Rooted in people’s preoccupation with how they are perceived and evaluated, shame and guilt are self-conscious emotions that play adaptive roles in social behavior, but can also contribute to psychopathology when dysregulated. Shame and guilt-proneness develop during childhood and adolescence, and are influenced by genetic and environmental factors that are little known to date. This study investigated the effects of early traumatic events and functional polymorphisms in the brain-derived neurotrophic factor (BDNF) gene and the serotonin transporter gene promoter (5-HTTLPR) on shame and guilt in adolescents. A sample of N = 271 healthy adolescents between 14 and 17 years of age filled in measures of early traumatic events and proneness to shame and guilt, and were genotyped for the BDNF Val66Met and 5-HTTLPR polymorphisms. Results of moderator analyses indicated that trauma intensity was positively associated with guilt-proneness only in carriers of the low-expressing Met allele of BDNF Val66Met. This is the first study that identifies a gene-environment interaction that significantly contributes to guilt proneness in adolescents, with potential implications for developmental psychopathology. Shame and guilt are negative self-conscious emotions , typically experienced in situations of failure or in which behavioral standards are violated ,2. WhileResearch on shame and guilt has mainly focused on childhood ,6, and rOccasional feelings of shame and guilt are functional and serve social goals . For exaHowever, problems can arise when people's lives are pervaded by shame and guilt, and over the long run, proneness to shame and guilt may play an important role in psychopathology. Studies have consistently linked shame-proneness in children and adolescents with anxiety, depression, eating disorders, externalizing symptoms and delinquent behavior ,21. DataConsidering the links between shame and guilt-proneness and child and adolescent psychological problems, recent research has focused on factors such as early trauma that may contribute to these emotional dispositions.Early adverse experiences may have lasting effects on people's emotional responses ,28, inclThe relation between guilt-proneness and early adverse experiences is less consistent. However, a study by Stuewig and McCloskey  found anEarly experience may have different emotional outcomes depending on genetic dispositions –43. The The goal of the current study was to explore gene-environment interactions in adolescent shame and guilt-proneness. While the potential influence of early trauma on shame and guilt-proneness has been examined to some extent, candidate genes that may contribute to shame and guilt-proneness by increasing susceptibility to environmental adversities have not been studied until now. However, shame and guilt-proneness have been linked with depression and anxiety ,21, and Although not all studies reported positive or convergent findings, meta-analyses support the view that carrying the low-expressing alleles of the Val66Met polymorphism in the brain-derived neurotrophic factor (BDNF) gene  and the These genetic polymorphisms are biologically plausible candidates for emotional development and psychopathology considering their involvement in neural plasticity ,49 and sRecent studies have also approached some of the psychological mechanisms that may be involved in the influence of BDNF Val66Met and 5-HTTLPR on emotional dispositions. It was found that children carrying the BDNF Met allele, whose mothers suffered from depression , as wellThis study is based on a sample of N = 271 adolescents (165 girls) aged between 14 and 17 years . They were all Caucasians and Romanian was their first language. None of the participants reported diagnosed neuropsychiatric disorders or use of psychoactive medication. Before the study, the procedure was explained to participants and their parents, and participants' assent and written parental consent were obtained. The study followed the recommendations of the Declaration of Helsinki regarding participant safety  and was DNA was extracted from buccal epithelial cells using the MasterPure Complete DNA & RNA Purification Kit  and kept at -20°C.BDNF Val66Met (rs6265) genotyping was performed using tetra primer amplified refractory mutation system (ARMS)-PCR method, as previously described . This poA and LG [G (coded S'), compared to the LA allele (coded L') [GLG and SLG), S'L'  and L'L' (LALA).Using a previously described protocol ,66, 5-HToded L') . The triTest of Self-Conscious Affect for Adolescents (TOSCA-A) [You and your friend are talking in class, and you get in trouble) is followed by a list of possible responses . Participants rate the likelihood of each response on a scale ranging from 1  to 5 (very likely). Similar to reliability coefficients reported in previous studies [TOSCA-A) . Shame-p studies , ChronbaChildhood Traumatic Events Scale (CTES) [e (CTES)  is a sele (CTES) ,73.The depression and anxiety scales from the Depression Anxiety Stress Scales  were useIn order to determine whether genotype frequencies in our sample differed from the general population, departures from the Hardy-Weinberg equilibrium were examined . Skewnes2, indicating the ratio of systematic variance accounted for by the moderator relative to unexplained variance in the criterion [Gene-environment interactions were analyzed by multiple regressions , using triterion . While ariterion .We report significant effects at the traditional level of significance (p < 0.05). However, considering that the effects of the two genotypes were investigated in three regression models, which may have inflated the type I error, the survival of significant effects at a Bonferroni-corrected (p/3) level of significance (p < 0.017) is also reported.2 = 3.380, p > 0.05). Due to the low number of Met homozygotes, they were included together with heterozygotes in a Met carrier group and compared to Val homozygotes.BDNF Val66Met genotyping indicated that there were 171 participants with Val/Val genotype, 82 with Val/Met genotype and 18 with Met/Met genotype. These genotypes were in Hardy-Weinberg equilibrium .Triallelic 5-HTTLPR genotyping indicated that there were 65 participants with L'L' genotype, 110 with S'L' genotype, and 68 with S'S' genotype. Due to insufficient DNA, 5-HTTLPR genotyping could not be performed for 28 participants. Considering that the number of girls and boys excluded was similar , the sex ratio in the remaining sample was not affected. In addition, shame and guilt proneness and trauma intensity were not significantly different in excluded participants compared to the rest of the sample . The distribution of 5-HTTLPR genotypes in this sample was in Hardy-Weinberg equilibrium (χMost participants (62%) reported at least one traumatic event. Death of family or close friend was the most frequent trauma (39.48%), followed by severe illness or injury (22.87%), other traumatic event not specified (22.14%), major parental conflicts or separation (18.08%), physical abuse (8.11%) and sexual abuse (1.47%). Death of family or close friend had the highest traumatic intensity , followed by other traumatic events not specified , major parental conflicts or separation , sexual abuse , physical abuse  and severe illness or injury .BDNF Val66Met and 5-HTTLPR (dummy coded) were separately tested as potential moderators in the relations between mean trauma intensity as predictor and shame and guilt-proneness as outcomes. Considering that girls reported higher levels of shame-proneness  and guilt-proneness  compared to boys, sex was included as covariate in all analyses.Guilt-proneness , but not shame-proneness was significantly skewed. Normalized  scores were therefore used in all analyses on the former outcome.None of the participants reported depression symptoms  and anxiety symptoms  over the clinical cut-offs, and these variables were therefore not used as covariates in the moderation analyses. However, shame-proneness significantly correlated with both depression  and anxiety , whereas guilt-proneness only correlated significantly with anxiety .Results of moderator analyses with shame-proneness as outcome  indicate2 = 0.022). Slope analysis indicated that trauma intensity was positively related to guilt in BDNF Met carriers , but not Val homozygotes  (= 0.538) . The mai= 0.538) . Trauma = 0.538) , but theWe investigated shame and guilt-proneness in relation to each type of traumatic event. Participants who reported experiences of severe illness or injury had increased shame-proneness compared to those who did not have such experiences . There were no other significant differences between participants who reported other types of traumatic events  and those who did not.The main finding of this study is that the intensity of early trauma in adolescents is positively related to guilt-proneness only in carriers of the low-expressing Met allele of BDNF Val66Met. Other positive relations between the 5-HTTLPR S'S' genotype and shame-proneness, and trauma intensity and guilt proneness, as well as the interaction between BDNF Val66Met and early trauma on shame-proneness were also found, but these effects did not survive at the level of significance corrected for multiple testing and are thus less reliable.This is the first gene-environment interaction study on shame and guilt-proneness, but it adds to a recent line of research that has linked early trauma and BDNF Val66Met to negative self-evaluation. For example, maternal depression has been associated with a memory bias toward negative self-descriptive traits in children carrying the Met allele of BDNF Val66Met . Using aThe present finding raises the question of the neural mechanisms by which the BDNF Val66Met and early trauma interaction influences guilt-proneness in adolescents. Recent neuroimaging work suggested that the hippocampus plays a central role in the neural pathway that links BDNF Val66Met and emotional vulnerability following early trauma. A history of early trauma in adults carrying the BDNF Met allele is associated with reduced hippocampal gray matter and depression . In addiTrauma intensity in adolescents was not related to shame-proneness, but was positively related to guilt-proneness. However, this effect was significant in only one of the models  and did not survive at the corrected level of significance. This is in line with recent evidence suggesting that childhood trauma influences shame and guilt-proneness only indirectly. In a study conducted by Stuewig and McCloskey , the autThere was only one main effect of 5-HTTLPR, with higher shame-proneness in adolescents with the S'S' genotype. However, this effect did not survive the correction for multiple testing. Failing to find an interaction between 5-HTTLPR and early trauma in relation to shame and guilt may seem surprising considering the literature supporting the effect of this interaction on depression and anxiety . HoweverThe implications of these results are related to the possibility of targeting shame and guilt-proneness in prevention programs aiming to reduce the risk of psychopathology in adolescents . Based oThis study has at least three limits. First, the sample is small in light of estimated sizes that would be needed in gene candidate studies focused on functional polymorphisms with small effects . TherefoIn conclusion, our results indicate that BDNF Val66Met is a significant moderator of the relation between early trauma and guilt-proneness in healthy adolescents. Should these results be replicated in independent samples, future studies might focus on the neural mechanisms underlying the effect of this gene-environment interaction on proneness to negative self-conscious emotions and their impact on developmental psychopathology.


Opinion: NoActive surveillance (AS) is a management strategy for early-stage prostate cancer (PCa) designed to balance early detection of aggressive disease and overtreatment of indolent disease . It is aActive surveillance consists of close observation via a regimen of periodic PSA measurements, digital rectal examinations and serial prostate biopsies, with the goal of offering curative therapy in the event of disease progression or reclassification . DespiteA number of studies also support the role of surgery in men in this intermediate risk group. The PIVOT study found men with intermediate-risk tumours who underwent radical prostatectomy  had a 31% relative reduction in all-cause mortality, as compared with those assigned to observation  . There wKlotz et al. performed a large cohort Canadian study with 993 patients and up to 16 years of follow-up, with 25% of the patients fulfilling the D’Amico criteria for intermediate risk .Although the lifetime risk of receiving a diagnosis of prostate cancer is about 17%, the risk of dying from the disease is approximately 3%, suggesting that conservative management may be appropriate for select men . Thus, tWhen managed with non-curative intent, intermediate-risk PCa is associated with 10-year and 15-year prostate-cancer-specific mortality rates of 13 and 19.6% . Thus, mAt least 50%-60% of individuals diagnosed with PCa ultimately die of other causes, and as a result, AS has become a chosen management option for low risk PCa patients . The lat


Central to the development of new treatments for human immunodeficiency virus 1 (HIV-1) is a more thorough understanding of the viral life cycle and the cellular cofactors upon which this depends. Targeting cellular proteins and their interaction with HIV-1 has the potential to reduce the problem of emerging viral resistance to drugs as mutational escape is more difficult. We performed a short interfering RNA (siRNA) library screen targeting 59 cellular RNA helicases, assessing the effect on both viral capsid protein production and infectious virion formation. Five RNA helicases were identified which, when knocked down, reproducibly decreased infectious particle production: DDX5, DDX10, DDX17, DDX28 and DDX52. Two of these proteins (DDX5 and DDX17) have known roles in HIV-1 replication. A further helicase (DDX10) was a positive hit from a previous genome-wide siRNA screen; however, DDX28 and DDX52 have not previously been implicated as essential cofactors for HIV-1. Given the high propensity of human immunodeficiency virus 1 (HIV-1) to mutate, drug resistance is a serious and growing threat, necessitating the ongoing development of new anti-retroviral agents . One posAmongst the cellular proteins identified by the three genome-wide siRNA screens were eight RNA helicases. The RNA helicases have a wide range of functions and are crucial for almost every aspect of RNA metabolism . In addiWe used a library consisting of three independent siRNAs against 59 RNA helicases to identify additional helicases with a role in HIV-1 replication and identify potential novel antiviral targets. Our focused RNA helicase siRNA library allowed for multiple rounds of screening in different formats using individual siRNAs rather than siRNA pools, hopefully allowing us to reconcile differences seen in the results of the genome-wide siRNA screens.The first round of screening was performed in triplicate experiments each in triplicate wells in 96-well plates in HeLa cells to overcome difficulties associated with achieving effective siRNA-mediated knockdown in T-cells. We initially used a pseudotyped HIV-1 system, co-transfecting cells with an envelope-deleted HIV-1 plasmid, a vesicular stomatitis virus (VSV) glycoprotein plasmid and a specific siRNA using Lipofectamine 2000 (Invitrogen). At 96 h post-transfection, samples were collected for capsid p24 (CA-p24) ELISA, intracellular CA-p24 ELISA and assessment of infectious virion production. The infectivity assay utilized TZM-bl cells – a HeLa-derived cell line expressing the receptors necessary for HIV infection and a luciferase gene under the control of a HIV-1 LTR. The assay was optimized to ensure the luminescence produced in response to infection was within the linear range. Included in each experiment were a non-targeting negative control siRNA (siCon) and siRNA against DDX3 as a positive control (siDDX3).siRNAs were considered to be of interest if they decreased all three measured parameters by >20 %. Also of interest were siRNAs which had no effect on intracellular CA-p24, but did decrease extracellular CA-p24 and infectious virion production, as this could represent a viral assembly defect. Of the 177 siRNAs screened in the first round, 43 fulfilled these criteria. The remaining siRNAs either resulted in no change in the measured parameters or the results were too variable to be reliably interpreted (coefficient of variance >30 % for two of the three measured parameters or >50 % for one parameter). We considered a particular helicase to be of interest if two of the three siRNAs against it resulted in a positive phenotype or if one siRNA gave a positive phenotype and the others were too variable to be interpreted. Overall, 16 of the 59 helicases were taken forward to the second round of screening.In the second round of screening, the siRNAs of interest were screened similarly in triplicate utilizing the pseudotyped virus system in HeLa cells in 24-well plates with the same three measures of viral replication assessed. Overall, the results in this plate format were less variable than seen in 96-well plates, with good reproducibility between the two plate formats. Only six of the 48 siRNAs rescreened gave a different result in the second round of screening compared with the first round. All helicases taken forward to the final stage of screening had reproducible results with at least two of the three siRNAs. We selected the eight helicases whose knockdown had the largest impact on viral replication for further study.To determine whether the results seen were specific to the pseudotyped virus system, the siRNAs against the helicases of interest  were screened again in triplicate experiments in both 96- and 24-well plates using WT HIV-1 (pLAI). Overall, the effects seen with the WT virus in 96-well plates were less marked than seen with the pseudotyped virus. This may be due to WT viral replication being more robust than pseudotyped replication or effects on the VSV component. The difference between the two systems was most notable in the case of DDX18, DDX19 and DDX56, which were therefore not studied further. The remaining 15 siRNAs were screened again in 24-well plates using WT HIV and a sequential transfection approach, whereby the siRNAs were transfected initially, followed 24 h later by the viral plasmid. The results of the supernatant CA-p24 ELISA and infectivity assay are shown in Compared with siCon, the pool of DDX3 siRNAs significantly decreased CA-p24  productiInterestingly, the siRNA effects were generally greater on viral protein production than on infectious virus production. For example, the three siRNAs against DDX52 caused a modest reduction in CA-p24 production; however, there was little difference in the infectivity of the virions produced from these cells. Possible explanations for this include the siRNAs causing fewer virions to be produced, but for these virions to be more infectious. Alternatively, these results may represent a reduction in the amount of free CA-p24 or abnormal virions released from cells.Only the DDX52 siRNAs gave a consistent phenotype with all three siRNAs. For the other four helicases, two of the three siRNAs significantly impacted on viral replication, as had been seen at all previous stages of screening. This difference in effect of siRNAs against the same target may represent differing levels of knockdown of the target protein achieved by the individual siRNAs.To assess siRNA-mediated knockdown, Western blot analysis was performed on lysates of siRNA-treated HeLa cells. All the siRNAs against DDX5 , DDX10 , DDX17  and DDX2Due to the lack of a good antibody, a Western blot for DDX52 is not shown. With the exception of DDX28 the knockdown achieved with each siRNA is shown in duplicate as compared with the baseline expression of the helicase. The DDX17 blot  shows twTo rule out siRNA-induced cell death being non-specifically responsible for the effects seen on viral replication, we performed a luminescence-based cell viability assay . This assay was performed in the absence of virus production, with puromycin included as a positive control for cell death. As can be seen in Comparing the results in Overall, our siRNA library screen identified five helicases which when knocked down reproducibly reduced infectious virion production, suggesting a likely role in HIV-1 replication. Although our screen was performed in HeLa cells, all five helicases are thought to be expressed in cell types relevant to HIV-1 replication (Helicases with published roles in HIV-1 replication were not taken beyond the first round of screening. However, the roles of DDX5 and DDX17 in viral replication were published during the course of this work, and so they were included as further positive controls. These two proteins are highly related and can exist as homo- or heterodimers . DDX17 hDDX5 has also been shown to interact with Rev (Comparison of the results of our siRNA library screen with previously published genome-wide siRNA screens reveals only one overlap: DDX10 . The samExperiments to clarify the roles of these helicases are ongoing, including whether the effects can be reproduced in T-cells and at what stage of the viral life cycle the helicases are being utilized.


Chromatin remodelling is integral to the formation of long-term memories. Recent evidence suggests that histone modification may play a role in the persistence of memories associated with drug use. The present series of experiments aimed to examine the effect of histone deacetylase (HDAC) inhibition on the extinction and reinstatement of nicotine self-administration. Rats were trained to intravenously self-administer nicotine for 12 days on a fixed-ratio 1 schedule. In Experiment 1, responding was then extinguished through removal of nicotine and response-contingent cues. After each extinction session, the HDAC inhibitor, sodium butyrate (NaB), was administered immediately, or six hours after each session. In Experiment 2, response-contingent cues remained available across extinction to increase rates of responding during this phase, and NaB was administered immediately after the session. Finally, in Experiment 3, the effect of NaB treatment on extinction of responding for sucrose pellets was assessed. Across all experiments reinstatement to the cue and/or the reward itself was then tested. In the first experiment, treatment with NaB significantly attenuated nicotine and nicotine + cue reinstatement when administered immediately, but not six hours after each extinction session. When administered after cue-extinction (Expt. 2), NaB treatment specifically facilitated the rate of extinction across sessions, indicating that HDAC inhibition enhanced consolidation of the extinction memory. In contrast, there was no effect of NaB on the extinction and reinstatement of sucrose-seeking (Expt. 3), indicating that the observed effects are specific to a drug context. These results provide the first demonstration that HDAC inhibition facilitates the extinction of responding for an intravenously self-administered drug of abuse and further highlight the potential of HDAC inhibitors in the treatment of drug addiction. Tobacco addiction is a major cause of preventable ill health and death in western countries. Unfortunately, quitting is difficult, and among those that do quit, relapse is common . A majorLearning to overcome cravings and remain resistant to relapse is crucial to achieving long-term abstinence. This process can be accurately modelled in animals via the extinction and reinstatement paradigm . In thisIt is now well established that, like long-term memory formation, the encoding of long-term extinction memories requires active gene transcription . IncreasThere are numerous epigenetic changes that have been investigated with respect to animal behaviour. Of these, histone acetylation is the most widely studied regarding both long-term memory formation and drug addiction. Through controlling accessibility of transcription enzymes to DNA, histone acetylation facilitates  or silences  gene expression and any resultant changes which may be crucial to the persistence of memories across time .Early evidence for the role of histone acetylation in the extinction of conditioned behaviour comes from Pavlovian conditioned fear in rats, where administration of an HDAC inhibitor prior to each extinction session enhances long-term extinction memory formation –9. More Extending the results of Pavlovian conditioning to an instrumental paradigm is particularly important in the study of drug addiction, as this type of procedure has higher face validity, more closely approximating drug taking behaviour in humans . To thisTherefore the present study aimed to specifically address the involvement of histone acetylation in the extinction of nicotine self-administration and the subsequent susceptibility to relapse. First, the HDAC inhibitor, sodium butyrate (NaB), was administered immediately after each extinction session to facilitate the encoding of extinction memories. An additional group received NaB six hours after the end of the session to determine if any observed effect was specifically due to memory consolidation, or rather occurred simply as a consequence of NaB exposure (Experiment 1). A cue-extinction procedure was then utilised to increase responding across extinction sessions, maximising the possibility of detecting treatment effects across this period when low rates of responding are typically observed (Experiment 2). Finally, the effect of NaB on extinction and reinstatement of sucrose-seeking was examined to determine if any detected effects were specific to nicotine-seeking, or are a property of reward-related learning more generally (Experiment 3).ad libitum access to food and water until two days prior to self-administration when food was restricted to 20 g/rat/day [Male Sprague Dawley rats  were housed four per cage on a 12 h reverse light/dark cycle (lights off at 7 a.m). Rats in all experiments received /rat/day . Rats weth ed, 2013). Surgeries were performed under isoflurane anaesthesia, and all efforts were made to minimise the number of animals used and their suffering.All procedures were approved by the Animal Care and Ethics Committee of The University of New South Wales (protocol number 12/155B) and the Macquarie University Animal Ethics Committee (protocol number 2011/007) and were conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes  were dissolved in sterile saline (0.9% NaCl). Nicotine solutions for subcutaneous (s.c.) priming injections were pH adjusted to 7.4 using sodium hydroxide. All doses of nicotine are reported as free base.Two weeks after arrival, rats in Experiments 1 and 2 underwent surgery for the implantation of an intravenous catheter as described previously .All self-administration studies were carried out in 8 standard self-administration chambers  housed within wooden sound attenuation boxes . ChamberTraining began with two daily 1 hr habituation sessions with nose-pokes covered and house-light on. On the third day, nose-pokes were revealed and 12 daily 1 hr nicotine-self administration sessions commenced. Each response on the active nose-poke resulted in an infusion of nicotine (30 μg/kg/100 μL over 3 s), presentation of a cue-light within the nose-poke hole (3 s) and extinction of the house-light (20 s). Responses during the 20 s time-out or on the inactive nose-poke were recorded but were of no consequence. Rats were considered to have acquired self-administration if they achieved a minimum of six infusions/session across the last three sessions and showed a preference for the active nose-poke (2 active: 1 inactive).Forty two rats were used to examine the effect of NaB treatment on the extinction and reinstatement of nicotine-seeking. A schematic representation of the experimental design can be seen in Following the conclusion of self-administration training, daily extinction sessions commenced by removing nicotine and its associated cues. Based on performance during the last three days of acquisition (average active nose-pokes), rats were allocated to one of three treatment groups. Group ‘NaB’ and ‘NaB + 6 h’ received an intraperitoneal (i.p.) injection of the HDAC inhibitor, NaB (100 mg/kg at 1 ml/kg), immediately or six hours after each extinction session, respectively. This dose was selected as it has been shown to modulate cocaine-induced behavioural changes without affecting locomotor activity . Group ‘Once rats had achieved the extinction criteria, reinstatement was assessed during three tests each separated by a minimum of two re-extinction sessions. NaB or saline injections were again administered across re-extinction.Testing began with a drug-free cue-reinstatement test where response-contingent cues were available throughout the session. This was followed by a nicotine-primed reinstatement test  and then a combined nicotine + cue reinstatement test (nicotine prime plus response-contingent cue presentation). NaB was not administered following reinstatement tests.In Experiment 1 it was possible that the effect of NaB on responding across extinction was not detected due to low levels of nose-poking. Experiment 2 addressed this possibility with 24 additional rats that continued to receive the response-contingent cue across extinction, increasing responding across this phase.Conditions during extinction were identical to those during initial self-administration, except that saline was substituted for nicotine (i.e. cues remained available in a response-contingent manner). Immediately following each extinction session, rats received an i.p injection of either NaB or saline. Once the extinction criteria were achieved or a maximum of 18 extinction sessions elapsed, a single nicotine + cue reinstatement test was conducted as described above.To determine whether the effects of NaB were specific to the extinction and reinstatement of drug-seeking, Experiment 3 examined the effect of NaB on the extinction and reinstatement of sucrose-seeking.Parameters for sucrose self-administration were identical to those for nicotine with three exceptions: (1) rats did not undergo surgery; (2) active nose-pokes were reinforced with a 45 mg sucrose pellet  delivered into the magazine; and (3) sessions lasted until a maximum of 30 pellets were earned or 30 minutes had elapsed.Following acquisition, rats treated with either NaB or saline (immediately following each extinction session) underwent the same extinction/reinstatement procedure described in Experiment 1. Food-primed reinstatement tests involved placing 3 sucrose pellets in the magazine prior to test.For all experiments, active and inactive nose-pokes across the last 3 days of acquisition were analysed using a mixed model ANOVA with the between-subjects factor of treatment , and the within-subjects factor of nose-poke (active verses inactive).Total active and inactive responses during the first six days of extinction were analysed using a mixed model ANOVA with the between-subjects factor of treatment and the within-subjects factors of session and nose-poke. As the greatest effect of NaB was expected on the first session after treatment , a plannActive and inactive responses during reinstatement were analysed using planned orthogonal contrasts, with the between-subjects factor of group, and the within subjects factor of nose-poke . As respTwelve rats were excluded from data analysis due to a failure to satisfy the acquisition criteria or loss of catheter patency . One rat (NaB) was excluded due to responding more than 2 standard deviations above the mean on reinstatement tests.1,26 = 33.15, p < 0.001; At the conclusion of training rats clearly discriminated the drug source , indicated that this reduction differed among treatment groups, however, this appears to be due to high levels of inactive responses in the NaB + 6 h condition only , and this difference was specific to responding on the active nose-poke . Active and inactive responding were equivalent amongst groups Saline and NaB + 6 h (Fs<1).The nicotine + cue reinstatement test produced a similar result . TreatmeTherefore, when administered immediately, but not six hours following extinction, NaB attenuated reinstatement by nicotine alone, or in combination with response-contingent cues.Seven rats were excluded from data analysis due to a failure to satisfy the acquisition criteria or loss of catheter patency .1,15 = 30.64, p < 0.001, Responding across the last 3 days of training was greater on the drug-paired nose-poke .When cues were available to elevate responding across extinction an effect of NaB was now observed . However, it must be noted that the interpretation of this data is limited given the group differences in the number of extinction days received, and that several saline animals failed to extinguish at all prior to test.1,22 = 193.60, p < 0.001, All rats rapidly learned to nose-poke for sucrose pellets, resulting in a strong preference for the active nose-poke  on reward-related learning. This finding is consistent with previous research demonstrating that increasing histone acetylation decreases cocaine and ethanol, but not sucrose, self-administration in rats , 22, 23.The results of the present series of experiments support the growing body of research suggesting that HDACs are crucial negative regulators of long-term memory , and thaIn summary, this study shows for the first time that treatment with the HDAC inhibitor, NaB, facilitates the extinction of nicotine-seeking in a manner that provides resistance to reinstatement. It is also the first study demonstrating that increasing histone acetylation during memory consolidation facilitates the extinction of an intravenously self-administered drug, a finding not observed when rats respond for a natural reward. Results of the present series of experiments provide further support for the role of HDACs as negative regulators of the extinction of drug-associated memories.S1 FigNote: SA sessions in Experiment 3 lasted until a maximum of 30 pellets were earned or 30 minutes had elapsed.Locomotor activity during acquisition of self-administration (IVSA/SA) and the first six days of extinction (E1–E6) for rats treated with sodium butyrate (NaB) or saline in (A) Experiment 1, (B) Experiment 2, and (C) Experiment 3. (TIF)Click here for additional data file.


Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish  Drosophila larvae, Caenorhabditis elegans). However, invertebrate lineages such as cephalopod mollusks exhibit morphological and behavioural complexity that parallels vertebrates, thus providing the opportunity to study the developmental genetics of organ systems that are convergent with analogous vertebrate structures. For example, amongst a handful of cartilage-bearing invertebrate lineages, cephalopod mollusks possess cellular cartilages that are remarkably similar to vertebrate cartilages in terms of histology and developmental patterns Cole and Hall  7] [7]Droso [7]Drosoand Hall ; 9]; , ). ). DrosopOne group of genes that are known to play important roles in tissue specification and differentiation are members of the SOX family of transcription factors. The Sox-family genes are widely expressed throughout development and the different family groups show divergent functions in a broad range of tissue types. Here we describe for the first time the characterization of two of the Sox family groups in a cephalopod mollusk: SoxB and SoxE.Dugesia japonica . W. WDrosopCephalopods are highly intelligent invertebrate animals and as such require special consideration  41]. No. No41]. Sepia officinalis females maintained in aquaria were obtained from the Animal Services of the Stazione Zoologica ‘Anton Dohrn di Napoli (SZN). Fertilized eggs were kept in a closed salt water aquarium, as described in , , ,55, 8],,8], and Sof-SoxB1 is expressed in all developmental stages examined. At the onset of organogenesis (stage E18) Sof-SoxB1 is expressed within the overlying epithelium of the entire embryo  at this stage, specifically within the supraesophageal mass  on frozen sections. No expression was found within the mantle cavity, including the stellate ganglia, at any stage investigated. At stage E24 expression is detected in the outer epithelial layer of the mantle, and within the epithelium of the anus an;  and the eal mass , in a feeal mass , in the eal mass , within eal mass . By stageal mass  and throeal mass . These eeal mass , with theal mass . At stageal mass , whereaseal mass .Sof-SoxB2 within whole mount preparations is similar to the Sof-SoxB1 gene, both genes showing expression within the outer epithelia of stage E24 embryos, masking expression domains from deeper within the embryo. Thus, we examine the spatial dynamics of Sof-SoxB2 gene expression on serially sectioned embryos from stages E24-E27. Sequence alignment between the two cuttlefish SOXB genes confirms significant sequence divergence within the gene fragments used as in situ probes, thus we are confident that the gene expression patterns described for these two related genes accurately reflects gene specific expression and any potentially overlapping expression domains express both genes.Expression of Sof-SoxB1 gene expression, Sof-SoxB2 expression is found within the epithelium covering the entire animal, including the head and brachial arms, and the entire mantle  from the European cuttlefish, et al have recently characterized the development of sensory epithelium and peripheral nervous system in the European cuttlefish, through the expression of the Sof-pax3/7 and Sof-elav1 genes. We show here that while Sof-SoxB1 is initially expressed more broadly in the ectoderm with respect to Sof-pax3/7, which expands to encompass the entire outer epithelium  3] [3]Sof-S [3]Sof-SSof-SoxE does not appear to be involved in chondrogenesis, it is involved in vasculargenesis of the circulatory system; Sof-SoxE is expressed in the cells that form the architecture of the developing peripheral circulatory system throughout the body at the on-set of vessel formation. We infer that these are blood vessels based upon these criteria: the main vessel tracks in the head correspond to locations of major blood vessels well described in adult cuttlefish , , , 10], , , 12], , . Here we


Tobacco is a leading environmental factor in the initiation of respiratory diseases and causes chronic obstructive pulmonary disease (COPD). Suppressor of cytokine signaling (SOCS) family members are involved in the pathogenesis of many inflammatory diseases and SOCS-3 has been shown to play an important role in the regulation, onset and maintenance of airway allergic inflammation indicating that SOCS-3 displays a potential therapeutic target for anti-inflammatory respiratory drugs development. Since chronic obstructive pulmonary disease (COPD) is also characterized by inflammatory changes and airflow limitation, the present study assessed the transcriptional expression of SOCS-3 in COPD.Real-time PCR was performed to assess quantitative changes in bronchial biopsies of COPD patients in comparison to unaffected controls.SOCS-3 was significantly down-regulated in COPD at the transcriptional level while SOCS-4 and SOCS-5 displayed no change.It can be concluded that the presently observed inhibition of SOCS-3 mRNA expression may be related to the dysbalance of cytokine signaling observed in COPD. Chronic obstructive pulmonary disease (COPD) is currently estimated to be the 3rd most common cause of death in 2020 . The disCOPD is related to tobacco smoke ,10 and aThe expression of SOCS-3 can be induced transiently by a large number of both inflammatory and anti-inflammatory cytokines such as interleukin (IL)-3, IL-6, IL-10 interferon or interferon gamma (IFN-gamma) . It has H2-mediated responses in bronchial asthma.The inhibition of cytokine signaling via the action of SOCS may also play an important role in the pathophysiology of chronic obstructive airway diseases  and a stSince there are no data available on the expression of this important cytokine signal inhibitor in COPD, the aim of the present study was to address the transcriptional expression level of SOCS-3 along with SOCS-4 and SOCS-5 in bronchial tissues of a previously characterized cohort of COPD patients ,22.1) was over 90% . Bronchial mucosal biopsies were obtained by routine fiberoptic bronchoscopy as described previously [Transcriptional expression of SOCS-3, SOCS-4 and SOCS-5 was assessed in bronchial biopsies of a previously characterized cohort of nine COPD patients . The pateviously . All subThe morphology of the tissues was assessed as previously described using routine histology ,27. The Total RNA was isolated from the bronchial biopsies as previously described . In brieThe quantitative assessment of SOCS transcripts was conducted by the use of the ABI Prism 7700 Sequence Detection system and the Taqman PCR Reagent Kit  according to the manufacturer’s protocols. For sequence-specific detection, established SOCS primer pairs were used Table . An amplAll data was analyzed using Graph Pad PRISM program. The results are expressed as mean ± SEM and tested for significant differences using the one-way ANOVA and Bonferroni`s Multiple Comparison tests.1 was below 80% of the norm , and typical chronic symptoms including cough, dyspnea, and sputum production. The histology revealed chronically inflamed tissues with typical signs of COPD-like lesions. In the submucosal layers, inflammatory cells were present which reached to submucosal glands .The mRNA levels of the different SOCS-3, -4 and 5 that inhibit cytokine signaling were assessed in the COPD tissues and compared to controls. After the presence of all transcripts was shown by qualitative PCR (data not shown), quantitative online PCR was performed in the bronchial biopsies obtained from patients with COPD and healthy controls and significant differences in gene expression were found.While online PCR for the two molecules SOCS-4 and SOCS-5 did not reveal a significant expression difference, the expression level of SOCS-3 significantly differed between the two groups of COPD and control tissues: In controls, the δδ-Ct-values of SOCS-3 mRNA expression was −3,99325 +/− 1,525749 indicating a high level of gene expression. By contrast, in COPD tissues, the δδ-Ct-values were −0,6110268 +/− 1,289377 with a p value of 0.0012 indicating a highly significant downregulation of transcriptional SOCS-3 expression  and Salmeterol (SAL) on SOCS expression since they are commonly used in combination therapy for patients with COPD . They evH1- and TH2-mediated effects. A hallmark study focused on the expression and function of SOCS-3 in allergic bronchial asthma since the functional relevance of SOCS-3 in the allergic, TH2-mediated immune response was not clear [H2-mediated allergic disease. Since the T cell-constitutive expression of SOCS-3 in an animal model led to an increase in airway hyperreactivity it was suggested that a TH2-specific expression of SOCS-3 plays an important role in the disease [This transcriptional down-regulation presently observed for COPD might have an impact on the balance of cytokines that determine general immune responses and the onset of Tot clear . It was  disease  and that1 and more prevalent in COPD patients with chronic cough and sputum production [In contrast to the increased expression in bronchial asthma, we here found a transcriptional down-regulation of SOCS-3 in COPD. In this respect, there are major differences in the cellular inflammation between COPD and asthma. While mast cells and eosinophils play a prominent role in allergic asthma, the major inflammatory cell types in COPD are macrophages and neutrophils -46 and aoduction . Lymphocoduction ,49 but toduction -46 and toduction  in controduction ,52.Similar to these differences in inflammatory cell populations that was demonstrated for asthma and COPD in the past years, a different expression pattern of cytokines and cytokine signaling inhibitors may be present in asthma and COPD. To this extend, we here shown that SOCS-3 is transcriptionally downregulated in COPD and therefore shows an expression pattern in COPD reciprocal to that in asthma, in which the molecule was shown be upregulated .A further allergic disease was also characterized to have an expression level of SOCS-3 contrary to the presently identified COPD profile . It was It would be interesting to study the functional role of SOCS-3 using an animal model of experimental COPD and different approaches to mimic COPD have been developed in the past but are limited in comparison to models of allergic asthma since they usually do not mimic all major features of human COPD.Depending on the duration and intensity of exposure, noxious stimuli such as tobacco smoke, nitrogen dioxide, or sulfur dioxide could be used to induce signs of chronic inflammation and airway remodeling wile emphysema could be achieved by combining such an exposure with the instillation of tissue-degrading enzymes. However, this such studies can not be realized at the moment since mice either constitutively expressing or lacking the SOCS-3 gene have a defect in fetal liver erythropoiesis or placental function, both leading to embryonic lethality ,54. In fIn conclusion, the present studies revealed a direct link between COPD and alterations in the transcriptional regulation of SOCS-3 that was demonstrated to play a major role in bronchial asthma. The present results indicate that the regulation of SOCS may differ in COPD compared to asthma and suggest that these cytokine signaling inhibitors also play a role in pathomechanisms underlying the inflammatory changes in COPD.The authors declare that they have no competing interests.JS, FS, CP, QTD, DQ, DAG have made substantial contributions to the conception and design of the study, acquisition of the data and interpretation. They have been involved in drafting and revising the manuscript. All authors have read and approved the final manuscript.


Control of the lumbar spine requires contributions from both the active and passive sub-systems. Identifying interactions between these systems may provide insight into the mechanisms of low back pain. However, as a first step it is important to investigate what is normal. The purpose of this study was to explore the relationships between the lumbar inter-vertebral range of motion and paraspinal muscle activity during weight-bearing flexion in healthy controls using quantitative fluoroscopy (QF) and surface electromyography (sEMG). Contemporaneous lumbar sEMG and QF motion sequences were recorded during controlled active flexion of 60° using electrodes placed over Longissimus thoracis pars thoracis (TES), Longissimus thoracis pars lumborum (LES), and Multifidus (LMU). Normalised root mean square (RMS) sEMG amplitude data were averaged over five epochs, and the change in amplitude between epochs was calculated. The sEMG ratios of LMU/LES LMU/TES and LES/TES were also determined. QF was used to measure the maximum inter-vertebral range of motion from L2-S1, and correlation coefficients were calculated between sEMG amplitude variables and these measurements. Intra- and inter-session sEMG amplitude repeatability was also assessed for all three paraspinal muscles. The sEMG amplitude measurements were highly repeatable, and sEMG amplitude changes correlated significantly with L4-5 and L5-S1 IV-RoMmax (r = −0.47 to 0.59). The sEMG amplitude ratio of LES/TES also correlated with L4-L5 IV-RoMmax (r = −0.53). The relationships found may be important when considering rehabilitation for low back pain. Optimal control of the spine during voluntary trunk bending requires fine-tuned coordination of numerous trunk muscles . This dyet al. (1998) investigated the reduction in lumbar muscular activity during full flexion (flexion relaxation) and spinal kinematics at an inter-vertebral level [In order to directly investigate the passive and active sub-systems of the spine, there have been many efforts to concurrently measure spinal kinematics and muscle activity ,10,11,12Study of the integrated function of the joints and muscles of the spine requires contemporaneous multi-level kinematic and electromyographic monitoring throughout the motion. This is necessary to incorporate timing, magnitude, and segmentation in the two systems to characterise control. Multi-level surface electromyography fulfils these requirements for motor control and quantitative fluoroscopy measures a range of continuous inter-vertebral motion variables . ContempIn order to investigate relationships between segmental kinematics and local muscle activity, suitable variables from each must be identified. While responses to perturbation , and theTo investigate the relationships between lumbar muscle activity and inter-vertebral restraint during bending requires access to the maximum IV-RoM (IV-RoMmax). Continuous intervertebral rotation data allows both temporal comparisons with other variables and the actual maximum IV-RoM (IV-RoMmax), rather than IV-RoM at the limit of voluntary trunk bending, to be extracted. Recording in the standing orientation allows these comparisons.et al. (2015) suggest that there are only subtle differences between various low back patient groups and healthy controls in terms of paraspinal muscle activity and regional lumbar movement [Sanchez-Zuriaga movement . This memovement  and so iThe development of QF techniques has seen its use in LBP research become more common ,21,22. IThe purpose of this study was to quantify the relationships between IV-RoMmax during flexion of the lumbar spine with the accompanying paraspinal muscle activity.To determine whether ratios of inter-level lumbar paraspinal sEMG amplitudes are related to the IV-RoMmax at lumbar inter-vertebral levels.To determine whether changes in sEMG amplitudes during different phases of the forward bending cycle are related to IV-RoMmax at lumbar inter-vertebral levels.To determine the inter- and intra-session reliability and agreement of normalised sEMG amplitudes during weight-bearing sagittal flexion and return.The eligibility criteria for the study are shown in Lumbar spine fluoroscopic images were collected at 15 Hz using a Siemens Arcadis Avantic VC10A digital fluoroscope (CE0123) and an upright motion frame, which stabilised participants and guided their bending motion. Participants were asked to stand with their right side against the motion frame , and folBefore image acquisition commenced, participants were taken in 20° increments through to the full 60° to ensure that they were able to tolerate the motion. The movement of the motion frame was recorded by electronic feedback from its motor drive and synchronised with the fluoroscopic imaging. To avoid bending at the hip joints, the pelvis was stabilised using a belt secured around the anterior superior iliac spine and secured to a bracing pad placed against the lower sacral segments. A lead apron was worn to shield the gonads. Flexion and return sequences were then transferred to a desktop computer for analysis using bespoke image processing codes written in Matlab  . The verThe data extracted comprised the continuous inter-vertebral angle in flexion and the IV-RoMmax. IV-RoMmax for each inter-vertebral level  was calculated as the maximum angular range reached at any point throughout the 60° trunk flexion and return cycle.Prior to the commencement of data collection, participants lay prone in order for 12 electrode sites to be marked on their backs with a skin pencil. In preparation for this, the skin over their lower backs was prepared for sEMG electrode application by light abrasion, cleaning with an alcohol swab, and when necessary, shaving of the area. Disposable pre-gelled self-adhesive Ag-AgCl electrodes were then applied over three bilateral muscle groups with a 20 mm centre-to-centre inter-electrode distance as follows: Thoracic erector spinae (TES)  , the lumAlthough cross talk from multiple muscles will inevitably contribute to the signal recorded at each electrode site, cross-sections of the spine at each electrode site showed that the muscles that will predominate at T9 (TES) and L2 (LES) is longissimus thoracis, and at L5 (LMU) multifidus . Three BElectrode application accuracy is dependent on the subjective identification of bony anatomical landmarks, and current methods used are therefore limited by human subjectivity and variation in individual anatomy ,33,34,35The sEMG signal data were recorded at a sampling rate of 2000 Hz using a common-mode rejection ratio (CMRR) of 110 dB and an input impedance of 1000 MOhms. The six signals were band pass filtered at 10–500 Hz and full wave rectified. The root mean square (RMS) amplitude was calculated for individual participant cycles and normalised during post-processing to sub-maximal voluntary contractions expressed as a percentage of the sMVC.When data collection had been completed, and in order to provide a sub-maximal reference contraction (sMVC) , particiThe QF motion frame controller recording and the sEMG data recording were co-ordinated using a trip switch attached to the motion arm of the frame. This registered a data point on the sEMG timeline .A separate convenience sample of 10 participants was used for the sEMG amplitude intra- and inter-subject repeatability studies. These studies were done without QF imaging. The acquisition cycle was repeated four times  at baseline and follow up. Intra-session results compared cycles 1 and 2 (of the four), whereas inter-session results were calculated as an average of the four mean (left and right) normalised amplitudes recorded over the cycle duration. All analysis was conducted by ADR.sEMG ratios ,39 were p values < 0.05) were further analysed using simple linear regression. Intra-subject reliability and agreement of the mean normalised RMS sEMG amplitudes throughout the flexion and return cycle were assessed using intra-class correlations  [All data were tested for normality using the Shapiro-Wilk test. Relationships between IV-RoMmax and sEMG ratios and changes from normally distributed data were analysed using the Pearson product-moment correlation coefficient, and non-normal data using the Spearman’s Rank Correlation. Significant relationships  , and theCC 3, 1) . StatistTwenty males with no history of low back pain over the previous year consented to participate. Failed template tracking occurred in two participants’ sequences, and their QF and sEMG data were therefore discarded. The mean (SD) age, height, and body mass Index (BMI) were 27.6 years (4.4), 1.8 m (0.06), and 24 (2.2), respectively. Average radiographic exposure factors for the group were recorded as 79.7 kV SD (5.4) and 55.4 mA SD (3.4). The mean effective dose was calculated using ICRP103 conversion software PCXMC (Monte Carlo Simulation Package) , and wasIntra- and inter-session reliability and agreement for normalised muscle activity during the bending sequence was high for all muscle levels . The higA summary of all correlations between changes in muscle activity and IV-RoMmax is given in . Signifi2 values range from 0.177 to 0.247.All significant correlations were further analysed using simple linear regression. The effects of muscle activity changes on IV-RoMmax are shown in . The tabp = 0.13). The correlations between sEMG ratios and IV-RoMmax at all inter-vertebral levels are shown in . The onlIt is recommended that any procedures to be used in EMG studies should undergo reliability testing . In thisThe results demonstrate that changes in activity of TES, LES, and LMU at various stages of the forward bending cycle, can all be to some degree related to the IV-RoMmax at lower lumbar levels (L4-5 and L5-S1). It has been suggested that intersegmental forces maintain or decrease inter-vertebral motions ,48, it wOf particular interest is the apparent shift in effect between TES and LES on the IV-RoMmax of L5-S1 . As LES When considering the LES to be local  and TES to be global  , then thThere are many correlations that approach significance , and thePrevious work has indicated a clear distinction between the kinematic behaviour of the upper and lower sections of the lumbar spine . It was et al. (2012), who also demonstrated a significantly higher LES/TES ratio in LBP patients during gait [The ratio of lumbar erector spinae over thoracic erector spinae activity has been investigated in several previous studies ,54,55,56et al. also investigated this muscle activation imbalance in varsity athletes, and while maintaining that there was indeed a relationship between muscle imbalance between levels and LBP, the authors also found that in some individuals with a history of LBP, TES activity could be dominant [Reeves dominant . The autdominant . CrucialThe results of this study highlight that the ratio of LES/TES can vary in a population with no long term history of low back pain, and would appear to relate to variations in inter-vertebral mechanics in such a population. It has been proposed that lumbar inter-segmental movement is also influenced by spinal morphology , but theIt has been suggested that muscle imbalance between levels does not cause low back injury . It is aIn a LBP free population sample, it might be assumed that variations in muscle activity patterns do not represent adaptations to pain. However, that is not to say that particular activity patterns and thus kinematic behaviours may not be risk factors for future LBP episodes. It also questions the conclusions of studies that compare LBP population groups with healthy pain free controls, as muscle activity patterns may not be adaptations to the episode.It is suggested that achieving sufficient stability is a moving target, and that no single muscle can therefore be considered the best stabiliser, as the most important muscle is transient dependent on the task . The resi.e., young healthy male adults) and so the results are not generalizable to other groups. It is anticipated that variations in kinematic and morphological parameters that are associated with age related change and gender would also affect IV-RoMmax, and therefore also warrant further investigation. Future investigators may also wish to incorporate measurements such as thoracic kyphosis and pelvic incidence in order to gain insight into changes in kinematic behaviour beyond the lumbar spine.Finally, it is a limitation of this study that people with non-specific low back pain were not included, yet it would be important to know to what extent these relationships, which are consistent with maintaining appropriate restraint on vertebrae during bending, are disrupted in patients. If so, this would point to a potential route for patient stratification based on biomechanics. Such studies are now warranted. The study also only investigates a narrow population (This study found weak to moderate but significant correlations between both muscle activity changes and ratios and IV-RoMmax at various inter-vertebral levels. Of particular interest was the correlation between decreased LMU and increased IV-RoMmax at L5-S1 in the latter stages of flexion, the apparent co-dependency between LES and TES during early to mid-flexion, and the effect of the LES/TES ratio on the IV-RoMmax at L4-L5. These relationships, when combined with other influencing factors, may be important when specific inter-vertebral levels are considered to be sources of pain generation and when considering rehabilitative or surgical planning. Multivariate investigations in larger samples are warranted, potentially leading to longitudinal outcome studies in LBP groups.


In order to grow, tumors need to induce supportive alterations in the tumor-bearing organ, by us named tumor instructed normal tissue (TINT) changes. We now examined if the nature and magnitude of these responses were related to tumor size and aggressiveness. Three different Dunning rat prostate tumor cells were implanted into the prostate of immune-competent rats; 1) fast growing and metastatic MatLyLu tumor cells 2) fast growing and poorly metastatic AT-1 tumor cells, and 3) slow growing and non-metastatic G tumor cells. All tumor types induced increases in macrophage, mast cell and vascular densities and in vascular cell-proliferation in the tumor-bearing prostate lobe compared to controls. These increases occurred in parallel with tumor growth. The most pronounced and rapid responses were seen in the prostate tissue surrounding MatLyLu tumors. They were, also when small, particularly effective in attracting macrophages and stimulating growth of not only micro-vessels but also small arteries and veins compared to the less aggressive AT-1 and G tumors. The nature and magnitude of tumor-induced changes in the tumor-bearing organ are related to tumor size but also to tumor aggressiveness. These findings, supported by previous observation in patient samples, suggest that one additional way to evaluate prostate tumor aggressiveness could be to monitor its effect on adjacent tissues. Prostate cancer is a common, generally multifocal, disease with variable behavior ranging from harmless to lethal. Unfortunately our ability to safely diagnose cancer and then differentiate the aggressive from and the non-aggressive forms are limited . Many prNumerous studies have shown that the normal prostate tissue in cancer patients is altered and that detection of such changes  can possibly be used to indicate that cancer is present elsewhere in the organ and perhaps also be used to evaluate its future behavior , 3, 5, 6tumor instructed (and thus indicating) normal tissue  A, 6 A3, 6The fast growing locally aggressive and metastatic MatLyLu tumors appear to be particularly effective in recruiting macrophages, of which many are of the tumor-stimulating M2 phenotype (CD163 positive), to the tumor, to the invasive front and to the surrounding tumor-bearing organ. Depletion of these macrophages reduced tumor growth, vascular densities and micro- and macro-vascular BrdU labeling in prostates implanted with AT-1 tumors , 15. It In this study we examined the MaLyLu model up to 10 days after tumor cell implantation. At this time-point metastases cannot be detected in lymph nodes or in lungs but such metastases can be seen one week later (own unpublished observations). It is therefore possible that the requirements for metastatic seed of tumor cells are prepared for already at 10 days. One cell-type involved in facilitating vascular invasion, extravasation and colonization of tumor cells is macrophages , 40. FutMast cells were attracted to the prostate tissue surrounding Dunning tumors, and inhibition of mast cells function retarded vascular and tumor growth in the AT-1 model . Mast ceIn summary, in this paper we demonstrate that implantation of tumor cells into the rat prostate results in adaptive, presumably tumor promoting, changes in the tumor-bearing organ. Some of these adaptive TINT changes appear to be mainly related to the tumor size whereas others are also related to the growth and metastatic potential of the growing tumor. Several investigators have already described changes in the tumor-bearing human prostate related to the presence, and for some factors also to the grade and distance to tumors present elsewhere in the organ , 3, 5, 6


Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for  Vibrio fischeri [luxI, encoding for an autoinducer synthase, responsible for the production of the small signalling molecule N-3-oxohexanoyl-L-homoserine lactone (AHL), and luxR, encoding for the LuxR transcription factor that is activated by AHL. The activated complex LuxR-AHL binds a DNA domain called lux box, located upstream of the Plux promoter, thus activating the transcription of downstream genes, involved in bioluminescence, in a concentration-dependent fashion [Quorum sensing (QS) is a cell-to-cell communication mechanism exploited by Gram-negative and Gram-positive bacteria to implement a coordinated and density-dependent control of gene expression –4. The efischeri . Here, t fashion .QS elements have been widely used in synthetic biology to control the behaviour of single cells and bacterial populations via ad-hoc constructed gene networks. Such elements have been exploited to construct exogenously inducible input devices to regulate gene expression in several synthetic devices, such as a lysis device , logic glux and las systems [Synthetic circuits have also been constructed with the aim of improving the knowledge on the behaviour of natural QS systems , 24. Nat systems .In order to improve the knowledge on the quantitative behaviour of QS elements and to expand the list of available parts, novel synthetic components were designed and built up starting from known regulatory elements. Promoters represent key regulatory elements in gene network design. The design of their response logic and the tuning of their activity in a rigorous fashion is necessary in order to meet the specifications derived from circuit design.Constitutive active variants of the LuxR transcription factor were constructed by mutagenesis or truncation to create an AHL-independent LuxR-inducible promoter, functioning as an amplifier that can be re-used in different synthetic networks .QS-based synthetic promoters have been designed to be responsive either to multiple autoinducer molecules  or to adesaR promoter, repressible by the EsaR-AHL complex, to obtain a set of QS-inducible systems with disparate gene expression levels, regulatory range, AHL sensitivity and timing [lsrR genes (starting from the E. coli master QS regulator) able to show novel activation and repression logic [Many efforts have been carried out to create promoters able to sense two or more inputs. Tamsir et al. constructed tandem promoters composed by the sequence of two promoters with the same orientation, behaving as an OR gate with two QS signalling molecules (or other molecules) as inputs , while Bd timing . They ald timing . The twoon logic .lux box, AraC, LacI and TetR operator sites) in the same promoter, in the distal, core or proximal position, obtaining a library of mixed-regulated promoters, responding to one or more stimuli and exhibiting different activation logics [Cox et al. randomly combined different transcription factor binding sites  the E. coli consensus -35 (TTGACA) and -10 (TATAAT) promoter regions. This promoter exhibited an inverted logic, compared to the wild type Plux promoter, being repressed by the LuxR-AHL complex.Egland and Greenberg  reportedThere is no accordance between the findings of Cox et al.  and EglaSerious efforts have been performed in the development of predictive tools to relate promoter strength to the DNA sequence, but no strategy has shown to fully capture this cryptic relation . In factSeveral techniques for libraries generation have been exploited, such as random mutagenesis of existing promoters via error-prone PCR , PCR witIn this work, we report the generation of a library of LuxR-repressible promoters see , inspireThe obtained synthetic promoters, constituting the library, are available in the Registry of Standard Biological Parts (Registry) with BioBrick codes from BBa_J107100 to BBa_J107111 and all the documentation and experience has been shared in this repository .E. coli (Invitrogen) were used for parts cloning and tests. Plasmids were propagated in recombinant strains grown in selective LB medium [TOP10 B medium . TOP10 bApart from the newly synthesized promoters, whose construction is described below, all the parts and plasmids used in this study were assembled from basic or composite parts from the Registry 2009 or 2010 DNA Distribution , accordiDNA-modifying enzymes were purchased from Roche. DNA purification kits were purchased from Macherey-Nagel. All parts were verified through sequencing, performed via the BMR Genomics DNA analysis service . AHL  was dissolved in deionized water to prepare a 2 mM stock, routinely stored at -20°C.The resulting library includes 12 members. lux box) for the binding of the LuxR-AHL activated complex in the core region, between the -35 and -10 sequences [lux box was maintained unaltered.The LuxR-repressible promoters are characterized by the presence of a 20-bp DNA domain  were included in the consensus determination. The resulting consensus sequence is reported in The Anderson constitutive promoter collection  includesreenberg . The Andlux box sequence is present in the ‘core’ position, to provide the AHL-repressible behaviour. However, this part cannot be readily assembled upstream of a gene expression system composed by an RBS-gene-Terminator sequence because the promoter ends with the -10 region. For this reason, the transcription start site (+1) would lie after the beginning of the RBS sequence and the assembly of a spacer between promoter and RBS would be required to properly express the downstream gene. To overcome this problem, a new LuxR-repressible promoter was constructed via oligonucleotide annealing, according to the Anderson library sequence and with the -35 and -10 sequences of BBa_R0061 and BBa_J23119. Two oligonucleotides (sense—J107100_FWD and anti-sense—J107100_REV) were designed according to the structure of the BBa_J23119 promoter  and -10 (TATAAT) regions as the BBa_J23119 promoter of the Anderson collection and the lux box in the core region, overlapping for one nucleotide with both the -35 and -10 sequences  with P_LIB_FWD and P_LUXH_REV primers see , to remoE. coli TOP10 and the grown colonies were selected according to a visual inspection of RFP expression: transformation plates with colonies bearing low-copy plasmids were used to try to select phenotypes only for very strong promoters and not to give a metabolic burden impairing cell growth, while plates with colonies bearing high-copy plasmids should allow the selection of medium to weak promoters. The selected clones were assayed as described in the next section both in the constitutive and regulated conditions. Then, only the clones with detectable fluorescence and which showed repression upon AHL induction were considered. All the promising candidates in the high-copy condition were then transferred in the low-copy plasmid pSB4C5 and all the resulting promoters in the low-copy condition were sequence-verified. Promoters with unique sequences were selected as members of the library. The members of the LuxR-repressible promoter family obtained via the first PCR round with degenerate primers are named BBa_J1071xx, with xx = 01,02,…,10.The obtained parts in both the low- and high-copy plasmids were transformed in The library members were characterized in different contexts. The ‘constitutive’ mode indicates the design that does not include a LuxR expression cassette , while tThe constitutive version of the library, obtained as described above, is shown in E. coli TOP10 cells bearing the RFP-LC library members with the pLuxR-MC plasmid, which is the pSB3K3 plasmid containing the LuxR expression cassette BBa_S03119 as insert. This configuration is named ‘RFP-LC-LUXR’  . Like inGFP was assayed as a different reporter. The BBa_E0240 GFP generator  was XbaI/PstI-digested and inserted in both the SpeI/PstI-digested RFP-MC and RFP-MC-LUXR constructs (thus replacing the RFP expression system with a GFP expression system). These contexts are named GFP-MC and GFP-MC-LUXR, respectively . Here, tLlacO1, PlacIQ and PR promoters, instead of the members of the Anderson library (in this case with the standard DNA scar 'TACTAGAG').The Anderson promoter library members were used to investigate an experimentally observed and previously uncharacterized repressible behaviour of BBa_J23101 by the LuxR-AHL complex. All the sequence-verified Anderson promoters available in the Registry  on the activity of the BBa_J107100 promoter was assayed by constructing redLC (BBa_J107120), redLC-luxRup (BBa_J107121), redLC-luxRdown (BBa_J107122), redLC-luxRMC (BBa_J107120 co-transformed with BBa_S03119), greenMC (BBa_J107123), greenMC-luxRup (BBa_J107124) and greenMC-luxRdown (BBa_J107125) parts. These constructs were submitted to the Registry and are described in 4, 0.1 mM CaCl2, 0.2% casamino acids and 0.4% glycerol), incubated at 37°C, 220 rpm for 16 hours. Cultures were 100-fold diluted in 200 μl of selective M9 in a 96-well microplate. When required, 2 μl of properly diluted AHL were added into the plate wells to reach the desired final inducer concentration. Deionized water was used to prepare the AHL dilutions. No water was added to the wells where AHL-inducible cultures were assayed without inducer. The plate was then incubated in the Infinite F200 microplate reader (Tecan) and the following kinetic cycle, programmed with the i-control software (Tecan), was used to assay the cultures: every 5 min, 15 s linear shaking (3 mm amplitude), 5 s wait, optical density at 600 nm (OD) measurement, fluorescence measurement. The total duration of the kinetic cycle was about 18 hours. RFP detection was carried out with the 535/620 nm filters for excitation/emission and acquisition gain as appropriate, ranging from 50 to 100. GFP-expressing cells were assayed through the same procedure, but using the 485/540 nm filters for fluorescence acquisition. In every experiment, 200 μl of M9 supplemented medium and a non-fluorescent TOP10 culture were also included to measure the background of OD and fluorescence, respectively [Glycerol stocks were streaked on LB agar plates supplemented with the proper antibiotics. Plates were incubated at 37°C for 16 hours. Single colonies were picked and used to inoculate 1 ml of selective M9 supplemented medium  and Relative Promoter Units (RPUs) during the exponential growth phase for each culture. OD, RFP and GFP were background-subtracted as previously described [ln(OD) time series in our experiments. The slope m of this line was computed as the average of the numeric time derivative of ln(OD) in the exponential phase to yield the cell growth rate. Doubling time was computed as ln(2)/m.Raw absorbance and fluorescence time series were processed to obtain doubling time, average RFP and GFP synthesis rate per cell . Relative units, to express the activity of promoters in standardized measurement units, were computed as RPUx = Scell,x/Scell,ref, where x is the culture of interest and ref is the culture bearing the standard reference promoter (BBa_J23101) with RFP or GFP, in the same copy number conditions and with identical measurement system (and DNA junction between promoter and downstream reporter expression system) as the culture of interest, and acquired with the same gain factor.A signal proportional to the RFP or GFP synthesis rate per cell was computed as the numeric time derivative of fluorescence, divided by OD over time. This signal was averaged over the exponential growth phase to obtain the mean M is the switch point of the Hill function, δ+VMAX is the maximum activity in absence of AHL and η is the Hill coefficient. Y is the RPU of the system and 'A' is the AHL concentration in the culture medium.Repression curves were fitted with the Hill equation The coefficient of variation (CV) of the estimated parameters was computed via a Monte Carlo simulation method. In particular, 10,000 synthetic datasets were generated by adding Gaussian noise . Least square optimization was performed using the Matlab Flow cytometric data from single-cell experiments, in FCS format, were exported and processed using an FCS data reader package  via MatScell. Cell-to-cell variability (in terms of CV) was also calculated for every experiment.RPUs were calculated as described above, by using the background-subtracted fluorescence values instead of Although the originally proposed formulas for RPU computation include the growth rate , here itStatistical analysis was performed as reported in : the ANOAll the promoters were initially present in low-copy (LC) or high-copy (HC) vector with an RFP expression cassette downstream, thus enabling quantitative measurements via reporter protein. A total of 90 clones were selected and screened by measuring constitutive RFP expression and repression capability in the regulated mode (promoters 1–90). At the end of this screening, the functional promoters in high-copy were transferred into the pSB4C5 low-copy vector.A total of 28 candidates, all in low-copy plasmid, were obtained and sequenced via the VF2 and VR primers . 10 uniqThe screening procedure for library generation was based on fluorescence measurements in single clones without replicates, in heterogeneous plasmid measurement systems (LC and HC). In order to fully characterize the library performance, the promoters were then tested in different contexts and with a suitable number of biological replicates.All the 11 members of the library of repressible promoters have been assembled in the six contexts represented in The constitutive activity is shown in LtetO1 promoter (according to unpublished experiments of our group). For the promoters with activities that are significantly different among the three measurement systems , CV was computed. Promoters with RPU values greater than 0.1 always have a CV lower than 61%, while CV is much higher for the two weakest promoters, probably because of the high noise levels affecting measurements. Considering library members with RPU>0.1, 7 out of 9 promoters show a statistical difference among the tested conditions. CV entities are consistent with previous studies of our group on context-dependent variability of individual promoters [The strongest promoters have RPU activities comparable to the widely used Promoters , 49, wheromoters , 50, 51.MAX is highly correlated with the constitutive strength of promoters  involving devices with the wild type Plux inducible promoter, where the luxR gene was expressed at high levels by the PLtetO1 promoter (like in this study) in the MG1655 strain [LlacO1 promoter in the MG1655-Z1 strain [MAX values . This might be due to cell overburdening or nonlinear saturation phenomena occurring in the cell, related to copy number increase [MAX values in the regulated low-copy context  did not show a significant context-dependent variation. On the other hand, the use of GFP or RFP as reporter system did not result in important differences in promoter activity.Single-cell measurements have been performed in order to evaluate the library activity with a different measurement system, and to evaluate if fluorescence distributions in cell populations have a unimodal behaviour. Library members have been assayed in constitutive and regulated modes, in the GFP-MC context. Regulated modes were assayed in the full (100 nM), intermediate (5 nM) and no induction conditions. Fluorescence distributions are shown in The 11-member library obtained so far shows an uncovered constitutive activity range between about 0.7 and 2 RPUs see . PromoteIn order to investigate the effects of the presence of flanking sequences on promoter activity and to evaluate their impact in different configurations, seven recombinant strains have been constructed see , by varyThe activity of the LuxR-repressible promoter was measured in absence of AHL (maximum activity) as described in the Methods section and the results were used to perform ANOVA tests. Significant differences in promoter activities have been observed  between the four strains expressing RFP in LC plasmid see , howeverIn the GFP expressing strains, ANOVA test showed that a significant difference is present between promoter activities  and post-hoc comparisons showed a clear separation between the device without the LuxR expression cassette (greenMC) and the ones with the LuxR expression cassette, upstream or downstream of the promoter (greenMC-luxRup and greenMC-luxRdown). In the latter case, promoter activity is reduced by about 2-fold. This result suggests that the presence of an additional expression cassette in MC plasmid can be responsible of the overload of cell machinery, thus causing a diminished promoter activity, even though no effect on the doubling times was observed , PlacIQ  and PR  in order to validate their behaviour in presence of the LuxR-AHL complex.This phenomenon has been further investigated by characterizing all the available promoters from the Anderson library, in LC plasmids in presence of LuxR from the pLuxR-MC plasmid. Other promoters were also included in this analysis: PThe results of a preliminary test are reported in To our knowledge, such phenomena were not reported in literature, and additional efforts should be required to elucidate the underlying mechanism, especially when such promoters are used in complex gene networks where their specific constitutive activity is crucial to enable the correct functioning. This effect has been documented in the Experience section of the BBa_J23101, BBa_J23102 and BBa_J23104 Registry pages.Naturally-occurring cell-to-cell communication mechanisms are widely used as a source of genetic elements to engineer biological systems that show disparate complex functions . In ordeAt the state of the art of synthetic biology, the cryptic relation occurring between promoter nucleotide sequence and strength has not been fully captured, thereby limiting the availability of predictive tools to drive the de-novo design process. The creation of libraries of graded-strength promoters can face such limitations by providing synthetic biologists with a catalogue of parts to choose the one with the desired behaviour.E. coli. The LuxR-repressible behaviour depends on the presence of a lux box  in the core sequence of all the promoters, while the graded strength is obtained by varying the nucleotide sequences of the -35 and -10 regions, according to the consensus determined from an existing constitutive promoter library .lux box in the core sequence was inspired by the work of Egland and Greenberg [The construction of promoters with the reenberg , who valreenberg . Consistin vivo via two different instruments: a microplate reader to perform population-based acquisitions and a flow cytometer to provide single-cell data. Since all the obtained promoters can be used as constitutive or repressible elements, their activity in absence or presence of LuxR was measured, respectively, to provide data on the use of the promoters in both contexts.The availability of catalogues of quantitatively characterized parts can support the rational design of biological systems by enabling the choice of suitable components for the correct functioning of the target system, which can be studied, for instance, via mathematical modelling tools. A central problem in synthetic biology is the context-dependent variability of parts performance when they are used in different circuits or conditions. For this reason, the characterization of parts activity in different contexts and measured via different instruments can strongly support the re-use of genetic parts and could increase the probability of obtaining the expected quantitative behaviour in the designed system. To further support this process, standardized units are also crucial for the re-use of components in different laboratories where different measurement equipment is available. In light of the mentioned requirements, we carried out a multi-faceted characterization of our new library of components by reporting promoter activities in RPU. Two reporter measurement systems (RFP and GFP) were tested downstream of the promoters, in plasmids that are maintained at two different per-cell copy number, and measurements were carried out The final LuxR-repressible promoter library described in this work is composed by 12 members spanning an activity range from about 0.03 to about 3 RPUs and with about 42% mean spacing between the activities of adjacent members. All of them can be tightly repressed by the LuxR-AHL complex, as demonstrated by dose-response experiments.One of the 12 final members (BBa_J107100) did not result from the random screening process, but it was independently designed according to the -35 and -10 sequences of BBa_R0061, a LuxR-repressible promoter present in the Registry, that was designed according to the promoter constructed by Egland and Greenberg . BecausePromoter activity in the constitutive mode, i.e., without a source of LuxR, showed a reasonably contained context-dependent variability among the three biological measurement systems used, with a CV not greater than 31% for promoter members with activity higher than 0.4 RPU. This variability value indicates the expected activity deviation that can be observed when re-using the promoter library members in a new context, such as a gene regulatory network or an expression cassette for a different gene. Although we studied different parts with different measurement systems, the CV among the tested contexts was consistent with our previous works, where promoters with a similar RPU range was investigated , 49. In MAX parameter) which was consistent with their constitutive activity, as expected, but was also dependent on the copy number context. In fact, VMAX in medium-copy (with both RFP and GFP reporter systems) was 2-fold lower than in the low-copy context, measured via RFP. The KM parameter was highly consistent with the one of the wild type Plux promoter when characterized in presence of the same LuxR generator device [For promoters in the regulated mode, i.e., in presence of LuxR, the AHL-RPU data were fitted with a Hill function and the resulting parameters can be used to summarize the quantitative behaviour of the repressible devices. Although some of the promoters showed fitting problems due to their low activity, the overall distribution of the estimated parameters did not have a clear strength- and context-dependent behaviour, except the maximum activity of promoters  in low- (with RFP) and medium- (with GFP) copy contexts with the LuxR expression cassette in different positions: upstream or downstream in the same plasmid, in a different plasmid (only for the low-copy with RFP context), or absent. The results clearly demonstrated that the above observed activity difference was due to the presence of two expression cassettes in a medium-copy plasmid, probably causing a metabolic overload in the host cell, while no significant upstream sequence-dependent effect could be detected when the LuxR expression cassette was present upstream of the reporter expression cassette driven by BBa_J107100.Flow cytometry was used to measure the activity of promoters in one of the above tested contexts  via different measurement equipment. All the tested constructs showed a unimodal fluorescence distribution, whose arithmetic mean was highly consistent with the one obtained via population-based measurements. Single-cell data also gave the occasion to estimate cell-to-cell variability of clones. Excluding promoters with RPU<0.1, an average 55% CV was observed, independently from promoter activity, presence of LuxR expression cassette, and AHL addition. The quantification of cell-to-cell variability has an important impact on the bottom-up design of synthetic networks from basic modules, since it can be used to model and predict interconnection effects that cannot be trivially obtained from population-based measurements . HoweverlacIQ, PLlacO1 and PR promoters. This phenomenon should be taken into account when these parts are used as constitutive promoters in circuits containing the luxR gene and AHL is present, since unpredictable effects may occur, due to a non correct evaluation of their transcriptional strength. Although the maximum repression entity was obtained at 20 μM AHL, which is a very high concentration, it is worth noting that commonly used AHL levels (10 nM to 1 μM) also resulted in a non-null repression.In addition, an undocumented LuxR-dependent repression of three members of the widely used Anderson promoter collection  in presence of AHL was reported in this study. This repression entity was 78%, 28% and 49%, respectively, at 20 μM AHL and cannot be explained by cell growth variation, which is almost unaffected (data not shown). Other 9 tested members of the Anderson collection were unaffected, as well as the three popular PE. coli and other organisms [LtetO1 strong promoter and also with the activity of BBa_J23119 constitutive promoter [Considering the initial 11-member library construction process, the number of obtained members (10 unique clones of graded strength) is consistent with other works in rganisms , 54, altrganisms , 40, 55 rganisms , 39. Ourpromoter , whose -The 'efficiency' of a library generation process is a controversial term, since different works provide it according to different definitions, such as the percentage of final unique members over the initial colonies, or the percentage of colonies with visible fluorescence over the total number of colonies on the transformation plate , 56 and Regulated promoters such as the ones here constructed are widely used in biotechnology and synthetic biology research for several tasks, such as driving the expression of recombinant proteins, optimizing industrially-attractive metabolic pathways and constructing complex circuits with customized behaviour. The graded strength can be exploited to optimize the process yield, in concert with other factors such as plasmid copy number, RBS and codon optimization. The repressible behaviour of our novel components (as well as the inducible behaviour of the currently existing QS-based parts) enables the control of gene expression timing. AHL can be exogenously added or provided via other QS elements  to engineer complex, coordinated and unnatural biological functions. We expect that the herein reported library will be used in different QS-based synthetic circuits or as tunable (or constitutive) promoters in simple gene expression systems. The provided characterization data will strongly support the use of such elements in the circuit of interest according to design specifications, which can also be met with the help of mathematical models where the quantitative features of the reported promoters can be included . The libS1 FigPanels A, B and C show the results in the RFP-LC, RFP-MC and GFP-MC contexts, respectively. The doubling time of TOP10 bearing the BBa_J23101 is also shown in each context. Bars represent the average value, while error bars represent the 95% confidence intervals of the mean value, computed on at least three biological replicates. Results show that no significant promoter activity-dependent trend is present and, for each context, the doubling times are comparable between constitutive and regulated mode and with the strains bearing the BBa_J23101 promoter. We only considered promoters in the regulated mode without AHL, since we found that, given a context, RFP expression never exerted a statistically significant AHL-dependent doubling time increase .(TIFF)Click here for additional data file.S2 FigEach panel shows the curves corresponding to the promoter indicated by the number. Solid lines are the fitted curves, while circles represent the average of three experimental replicates and error bars represent the 95% confidence intervals. Blue indicates the LC-RFP context, red the MC-RFP context and green the MC-GFP context.(TIFF)Click here for additional data file.S3 FigA) Constitutive activity of the 12-member promoter library in the RFP-LC context, showing that promoter 122 (green bar) has successfully filled an activity gap between promoters 2 and 35. B) AHL-dependent Hill repression curve of promoter 122 in the regulated mode. Estimated parameters are reported with their 95% confidence intervals. Bars (panel A) and circles (panel B) represent the average value, while error bars represent the 95% confidence intervals of the mean value, computed on at least three biological replicates. The average doubling time of TOP10 bearing promoter 122 in the RFP-LC context was 89.7 min  and 84.7 min  in the constitutive and regulated (without AHL) modes, respectively. As observed in the other promoters, no significant AHL-dependent variation in doubling time was present in the regulated mode .(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 FigLlacO1, PlacIQ and PR). Promoters were assembled in LC plasmids and were co-transformed with pLuxR-MC (including a LuxR expression cassette in the pSB3K3 vector). Blue bars represent the activity when no AHL was added to the medium, while green bars represent the repressed activity (AHL 20 μM was added to the medium). Only BBa_J23101, BBa_J23102 and BBa_J23104 showed repression and, for them, percent repression is reported. B) Repression curves for the three promoters studied. Differently from the library members, for the used AHL concentrations these promoters do not show a full-repression upon induction.A) Constitutive and repressed activity of all available sequence-verified promoters from the Anderson promoter collection, and three additional popular promoters (P(TIFF)Click here for additional data file.