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Drawing on collective experience from ten collaborative research projects focused on the Global South, we identify three major challenges that impede the translation of research on sustainability and resilience into better-informed choices by individuals and policy-makers that in turn can support transformation to a sustainable future. The three challenges comprise: (i) converting knowledge produced during research projects into successful knowledge application; (ii) scaling up knowledge in time when research projects are short-term and potential impacts are long-term; and (iii) scaling up knowledge across space, from local research sites to larger-scale or even global impact. Some potential pathways for funding agencies to overcome these challenges include providing targeted prolonged funding for dissemination and outreach, and facilitating collaboration and coordination across different sites, research teams, and partner organizations. By systematically documenting these challenges, we hope to pave the way for further innovations in the research cycle., (© 2024. The Author(s).)

Background: Falls represent the leading cause of nonfatal unintentional injuries among children in the United States. Although unintentional injury risks have been studied, neighborhood impact on falls remains underexplored. This study examined the association of neighborhood attributes with rates of fall-related injuries., Methods: This is a retrospective study of children who presented to emergency departments within a statewide hospital network for fall-related injuries between 2005 and 2014. Patients' home addresses were geocoded to identify US Census block groups (BGs). Average annual fall rates were computed for each BG. A neighborhood risk index was constructed using 8 socioeconomic BG measures (education, crowding, vacancy, renter occupancy, poverty, family structure, race/ethnicity, and housing age). Public outdoor recreational facilities in each BG were enumerated. Linear regression analysis was used to assess the association of neighborhood risk and recreational facilities with fall rates., Results: From 2005 to 2014, there were 139,986 unintentional injury emergency department visits; of these, 42,691 (30%) were for falls. The largest proportion of falls were among males (58%), children ages 1 to 4 years (39%), non-Hispanic whites (59%), and children with public health insurance (53%). Higher quintiles of neighborhood risk were associated with higher annual fall rates compared to the lowest quintile of risk: quintile 2, β = 0.44, 95% confidence interval (CI), 0.20-0.68; quintile 3, β = 0.85, 95% CI, 0.61-1.10; quintile 4, β = 1.11, 95% CI, 0.85-1.37; quintile 5, β = 1.57, 95% CI, 1.29-1.85. The presence of public outdoor recreational facilities was not associated with fall rates (β = 0.01; 95% CI, -0.14 to 0.15)., Conclusion: Neighborhood-level socioeconomic characteristics are associated with higher fall-related injuries. Injury prevention programs could be tailored to address these neighborhood risks., (Copyright © 2018. Published by Elsevier Inc.)

Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection., Importance: Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue have not been defined. While bnAbs are highly effective against cell-free virus, they are not induced by current vaccine candidates. However, nnAbs, readily induced by vaccines, can trigger antibody-dependent cellular effector functions, through engagement of their Fc-gamma receptors. Fc-mediated antiviral activity has been implicated as a secondary correlate of decreased HIV-1 risk in the RV144 vaccine efficacy trial, suggesting that protection might be mediated in the absence of classical neutralization. To aid vaccine design and selection of antibodies for use in passive protection strategies, we assessed a range of bnAbs and nnAbs for their potential to block ex vivo challenge of mucosal tissues. Our data clearly indicate the superior efficacy of neutralizing antibodies in preventing mucosal acquisition of infection. These results underscore the importance of maintaining the central focus of HIV-1 vaccine research on the induction of potently neutralizing antibodies., (Copyright © 2016 Cheeseman et al.)

The functionally generated path (FGP) is a static representation of the opposing cusps' dynamic eccentric movements from a centric position to achieve optimal articulation and occlusal harmony. When understood and appreciated, use of the FGP technique is a straightforward and practical method to achieve harmonious occlusal anatomy of restorations with the anterior determinant/anterior guidance, the posterior determinant/condylar guidance, existing occlusal and cuspal anatomy, and the neuromuscular system. Although the FGP technique is normally used in the fabrication of maxillary posterior indirect restorations, it is described and applied here in the fabrication of mandibular posterior restorations that maintained the patient's bilateral group function occlusion while eliminating the nonworking side and protrusive interferences. This novel procedure involved the use of a stone crib to intraorally construct a stone core that captured the FGP recording while simultaneously indexing to the contralateral and ipsilateral mandibular dentition. This technique lends additional stability to the stone core to minimize error during the mounting process., (© 2012 by the American College of Prosthodontists.)

Background: Interactions between the HIV-1 envelope glycoprotein (Env) and its primary receptor CD4 are influenced by the physiological setting in which these events take place. In this study, we explored the surface chemistry of HIV-1 Env constructs at a range of pH and salinities relevant to mucosal and systemic compartments through electrophoretic mobility (EM) measurements. Sexual transmission events provide a more acidic environment for HIV-1 compared to dissemination and spread of infection occurring in blood or lymph node. We hypothesize functional, trimeric Env behaves differently than monomeric forms., Results: The dynamic electrophoretic fingerprint of trimeric gp140 revealed a change in EM from strongly negative to strongly positive as pH increased from that of the lower female genital tract (pHx) to that of the blood (pHy). Similar findings were observed using a trimeric influenza Haemagglutinin (HA) glycoprotein, indicating that this may be a general attribute of trimeric viral envelope glycoproteins. These findings were supported by computationally modeling the surface charge of various gp120 and HA crystal structures. To identify the behavior of the infectious agent and its target cells, EM measurements were made on purified whole HIV-1 virions and primary T-lymphocytes. Viral particles had a largely negative surface charge, and lacked the regions of positivity near neutral pH that were observed with trimeric Env. T cells changed their surface chemistry as a function of activation state, becoming more negative over a wider range of pH after activation. Soluble recombinant CD4 (sCD4) was found to be positively charged under a wide range of conditions. Binding studies between sCD4 and gp140 show that the affinity of CD4-gp140 interactions depends on pH., Conclusions: Taken together, these findings allow a more complete model of the electrochemical forces involved in HIV-1 Env functionality. These results indicate that the influence of the localized environment on the interactions of HIV with target cells are more pronounced than previously appreciated. There is differential chemistry of trimeric, but not monomeric, Env under conditions which mimic the mucosa compared to those found systemically. This should be taken into consideration during design of immunogens which targets virus at mucosal portals of entry.

Impairment of adipogenesis contributes to the development of obesity-related insulin resistance. The current in vitro approaches for its assessment represent crude estimates of the adipogenic potential because of the disruption of the in vivo microenvironment. A novel assessment of in vivo adipogenesis using the incorporation of the stable isotope deuterium ((2)H) into the DNA of isolated adipocytes and stroma-vascular fraction from adipose tissue has been developed. In the current study, we have refined this technique by purifying the adipocytes via a negative immune selection and sorting the plastic adherent stroma-vascular (aSV) subfraction (using 3 h culture) that contains mostly adipocyte progenitor cells and ∼10% of small adipocytes. Using a 3-week 8% (2)H(2)O ingestion with a high-fat diet (HFD) or HFD plus pioglitazone (HFD-P), we demonstrate that the fractions of new aSV cells (f(aSV)) and immunopurified adipocytes (f(AD)) (the ratio of their (2)H-enrichment of DNA to the maximal (2)H-enrichment of DNA of bone marrow reference cells) recapitulate the known hyperplastic mechanism of weight gain with pioglitazone treatment. We conclude that f(aSV) and f(AD) are reliable indices of in vivo adipogenesis. The proposed method represents a valuable tool for studying the effect of interventions (drugs, diets, and exercise) on in vivo adipogenesis.

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptor's activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

Isolates of Alternaria dauci causing Alternaria leaf blight (ALB) were collected from commercial carrot (Daucus carota var. sativus) fields in northeastern North America during 2004. Twenty-two isolates representing a range of genetic diversity were analyzed for their aggressiveness on three commercial carrot varieties (Bolero, Enterprise, and Heritage) varying in disease susceptibility as well as their in vitro response to three fungicides (azoxystrobin, chlorothalonil, and boscalid) commonly used for ALB control. Severity of leaf and petiole blight and leaf chlorosis varied among isolates and carrot varieties in each of three experiments. Visible differences in disease severity, which ranged from 10.9 to 45.1% of the leaf area affected, were apparent 16 days after inoculation. Intensity of chlorosis correlated strongly with blight severity among all isolates. Significant differences were noted among carrot varieties in response to ALB. These varieties may prove useful as differentials capable of distinguishing isolates because variety by isolate interactions were detected. Inhibition of conidial germination ranged from 0.01 to 0.37 μg/ml for azoxystrobin, 0.009 to 0.08 μg/ml for chlorothalonil, and 0.09 to 0.59 μg/ml for boscalid. On average, isolates were more sensitive to chlorothalonil than to azoxystrobin and boscalid. No significant correlation was noted between fungicide sensitivity and aggressiveness. These data provide evidence for phenotypic diversity among A. dauci isolates collected from areas of commercial carrot production.

Several metabolic abnormalities are associated with relative excess or deficiency of adipose tissue. Identifying the regulators of adipogenic differentiation is critical for its successful manipulation. Ad36, a human adenovirus, is a novel factor that promotes adipogenesis. We exploited the adipogenic potential of Ad36 to reveal exogenous modifiers of adipogenesis in rodent preadipocyte cell line in the presence or absence of differentiation inducers methyl-isobutyl-xanthine, dexamethasone, and insulin (M, D, and I; MDI). A nonadipogenic human adenovirus Ad2 was used as a negative control for viral infection. First, we confirmed that, Ad36, but not Ad2, increases lipid accumulation in the presence or absence of MDI. Time-course studies for expression of key genes of adipogenic cascade showed that it is Ad36, but not Ad2, which downregulated preadipocyte marker gene Wnt10b, and upregulated expression of early (C/EBPDelta and C/EBPbeta), intermediate (PPARgamma2), and late genes (aP2 and G3PDH) of adipogenic cascade even in the absence of MDI. In the presence of MDI, onset of expression of adipogenic genes coincided for Ad36 and control groups, but the expressions were significantly greater for the Ad36 group. Next, we observed that attenuation of Ad36 mRNA expression by an antiadenoviral agent reduced 3T3-L1 differentiation, indicating that viral mRNA expression is required for the process. Furthermore, with or without MDI or its components, Ad36 significantly increased lipid accumulation in 3T3-L1 cells. Cell confluency at the time of Ad36 infection positively influenced lipid accumulation. The results reveal that Ad36 is an MDI-independent exogenous regulator of the adipogenic process. Elucidating the molecular pathways involved may reveal novel regulatory controls of adipogenesis.

Obesity and nutrient homeostasis are linked by mechanisms that are not fully elucidated. Here we describe a secreted protein, adropin, encoded by a gene, Energy Homeostasis Associated (Enho), expressed in liver and brain. Liver Enho expression is regulated by nutrition: lean C57BL/6J mice fed high-fat diet (HFD) exhibited a rapid increase, while fasting reduced expression compared to controls. However, liver Enho expression declines with diet-induced obesity (DIO) associated with 3 months of HFD or with genetically induced obesity, suggesting an association with metabolic disorders in the obese state. In DIO mice, transgenic overexpression or systemic adropin treatment attenuated hepatosteatosis and insulin resistance independently of effects on adiposity or food intake. Adropin regulated expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor gamma, a major regulator of lipogenesis. Adropin may therefore be a factor governing glucose and lipid homeostasis, which protects against hepatosteatosis and hyperinsulinemia associated with obesity.

The nuclear hormone receptors liver X receptor alpha (LXRalpha) and LXRbeta function as physiological receptors for oxidized cholesterol metabolites (oxysterols) and regulate several aspects of cholesterol and lipid metabolism. Seladin-1 was originally identified as a gene whose expression was down-regulated in regions of the brain associated with Alzheimer's disease. Seladin-1 has been demonstrated to be neuroprotective and was later characterized as 3beta-hydroxysterol-Delta24 reductase (DHCR24), a key enzyme in the cholesterologenic pathway. Seladin-1 has also been shown to regulate lipid raft formation. In a whole genome screen for direct LXRalpha target genes, we identified an LXRalpha occupancy site within the second intron of the Seladin-1/DHCR24 gene. We characterized a novel LXR response element within the second intron of this gene that is able to confer LXR-specific ligand responsiveness to reporter gene in both HepG2 and human embryonic kidney 293 cells. Furthermore, we found that Seladin-1/DHCR24 gene expression is significantly decreased in skin isolated from LXRbeta-null mice. Our data suggest that Seladin-1/DHCR24 is an LXR target gene and that LXR may regulate lipid raft formation.

Cholesterol is required for normal cellular and physiological function, yet dysregulation of cholesterol metabolism is associated with diseases such as atherosclerosis. Cholesterol biosynthesis is regulated by end product negative feedback inhibition where the levels of sterols and oxysterols regulate the expression of cholesterologenic enzymes. Sterol regulatory element-binding protein-2 is responsive to both sterols and oxysterols and has been shown to mediate the transcriptional response of the cholesterologenic enzymes to these lipids. Here, we show that the nuclear hormone receptor for oxysterols, the liver X receptor alpha (LXRalpha), regulates cholesterol biosynthesis by directly silencing the expression of two key cholesterologenic enzymes (lanosterol 14alpha-demethylase (CYP51A1), and squalene synthase (farnesyl diphosphate farnesyl transferase 1)) via novel negative LXR DNA response elements (nLXREs) located in each of these genes. Examination of the CYP51A1 gene revealed that both the SRE and nLXRE are required for normal oxysterol-dependent repression of this gene. Thus, these data suggest that LXRalpha plays an important role in the regulation of cholesterol biosynthesis.

Objective: Experimental infection of rats with human adenovirus type 36 (Ad-36) promotes adipogenesis and improves insulin sensitivity in a manner reminiscent of the pharmacologic effect of thiozolinediones. To exploit the potential of the viral proteins as a therapeutic target for treating insulin resistance, this study investigated the ability of Ad-36 to induce metabolically favorable changes in human adipose tissue., Research Design and Methods: We determined whether Ad-36 increases glucose uptake in human adipose tissue explants. Cell-signaling pathways targeted by Ad-36 to increase glucose uptake were determined in the explants and human adipose-derived stem cells. Ad-2, a nonadipogenic human adenovirus, was used as a negative control. As a proof of concept, nondiabetic and diabetic subjects were screened for the presence of Ad-36 antibodies to ascertain if natural Ad-36 infection predicted improved glycemic control., Results: Ad-36 increased glucose uptake by adipose tissue explants obtained from nondiabetic and diabetic subjects. Without insulin stimulation, Ad-36 upregulated expressions of several proadipogenic genes, adiponectin, and fatty acid synthase and reduced the expression of inflammatory cytokine macrophage chemoattractant protein-1 in a phosphotidylinositol 3-kinase (PI3K)-dependent manner. In turn, the activation of PI3K by Ad-36 was independent of insulin receptor signaling but dependent on Ras signaling recruited by Ad-36. Ad-2 was nonadipogenic and did not increase glucose uptake. Natural Ad-36 infection in nondiabetic and diabetic subjects was associated with significantly lower fasting glucose levels and A1C, respectively., Conclusions: Ad-36 proteins may provide novel therapeutic targets that remodel human adipose tissue to a more metabolically favorable profile.

Objective: Human adenovirus type 36 (Ad-36) increases adiposity but improves insulin sensitivity in experimentally infected animals. We determined the ability of Ad-36 to increase glucose uptake by human primary skeletal muscle (HSKM) cells., Research Design and Methods: The effect of Ad-36 on glucose uptake and cell signaling was determined in HSKM cells obtained from type 2 diabetic and healthy lean subjects. Ad-2, another human adenovirus, was used as a negative control. Gene expression and proteins of GLUT1 and GLUT4 were measured by real-time PCR and Western blotting. Role of insulin and Ras signaling pathways was determined in Ad-36-infected HSKM cells., Results: Ad-36 and Ad-2 infections were confirmed by the presence of respective viral mRNA and protein expressions. In a dose-dependent manner, Ad-36 significantly increased glucose uptake in diabetic and nondiabetic HSKM cells. Ad-36 increased gene expression and protein abundance of GLUT1 and GLUT4, GLUT4 translocation to plasma membrane, and phosphatidylinositol 3-kinase (PI 3-kinase) activity in an insulin-independent manner. In fact, Ad-36 decreased insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and IRS-1-and IRS-2-associated PI 3-kinase activities. On the other hand, Ad-36 increased Ras gene expression and protein abundance, and Ras siRNA abrogated Ad-36-induced PI 3-kinase activation, GLUT4 protein abundance, and glucose uptake. These effects were not observed with Ad-2 infection., Conclusions: Ad-36 infection increases glucose uptake in HSKM cells via Ras-activated PI 3-kinase pathway in an insulin-independent manner. These findings may provide impetus to exploit the role of Ad-36 proteins as novel therapeutic targets for improving glucose handling.

The nuclear hormone receptors, REV-ERBalpha [NR1D1] and REV-ERBbeta [NR1D1], were recently demonstrated to be receptors for the porphyrin, heme. Heme regulates the ability of these receptors to repress transcription of their target genes via modulation of the affinity of the receptor's ligand binding domain for the corepressor, NCoR. The REV-ERBs function as critical components of the mammalian clock and their expression oscillates in a circadian manner. Here, we show that in NIH3T3 cells intracellular heme levels also oscillate in a circadian fashion. These data are the first to show the temporal relationship of intracellular heme levels to the expression of its receptor, Rev-erbalpha, and suggest that the rapid oscillations in heme levels may an important component regulating REV-ERB transcriptional activity.

Objective: Understanding the regulation of adipocyte differentiation by cellular and extracellular factors is crucial for better management of chronic conditions such as obesity, insulin resistance and lipodystrophy. Experimental infection of rats with a human adenovirus type 36 (Ad-36) improves insulin sensitivity and promotes adipogenesis, reminiscent of the effect of thiozolinediones. Therefore, we investigated the role of Ad-36 as a novel regulator of the adipogenic process., Design and Results: Even in the absence of adipogenic inducers, infection of 3T3-L1 preadipocytes and human adipose-derived stem cells (hASC) by Ad-36, but not Ad-2 that is another human adenovirus, modulated regulatory points that spanned the entire adipogenic cascade ranging from the upregulation of cAMP, phosphatidylinositol 3-kinase and p38 signaling pathways, downregulation of Wnt10b expression, and increased expression of CCAAT/enhancer binding protein-beta and peroxisome proliferator-activated receptor gamma2 and consequential lipid accumulation. Next, we identified that E4 open reading frame (orf)-1 gene of the virus is necessary and sufficient for Ad-36-induced adipogenesis. Selective knockdown of E4 orf-1 by RNAi abrogated Ad-36-induced adipogenic signaling cascade in 3T3-L1 cells and hASC. Compared to the null vector, selective expression of Ad-36 E4 orf-1 in 3T3-L1 induced adipogenesis, which was abrogated when the PDZ-binding domain of the protein was deleted., Conclusion: Thus, Ad-36 E4 orf-1 is a novel inducer of rodent and human adipocyte differentiation process.

Type I human hepatic 3alpha-hydroxysteroid dehydrogenase (AKR1C4) plays a significant role in bile acid biosynthesis, steroid hormone metabolism, and xenobiotic metabolism. Utilization of a hidden Markov model for predictive modeling of nuclear hormone receptor response elements coupled with chromatin immunoprecipitation/microarray technology revealed a putative binding site in the AKR1C4 promoter for the nuclear hormone receptor known as liver X receptor alpha, (LXRalpha [NR1H3]), which is the physiological receptor for oxidized cholesterol metabolites. The putative LXRalpha response element (LXRE), identified by chromatin immunoprecipitation, was approximately 1.5 kilobase pairs upstream of the transcription start site. LXRalpha was shown to bind specifically to this LXRE and mediate transcriptional activation of the AKR1C4 gene, leading to increased AKR1C4 protein expression. These data suggest that LXRalpha may modulate the bile acid biosynthetic pathway at a unique site downstream of CYP7A1 and may also modulate the metabolism of steroid hormones and certain xenobiotics.

The nuclear receptors REV-ERBalpha (encoded by NR1D1) and REV-ERBbeta (NR1D2) have remained orphans owing to the lack of identified physiological ligands. Here we show that heme is a physiological ligand of both receptors. Heme associates with the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry and enhances the thermal stability of the proteins. Results from experiments of heme depletion in mammalian cells indicate that heme binding to REV-ERB causes the recruitment of the co-repressor NCoR, leading to repression of target genes including BMAL1 (official symbol ARNTL), an essential component of the circadian oscillator. Heme extends the known types of ligands used by the human nuclear receptor family beyond the endocrine hormones and dietary lipids described so far. Our results further indicate that heme regulation of REV-ERBs may link the control of metabolism and the mammalian clock.

Fixed sample-size plans for monitoring Plutella xylostella (L.) (Lepidoptera: Plutellidae) on broccoli and other Brassica vegetable crops are popular in Australia for their simplicity and ease of application. But the sample sizes used are often small, approximately 10-25 plants per crop, and it may be that they fail to provide sufficient information upon which to base pest control decisions. We tested the performance of seven fixed sample-size plans (10, 15, 20, 30, 35, 40, and 45 plants) by resampling a large data set on P. xylostella in commercial broccoli crops. For each sample size, enumerative and presence-absence plans were assessed. The precision of the plans was assessed in terms of the ratio of the standard error to the mean; and at least 45 and 35 samples were necessary for the enumerative and presence-absence plans, respectively, to attain the generally accepted benchmark of < or = 0.3. Sample sizes of 10-20 were highly imprecise. We also assessed the consequences of classifications based on action thresholds (ATs) of 0.2 and 0.8 larvae per plant for the enumerative case, and 0.15 and 0.45 proportion of plants of infested for the presence-absence case. Operating characteristic curves and investigations of the frequency of correct decisions suggest improvements in the performance of plans with increased sample size. In both the enumerative and presence-absence cases, the proportion of incorrect decisions was much higher for the lower of the two ATs assessed, and type II errors (i.e., failure to suggest pest control upon the AT is exceeded) generally accounted for the majority of this error. Type II errors are the most significant from a producer's standpoint. Further consideration is necessary to determine what is an acceptable type II error rate.

Foliar diseases of carrot caused by Alternaria dauci and Cercospora carotae occur every year in Wisconsin, requiring repeated foliar fungicide applications to minimize defoliation and yield reduction. Improved timing of fungicide applications combined with the use of disease resistant cultivars offer growers a means to improve disease control with fewer fungicide inputs compared with the current strategy of weekly fungicide applications to a susceptible cultivar. Field experiments in 2002 to 2004 examined fungicide application schedules indicated by a disease forecasting model that calculated the duration of environmental conditions favorable for A. dauci and C. carotae (adaptation of TOM-CAST) on two carrot cultivars differing in susceptibility to these foliar diseases. All fungicide programs were initiated at a 1% disease severity threshold determined by scouting. Intervals for weather-based spray programs were based on in-canopy leaf wetness and temperature data. Fungicide sprays were applied according to 15 and 20 disease severity value (DSV) application thresholds, and were compared with a weekly spray program and an untreated control. Results of this trial demonstrated that fungicide sprays made according to weather data may reduce fungicide inputs in most years compared with current industry-standard, calendar-based spray programs. Host susceptibility affected the efficacy of weather-based spray programs, resulting in longer spray intervals and fewer fungicide applications on the resistant cultivar Bolero when compared with the susceptible cultivar Fontana. Fungicide spray programs based on TOM-CAST diminished fungicide inputs by 30 to 50%, compared with the weekly spray program, by lengthening intervals between applications without compromising disease control or root yield.

The melanin biosynthetic pathway from tyrosine is a potential target for combating malignant melanoma. N-Acetyl-4-S-cysteaminylphenol 1 is a previously synthesized analogue of tyrosine that probably acts by this pathway. It interferes with cell growth and proliferation via selective oxidation in melanocytes to an oquinone that can alkylate cellular nucleophiles. We previously synthesized a range of analogues of the original lead compound 1 most of which displayed greater cytotoxicity than 1. Eighteen new analogues with the amide group reversed have now been synthesized and tested for antimelanoma activity. Most of these reverse amides showed greater cytotoxicity than N-acetyl-4-S-cysteaminylphenol towards five representative melanoma cell lines. The highest cytotoxicity was observed for the piperidine and hexamethyleneimine derivatives 7, 8, 12, 13, and 17 and the catechol 18. The most active compound, 7, had cytotoxicity comparable to cisplatin against the five melanoma cell lines. The moderate activity of 7 and 18 against SK-Mel-24 (non-tyrosinase containing) and an ovarian cell line suggests that interference with the melanin pathway may not be the only mode of action of these compounds. Assays of some of the compounds as substrates for tyrosinase showed that the catechol 18 was the best substrate and that the piperidine derivative 7 was the best substrate of the phenolic compounds synthesized.

Interference with the biosynthetic pathway to melanin may be a useful means for developing new chemotherapeutic drugs to combat malignant melanoma. N-Acetyl-4-S-cysteaminylphenol (1) is an analogue of tyrosine that is involved in the pathway to melanin. It is probably oxidized selectively in melanocytes to an o-quinone that can alkylate thiol groups on important cellular enzymes, resulting in interference with cell growth and proliferation. We previously synthesized a range of more lipophilic analogues of 1 by independently varying the acyl portion and introducing substitution alpha to the nitrogen. Most of the new compounds displayed greater cytotoxicity than the original lead compound 1. We also made a series of tertiary amides that again showed higher cytotoxicity than 1. In this work three new acetamides and two new cyclohexanecarboxamides containing 4-S-cysteaminylphenol were prepared incorporating both substitution alpha to the nitrogen and different substituents on the nitrogen of the amide in each compound to increase lipophilicity and to reduce further the possibility of hydrolysis of the amides. Most of the new tertiary amides showed greater cytotoxicity towards five representative melanoma cell lines than the parent secondary amide. The highest cytotoxicity against these five cell lines with IC50 values of 1-15 nicroM, comparable to cisplatin, was observed for N-[2[(4-hydroxyphenyl)thio]-1,1-dimethylethyl]-N-methylcyclohexanecarboxamide (8c). The IC50 values of 14.5 and 5.4 microM for this compound against SK-Mel-24 (not containing tyrosinase) and an ovarian cell line, respectively, suggest that interference with the melanin pathway may not be the only mode of action of this new compound. The cyclohexanecarboxamides were better substrates for mushroom tyrosinase (EC 1.14.18.1) than the acetamides.

The p53 tumor suppressor protein is phosphorylated and activated by several DNA damage-inducible kinases, such as ATM, and is a key effector of the DNA damage response by promoting cell cycle arrest or apoptosis. Deregulation of the Rb-E2F1 pathway also results in the activation of p53 and the promotion of apoptosis, and this contributes to the suppression of tumor development. Here, we describe a novel connection between E2F1 and the ATM DNA damage response pathway. In primary human fibroblasts lacking functional ATM, the ability of E2F1 to induce the phosphorylation of p53 and apoptosis is impaired. In contrast, ATM status has no effect on transcriptional activation of target genes or the stimulation of DNA synthesis by E2F1. Cells containing mutant Nijmegen breakage syndrome protein (NBS1), a component of the Mre11-Rad50 DNA repair complex, also have attenuated p53 phosphorylation and apoptosis in response to E2F1 expression. Moreover, E2F1 induces ATM- and NBS1-dependent phosphorylation of the checkpoint kinase Chk2 at Thr68, a phosphorylation site that stimulates Chk2 activity. Delayed gammaH2AX phosphorylation and absence of ATM autophosphorylation at Ser1981 suggest that E2F1 stimulates ATM through a unique mechanism that is distinct from agents that cause DNA double-strand breaks. These findings identify new roles for several DNA damage response factors by demonstrating that they also participate in the oncogenic stress signaling pathway between E2F1 and p53.

The biosynthetic pathway to melanin is a realistic target for therapeutic intervention in the development of new drugs to combat malignant melanoma. N-Acetyl-4-S-cysteaminylphenol (1) is an analogue of a biosynthetic intermediate in the pathway to melanin. It probably acts as a prodrug and is oxidized selectively in melanocytes to an o-quinone, which can alkylate cellular nucleophiles resulting in interference with cell growth and proliferation. We previously synthesized a range of more lipophilic analogues of 1 by varying the acyl portion and introducing substitution alpha to the nitrogen. Most of the new compounds displayed greater cytotoxicity than the original lead compound 1. We have now prepared 12 new compounds with varying acyl portions and three different substituents on the nitrogen of the amide in order to increase lipophilicity and to reduce the possibility of hydrolysis of the amides. Most of the tertiary amides showed greater cytotoxicity towards five representative melanoma cell lines than the parent secondary amide. The highest cytotoxicity, comparable to cisplatin, was observed for the benzyl substituted compounds 4, 8, 12, and 16 and the cyclohexylacetamide derivatives 13-15 against these five cell lines. The moderate activity of 13-16 against SK-Mel-24 (non-tyrosinase containing) and an ovarian cell line suggests that interference with the melanin pathway may not be the only mode of action of these new compounds.

Previous studies have demonstrated both oncogenic and tumor suppressive properties for the E2F1 transcription factor. In this study, E2f1-null mice were crossed with transgenic mice expressing Myc under the control of an epithelial-specific keratin 5 promoter to determine whether the absence of E2F1 would modulate the oncogenic activity of Myc. Inactivation of E2f1 was found to significantly accelerate tumor development in keratin 5 Myc transgenic mice. Acceleration of tumorigenesis occurred despite the fact that apoptosis levels were increased in transgenic tissue and tumors null for E2f1, whereas Myc-induced proliferation was unaffected by the status of E2f1. These findings provide new insight into the tumor suppressive activity of E2F1 and identify for the first time a specific oncogenic alteration that cooperates with the loss of E2F1 in tumorigenesis.

The ARF tumor suppressor participates in a p53-dependent apoptotic pathway that is stimulated in response to some oncogenic stimuli. The E2F1 transcription factor is a critical downstream target of the Rb tumor suppressor and, when active, can promote proliferation as well as apoptosis. The finding that E2F1 transcriptionally regulates the ARF gene has led to the suggestion that ARF contributes to E2F1-induced apoptosis. Counter to this hypothesis, this study demonstrates not only that ARF is unnecessary for E2F1 to induce apoptosis but also that inactivation of ARF actually enhances the ability of E2F1 to promote apoptosis. Inactivation of ARF also cooperates with E2F1 activity to promote entry into the S phase of the cell cycle. This relationship between ARF and E2F1 is demonstrated in transgenic epidermis in vivo and in mouse embryo fibroblast cultures in vitro. In contrast, the ability of Myc to induce apoptosis is diminished in the absence of ARF. E2F1 induces the accumulation of p53 in the absence of ARF, and this is associated with the phosphorylation of p53 on several residues. These findings demonstrate that ARF is a negative regulator of E2F1 activity and is not required for E2F1-induced apoptosis.

The effect of overexpressing the antiapoptotic protein BclXL in a human ovarian carcinoma cell line has been investigated in terms of sensitivity to the 2 major drugs used to treat this disease, paclitaxel and cisplatin. Stable transfection of BclXL into CH1 cells, which are relatively sensitive to cisplatin, resulted in around 2.7-fold higher expression in comparison with empty vector controls. However, this level of overexpression did not result in significant resistance in vitro to paclitaxel or cisplatin at the 50% inhibition level, using either short-term (4-day) growth inhibition or longer term colony-forming assays. By contrast, parallel subcutaneous xenograft models of these isogenic ovarian carcinoma cells in vivo, differing only in BclXL status, showed that this low-level BclXL overexpression conferred significant resistance to both paclitaxel and cisplatin in comparison with parent, nontransfected tumours. Whereas parent non-BclXL transfected tumours were highly responsive, with the disappearance of tumours for at least 50 days post treatment, tumours overexpressing BclXL grew back after 30 and 20 days after treatment with paclitaxel and cisplatin, respectively. These differences in responsiveness to paclitaxel in vivo were not attributable to any significant changes in the delivery of drug to the tumour. These data suggest that the responsiveness of ovarian cancer to paclitaxel and cisplatin in vivo, and therefore perhaps clinically, is influenced by levels of the antiapoptotic protein BclXL. Such effects may be missed in vitro when using short-term growth inhibition or clonogenic assays., (Copyright 2001 Wiley-Liss, Inc.)

With recent advances in dental technology, titanium is currently used for fabrication of crowns, fixed partial dentures, implant frameworks, and removable partial denture frameworks. The use of titanium-aluminum-vanadium (Ti-6Al-4V) alloy assumes that it imparts similar anti-corrosion characteristics to the commercially pure titanium. This clinical report describes a patient who experienced discoloration of a Ti-6Al-4V alloy removable partial denture.

N-Acetyl-4-S-cysteaminylphenol 1 is an analogue of a biosynthetic intermediate in the pathway to melanin. It is probably oxidized to an o-quinone which can alkylate cellular nucleophiles resulting in interference with cell growth and proliferation. It is reported to have useful anti-melanoma activity. We previously synthesized a range of analogues of 1 by varying the acyl portion of the amide. A modest increase in melanoma activity against six melanoma cell lines for these analogues could be correlated with increased lipophilicity. Thirteen new analogues of 1 containing two methyl groups at the alpha-position of the amino component and various acyl groups have now been prepared and assessed for anti-melanoma activity against six human melanoma cell lines. Most of the new compounds displayed greater cytotoxicity than the lead compound 1. The highest cytotoxicity against the cell lines was observed for the cyclohexylacetamide 11 followed by the cyclohexylcarboxamide 10 and the 2,2-dimethylpropanamide 6. The IC50 values of the most cytotoxic compound 11 against the cell lines were comparable with those of cisplatin. Small variations in the acyl components of these analogues, such as reducing the ring size, lengthening the carbon chain and reducing the amount of chain branching, resulted in a considerable loss of cytotoxicity. The moderate activity of 6, 10 and 11 against SK-Mel-24 cells (non-tyrosinase containing) and an ovarian cell line suggests that interference with the melanin pathway may not be their only mode of action.

N-Acetyl-4-S-cysteaminylphenol (1) has been shown by Jimbow and co-workers to possess useful antimelanoma activity. It is an analogue of a biosynthetic intermediate in the pathway to melanin and probably acts as a prodrug, being oxidized to an o-quinone which reacts with essential enzymes containing sulphydryl groups resulting in interference with cell growth and proliferation. We have synthesized a range of analogues of 1 by varying the acyl portion of the amide with the intention of increasing the lipophilicity of the compounds. A modest increase in melanoma activity against six melanoma cell lines for these analogues could be correlated with increased lipophilicity. The most active of these compounds, N-[2-[(4-hydroxyphenyl)thiol]ethyl]cyclohexanecarboxamide (9), showed promising selectivity (lack of cytotoxicity) on the non-melanotic cell line SK-Mel-24 and on an ovarian cell line. A significant improvement in antimelanoma activity was observed with a new type of analogue containing three carbon atoms between the sulphur and nitrogen. The most active of these new analogues, N-[3-[(4-hydroxyphenyl)-thiolpropyl]-1-cyclohexanecarboxamide (15), had activity comparable to cisplatin against several cell lines and low cytotoxicity towards the non-melanotic cell line.

Background: To our knowledge, 17-allylamino,17-demethoxygeldanamycin (17AAG) is the first inhibitor of heat shock protein 90 (Hsp90) to enter a phase I clinical trial in cancer. Inhibition of Hsp90, a chaperone protein (a protein that helps other proteins avoid misfolding pathways that produce inactive or aggregated states), leads to depletion of important oncogenic proteins, including Raf-1 and mutant p53 (also known as TP53). Given its ansamycin benzoquinone structure, we questioned whether the antitumor activity of 17AAG was affected by expression of the NQO1 gene, which encodes the quinone-metabolizing enzyme DT-diaphorase., Methods: The antitumor activity of 17AAG and other Hsp90 inhibitors was determined by use of a sulforhodamine B-based cell growth inhibition assay in culture and by the arrest of xenograft tumor growth in nude mice. DT-diaphorase activity was determined by use of a spectrophotometric assay, and protein expression was determined by means of western immunoblotting., Results: In two independent in vitro human tumor cell panels, we observed a positive relationship between DT-diaphorase expression level and growth inhibition by 17AAG. Stable, high-level expression of the active NQO1 gene transfected into the DT-diaphorase-deficient (by NQO1 mutation) BE human colon carcinoma cell line resulted in a 32-fold increase in 17AAG growth-inhibition activity. Increased sensitivity to 17AAG in the transfected cell line was also confirmed in xenografts. The extent of depletion of Raf-1 and mutant p53 protein confirmed that the Hsp90 inhibition mechanism was maintained in cells with high and low levels of DT-diaphorase. 17AAG was shown to be a substrate for purified human DT-diaphorase., Conclusion: These results suggest that the antitumor activity and possibly the toxicologic properties of 17AAG in humans may be influenced by the expression of DT-diaphorase. Careful monitoring for NQO1 polymorphism and the level of tumor DT-diaphorase activity is therefore recommended in clinical trials with 17AAG.

The mechanisms by which cis-diamminedichloroplatinum(II) (cisplatin) is transported across the plasma membrane (i.e., passive diffusion versus active transport) were investigated in the 41M and CH1 human ovarian carcinoma cell lines and their acquired cisplatin-resistant variants 41McisR6 and CH1cisR6, respectively. Intracellular cisplatin accumulation was significantly reduced (4.0 +/- 1.7-fold) in the parental 41M line at 4 degrees C when compared to incubations at 37 degrees C. However, no significant differences in platinum uptake were observed in the 41McisR6 and in the CH1 pair of lines at 4 degrees C versus 37 degrees C. Similarly, in the presence of ouabain (an inhibitor of Na+,K+-ATPase), there was a marked reduction (2.0 +/- 0.4-fold) in drug accumulation in the sensitive 41M cells only, and no changes in drug uptake were observed in the other cell lines in the absence or presence of ouabain. Platinum accumulation was significantly enhanced in all cell lines in the presence of metabolic inhibitors (NaF and NaN3). These results suggest that in the parental 41M cell line, cisplatin transport may occur via passive diffusion and active/facilitated transport, whereas in the resistant 41McisR6 variant, cisplatin enters cells by passive diffusion only. The orally active drug bis-acetato-ammine-dichloro-cyclohexylamine platinum(IV) (JM216) is a lipophilic platinum(IV) complex that has been shown to circumvent cisplatin resistance in the 41McisR6 by increasing drug uptake. Across the entire range of concentrations used (5-50 microm), intracellular accumulation of JM216 was significantly reduced in 41M and 41McisR6 cells (3.5 +/- 0.7-fold; P < 0.01), and in CH1 and CH1cisR6 cells (14.2 +/- 6.0-fold; p < 0.01) at 4 degrees C when compared to incubations at 37 degrees C. No significant difference in JM216 uptake was observed in the 41M pair of lines in the absence or presence of ouabain. Additional studies have revealed that the fold reduction observed in cis-ammine(cyclohexylamine)dichloroplatinum(II) (JM118) accumulation in the 41M and 41McisR6 cells at 4 degrees C (3.7 +/- 1.9) reflects similar fold reductions to those observed with JM216 uptake at 4 degrees C. These results suggest that the mechanism of JM216 transport across cell membranes is through passive diffusion, predominantly as a result of its enhanced lipophilicity. Notably, an overexpression of a Mr 36,000 plasma membrane protein was observed in the 41McisR variants when compared to the sensitive 41M line. Increased levels of this Mr 36,000 protein may relate to the observed reduction in active transport of cisplatin in the 41McisR6 variant. Tyrosine phosphorylation of the Mr 36,000 protein appeared to be greater in the resistant 41McisR6 variant than in the parental 41M line. In addition, the constitutive levels of the Mr 36,000 protein in the CH1 pair of lines and in two acquired JM216-resistant variants (41M/JM216R and CH1/JM216R), where resistance in these cell lines is not mediated through reduced drug uptake, were similar to those observed in their respective parental lines. These results suggest that the overexpression of this Mr 36,000 protein in the acquired cisplatin-resistant subline 41McisR6 may play a significant role in cisplatin uptake in resistant cells exhibiting reduced drug accumulation as a major mechanism of cisplatin resistance.

Uninoculated roots of pea (Pisum sativum) and French bean (Phaseolus vulgaris) have been shown to exude a number of antifungal compounds when grown in a non-sterile aqueous aerated medium. These have been identified and their possible importance in relation to disease resistance is discussed.