Age-related macular degeneration (AMD) is one of the main socioeconomical health issues worldwide. AMD has a multifactorial etiology with a variety of risk factors. Smoking is the most important modifiable risk factor for AMD development and progression. The present review summarizes the epidemiological studies evaluating the association between smoking and AMD, the mechanisms through which smoking induces damage to the chorioretinal tissues, and the relevance of advising patients to quit smoking for their visual health. Age-related macular degeneration (AMD) is a macular neurodegenerative disease that nowadays constitutes one of the main socioeconomical health issues worldwide, afflicting the elderly population. The exponentially increasing prevalence of AMD is linked to progressive aging of the population, and it is the leading cause of legal blindness in the developed world –6. It isOn the other hand, the presence or absence of CNV makes the distinction between neovascular AMD (presence of macular fluid and/or hemorrhage secondary to CNV) and atrophic or nonneovascular AMD (presence of any other AMD sign except for CNV). Neovascular AMD accounts for the most cases of severe vision loss, although the atrophic form is the most frequent presentation of the disease –10.A variety of risk factors for AMD have been described. However, the evidence and strength of such associations are widely variable, probably due to the difficulty of measuring some of these factors in clinical practice , 12. AdvRisk factors may be classified as modifiable and nonmodifiable . They caThe purpose of this review is to analyze the current scientific evidence of smoking as an independent risk factor in AMD and the relevance of advising patients to quit smoking for their visual health.A systematic review of all of the peer-reviewed articles indexed in PubMed about smoking and age-related macular degeneration was performed. The analyzed data were summarized classifying them into four main headings: reported epidemiological association between smoking and AMD; studied mechanisms for toxic damage to the retina and choroid induced by smoking; smoking and biomarkers in AMD; and treatment considerations for AMD and smoking.Smoking is a major modifiable risk factor for AMD. The association between smoking and AMD has been consistently demonstrated in many epidemiological studies carried out within different populations in the last decades confirming previous clinical impressions. Cross-sectional studies and prospective cohort studies have described the natural history of the disease and its associations with risk factors, where smoking has been the most consistent factor associated with geographic atrophy and neovascular AMD.Cross-sectional studies examining the association between smoking and AMD include two American, three European, and two large Australian populations. Further studies also provided additional information about smoking as a risk factor for AMD in different ethnic groups and geographic areas . PopulatThe Beaver Dam Eye Study recruited 4771 patients from Beaver Dam  from 1988. After controlling subjects for age and passive smoking, higher rates of neovascular AMD in current-smokers compared to those who had never smoked independently of gender were evidenced .More recently, the study on the large Beaver Dam Offspring Study (BOSS) cohort found a prevalence of AMD of 3.4%. After controlling subjects for age and gender, a history of current-smoking and greater numbers of pack-years smoked were associated with early AMD .The Rotterdam Study is a single-center prospective study of the population aging over 55 years in Rotterdam (The Netherlands). A total number of 6251 participants were included from 1990 until 1993. Current- and former-smoking was associated with an increased risk of neovascular AMD. This increased risk was present up to 20 years after cessation of smoking. This association was not present in persons aging 85 years and older .The Blue Mountains Eye Study was conducted in Sydney  between 1992 and 1994. A total number of 3654 patients were included. A highly significant association between current-smokers and geographic atrophy and neovascular AMD was evidenced . MitchelPooled data from the aforementioned trials from three different continents provided strong and consistent evidence that tobacco smoking is the main known preventable exposure associated with any form of AMD. Depending on the type of AMD, current-smokers have 2- to 4-fold increase in risk for AMD when compared to patients that never smoked .The Age-Related Eye Disease Study (AREDS) is a longitudinal multicenter study of the natural history of AMD and cataract in 4757 participants recruited between 1992 and 1998. One of the main findings of the AREDS was that smoking was associated with all of the three more severe AMD groups .The EUREYE Study is a cross-sectional study that included 4,750 participants in 2003 from seven centers across Europe. Current-smokers had increased odds of any type of late AMD, and bilateral AMD was also more likely to have a history of heavy smoking. The attributable fraction for AMD due to smoking was 27%.The Eye Disease Case-Control Study of risk factors for neovascular AMD studied 421 cases and 615 controls between 1986 and 1990, finding a strong association between current cigarette smoking and incidence of AMD .The POLA (Pathologies Oculaires Liées à l'Age) Study included 2196 participants in France. Current- and former-smokers were found to have an increased prevalence of late AMD. This risk was higher in those patients who smoked 20 pack-years and more . The oddOther recent case-control studies show a significant association between both current- and former-smokers and early AMD and polypoidal choroidal vasculopathy in Asian patients , 35.A novel population of monozygotic twins with discordant phenotypes of AMD was studied. Smokers had a twice-fold increased risk of AMD implicating that factors other than DNA sequence are involved in the etiology of AMD.Prospective cohort study design is most suitable in order to demonstrate that smoking precedes AMD development. There are three key cross-sectional studies that were subsequently extended into longitudinal studies. The length of the follow-up period is the strength of two of them.At 5-year followup, the Beaver Dam Eye Study reported that men who smoked greater amounts of cigarettes were more likely to develop early AMD. These results were confirmed over a 10-year followup and also evidenced that they were more likely to have progression of the disease , 37.The 5-year incidence of any late AMD lesions found in current, past, or never-smokers in the Blue Mountains Eye Study was 3.1%, 1.2%, and 1.4%, respectively. After adjusting for age, current-smokers had an increased risk of incident geographic atrophy  and any late lesions  . The lonThe Physicians' Health Study  and the The AREDS cohort with a median followup of 6.3 years estimated that a larger amount smoked was statistically significantly associated with the incidence of geographic atrophy due to AMD . More reThe risk of smoking has also been estimated in a meta-analysis from six prospective cohort studies, five case-control studies, and five cross-sectional studies. Significant increases in AMD risk were seen for current- versus never-smokers. The odds ratio for case-control studies was 1.78 (95% CI: 1.52–2.09), and it was 3.58 (95% CI: 2.68–4.79) for cross-sectional studies. The relative risk (RR) obtained through analysis of prospective cohort studies was 1.86 (95% CI: 1.27–2.73) . SimilarSeveral studies have investigated the dose-response effect by comparing different levels of smoking classified as pack-years, and most of them confirmed a dose-response effect. The Rotterdam Study and the POLA Study found an increased risk of neovascular AMD in persons who had smoked 10 or more pack-years  and 20 or more pack-years  , 33. TheExsmokers still have an increased risk of developing AMD compared with never-smokers. The Rotterdam Study was the first to reveal that the increased risk of neovascular AMD remained present up to 20 years after cessation of smoking , which wCigarette smoke is known to contain an abundant number of toxic compounds. Some of them are known to be either toxic or mutagenic . Its patα-adrenergic stimulation which may impair blood flow through the choroid [Cigarette smoke promotes pathophysiological processes that contribute to atherosclerosis, including thrombosis, vascular inflammation, and endothelial dysregulation . Nicotin choroid .Nornicotine, a metabolite of nicotine catalyses, can lead to the accumulation of lipofuscin and, therefore, also contribute to the formation of drusen in RPE. Nornicotine can catalyze the alkene isomerization of key retinal intermediates through iminium-ion formation and disrupt proper retinoid homeostasis, revealing an underlying molecular mechanism for tobacco-dependent pathologies, particularly AMD .Cigarette smoke also contains dioxins that are present primarily in the gaseous phase. Most of the toxic effects of dioxins are mediated by the cytosolic dioxin receptor known as aryl hydrocarbon receptor (AhR) , 55. DioThe oxidative damage to the RPE contributes to the development and progression of AMD, and the alterations in the metabolic support of the RPE cause apoptosis of the photoreceptors , 58. Cigin vitro and in vivo findings suggest that HQ-induced oxidative damage is unequivocally associated with an imbalance between VEGF and PEDF [The RPE cells provide support for the structure and function of the outer retina by secreting several cytokines including monocyte chemoattractant protein-1 (MCP-1) , 64. RPEand PEDF  leading and PEDF . RPE celand PEDF –67.The exposure to cigarette smoke and HQ results in RPE membrane blebbing and sub-RPE deposits in mice. In cultured RPE cells, HQ-induced oxidative injury results in reorganization of actin cytoskeleton and blebs formation important for accumulation of deposits –67. Aftein vitro models [in vitro, cell death, significantly reduces viability in both ARPE-19 cells and primary RPE cells, via alterations in mitochondrial integrity, and increases lipid peroxidation [β-galactosidase  cells [β-Gal activity in primary cultured RPE cells [in vivo in RPE cells of old primate eyes [Cigarette smoke extract (CSE) is widely used for o models –70. CSE xidation . Lower cl) cells . SublethPE cells  and in vate eyes .Other molecules are involved in oxidative damage. Exposure of primary human RPE cells to CSE may also lead to significant elevations of Apo J, CTGF, and fibronectin expression, which are senescence-associated biomarkers . All thrOxidative stress is thought to be essential in lipofuscin and drusen formation , 78. AcrPolycyclic aromatic hydrocarbons (PAHs) are one of the most toxic compounds in cigarette smoke . They foin vitro studies of cigarette smoke effect in RPE cells. These cells are treated with different toxic substances derived from cigarette smoke such as HQ [p-dioxin (TCDD) [in vitro experiments to study the effect of exogenous B(a)P [ARPE-19 cells, a spontaneously arising human RPE cell line , are widch as HQ , acroleich as HQ , 85, CSEch as HQ , 72, B . Recentln (TCDD)  and CSE us B(a)P . Other tus B(a)P , 65.in vitro models, the same toxic compounds are used in animals to investigate the effect of cigarette smoke in the retina. C57BL/6-pigmented mice are the most widely used in the literature. Hence, these mice receive HQ orally in drinking water for a period of time [As in  of time  and are  of time . On the  of time , 86, 87. of time .The RPE constitutes a cell monolayer that is crucial to maintain a normal photoreceptor function. RPE cell apoptosis and basal deposits, or accumulations of heterogeneous debris in Bruch's membrane, are two critical histopathologic changes that are well recognized to occur during the development of early AMD . FujiharIn summary, the most important alterations observed are Bruch's membrane thickening, mild basal deposits and enlargement, and loss of basolateral infoldings, which are an established marker of epithelial cell injury. The formation of vacuoles is a second sign of RPE damage.In order to look for the most appropriate therapies and to individualize lifestyle recommendations, it is ideally necessary to integrate the different clinical features, the habits, and, if available, the biomarkers of a certain disease. A biomarker is a characteristic objectively measured and evaluated as indicator of physiologic/pathologic processes or pharmacologic responses.P = .02), so the highest levels of CRP seem to increase the risk of AMD independently of smoking [Different biomarkers have been studied in AMD patients –91. Howe smoking .In a subsequent study, Seddon et al. found that smoking had a positive association with some proinflammatory cardiovascular disease biomarkers such as CRP, interleukin 6, soluble tumor necrosis factor alpha receptor 2, soluble intercellular adhesion molecule-1 (ICAM-1), and apolipoprotein B (ApoB) but not with vascular cell adhesion molecule-1 (VCAM-1) or lipoprotein(a) in nonexudative AMD .Gibson et al. assessed the levels of plasmatic complement component C1 inhibitor (C1inh), and they found that C1inh levels were higher in smokers compared to nonsmokers . These rOther biomarkers have been studied separately in smokers versus nonsmokers and AMD patients versus normal patients and a parallel increase  and decrease  of considered markers have been found in both kinds of studies , 94–98.There is limited information about the specific treatment of dry and wet AMD in smokers. The use of antioxidant supplementation consisting of vitamin C (500 mg), vitamin E , beta-carotene (15 mg), zinc (80 mg), and copper (2 mg) demonstrated reduction of the risk of progression to advanced dry AMD in the AREDS Study with an average followup of 6.3 years. Some evidence suggested that smokers taking beta-carotene supplementation had an increased risk of lung cancer , 100. HoMore recently, the AREDS2 Study showed that the addition of omega-3 fatty acids and/or lutein+zeaxanthin to the the original AREDS formulation only reduces by 10% the risk of progression to advanced dry AMD or neovascular AMD . MoreoveIn addition, the influence of smoking on the visual outcomes in cases of neovascular AMD treated with intravitreal vascular endothelial growth factor inhibitors has also been analyzed. Lee et al. evidenced that smokers had a statistically significant higher risk (7-fold increase) for a poor response to intravitreal therapies , whereasAge-related macular degeneration (AMD) is the commonest cause of irreversible visual loss in the Western World –6. AMD iAmong them, cigarette smoking is a proven risk factor for both development and progression of AMD –43, as wRecently, genetic testing has arisen as an option to provide patients with a certain risk profile based on their own genetic phenotypes in the high-risk genes for AMD –108. ThiIn the situation described above, we believe that institutional support to disseminate the relevance of cigarette smoking in terms of visual health is warranted. Very few countries show health warnings on cigarette packets related to this issue (“SMOKING CAUSES BLINDNESS”), whereas several other health issues warnings are usually included.


The promyelocytic leukemia tumor suppressor gene (PML) critically regulates several cellular functions that oppose tumorigenesis such as oncogene-induced senescence, apoptosis, the response to DNA damage and to viral infections. PML deficiency occurs commonly in a broad spectrum of human cancers through mechanisms that involve its aberrant ubiquitination and degradation. Furthermore, several viruses encode viral proteins that promote viral replication through degradation of PML. These observations suggest that restoration of PML should lead to potent antitumor effects or antiviral responses. In this review we will summarize the mechanisms involved in PML degradation with the intent to highlight novel therapeutic strategies to trigger PML restoration. As a result, all of the PML isoforms contain a conserved N-terminal region consisting of the RING, B-boxes, coiled-coil (RBCC) motifs, and the SUMO Binding Domain (SBD), and they all differ in the central and C-terminal regions , also called PML Oncogenic Domains (POD), Nuclear Domain 10 (ND10), or Kremer bodies , apoptosis and cellular senescence, tumor suppression, viral pathogenicity, and DNA repair  pathway in human fibroblasts  mediates selective uploading of the PML mRNA onto polyribosomes and its selective translation, leading to p53 independent PML upregulation. These data suggest that selective protein translation plays an important role in mediating an efficient oncogene-induced replicative senescence response , AKT, and the protein phosphatase 2a (PP2a) at mitochondria-associated membranes (MAMs). Here PML regulates IP3R-mediated Ca2+ release from the endoplasmic reticulum with the consequent induction of apoptosis  and hypoxia-inducible factor 1 (HIF1). PML achieves this effect by repressing HIF1 transcriptional activity, and controlling HIF1 accumulation by the negative regulation of the AKT-mTOR pathway , homologous recombination repair (HR), as well as the alternative lengthening of telomerases (ALT)  has been shown to phosphorylate PML on several residues. These modifications have been implicated in the degradation of PML upon treatment with arsenic trioxide (ATO)  inhibitor trichostatin A (TSA), suggesting a role of PML acetylation in TSA-induced apoptosis  of APL is a reciprocal and balanced translocation giving rise to both PML-RARA and a RARA-PML fusion proteins. As a result of this translocation, both PML and RARA are reduced to hemizygosity. Extensive work from several laboratories has characterized the properties and mechanisms of action of PML-RARA  located in the PML degron, leading to PML ubiquitin-mediated degradation. Accordingly, PML mutants that cannot be phosphorylated by CK2 show a resistance to ubiquitin-mediated degradation and increased tumor suppressive functions. In this process, the activity of the p38 MAPK is required in order to activate CK2, and pharmacological inhibition of CK2 enhances the PML tumor suppressive activity in vitro and in vivo. However, some links are missing from this working model. For example, the ubiquitin E3-ligase involved in the PML ubiquitination is still unknown. Moreover, the initial report, did not determine whether SUMOylation, which has been involved in PML degradation in cells exposed to ATO, is involved in CK2 mediated PML degradation.This work described how oncogenic stress through the activation of CK2 is able to induce PML degradation in cancer cells both SUMOylation has been implicated in the regulation of both PML and PML-RARA  involved in the final process of PML ubiquitination and degradation in cancer cells is still unknown and future investigations should be directed to its/their identification. Finally, it is not clear yet if the ubiquitination occurs on PML itself, or on the SUMO chains as described for PML-RARA , a mediator of IFN-induced cell death. The KLHL20-dependent DAPK ubiquitination is suppressed in cells stimulated with IFN, which induce the KLHL20 sequestration into the PML-NBs  and the Peptidyl-prolyl cis/trans isomerase Pin1 to mediate PML degradation in hypoxic conditions. Under hypoxia, HIF1 induces the transcription of KLHL20, increasing its accumulation into PML-NBs. In this scenario, two different post-translational modifications of PML make possible its interaction with KLHL20: first, PML isoform I (PML-I) is phosphorylated on serine 518 by CDK1 and CDK2. Second, phosphorylated PML-I undergoes Pin1-dependent prolyl-isomerization. Notably, the authors reported that hypoxia-dependent PML degradation, not only attenuates PML tumor suppressor activity, but also participates to a feedback mechanism that maximizes the production of HIF1 during hypoxic stress. These data are consistent with the observation that in human prostate cancers PML inversely correlates with the over expression of HIF1, KLHL20, and Pin1  exist; thus, this strategy could be tested in clinic.E6AP was the first mammalian ubiquitin E3-ligase to be identified. Initially, E6AP was identified as the ubiquitin E3-ligase that cooperates with the human papillomavirus (HPV) protein E6 to promote p53 degradation  led to the rapid disruption of the PML-NBs . In this case, expression of BZLF1 alone is sufficient to induce PML-NBs disruption and the loss of the SUMOylated forms of PML. Interestingly, BZLF1 can be SUMOylated, and it has been reported that BZLF1 competes with PML for its SUMOylation. This observation suggests that if a cellular component of the SUMOylation machinery is limiting, BZLF1 could inhibit PML SUMOylation by the direct competition with the limiting factor (Adamson and Kenney, Taken together, these data suggest that the disruption of PML-NBs and the degradation of PML are an important step for the lytic cycle of all herpes virus (Adamson and Kenney, Other viral infections have also been association with the disruption of PML-NBs Everett, . For exaThe antiviral activity of PML resides on its ability to re-organize the PML-NBs in order to activate an efficient antiviral response. For this reason, it has been suggested that the ability of certain virus to interact with the PML-NBs formation inducing PML down-regulation is a strategy to evade the antiviral effect of PML (Geoffroy and Chelbi-Alix, in vivo. We reason that these studies will most likely benefit from the availability of faithful and genetically defined mouse cancer models and correlative studies in primary human samples. We are looking forward to these developments with the expectation that pharmacologic targeting of the networks that control PML degradation will lead to specific therapies for cancer and for several viral infections.Promyelocytic leukemia tumor suppressor gene is a key tumor suppressor and its inactivation through aberrant degradation has been found in several human cancers types. Moreover, PML downregulation plays an important role in promoting viral infections. A large body of literature indicates that PML is rarely mutated and its downregulation occurs predominately at the post-translational level. Several pathways have been reported to trigger PML ubiquitination and degradation Figure . This siThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alcohol problems are a serious public health concern, and few problem drinkers ever seek treatment. The Internet is one means of promoting access to care, but more research is needed to test the best types of interventions to employ. Evaluation of Internet-based interventions that contain a variety of research-validated cognitive-behavioral tools, which have been shown to be helpful to those with more severe alcohol concerns, should be a priority.To evaluate whether providing access to an extended Internet intervention for alcohol problems offers additional benefits in promoting reductions in alcohol consumption compared with a brief Internet intervention. The hypothesis for the current trial was that respondents who were provided with access to an extended Internet intervention (the Alcohol Help Center [AHC]) would display significantly improved drinking outcomes at 6-month follow-up, compared with respondents who were provided with access to a brief Internet intervention (the Check Your Drinking [CYD] screener).A single-blinded randomized controlled trial with a 6-month follow-up. A general population sample of problem drinkers was recruited through newspaper advertisements in a large metropolitan city. Baseline and follow-up data were collected by postal mail.P = .046).A volunteer sample of problem drinkers of legal drinking age with home access to the Internet were recruited for the trial. Of 239 potential respondents recruited in 2010, 170 met inclusion criteria . Follow-up rates were 90.0% (153/170) with no adverse effects of the interventions reported. A repeated-measures multivariate analysis of variance of the outcome measures using an intent-to-treat approach found a significantly greater reduction in amount of drinking among participants provided access to the AHC than among participants provided access to the CYD  Alcohol is the third-leading cause of preventable death . UnfortuInternet-based interventions have been identified as one promising option. Several reviews have concluded that there is a fast-developing evidence base for the efficacy of these interventions -8, partiIn this paper we report results of a randomized controlled trial testing the added benefit of providing access to such an extended Internet-based intervention, which contained an extensive array of cognitive-behavioral tools for problem drinkers (the Alcohol Help Center [AHC]), over the provision of a brief, personalized feedback Internet-based intervention (the Check Your Drinking [CYD] screener). Both of these Internet-based interventions are available free of charge on the Internet, making evaluations of their use of immediate benefit to problem drinkers. In addition, the CYD has already been subjected to four randomized controlled trials, in which the provision of this brief intervention yielded reductions in alcohol consumption among participants in a variety of different settings -12. In tParticipants were recruited through newspaper advertisements in metropolitan Toronto, Canada (May to September 2010) asking for current drinkers interested in helping “in the process of developing and evaluating Internet-based interventions for alcohol users.” Interested potential participants called the telephone number provided and left their name and address to be sent a consent form and a baseline questionnaire by postal mail. The questionnaire contained a graphic describing a standard drink .Those returning the signed consent form and the completed baseline questionnaire were randomly assigned into one of two conditions: to be provided access to the brief Internet-based CYD brief personalized feedback intervention or to be provided access to the extended Internet-based AHC. Access was provided by sending each participant a unique password to be entered into a study-specific online portal . Random numbers where generated in blocks of two using an online random numbers generator  by the author. The random numbers list was used sequentially by a research assistant, who sent out the relevant intervention letter to participants in the order that their consent forms were returned. Participants were blind to the different experimental conditions in the study. Participants were followed up at 6 months and were compensated Can $20 for completing the follow-up questionnaire . Follow-ups were conducted from November 2010 to June 2011. If participants did not return their original follow-up questionnaire within 1 month, they were sent a second questionnaire by postal mail. The research assistant in charge of follow-up was not blind to experimental condition, as the follow-up questionnaires were slightly different . The trial was approved by the standing research ethics committee for the Centre for Addiction and Mental Health.As per protocol, the primary outcome measures were number of drinks consumed in a typical week, highest number of drinks on one occasion in the last 6 months, frequency of drinking, typical quantity of drinking on one occasion, and frequency of drinking 5 or more drinks on one occasion . These last 3 items were combined to form the Alcohol Use Disorders Identification Test-Consumption subscale (AUDIT-C) . For parResults from a related self-help trial  were useThe CYD screener (CheckYourDrinking.net) is a brief and personalized 18-item screener that has been designed to assess quantity and frequency of drinking, and the severity of drinking problems . FollowiDrinkWise [Alcohol and You, developed by the principal investigator of this study [The AHC (AlcoholHelpCenter.net) is a website that has been developed to contain the cognitive-behavioral, motivational, and relapse prevention components that have been found to be effective in well-validated self-help books and other brief interventions. Specific sources for the content include (1) components found in the self-help book rinkWise , (2) exeis study , and (3)is study . The maiis study , (2) an P > .05). Participants’ mean age was 45.2 (SD 12.2), 59.4% (101/170) were male, 58.8% (100/170) had some postsecondary education, 39% (67/170) were married or living with a partner, 55% (94/170) were employed full- or part-time, and more than half  had a yearly household income of Can $30,000 or more. Baseline drinking was heavy, with participants reporting an average AUDIT score of 22.1 (SD 7.6), typical weekly consumption of 31.7 (SD 18.4) drinks, and highest number of drinks consumed on one occasion of 13.7 (SD 7.2). A total of 71.8% (122/170) of participants used the password and logged in to their respective online intervention; however, all participants were included in the analysis in an intent-to-treat approach. Follow-up rates were good, with 90% (n = 153) completing the 6-month follow-up. There were no significant differences (P > .05) between experimental conditions, demographic characteristics, or drinking characteristics in the proportion of participants who completed the 6-month follow-up.A total of 170 participants met inclusion criteria for this trial. Bivariate comparisons compared baseline demographic and drinking characteristics between experimental conditions  and found no significant differences between them . In addition, there was a significant interaction between time and condition . Further univariate tests found that the time × condition interaction was not significant for the number of drinks in a typical week variable , approached significance for the AUDIT-C variable , and was significant for the highest number of drinks on one occasion variable . A multivariate analysis of variance was conducted and found a main effect for time  toward an additional benefit of the AHC over the CYD on the AUDIT-C scores, a scale that is a composite measure of hazardous drinking. Finally, participants in both groups reduced their drinking in a typical week substantially (by 4.9 drinks per week in the CYD condition and 6.3 drinks per week in the AHC condition). These reductions are similar in scope to that seen in an earlier randomized controlled trial comparing the CYD with a no-intervention control [The current trial found a small but significant additional reduction in drinking among participants who were provided with access to the extended Internet-based intervention (the AHC) as compared with participants receiving the brief Internet-based intervention (the CYD) at a 6-month follow-up. The difference specifically had to do with reductions in amount of alcohol consumed during the participants’ heaviest drinking occasion. Given the increased risks associated with heavy drinking situations , this reA strength, and a simultaneous limitation, of this trial was the severity of current drinking problems in the participants  . Thus, wDespite these limitations, the results of this trial are important, as they demonstrate the added benefit of an extended Internet-based intervention over a brief, personalized feedback intervention for problem drinkers. Systematic replications of these findings would allow the development of an adequate research base supporting this highly accessible and cost-effective means of providing assistance to problem drinkers who might otherwise receive no help for this important health concern.


Mycobacterium tuberculosis (MDR-TB) remain poor and this is compounded by high drug toxicity. Little is known about the influence of adverse drug reactions (ADRs) on treatment outcomes in South Africa.Globally treatment outcomes for multidrug-resistant We evaluated the impact of severe ADRs among a prospective cohort of MDR-TB patients in South Africa (2000–2004). The HIV-infected study participants were anti-retroviral naïve.Of 2,079 patients enrolled, 1,390 (66.8%) were included in this analysis based on known HIV test results (39.1% HIV-infected). At least one severe ADR was reported in 83 (6.9%) patients with ototoxicity being the most frequent ADR experienced (38.9%).We found that being HIV-infected but antiretroviral naïve did not increase occurrence of SADRs in patients on second-line anti-tuberculosis drugs. Early screening and proactive management of ADRs in this patient population is essential, especially given the rollout of decentralized care and the potential for overlapping toxicity of concomitant MDR-TB and HIV treatment. One of the challenges facing patients treated with second-line drugs (SLD) is the toxic nature of these drugs An ADR is defined as: any unintended adverse response to a drug occurring at a therapeutic dose and resulting in either death, drug withdrawal, change in the administration of the frequency or dose of the drug, or, that no action is required. The severity of these reactions can range from mild, no intervention required, to the more severe or life-threatening (SADR) where drugs need to be withdrawn, either completely from the regimen or with re-introduction once the SADR has subsided. Proactive and vigilant management of SADRs among patients treated with SLDs are especially important as the access of MDR-TB cases to anti-retroviral treatment (ART) continues to expand and guidelines now recommending treatment of MDR-TB patients with ART regardless of their CD4 count We conducted a prospective DOTS-plus study to examine treatment outcomes among a large cohort across South Africa This study was approved by the South African Medical Research Council's Ethics Committee, the nine Provincial Research Committees  and the Johns Hopkins University School of Public Health Institutional Review Board. Written informed consent was obtained from each study participant before enrolment and the informed consent process was approved by the respective ethical/review boards. Members of the clinical teams at the treatment centres, or the site-specific research coordinator were trained and capacitated to invite patients to participate in the study, obtain informed consent, complete the documentation as well as to countersign and provide MRC with the completed case record forms (CRFs). Data capturing from the case record forms was not done on those patients for whom a signed consent form was not available.Between 2000 and 2004 adults, 18 years and above who presented with a culture proven bacteriological diagnosis of MDR-TB were consecutively enrolled in the DOTS-Plus study Since 2000, most of the MDR-TB treatment centres in South Africa have used standardised programmatic management of MDR-TB (DOTS-Plus) The CRF included an ADR screening tool which as well as listing the 16 most commonly identified drug reactions related to SLD treatment , asked fData were entered into EpiData®, version 6.1 and analyzed in Stata®, version 11. Frequencies were used to compare differences in demographic and baseline clinical data between patients experiencing SADRs and those not as well as for describing the frequencies and range of SADRs. Significance of differences were tested by chi square or Fischer's exact.A total of 2079 patients were enrolled in the DOTS-Plus cohort. Of these, 1390 (66.8%) had a known HIV test result  and did While the majority of patients 63/83 (75.9%) experienced one SADR during their duration of treatment, 15/83 (18%) experienced two SADRs while five patients (6.0%) experienced a total of three. Decreased hearing, leading to withdrawal of the injectable was the most frequent reaction observed 42/108 (38.9%), followed by psychosis in 16/108 (14.8%), and the remainder of the 14 reactions being reported less than ten times .Cycloserine/terizidone, together with ofloxacin were the drugs implicated in those patients in whom psychotic side effects were experienced. Of the 16 SADRs which were psychotic in nature, 8 were observed during the intensive phase while the remainder were experienced during the continuation phase. The majority of all the other side effects occurred during the first four months of treatment . Fifty sThis is the first evaluation of SADRs among a cohort of HIV-infected MDR-TB patients, on standardised treatment regimens in South Africa in the absence of ARV therapy per se predisposes a patient to experience SADRs; and this study confirms that the rate and range of SADRs amongst HIV-infected ART naïve and uninfected patients are similar. However, HIV-uninfected cases experienced slightly more, (1.35) serious adverse drug reactions on average, compared to the 1.25 in HIV-infected cases.We do not believe that being HIV-infected There were relatively fewer SADRs experienced in our cohort compared to other MDR-TB treatment cohorts We identified SADRs through passive screening, which could be prone to clinical subjectivity or patient self-reporting/tolerance levels. The management of adverse drug reactions also depended on the clinician's access to other screening tests, e.g. audiology, ancillary treatment and other patient psychological support systems. The second most common adverse drug reactions were the psychotic episodes 16/108 (14.8%), and in contrast to the other drug reactions, occurred as often after the first four months of treatment as it did during the last 16 months.We note several limitations in our findings. We have reported a relatively small number of ADR's compared to other studies but this could be due to the fact that we were only reporting on SADRs. We also may have missed a range of drug reactions that showed association with baseline clinical factors or with specific treatment outcomes. Furthermore, it could not be guaranteed that clinicians from all the MDR-TB centres were consistent in their reporting of SADRs despite close monitoring, ongoing education and encouragement to report ADRs. Furthermore, excluding a number of MDR-TB patients without any HIV information may have biased our results. Access to CD4 counts of the HIV-infected cases could have improved the description of the cohort and allowed us to explore those associations. During the DOTS-Plus study period the case loads in the MDR-TB treatment centres were much lower than they have been in the past few years Towards the end of the DOTS-Plus study and onwards, HIV-infected MDR-TB cases had access to anti-retrovirals with overlapping toxicities of the two regimens being recognized as a challenge in universal access of combined therapy The observation that the majority of the SADRs occurred during the first four months of treatment has ramifications for MDR-TB management in South Africa as the country moves towards decentralised care with treatment of patients nearer to their homes and where they can receive support from the community in which they reside. Similarly, our findings showing that an HIV-uninfected case could experience more adverse drug reactions than an HIV-infected case, means that HIV-uninfected cases should not be neglected from screening.These models of care should include tools specific for the vigilant screening of serious adverse drug reactions by all clinicians as well as an appropriate and timely response once identified. An integral part of the successful management of adverse drug side effects within these models, especially for those co-infected, will be the improved treatment literacy of patients, and the equipping of close contacts and treatment supporters on providing the necessary treatment support to ensure case holding and in turn improved treatment outcomes.


Oral squamous cell carcinoma (OSCC) patients have a high mortality rate; thus, new clinical biomarkers and therapeutic options are needed. MicroRNAs (miRNAs) are short noncoding RNAs that regulate posttranscriptional gene expression and are commonly deregulated in OSCC and other cancers. MicroRNA-21 (miR-21) is the most consistently overexpressed miRNA in several types of cancer, and it might be a useful clinical biomarker and therapeutic target. To better understand the role of miR-21 in OSCC, paraffin-embedded tumor tissue samples from 86 patients with primary OSCC were analyzed by in situ hybridization. We found that miR-21 was primarily expressed in the tumor stroma and in some tumor-associated blood vessels with no expression in the adjacent normal epithelia or stroma. Using image analysis, we quantitatively estimated miR-21 expression levels specifically in the stroma of a cohort of OSCC samples. These miR-21 levels significantly correlated with disease free survival with the highest levels being located in the stroma. Stromal miR-21 expression was independently associated with a poorer prognosis, even after adjusting for clinical parameters  in a multivariate analysis. In summary, we have shown that miR-21 is located in the carcinoma cells, stroma and blood vessels of tumors, and its expression specifically in the stromal compartment has a negative prognostic value in OSCC. Oral squamous cell carcinoma (OSCC) is a serious disease with a five year overall survival of 60% MicroRNAs (miRNAs) are short, single stranded, non-coding RNAs involved in the posttranscriptional regulation of target messenger RNAs. One miRNA can target multiple different mRNAs, and their expression pattern may change in different disease states. miR-21 regulates cell proliferation, apoptosis and the epithelial to mesenchymal transition during neoplastic progression and is up regulated in a variety of cancers Different cancer-associated fibroblasts have been described, but the one that is most consistently shown to have an adverse effect on prognosis are myofibroblasts In this study, we demonstrate that OSCC miR-21 expression is predominately localized to tumor stromal cells with colocalization between miR-21 and α-smooth muscle actin. We further demonstrate that the levels of miR-21 significantly correlate with disease free survival.The Scientific Ethical Committee of the Capitol Region of Denmark, and the Data Protection Authority approved this study (ID nr: H-2-2012-050). Since a large group of patients had died or were severely ill, it was judged by the ethical committee that it would cause more distress than good to be informed about the project; it was therefore judged not to be necessary to obtain informed consent. Patient records were anonymized and de-identified prior to analysis. We selected archived tissue from 111 patients diagnosed with either tongue cancer or floor of the mouth cancer from 2000–2008 who were treated at Rigshospitalet Copenhagen, Denmark using the DAHANCA database Formalin-fixed paraffin-embedded (FFPE) tissue was cut into 5 µM sections under RNAse free conditions and mounted on Super Frost slides. For the detection of miR-21, the miRCURY LNA microRNA ISH optimization kit (FFPE)  was used according to the manufacturer’s recommendations. Briefly, slides were deparaffinized and rehydrated before being placed in a hybridizer . Tissue sections were treated with 15 mg/µl proteinase-K at 37°C for 8 minutes and hybridized with a digoxigenin (DIG)-labeled LNA probe at 55°C for 120 min. Slides were subsequently washed in decreasing concentrations of a saline-sodium citrate (SSC)-buffer at the hybridization temperature. Subsequently, DIG blocking reagent was applied for 15 min followed by incubation with an alkaline phosphatase-conjugated anti-DIG antibody. The probes were visualized with alkaline phosphatase substrate  over night at room temperature. Slides were counterstained with nuclear fast red  for 1 minute at room temperature, dehydrated in alcohol and mounted with Eukitt mounting medium . The double DIG-labeled miRCURY LNA miRNA detection probes used to detect miR-21 in this study were full-length 22-mers .We validated the specificity of the miR-21 expression by comparing the miR-21 expression in the samples to the expression of a nonspecific sequence (scrambled), which gave no expression , thus exImmunohistochemistry for cytokeratin was carried out using the Ventana Benchmark Ultra autostainer  with the optiView detection kit and the pan-cytokeratin antibody CK-WSS  in a 1:4000 dilution. The immunohistochemistry and in situ hybridization was performed on serial sections with a maximum of 5µm apart.A total of 10 samples were randomly chosen to undergo combined fluorescent in situ hybridization (FISH) and immunofluorescence (IF) staining. The protocol for combined FISH and IF was identical to the above-mentioned ISH with a few modifications Samples used for miR-21 in situ hybridization were scanned using the panoramic axioscan midi, . Samples used for cytokeratin immunohistochemistry were scanned using the Axio Scan.Z1 . The digital files were opened in the image analysis module Visiomorph in the Visiopharm software . The ISH and cytokeratin-stained sections were subsequently aligned using the image analysis module TissueAlign. TissueAlign is able to perform a cell-to-cell alignment of serial sections with any kind of staining, which enables so-called virtual double staining. We then manually delineated the entire tumor area on each patient sample as a region of interest, in which the subsequent analyses were performed. We used the cytokeratin stained sections to delineate tumor cells. We did this by setting a threshold for the brown, 3,3'-Diaminobenzidine (DAB), signal, to a level in which stroma was separated from the tumor, as indicated in Disease free survival was defined as time from operation to recurrence. Survival curves were plotted using the Cox regression survival curves, which show relationships between miR-21 expression and disease free survival (DSF). Multivariate Cox proportional hazard regression analysis was performed using the four prognostic factors that were found to be statistically significant in the univariate analysis followed by backward elimination. The proportional hazards assumption was tested using partial residuals from the variables plotted against time to recurrence. TBS, TBT, perineural invasion, stage and N-stage fulfilled the proportional hazards assumption. Statistical analysis was carried out using SPSS 20 with an overall significance level of P<0.05. P-values from the Cox regression analysis were from log-rank tests. Correlation analyses were performed using Spearmans rank correlation.We found little to no miR-21 stroma expression in 10 of the 86 samples as observed from the TBS results in The miR-21 expression was observed in stromal fibroblast like cells  and endoThere was a high degree of overlap between miR-21 and α-SMA staining . HoweverA significant correlation between miR-21 expression in the stroma (TBS) and gender was observed, in that males had significantly higher TBS values. In addition a correlation between high TBS and an increased risk of relapse was found . Also a High stromal miR-21 expression is associated with a decreased disease free survival We did not find any significant differences in tumor subsite with regard to clinical or pathological characteristics, and we therefore chose not to stratify survival analyses of clinical and pathological characteristics based on subsite. First, we examined the relationship of miR-21 expression and disease free survival and chose to divide patients into tertiles based on their TBS  in relation to clinical and histological characteristics . Only noWe also performed a multivariate Cox-regression analysis of disease free survival related to miR-21 expression levels in combination with other clinical parameters derived from the univariate analysis, which were shown to be of importance for DFS  and perfIn the present study, we correlated miR-21 expression specifically in the tumor stroma, measured as the product of miR-21 intensity and the area occupied by miR-21 , TBS, with patient survival and found that patients with high miR-21 expression have a shorter disease-free survival than patients who have low miR-21 expression. Clinically we observed a correlation between gender and miR-21 expression which have previously been observed in colon cancer The distribution of miR-21 expression varies with tumor type The TBS measurement used to quantitate miR-21 expression (DB/R) discriminates between stroma and tumor. To exclude the possibility that the poor prognosis seen in the patients overexpressing miR-21 was simply related to a larger stromal component of their tumors, we also investigated whether increasing stromal tumor content was related to a reduced disease free survival. This was however seen not to be related to a decreased disease free survival. In addition we were able to quantitate the amount of miR-21 specifically in the epithelial tumor cell compartment, to see if this was related to disease free survival. However, miR-21 expression in the tumor compartment was not related to a decreased disease free survival. Thus, it is interesting that the poor prognosis related to miR-21 expression reflects a pathological process in the stromal compartment. This observation is in agreement with previous findings, which show that high levels of myofibroblasts in the stroma of OSCCs are strongly associated with increased mortality from oral carcinomas regardless of disease stage alone was not sufficient to drive the differentiation toward myofibroblasts Myofibroblasts can be classified into two types. The first type is a protomyofibroblast, which is a partly differentiated fibroblast that contains actin stress fibers but no immunohistochemically detectable α-smooth muscle actin The present study builds on prior studies, which have demonstrated that neoplastic progression is not solely determined by the cancer cells but also by stromal processes surrounding the tumor Our data shows that miR-21 expression was in the center of the tumor and not at the invasive front. This finding is in accordance with previous studies on the distribution of myofibroblasts and fibroblast activating protein (FAP), another marker for activated fibroblasts, which is highly expressed within colon carcinomas and correlates with patient survival Histological investigation of tumors has shown that tumor budding, which are small clusters of tumor cells at the invasive front, is strongly associated with a poor patient survival In addition to its function as a biomarker for disease free survival in OSCC, miR-21 could potentially be exploited as a therapeutic target. Promising in vivo studies have shown that anti-miR-21 PNAs could potently reduce miR-21 expression in xenograft models of breast cancer, which resulted in a reduced cancer growth In conclusion, to our knowledge we have for the first time provided the location of miR-21 and related this location to the presence of myofibroblasts in OSCC. In addition, we have shown that the expression of miR-21 in OSCC does not vary significantly with tumor site (tongue cancer or floor of the mouth cancer). We also find that increased miR-21 expression in the tumor stroma may be used as an independent prognostic biomarker (hazard ratio 2.7) for disease free survival in OSCC.


Methionine Sulfoxide Reductase A (MsrA), an enzyme in the Msr gene family, is important in the cellular anti-oxidative stress defense mechanism. It acts by reducing the oxidized methionine sulfoxide in proteins back to sulfide and by reducing the cellular level of reactive oxygen species. MsrA, the only enzyme in the Msr gene family that can reduce the S-form epimers of methionine sulfoxide, has been located in different cellular compartments including mitochondria, cytosol and nuclei of various cell lines.In the present study, we have isolated a truncated form of the MsrA transcript from cultured mouse embryonic stem cells and performed eGFP fusion protein expression, confocal microscopy and real time RT-PCR studies.Results show a different expression response of this truncated transcript to oxygen deprivation and reoxygenation treatments in stem cells, compared to the longer full length form. In addition, a different subcellular localization pattern was noted with most of the eGFP fusion protein detected in the cytosol.One possibility for the existence of a truncated form of the MsrA transcripts could be that with a smaller protein size, yet retaining a GCWFG action site, this protein might have easier access to oxidize methionine residues on proteins than the longer form of the MsrA protein, thus having an evolutionary selection advantage. This research opens the door for further study on the role and function of the truncated MsrA embryonic mouse stem cells. Free radical damage of cellular components, including proteins, has long been recognized in physiological aging and disease conditions. One of the cellular defense mechanisms to reduce the oxidized residues in proteins, and thus restore their functions as well as reduce oxidative stress, relies on the methionine sulfoxide reductase (msr) family genes, of which three MsrB genes , have been identified in mammals . Only onAlthough only a single MsrA gene is found in mammals, the corresponding protein is found to localize in multiple cellular compartments . FurtherTo date, most of the studies on MsrA isoforms are focused on the 5' terminus where a mitochondrial signal peptide is alternatively presented in different isoforms dictating whether or not the protein products are localized in the mitochondria ,12,13. AWe have consistently found a MsrA transcript from alternative splicing at the 3' end, skipping exon 5, thus producing a shorter isoform with a presumably truncated protein product containing the conserved catalytic active site in cultured mouse embryonic stem cells. Due to the importance of anti-oxidative stress mechanisms in stem cells, we have performed studies on this isoform in its expression pattern in normal culture conditions and its response to oxygen depletion/reoxygenation conditions in mouse embryonic stem cells.The mouse embryonic stem (MES) cells (CCE-24) were routinely grown on 0.1% gelatin-coated dishes in Dubecco's Modified Eagle's medium (DMEM) containing 15% heat-inactivated fetal bovine serum , 10 ng/ml human leukemia inhibitory factor (LIF) , and monothioglycerol  at 4.5 × 10-4 M . Cells wThe anoxic treatment of mouse embryonic stem cells was performed by incubating the cells in an anaerobic chamber  supplied with 90% nitrogen gas, 5% hydrogen gas and 5% carbon dioxide at 37°C. Cells were removed after selected periods of treatment and incubated again in a regular cell culture incubator at 37°C for designated times.Cloning of the truncated form of MsrA cDNA and construction of MsrA-truncated-eGFP fusion expression plasmid followed methods routine in our laboratory and recently published .MsrA cDNA was amplified from the total RNA extracted from cultured embryonic stem cells using the primer pairs of: MsrA-for: 5'-cctggctgcggaggtggagaaac and MsrA-rev: 5'-atggccatcgggcaggaaactcc. The 744bp DNA band was gel purified and ligated into pGEM-T easy vector . After sequencing to confirm gene sequences and rule out mutations, PCR was performed again using the following primer pairs to amplify the truncated form of MsrA and introduce a BamHI cutting site at the 3' end (MsrA-for: 5'-cctggctgcggaggtggagaaac and MsrA-BamHI-rev: 5'-tggggccaaggatccgctttgaaagaacc). The amplicons were gel purified and ligated into pEGFP-C vectors  to construct the truncated MsrA-eGFP expression plasmid. The truncated MsrA cDNA in the final plasmid was sequenced again to confirm the open reading frame.After the MsrA-truncated-eGFP fusion expression plasmid was transfected into cells for three to five days and after confirmation of the GFP fluorescence signal by epi-fluorescent microscopy, 500 nM Mitotracker (Molecular Probe (Invitrogen), Carlsbad, CA) was diluted with complete culture medium and added to the cells. The cells were incubated for 30 minutes and checked under epi-fluorescence microscopy to confirm that they were well stained by the dye. The Mitotracker medium was discarded. Cells were washed with PBS and fixed in 1% Formaldhyde in PBS for 15 minutes at room temperature. Cells were rinsed with PBS twice and incubated in a 1/10000 dilution solution of DAPI (10 mg/ml) in PBS for 15-20 minutes. Cells were then mounted in the mounting solution Pro-Long , air-dried for 2 days in the dark, and coverslips were sealed to the slides with fingernail polish. Confocal microscopy was carried out using a Carl Zeiss confocal microscope at the University of Miami Diabetes Research Institute. Five micron Z-series were scanned for each sample.Real time RT-PCR experiments were performed according to our published procedures ,17. The MsrA-long form-for: 5'-TCTGGGTCTTGAAAGGAGTGTA;MsrA-long-form-rev: 5'-AGGTATTGCTGGTGGTAGTCTTC; Amplicon size: 395bp.MsrA-truncated-for: 5'-CGACCCGACCCAAGGTTCTTTCA;MsrA-truncated-rev: 5'-GCCATCGGGCAGGAAACTCCAG; Amplicon size:168bpβ-actin-for: 5'-CCACTGCCGCATCCTCTTCCTC;β-actin-for: 5'-CAGCAATGCCTGGGTACATGGTG; Amplicon size: 249bp.From our previous studies on MsrA, we haveBG970953.1) with the same intron splicing pattern as the truncated form of MsrA cloned from embryonic stem cells, indicating this isoform might not be stem cell specific. The comparison between the EST sequence and truncated MsrA is illustrated in Figure The RT-PCR products of the smaller band were recovered from agarose gels and ligated to the pGEM-T-easy vector . After DNA sequencing, the smaller form sequence was aligned and compared to the full length cDNA from the Genebank. A deletion of the fifth exon 113bp) was found in the smaller form which ends up with a frame shift in the sixth exon directly attached to the fourth that generated a new premature stop codon Figure . The totbp was foThe long form of the MsrA full length protein conjugated with the eGFP tag has been studied previously in our laboratory and was found to be predominantly mitochondria located with detectable signals from the cytosol . CurrentRecent studies from Lee et al.,  and PascOur studies using real time RT-PCR on the long form MsrA expression responses to oxygen deprivation and reoxygenation show that the expression levels decrease along with longer anoxia/reoxygenation treatment combinations Figure . HoweverIn summary, a c-terminal truncated form of MsrA has been cloned from mouse embryonic stem cells due to the skipping of exon 5 and subsequent frame shift in exon 6, generating a premature stop codon. The truncated protein shows a different subcellular localization and pattern of expression response to anoxia/reoxygenation treatment on the stem cells. Further study on the enzymatic activity of this peptide is needed to consider it as a functional isoform. One possibility for the necessity of having such a truncated form could be that with a smaller protein size and retained GCWFG active site, this protein might have easier access to the oxidized methionine residues on proteins with structure hindering the access for the long form protein, thus having an advantage to be evolutionarily selected and maintained. Since the truncated protein itself does not contain the c-terminal cysteines, whether the final relieving of the oxidation step needs the long form MsrA is unknown, although we did observe that heterodimers formed between long form and truncated proteins in Western blotting experiments.The authors declare that they have no competing interests.PJ was involved in the conception and design, provision of study materials, collection and assembly of data, data analysis and interpretation and manuscript writing for this paper. CZ participated in the conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of manuscript for publication. YJ - Provided study materials, collection of data, data analysis and interpretation and manuscript writing for the paper. KW was involved in the conception and design, financial support, administrative support, provision of study materials, collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript. XH Participated in the collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript. AK was involved in manuscript writing and data analysis. SL was involved in the conception and design, administrative support, data analysis and interpretation, manuscript writing and final approval of the manuscript. LL Oversaw the research including the conception and design, financial support, administrative support, provision of study materials, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript as well as serves as the Principal Investigator of laboratory.


The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. N-[2-(Fmoc)aminoethyl]glycine benzyl ester (Fmoc-AEG-OBn) backbone 1 has yet to be reported. Synthesis of the Fmoc-protected carboxylic acid backbone Fmoc-AEG-OH has been reported ..6 in 80%6 requires only aqueous workup, and the deprotection step requires simple concentration and removal of TFA via formation of an azeotrope with toluene. The alkylation to produce 9 and the Fmoc protection to give 1 require flash column chromatography, but a large difference in Rf between the products and impurities makes purification possible using only a silica plug.A key to the scalability of our synthetic route is the relatively facile purification of the synthetic intermediates and final product. The Boc protection step to give 1 is a key intermediate in the synthesis of Fmoc-protected PNA monomers having modified nucleobases. However, to date, a scalable and cost-effective synthetic route to this molecule has yet to be reported in the literature. An efficient synthesis of the Boc-protected backbone has been reported, but our attempts to utilize this synthetic route with Fmoc in place of Boc failed to give product, likely due to the instability of synthetic intermediate 8. Rather, synthesis of 1 can be initiated using a Boc protecting group, followed by a protecting group swap to provide the Fmoc-protected product. The first two steps of our synthetic route mirror those of the published synthesis for the Boc-protected monomer [4. We were able to perform this transformation by generating the free base of 4 at reduced temperature and in the presence of Fmoc-OSu, enabling Fmoc protection to effectively compete with cyclization, providing 1 in moderate yield.Fmoc-protected PNA backbone  monomer . However1. Using this route, we have rapidly synthesized 31 g of 1 using inexpensive starting materials and only minimal purification. The overall yield for our synthetic route is modest at 32%; however, the low cost of starting materials and ease of purification enable this synthesis to be tractable on a large scale. Having a convenient route to access 1 is anticipated to ease the synthesis of new Fmoc-protected PNA monomers, presumably furthering the exploration of PNA having unique modified nucleobases.In summary, we describe here a novel route to the PNA backbone Fmoc-AEG-OBn


Hillslope instability has been thought to be one of the most important factors for landslide susceptibility. In this study, we apply geomorphic analysis using multi-temporal DEM data and shake intensity analysis to evaluate the topographic characteristics of the landslide areas. There are many geomorphologic analysis methods such as roughness, slope aspect, which are also as useful as slope analysis. The analyses indicate that most of the co-seismic landslides occurred in regions with roughness, hillslope and slope aspect of >1.2, >30, and between 90 and 270, respectively. However, the intersection regions from the above three methods are more accurate than that derived by applying single topographic analysis method. The ground motion data indicates that the co-seismic landslides mainly occurred on the hanging wall side of Longmen Shan Thrust Belt within the up-down and horizontal peak ground acceleration (PGA) contour of 150 PGA and 200 gal, respectively. The comparisons of pre- and post-earthquake DEM data indicate that the medium roughness and slope increased, the roughest and steepest regions decreased after the Wenchuan earthquake. However, slope aspects did not even change. Our results indicate that co-seismic landslides mainly occurred at specific regions of high roughness, southward and steep sloping areas under strong ground motion. Co-seismic landslides significantly modified the local topography, especially the hillslope and roughness. The roughest relief and steepest slope are significantly smoothed; however, the medium relief and slope become rougher and steeper, respectively.The online version of this article (doi:10.1186/2193-1801-2-544) contains supplementary material, which is available to authorized users. It has been commonly accepted that steep topography are of high landslide frequency in active orogenic region. The co-seismic landslides usually occurred in active orogenic regions, which are one of the major secondary nature hazards related to strong earthquakes  data derived from 1:50,000 topographic maps. The analyses indicate that the co-seismic landslides are closely correlated to the topographic conditions as well as the shaking intensity. The topographic effects of the Wenchuan earthquake are also analyzed based on post-earthquake DEM data derived from stereo pair of IRS-P5 (Indian Remote Sensing Satellite) remote sensing images by comparing with pre-earthquake DEM.The Longmen Shan region is one of the steepest margins of the Tibetan Plateau accompany with almost 5000 m altitude decrease across the margin within 50 km distance  contours distribute in the region peripheral to the co-seismic surface rupture with the maximum value of 957.7 gal (Li et al. Co-seismic landslide is a kind of special landslide directly triggered by strong earthquake (Harp and Jibson Landslide occurrence shows clearly correlation with the topographic conditions. At all the three sites, over 50% landslides occurred in the derived roughness, aspect and hillslope areas over the threshold values, respectively Table . In ordePost-earthquake DEM analysis indicates the medium roughness and hillslope regions are becoming rougher and steeper after the Wenchuan earthquake. The roughest relief and steepest slopes are smoothed by the Wenchuan earthquake Figure . This inCollaborative topographic analyses are efficient in landslide susceptibility evaluation. The co-seismic landslides are related to the topographic roughness, hillslope and slope aspect. The topographic thresholds are >1.2, between 90 and 270, and 30, respectively. The co-seismic landslides are also directly related to PGA values, which is usually occurred on hanging wall within the up-down and horizontal PGA contour of 150 and 200 gal, respectively.Topographic conditions such as roughness and hillslope are controlled by tectonic event like the Wenchuan earthquake, however, slope aspect is not modified by such event.The roughest relief and steepest slope regions were smoothed after the Wenchuan earthquake, however the medium roughness and slope regions became rougher and steeper, respectively.Based on the data and analyses of the present study, we arrived at the following conclusions.


There is a broad range of non-invasive imaging/spectroscopic methodologies capable of reporting drug pharmacodynamics at the tumour site [The value of biomarkers that report on the effects of drugs at the target site – largely non-invasive/imaging biomarkers - is increasingly being recognised. Such biomarkers can inform whether the drug modulates the target in the desired manner, as well as the magnitude and duration of target modulation and recovery dynamics. Correlative investigations with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and 8Ffluorodin situ and finally to distant sites [Ras→Erk signal transduction can increase PC levels in cells [The ‘phosphatidylcholine transformation and progression signature’ (PTPS) is one such phenotype that can be exploited to assess target drug effect. This signature, which is fairly generic in tumours, has been described extensively for breast cancer -7, and tnt sites . Growth in cells , 12, 13.in cells , 12, 14,et al in this issue accords with the prevailing view on exploitation of PTPS for assessing drug effects. They examined the metabolic response of melanoma cells to inhibition of cell growth by a heat shock protein 90 (Hsp90) inhibitor. Using MRS, they provide proof that, concomitant with growth inhibition and induction of differentiation, the hsp90 inhibitor, 17-AAG, alters the PTPS of melanoma cells in a predictable manner, leading to no changes in PC and increases in GPC, free fatty acyl chains and cytoplasmic lipid droplets. These biochemical changes are consistent with normalization of PTPS. It is often unclear if cellular effects are chemotype-specific (related to the specific structure of drug) or target-specific. In this context they examined a structurally dissimilar Hsp90 inhibitor and showed that the effect was independent of specific Hsp90 inhibitor.The work presented by Beloueche-Babari et al offers several insights into the potential role of exploiting tumor PTPS normalization as a non-invasive biomarker of anticancer drug effect in general, and specifically, potential use of these methods in melanoma patients for assessing response to Hsp90 inhibitors, as well as other agents like MEK inhibitors that target associated pathways [in vivo application of PTPS normalization as a non-invasive biomarker that require further investigation. Standard 31P-MRS methods do not sufficiently resolve individual choline metabolite resonances. Instead composite peaks: phosphomonoesters (comprising PC and phosphoethanolamine) and phosphodiesters (comprising GPC and glycerophosphoethanolamine), are detected in humans MRS measurements [2-free fatty acyl chains that give rise to the MR-visible mobile lipids [et al provide a body of evidence, including inhibition of calcium-independent phospholipase A2, to support assessment of these lipid resonances as biomarkers of drug effect. It will be interesting to demonstrate how the free fatty acyl chain resonances change with Hsp90 inhibition in vivo in animal models of melanoma and in patients. Beloueche-Babari et al demonstrate that the time-dependent evolution of the drug-related changes on growth mirror changes in the biomarker signature. This is important in assigning pharmacological relevance to the biomarker signature. Investigation of the dose-dependence of these PTPS readouts will further support the pharmacological relevance of the biomarker. In this context it will be useful to know whether the spectroscopic readout evolves as all-or-nothing phenomena or are indeed dose-related; the latter will be amenable to investigation of drug effects in early clinical trials of future inhibitors.Imaging and spectroscopic methods that exploit the PTPS may have clinical value as target-tissue pharmacodynamic biomarkers in melanoma and other solid tumors. Activating mutations in BRAF occur in ~7% of all human cancers including melanoma, papillary thyroid and colon cancer; the V600E mutation is the most common mutation occurring in approximately 70% of melanoma patients . Activatpathways , 19. Theurements . Furtherurements . Alternaurements . 1H-MRS e lipids , 22. Belin vivo.In summary, normalization of tumor PTPS appears to be a realistic biomarker for non-invasive monitoring of the pharmacological action of Hsp90 inhibitors. The potential value of this biomarker should be tested


Identification of phosphorylation sites by computational methods is becoming increasingly important because it reduces labor-intensive and costly experiments and can improve our understanding of the common properties and underlying mechanisms of protein phosphorylation.A multitask learning framework for learning four kinase families simultaneously, instead of studying each kinase family of phosphorylation sites separately, is presented in the study. The framework includes two multitask classification methods: the Multi-Task Least Squares Support Vector Machines (MTLS-SVMs) and the Multi-Task Feature Selection (MT-Feat3).Using the multitask learning framework, we successfully identify 18 common features shared by four kinase families of phosphorylation sites. The reliability of selected features is demonstrated by the consistent performance in two multi-task learning methods.The selected features can be used to build efficient multitask classifiers with good performance, suggesting they are important to protein phosphorylation across 4 kinase families. Protein phosphorylation, one of the most important forms of post-translational modification of proteins, occurs on several different types of amino acid substrates. Serine (S) phosphorylation is the most common, followed by threonine (T) and tyrosine (Y). Histidine and aspartate phosphorylation may also occur, but mostly in prokaryotes as part of two-component signalling transduction systems  or rarelProtein kinases, which catalyze phosphorylation, play critical roles in the regulation of the majority of cellular pathways, including metabolism, signal transduction, transcription, translation, cell growth, and cell differentiation. Protein kinases account for approximately 2% of known human proteins, but they are responsible of phosphorylating approximate 30% of known human proteins . All proIn recent years, identification of phosphorylation sites by computational methods is becoming increasingly important, with the growing gap between protein sequences information and annotated phosphorylation information of proteins with known sequences. That is due to still lack of high throughput experimental methods for identifying the phosphorylation sites of proteins and current technologies are labor-intensive and costly. Besides predicting phosphorylation sites, computational approaches can also be used to discover the common and specific features of different kinase groups.A large number of computational tools for predicting phosphorylation sites have been reported . These mAll current kinase-specific phosphorylation prediction methods are single-task learning methods (STL) because they are trained independent from each other. Such methods are optimized on individual training datasets and thus the commonalities between different datasets are not considered. In this study, we use Multi-Task Learning (MTL) methods, instead of STL methods in previous studies, to investigate the kinase-specific phosphorylation sites by learning all STs simultaneously. Using a shared representation, MTL learns all participated STs of a problem by a global optimization approach based on an intuitive idea: the common knowledge shared by related STs in a specific domain helps improving the performance . It has In this study, we apply two MTL methods, namely the Multi-Task Least Squares Support Vector Machines (MTLS-SVMs) and the Multi-Task Feature Selection MT-Feat3) to the data of 4 kinase families with phosphorylation sites using datasets collected by Ji et al [ to the dAs results, we identify 18 non-redundant common features, which are deemed as important to protein phosphorylation across 4 kinase families. Compared to the initial set of 560 features, the number of features used in the new predictor is reduced by more than 96% without deteriorating the performance. Based on those selected features, future work can be done to reveal some common mechanisms of phosphorylation by different kinase groups.The dataset MetaPS06 used in this study was downloaded . It consIn this study, we use 560 features  of twenty amino acid residues. Among them, 544 features were obtained from AAindex database  and the ith peptide is represented by N features in the form A fixed length window is applied to scan a peptide sequence. The window size is optimized using odd numbers from 3 to 21. The average of features of all amino acids in a fixed window is assigned to the middle amino acid of the window. Thus the Support vector machines (SVMs) derive parameters of the maximum-margin to construct an optimized separating hyperplane. The optimization of SVM classifiers includes the selection of kernel, optimization of the kernel's parameters and soft margin parameter C.Random Forest (RF) is an ensemble machine learning method that utilizes many independent decision trees to perform classification or regression. Each of member trees is built on a bootstrap sample from the training data by a random subset of available variables.LS-SVMs can be considered as a variant of classical SVMs. LS-SVMs realize the optimization by solving a set of linear equations instead of a convex quadratic programming for SVMs. LS-SVMs perform training faster than SVMs without sacrificing generalization performance . The LS-i yis its corresponding label, N is the sample number, i eis the error, ϕ is the non-linear mapping function, γ and b are parameters to be fitted.where MTLS-SVMs is developed based on the mechanism of data amplification. An MTLS-SVMs classifier learns common parameters by integrating the sub datasets. It is obtained by solving a restricted optimization problem as below (Formula 2), and then the optimization problem can also be solved by solving linear equations.T is the task number, t Nis the sample number of the tth task, th task, th sample of the tth task, ti yis its corresponding label, ϕ is the non-linear mapping function, λ, γ and b are parameters to be fitted.where MT-Feats (Multi-Task Feature Learning and Selection) algorithm was derived from a MTL framework, which was designed to learn sparse representation shared cross STs from the training data . MT-FeatWe modify MT-Feats algorithms to solve classification problems, by using LS-SVMs as element classifiers. MT-Feat1 was developed for feature learning and MT-Feat3 was for feature selection. Both feature learning and feature selection learn common parameters by jointly regularizing a common term (Formula 3).W = UA, other symbols have the same meaning as those in formula 2. If the U is set as identity matrix, the "Feature learning" problem (MT-Feat1) is reduced to a "Feature selection" problem (MT-Feat3). Thus, MT-Feat3 is a special case of MT-Feat1 algorithm (See Formula 4). In this study, we only use MT-Feat3 for feature selection.Where Performance is measured by average accuracy (aveAc) which is described in formula 5.Where TP and TN denote the total number of correctly classified positive and negative samples across all the STs. FP and FN denote the total number of incorrect classified positive and negative samples across all the STs. Since the datasets are relatively balanced, the average accuracy is sufficient to measure the performance of various predictors.We use MTLS-SVMs and MT-Feat3 methods to build classifiers for predicting phosphorylation sites on 4 kinase family datasets. To compare the performance of MTLS-SVMs and MT-Feat3 methods with that of the STL method, LS-SVMs classifiers are also built using the save datasets. Five-fold cross validation and grid-fitting of parameters are used to estimate the performance of all classifiers with window size from 3 to 21  and subset 2 (26 features) using the uniform window size of 7 or the optimized window sizes, respectively, are listed in Table To investigate the relationship between selected features, we cluster features in the subset 1 Figure  and subsThe best aveAc of MTLS-SVMs with the subset 4 is 0.792, very close to that of MTLS-SVMs with total features (0.7936). The best aveAc of MTLS-SVMs with the subset 3 is 0.7455, which is slightly poorer than that of MTLS-SVMs with total features (0.7595) (Table In this study, we use a multi-task learning framework to investigate phosphorylation sites across 4 kinase family datasets. In this framework, MT-Feat3 is used to select some common features, which are then validated by MTLS-SVMs classifiers. Selected features are further reduced to 18 features after eliminating features with high correlation coefficients with outer features. These features are considered as important common features for further analysis of possible properties and mechanisms of protein phosphorylation.The authors declare that they have no competing interests.SG and SX developed the programs. SG and YF did the dataset construction and calculation, and drafted the manuscript. JF conceived of the project, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Description of selected features from AAIndex. Descriptions of AAIndex records corresponding to selected features in subset 1, 2, 3 and 4.Click here for file


This system is assembled in two synthetic, modular, broad-host range plasmids based on pBBR1 and RK2 origins of replication, allowing its utilization in an extensive number of gram-negative bacteria. We analyze the performance of this dual cassette in two hosts, Escherichia coli and Pseudomonas putida, by examining the induction properties of the lacIq-Ptrc expression system in the first host and the Pb promoter of the benzoate degradation pathway in the second host. By quantifying the bioluminescence signal produced through the expression of the lux genes, we explore the dynamic range of induction for the two systems (Ptrc-based and Pb-based) in response to the two inducers. In addition, by quantifying the fluorescence signals produced by GFP expression, we were able to monitor the single-cell expression profile and to explore stochasticity of the same two promoters by flow cytometry. The results provided here demonstrate the power of the dual GFP-luxCDABE cassette as a new, single-step tool to assess promoter properties at both the population and single-cell levels in gram-negative bacteria.A dual promoter probe system based on a tandem bi-cistronic GFP- Living organisms are equipped with complex machinery dedicated to interaction with the surrounding environment. In fact, significant portions of prokaryotic genomes encode genes and elements associated with the processes of signal transduction and gene regulation, and the number of these elements is relatively large for generalist organisms lux reporters lux systems works by converting cellular reducing power into a light signal that can be easily quantified lux-related reporters is nonexistent with fluorescence-based systems such as GFP, the readout of which is virtually independent on the metabolic status of the cell. Additionally, lux-based systems are not suitable for examining stochastic processes at the single-cell level, as the signal produced is diffusive lux) into a synthetic single transcriptional unit. A similar approach has been successfully applied for monitoring promoter activity in gram-positive bacteria lux cassette in gram-negative bacteria for transcriptional studies is uncertain, as it has been limited thus far to tagging cells for ecological experiments Currently, most available methodologies for assessing the effects of stochastic processes in single cells are based on the use of a fluorescent reporter gene  fused to the target promoter element gfp and luxCDABE systems that is suitable and optimized for promoter probing and parameterization in gram-negative bacteria. The system is implemented in two modular, synthetic broad-host range vectors based on the pBBR1 and RK2 origins of replication. As explained below, the DNA segment encoding the upstream 5′ boundary of the dual reporter genes was engineered with an expanded set of unique restriction sites that facilitate the cloning and analysis of a wide variety of promoters. These vectors enable examination of stochastic transcription phenomena in an extensive range of bacterial hosts E. coli.In this report, we present a new dual reporter cassette based on the tirlux operon from Photorhabdus luminescens (luxCDABE) that encodes all the enzymes necessary to convert cellular reducing power to a luminescent signal http://seva.cnb.csic.es, ori of replication, the antibiotic marker and the cargo segment) flanked by unique, unusual restriction sites that facilitate interchangeability between different components. Functional modules of SEVA plasmids are edited for erasing any of the restriction sites of the pUC18 polylinker as thus ease cloning operations -lux cassette system was assembled into two vectors, each harboring a kanamycin resistance marker and either a pBBR1- (in the case of pGLR1) or RK2- (for pGLR2) based origin of replication, both of which are known to replicate in a wide number of hosts lux genes are cloned between HindIII and SpeI restriction sites of the plasmid vectors, as in the lux-based promoter probe plasmid pSEVA226 tir gene, encoding a stable variant of GFP, was placed upstream of the lux operon between SphI and HindIII restriction sites. It is important to note that since this GFP is stable, the protein half-life will generally equal the doubling time of the cell when the accumulated reporter is diluted between the two progeny cells. It is also worth noting that both reporter systems use the same optimized ribosome-binding site . The resulting dual cassette is located downstream of an expanded multiple cloning site (MCS) composed of 12 unique restriction sites that are used for promoter cloning  was engineered as well for transient expression studies To create a new dual reporter cassette, we considered three fundamental elements. First, we used a previously described GFP variant with an optimized ribosome binding site GFPtir. Second, cloning . An idenPlexA of E. coli, which responds to exogenous damage to DNA PlexA was then transformed in E. coli strain MG1655 and the cells overlaid onto a plate with nalidixic-acid containing disks. After growth, the plate was then either exposed to blue light  or examined in the dark with a CCD camera for capturing light emission. The images shown in PlexA at a high level, which is bound by either growth inhibition or by virtually no activity of the same promoter. Furthermore, the two images of fluorescence and luminescence overlapped perfectly, thereby demonstrating their coincidence in time and space and the lack of internal promoters able to create an artifactual background. When a similar plate test was conducted with strain E. coli MG1655 (pGFLR1-PlexA), which expresses a short-lived GFP protein,  while Pb was analyzed in pGLR1 (pBBR1-based).One key requirement of any robust dual reporter is the good correspondence between the readouts stemming from each of the cognate gene products. To examine this issue in the GFP-GBF-lux cassette, we first investigated the effect of concatenating the gfp and the lux reporters into a single transcriptional unit. For this assessment, we compared the promoter output of the Ptrc-based system in E. coli with each reporter alone compared to the dual cassette. A schematic representation of the transcriptional fusions created is shown in E. coli strain CC118 was transformed with pGLR2-Ptrc (Ptrc::GFP-luxCDABE fusion), pLUX-Ptrc (Ptrc::luxCDABE) or pGFP-Ptrc (Ptrc::GFP) and assayed in M9 minimal medium with glucose and casamino acids in the presence of 1 mM of IPTG. As shown in gfp gene between the Ptrc promoter and the lux operon had a moderate positive effect on the bioluminescence signal, as the final promoter activity was ∼1.75 times higher in cells harboring the pGLR2-Ptrc plasmid than in cells harboring pLUX-Ptrc. Furthermore, a comparison of promoter activity using the fluorescent reporter shows no difference between plasmids pGLR2-Ptrc and pGFP-Ptrc  to this promoter. Transcription thus occurs when the inducer  binds to LacI and removes it from Ptrc, allowing initiation Pb, a transcriptional activator, BenR, triggers the promoter activity when bound to the inducer benzoate. This regulator belongs to AraC-family and works by recruiting RNAP to the target promoter luxCDABE reporter, we assayed both transcriptional repression and activation in the two model organisms. For these tests, overnight-grown cells were diluted in fresh media containing increasing concentrations of the specific inducer (IPTG or benzoate) and assayed in a multilabel plate reader, as described in Material and Methods. At time intervals of 30 min, the optical density at 600 nm and the luminescence were recorded. Ptrc-based construct, the promoter activity grew in response to the increased dosage of IPTG and reached maximal levels at concentrations equal to or above 250 µM . However, another relevant piece of information about gene regulation is the performance of the system at the single-cell level. In general, promoters in single cells behave in either a graded fashion or an all-or-none fashion vs. OFF luxCDABE reporter cassette as a tool for diagnosing the single-cell behavior of target promoters, we examined the expression profiles of the Ptrc- and Pb-based system in response to 1 mM of IPTG or benzoate, respectively, through the analysis of the GFP reporter. To this end, overnight-grown cells  were diluted in fresh media and allowed to grow until mid-exponential phase. At this point, each reporter strain was exposed to the specific inducer and incubated for several hours. At time intervals of 1 h, samples were taken and analyzed by flow cytometry as indicated in Material and Methods. As shown in PtrcPblux operon as described in the previous section whereby reporter is strongly activated during the first hours of induction. Finally, the results presented in luxCDABE reporter system for studying single-cell behavior in gram-negative bacteria.As shown in the previous section, the luxCDABE cassette is its implementation with broad-host range plasmid vectors based on pBBR1 and RK2, two of the most promiscuous replication origins -lux reporter, we compared the levels of noise generated during Pb activation in P. putida harboring the pGLR1-Pb vector vs. another reporter strain in which an equivalent Pb::GFP fusion is placed in monocopy in the chromosome of P. putida MEG3-PbPb promoter sequence fused to a bi-cistronic GFP-lacZ fragment that was used to investigate stochastic processes during benzoate degradation in P. putidaPb, as it is based on the pBBR1 origin that has a higher copy number per cell benR gene (encoding the activator for Pb) is placed in monocopy in the chromosome; thus, any interference occasioned by the presence of multiple target promoters should be enhanced. The induction experiments were performed in response to 1 mM of benzoate, and cells were analyzed by flow cytometry as before. As shown in P. putida harboring pGLR1-Pb presented a single-cell profile very similar to that from P. putida MEG3-Pb  medium or in M9 minimal medium supplemented with 0.4% (w/v) glucose, 0.1% (w/v) casamino acids, 2 mM MgSO4, 0.1 mM CaCl2 and 0.05% (w/v) vitamin B1. P. putida cells were incubated at 30°C in M9 minimal medium supplemented with 2 mM MgSO4 and 25 mM succinate as the sole carbon source. When required, kanamycin  or chloramphenicol  was added to the growth media.The strains and plasmids used in this work are listed in PstI/HindIII fragment into pSEVA236 (a promoter probe vector with the pBBR1 origin of replication), previously digested with the same enzymes. The resulting vector was named pGLR1 and has a tandem GFP-luxCDABE fragment downstream of a multiple cloning site (gfp lva variant  GCG CAA GCT TTT AAG CTA CTA AAG CGT AGT TTT CG -3′), generating a fragment of 788 bp. This fragment was then cloned as a PstI/HindIII fragment into pSEVA236, digested with the same enzymes as before. The resulting vector was named pGFLR1 and has, as previously described for pGLR1, the GFPlva-luxCDABE fragment located downstream of a multiple cloning site. The construction of pGLR1-PlexA involved the amplification of the 106 bp fragment PlexA promoter 5′-CCC TTC CAG AAT TCG ATA AAT CTC TGG-3′) and 3PLEX (5′-CCC GGA TCC TCC GCC CCC TGG GTG TAT ATA CAG-3′), with the amplified sequence cloned as an EcoRI/BamHI fragment into pGLR1. The same PlexA-containing EcoRI/BamHI fragment was cloned in pGFLR1, thus creating pGFLR1-PlexA. For the cassette encoding the stable GFP variant, the ∼6.6 Kb fragment of pGLR1 spanning the GFP-luxCDABE segment was excised with SphI/SpeI restriction enzymes and cloned into a pSEVA221  and 3-PB (5′-GCG CGG ATC CGG CCA GGG TCT CCC TTG-3′) and cloned as an EcoRI/BamHI fragment into pGLR2, generating the pGLR2-Pb plasmid. This construct was then mobilized into P. putida strain KT2440, which has all native regulatory elements necessary to trigger Pb promoter activity in response to benzoate Cloning procedures were performed as described previously ing site . The DNAPtrc). In one case, the gfp tir gene was cloned as a PstI/HindIII fragment into pSEVA224 Ptrc plasmid, while in the other case, the luxCDABE operon was cloned as a HindIII/SpeI fragment into the same pSEVA224, resulting in the pLUX-Ptrc plasmid. The resulting plasmids were introduced in E. coli CC118, and the resulting reporter strains were analyzed as described below.To check for potential interference between the two reporter systems when they were placed in tandem, two additional plasmids where constructed in which each reporter was placed alone under the control of an inducible promoter  into fresh medium with different concentrations of IPTG (in the case of the Ptrc-based system) or benzoate (for the Pb-based reporter). Microtest™ 96-well assay plates  were filled with 200 µL per well of diluted culture and placed into a Wallac Victor 2 Microplate Reader (Perkin Elmer). Plates were then incubated with shaking for several hours. At time intervals of 30 min, the optical density at 600 nm (OD600) and the luminescence or the fluorescence of each of the cultures were measured. Non-inoculated M9 medium was used as a blank for adjusting the baseline for measurements. Promoter activities were calculated by normalizing the reporter signals (luminescence or fluorescence) to the OD600 readings. Data processing was performed using MATLAB software (MathWorks).Emission of fluorescence and production of light in cells growing on Petri dishes were recorded with a luminometer VersaDoc imaging system Model 4000 (Bio Rad), and the images were captured and processed with the analysis software Quantity One 4.6.9 of the same brand. For quantitative promoter activity assays, single colonies of http://www.cyflogic.com/).Single-cell experiments were performed with a Gallios (Perkin Elmer) flow cytometer. To this end, GFP was excited at 488 nm, and the fluorescence signal was recovered with a 525(40) BP filter. Overnight-grown cells were diluted 1/20 in fresh M9 media containing the carbon source indicated in each case and incubated for 4–5 hours. After this pre-incubation, at the mid-exponential phase, the cells were split into two samples: one was induced by the corresponding compound and the other was used as a non-induced control. Cultures were then incubated with shaking in air at the appropriate temperature, and each hour after induction, an aliquot of each sample was stored on ice until analysis. For every aliquot, 15,000 events were analyzed. The data processing was performed using Cyflogic software . Next, individual fluorescence values were processed with the R package (http://www.r-project.org/) to calculate the variance (var) and mean values (mean) of the samples. Finally, the noise values for each experiment were determined simply by dividing the var by the mean values.The noise for the single-cell experiments was calculated as previously described