethylethyl)phenoxy]acetic acid] is a novel, orally active, phenolic antioxidant and anti-inflammatory compound with antirheumatic properties. To elucidate its anti-inflammatory mechanisms, we evaluated AGIX-4207 for a variety of cellular, biochemical, and molecular properties. AGIX-4207 exhibited potent antioxidant activity toward lipid peroxides in vitro and displayed enhanced cellular uptake relative to a structurally related drug, probucol. This resulted in potent inhibition of cellular levels of reactive oxygen species in multiple cell types. AGIX-4207 selectively inhibited tumor necrosis factor (TNF)-alpha-inducible levels of the redox-sensitive genes, vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, with less inhibition of E-selectin, and no effect on intracellular adhesion molecule-1 expression in endothelial cells. In addition, AGIX-4207 inhibited cytokine-induced levels of monocyte chemoattractant protein-1, interleukin (IL)-6, and IL-8 from endothelial cells and human fibroblast-like synoviocytes as well as lipopolysaccharide-induced release of TNF-alpha, IL-1beta, and IL-6 from human peripheral blood mononuclear cells. AGIX-4207 did not inhibit TNF-alpha-induced nuclear translocation of nuclear factor of the kappa-enhancer in B cells (NF-kappaB), suggesting that the mechanism of action is independent of this redox-sensitive transcription factor. Taken together, these results provide a mechanistic framework for understanding the anti-inflammatory and antirheumatic activity of AGIX-4207 and provide further support for the view that inhibition of redox-sensitive inflammatory gene expression is an attractive approach for the treatment of chronic inflammatory diseases.

Reactivity to smoking cues in adolescent cigarette smokers.

This study examined reactivity to smoking cues in adolescent smokers (n=12) and nonsmokers (n=32), between 14 and 19 years of age. Participants were presented with videotaped smoking and neutral cues in a counterbalanced order. Subjective and physiological responses to each cue type were obtained. Findings indicated that smokers reported greater desire to smoke cigarettes in response to smoking cues, relative to neutral cues, when the smoking cues were presented first. Smokers also reported greater dominance (i.e., sense of control) during smoking-cue presentations, but only when these cues were presented second. Finally, smokers' heart rate was faster during the initial portion of the smoking-related video, relative to the neutral cue. Overall, this study demonstrates the feasibility of conducting laboratory-based cue-reactivity studies with adolescent smokers. Findings suggest that adolescents smokers show similar patterns of responding to smoking cues as adult smokers, although effects were not particularly robust in this sample and subjective effects were dependent on cue order.

Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow.

Atherosclerosis is a focal inflammatory disease and preferentially occurs in areas of low fluid shear stress and oscillatory flow, whereas the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow. Sphingosine kinase-1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1 phosphate (S1P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, we demonstrated that exposure of human aortic endothelial cells to oscillatory flow (shear stress, +/-5 dyn/cm(2) for 48 h) resulted in a marked increase in SphK1 mRNA levels compared with endothelial cells kept in static culture. In contrast, laminar flow (shear stress, 20 dyn/cm(2) for 48 h) decreased SphK1 mRNA levels. We further investigated the role of SphK1 in TNF-alpha-induced expression of inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and VCAM-1 by using small interfering RNA (siRNA) specifically for SphK1. Treatment of endothelial cells with SphK1 siRNA suppressed TNF-alpha-induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF-alpha-induced cell surface expression of VCAM-1, but not ICAM-1, protein. Exposure of endothelial cells to S1P led to an increase in MCP-1 protein secretion and MCP-1 mRNA levels and activation of NF-kappaB-mediated transcriptional activity. Treatment of endothelial cells with the p38 MAPK inhibitor SB-203580 suppressed S1P-induced MCP-1 protein secretion. These data suggest that SphK1 mediates TNF-alpha-induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature.

Laminar flow induction of antioxidant response element-mediated genes in endothelial cells. A novel anti-inflammatory mechanism.

Atherosclerotic lesions preferentially develop in areas of the vasculature exposed to nonlaminar blood flow and low fluid shear stress, whereas laminar flow and high fluid shear stress are athero-protective. We have identified a set of genes including NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), ferritin (heavy and light chains), microsomal epoxide hydrolase, glutathione S-transferase, and gamma-glutamylcysteine synthase, whose expression is induced by exposure to prolonged physiological levels of steady laminar flow (shear stress = 20 dyn/cm(2)) in endothelial cells (EC). These genes contain an antioxidant response element (ARE) or ARE-like transcriptional regulatory sequence in their promoters and generally function to protect cells against oxidant stress. We demonstrate that exposure of EC to laminar flow activates ARE-mediated transcriptional activity. Mutation of the ARE from either the NQO1 or HO-1 promoter abolished laminar flow-induced NQO1 and HO-1 transcriptional activation. Expression of antisense Nrf2 (a transcriptional factor for ARE), a dominant negative Nrf2, or the cytoplasmic inhibitor of Nrf2 (Keap1/INrf2) inhibited laminar flow-induced NQO1 promoter activation in EC. In addition, expression of NQO1 or Nrf2 inhibited tumor necrosis factor-alpha-induced activation of VCAM-1 (vascular cell adhesion molecule-1) gene expression in EC. These data define the ARE as a novel endothelial shear stress response element. Furthermore, laminar flow activation of antioxidant genes via an ARE-dependent transcriptional mechanism may represent a novel athero-protective and anti-inflammatory mechanism in the vasculature.

MRK, a mixed lineage kinase-related molecule that plays a role in gamma-radiation-induced cell cycle arrest.

Mitogen-activated protein (MAP) kinase pathways are three-kinase modules that mediate diverse cellular processes and have been highly conserved among eukaryotes. By using a functional complementation screen in yeast, we have identified a human MAP kinase kinase kinase (MAPKKK) that shares homology with members of the mixed lineage kinase (MLK) family and therefore was called MRK (MLK-related kinase). We report the structure of the MRK gene, from which are generated two splice forms of MRK, MRK-alpha and MRK-beta, encoding for proteins of 800 and 456 amino acids, respectively. By using a combination of solid phase protein kinase assays, transient transfections in cells, and analysis of endogenous proteins in stably transfected Madin-Darby canine kidney cells, we found that MRK-beta preferentially activates ERK6/p38gamma via MKK3/MKK6 and JNK through MKK4/MKK7. We also show that expression of wild type MRK increases the cell population in the G(2)/M phase of the cell cycle, whereas dominant negative MRK attenuates the G(2) arrest caused by gamma-radiation. In addition, exposure of cells to gamma-radiation induces MRK activity. These data suggest that MRK may mediate gamma-radiation signaling leading to cell cycle arrest and that MRK activity is necessary for the cell cycle checkpoint regulation in cells.

A functional interaction between CD46 and DLG4: a role for DLG4 in epithelial polarization.

Using a yeast two-hybrid screen, we identified a physical interaction between CD46 and DLG4. CD46 is a ubiquitous human cell-surface receptor for the complement components C3b and C4b and for measles virus and human herpesvirus 6. DLG4 is a scaffold protein important for neuronal signaling and is homologous to the Drosophila tumor suppressor DLG. We show that an interaction between CD46 and DLG4 is important for polarization in epithelial cells. Specifically, we show (i) biochemical evidence for an interaction between CD46 and DLG4, (ii) that this interaction is specific for the Cyt1 (but not Cyt2) domain of CD46, (iii) that both CD46 and an alternatively spliced isoform of DLG4 are polarized in normal human epithelial cells, and (iv) that the polarized expression of CD46 in epithelial cells requires the DLG4-binding domain and alters with expression of a truncated form of DLG4. This is the first identification of a direct and cytoplasmic domain-specific interaction between CD46 and an intracellular signaling molecule and provides a molecular mechanism for the polarization of CD46. These data also indicate that, in addition to the known role for DLG4 in neuronal cells, DLG4 may be important for polarization in epithelial cells.