O-methyltransferase (COMT) enzyme is critical for the catabolic regulation of synaptic dopamine, resulting in altered cortical functioning. The COMT Val158Met polymorphism has been implicated in human mental illness, with Met/Met homozygotes associated with increased susceptibility to posttraumatic stress disorder (PTSD). Our primary objective was to examine the intermediate phenotype of fear inhibition in PTSD stratified by COMT genotype  and differential gene regulation via methylation status at CpG sites in the COMT promoter region. More specifically, we examined the potential interaction of COMT genotype and PTSD diagnosis on fear-potentiated startle during fear conditioning and extinction and COMT DNA methylation levels (as determined using genomic DNA isolated from whole blood). Participants were recruited from medical and gynecological clinics of an urban hospital in Atlanta, GA, USA. We found that individuals with the Met/Met genotype demonstrated higher fear-potentiated startle to the CS−  and during extinction of the CS+  compared to Val/Met and Val/Val genotypes. The PTSD+ Met/Met genotype group had the greatest impairment in fear inhibition to the CS− (p = 0.006), compared to Val carriers. In addition, the Met/Met genotype was associated with DNA methylation at four CpG sites, two of which were associated with impaired fear inhibition to the safety signal. These results suggest that multiple differential mechanisms for regulating COMT function – at the level of protein structure via the Val158Met genotype and at the level of gene regulation via differential methylation – are associated with impaired fear inhibition in PTSD.The catechol- Posttraumatic stress disorder (PTSD), a debilitating psychiatric illness precipitated by exposure to a traumatic event, occurs in approximately one-fourth of traumatized individuals , an enzyme that catabolizes catecholamines such as norepinephrine, epinephrine, and dopamine. COMT is primarily expressed in the prefrontal cortex , including a G/A substitution (rs4680) at codon 158 in membrane bound COMT (MB-COMT) and codon 108 in soluble COMT (S-COMT). This SNP results in a valine  to methionine (Met) substitution that affects the thermostability and activity of COMT. Met/Met homozygotes exhibit 35–50% less activity compared to Val/Val homozygotes  in response to a sudden acoustic stimulus, may be enhanced in Met/Met homozygotes as compared to Val carriers. This enhancement has been observed upon exposure to aversive imagery  using an established fear-potentiated startle paradigm . Prior to their participation, all participants provided written informed consents approved by the Emory University Institutional Review Board and the Grady Health Systems Research Oversight Committee. Participants were assessed for demographic data, trauma history  and exported for statistical analyses. The EMG signal was filtered with low- and high-frequency cutoffs at 28 and 500 Hz, respectively. The maximum amplitude of the eyeblink muscle contraction 20–200 ms after presentation of the startle probe was used as a measure of the acoustic startle response. The startle probe was a 106-dB (A) SPL, 40-ms burst of broadband noise with near instantaneous rise time, delivered binaurally through headphones.Startle response data were acquired at a 1000 Hz sampling frequency using the electromyography (EMG) module of the BIOPAC MP150 for Windows . The eyeblink component of the acoustic startle response was measured by EMG recordings of the right The startle session began with a habituation phase to reduce startle reactivity and familiarize the subjects to the CSs. The fear conditioning phase immediately followed habituation and consisted of three blocks with four trials of each type , for a total of 36 trials. As expected, the CS+ acquires excitatory properties, signals the presence of an aversive outcome, and is termed a danger signal whereas the CS− acquires inhibitory properties, signals the lack of an aversive outcome, and is termed a safety signal  was used during each acoustic startle session to record the participants’ expectancy of the US on each CS presentation . The assay call rate was 97.6%. Within and across plate duplicates were used for quality control. All duplicates were concordant. Genotypes for control samples (identified as those without PTSD) were in Hardy–Weinberg Equilibrium (p > 0.05).DNA was extracted from saliva in Oragene collection vials  using the DNAdvance kit . The COMT were selected from the HumanMethylation 450K BeadChip . Briefly, 1 μg of DNA converted with sodium bisulfite, amplified, fragmented, and hybridized on the HumanMethylation 450K BeadChip  according to the manufacturer’s instructions. Beta values were generated with Beadstudio. Samples with probe detection call rates <90% and those with an average intensity value of either <50% of the experiment-wide sample mean or <2000 arbitrary units (AU) were excluded from further analysis. One sample of male DNA was included on each BeadChip as a technical control throughout the experiment and assessed for reproducibility using the Pearson correlation coefficient. For each individual sample and CpG site, the signals from methylated (M) and unmethylated (U) bead types were used to calculate a beta value as β = M/(U + M). The methylation differences were calculated using generalized linear models in R adjusting for batch and position effects.Genomic DNA was isolated from whole blood stored in EDTA tubes using the Gentra Puregene Kit . DNA was quantified using PicoGreen , and the quality was checked on an agarose gel. CpG sites in COMT Genotype  and PTSD Diagnosis . The dependent variables were fear-potentiated startle and US-expectancy ratings. Fear-potentiated startle was calculated by subtracting the startle response magnitude during each CS presentation from the startle magnitude to the NA trials, in order to control for individual differences in startle magnitude. Repeated measures analyses of covariance (RM ANCOVA) were used to test the effect of Block (three levels) and Trial Type  during acquisition as within-subjects variables and Genotype and PTSD Diagnosis as between-subjects variables while co-varying for age, sex, race, and trauma history. Interaction effects of Block and Trial Type were followed up with RM ANCOVAs for comparing Trial Type within the last two Blocks of acquisition, when the fear conditioned effects are largest, as in our previous work  = 18.24, p < 0.001], main effect of PTSD Diagnosis , a significant two-way Block × Trial Type interaction , and a significant two-way Genotype × Trial Type interaction, F = 3.23, p < 0.05. We then performed another RM ANCOVA during late acquisition, with Trial Type as the within-subjects variable, and Genotype and PTSD Diagnosis as the between-groups variables. We found a significant main effect of PTSD Diagnosis, F = 6.80, p = 0.01, and an interaction effect of Genotype × Trial Type, F = 3.13, p < 0.05  = 17.47, p = 0.00007, and the Val/Val group, F = 21.58, p = 0.000008, but not in the Met/Met group, F = 0.05, ns. As seen in Figure F = 4.08, p < 0.05. Looking at late acquisition, we found significantly higher US expectancies on the CS+ than the CS− across all groups after controlling for age, sex, race, and trauma history, F = 24.60, p < 0.00001, but no main or interaction effects with Genotype or PTSD Diagnosis. This suggests that all groups were equally able to discriminate, at a conscious level, between the CS+ vs. CS− shapes.Repeated measures analyses of covariance of fear-potentiated startle with Block and Trial Type as within-subject factors and Genotype and PTSD Diagnosis as between-groups variables, with age, sex, race, and trauma history as covariates, revealed a significant main effect of Block [F,514 = 18F,514 = 18F = 5.94, p = 0.003, a main effect of PTSD Diagnosis, F = 13.39, p = 0.0003, but no interaction effect between Genotype and PTSD, F = 2.10, p > 0.1. In all genotype groups, fear to the CS− was higher in the PTSD+ group compared to the PTSD− group , or the Val/Val group (p < 0.05). As there were no significant differences between the two Val genotypes, the Val/Met and Val/Val groups were collapsed in the further analyses described below. When we examined fear-potentiated startle to the safety signal within each diagnostic group with the Val/Met and Val/Val genotypes collapsed, we found that it was highest in the PTSD+ Met/Met genotype carriers compared to Val carriers, F = 8.01, p = 0.006 ; the main effect of Genotype was still significant, F = 6.22, p = 0.002. Furthermore, fear-potentiated startle was again highest in the PTSD+ Met/Met group compared to Val allele carriers, F = 7.91, p = 0.006.We also followed up the above Genotype × Trial Type interaction effect by analyzing the effects of Genotype and PTSD Diagnosis within each Trial Type separately, while controlling for potential covariates. A univariate ANCOVA of fear-potentiated startle to the CS+ , with sex, age, race, and trauma levels entered as covariates did not show any main or interaction effects. However, the same analysis of fear-potentiated startle to the CS− revealed a significant main effect of Genotype, F = 29.87, p < 0.00001, and a significant interaction of Extinction and PTSD Diagnosis, F = 10.36, p < 0.00001, an interaction of Extinction and Genotype, F = 3.54, p < 0.05, as well as a three-way interaction of Extinction, Genotype, and PTSD Diagnosis, F = 3.98, p = 0.03. Figure F = 19.18, p = 0.00001  = 5.49, p = 0.02, with the PTSD+ Met/Met genotype group showing the highest levels of fear during extinction . Thus, we next examined COMT methylation with respect to CS− only.These results indicate that Met158 homozygotes have higher physiological fear responses to safety signals, and that this elevation may be further enhanced in those with PTSD. In our previous studies, fear-potentiated startle to the CS− during acquisition predicted the degree of CS+ extinction  = 6.78, p = 0.00004, but no effect of PTSD or interaction with PTSD. The main effect of genotype was significant at each site  = 13.25, p = 0.000004 and cg04856117, F = 10.30, p = 0.00006). Tukey HSD post hoc tests showed an effect of Met allele number on methylation at each site .The level of DNA methylation of 41 CpG sites that spanned the F = 9.66, p = 0.002, and methylation, F = 11.73, p = 0.001, in that participants with High methylation levels had greater levels of fear to the safety signal compared to participants with Low methylation levels  = 10.56, p = 0.001.In order to examine the interaction between methylation and PTSD with respect to fear inhibition, we used a median split to divide the subjects into those with High and Low methylation levels at this same CpG site, and performed a univariate ANCOVA with fear-potentiated startle to the CS− as the dependent variable and age, sex, race, and trauma as covariates. We found significant main effects of PTSD, s Figure . We agaiF = 5.354.57, p < 0.0001, with methylation alone accounting for 4.2% of the variance in startle to the safety signal after controlling for PTSD, Fchange = 8.68, p = 0.004. These analyses suggest that higher methylation levels are associated with higher fear to the safety signal, and impaired fear inhibition.Finally, in order to parse out the unique contribution of methylation to impaired fear inhibition, we conducted a stepwise regression analysis with fear-potentiated startle to the safety signal as the outcome variable, entering demographics in the first step, PTSD in the second step, and methylation at CpG site cg23061416 in the third step. The overall model was significant, 158Met polymorphism and conditioned fear responses in an inner-city population of traumatized individuals. We hypothesized that COMT Met/Met genotype, associated with less efficient COMT function in prefrontal cortex, would be related to impaired fear inhibition in PTSD subjects. The main findings of this study are as follows: (1) traumatized individuals with PTSD and the Met/Met genotype displayed higher fear to the CS−  as compared to carriers of the Val allele, (2) PTSD was associated with impaired fear extinction, which was most impaired in Met/Met subjects with PTSD, (3) the Met/Met genotype-associated with DNA methylation at four CpG sites; methylation at two of these sites was also associated with fear-potentiated startle to the CS−, and (4) stepwise regression analysis with fear-potentiated startle to the CS− as the outcome variable revealed that methylation accounted for a significant degree of the variance in startle to the CS− after controlling for trauma exposure. The results of this study suggest that, in addition to COMT Val158Met genotype, higher methylation at these CpG sites in the promoter region of COMT is associated with impaired fear inhibition in an established fear conditioning paradigm . In recent years, it has become increasingly evident that the etiology of many psychiatric disorders, including PTSD, involves the complex interaction between genetic and environmental factors and that the latter can influence epigenetic mechanisms including DNA methylation  and the development and severity of symptoms appears to be strongly influenced by gene × environment interactions  yielded 30 Met homozygotes with 9 also having a diagnosis of PTSD. This frequency of Met allele carriers is consistent with other studies of the COMT regulation may be an important mediator of fear processing, particularly fear inhibition. Thus multiple mechanisms of regulating neurotransmitter catabolism may interact in differentiating cognitive processes underlying PTSD following trauma exposure.In summary, we have shown in a highly traumatized sample at significant environmental risk for PTSD, that differential risk vs. resilience is, in part, mediated by multiple levels of molecular regulation on COMT function. Although our data do not directly address the downstream mechanism, multiple lines of evidence suggest that COMT regulates synaptic catecholamine levels, particularly dopamine, and that this can closely modulate cortical functioning. Our data suggest that at the level of protein function, as well as with differential epigenetic and transcriptional control, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The study investigated the effect of the transport industry on the transmission and spread of HIV/AIDS. Transport workers are highly mobile and spend long intervals away from the comforts of their homes. They are often involved in risky sexual behaviours that make them vulnerable to HIV infection, and so constitute carriers in the spread of the pandemic.The study entailed interviewing more than 1,000 long haul drivers and workers with the objective of inducing frank talk to assess their sexual habits enroute their long hauls. The limitation of this study was their insistence on anonymity to avoid adverse effects on their social status and marriage stability.More than 80% of interviewees had more than 20 female friends stationed at the villages on highways across the country. 60% of the promiscuous group knew about condoms but never used them. To them, what was the use doing it if you could not have the real thing. Sadly, some of the interviewees stated they had no other pleasurable indulgences in life other than sex, and if they were to die doing the only thing they enjoyed then who is complaining.The NURTW was advised:Create rest stations along the nation’s highways, with lodging, canteen, games, TV/Video sets, and other recreational facilities for drivers and motor boys.Provide GSM phone facilities for workers on long distance engagements to allow them keep in touch with their families.Organize seminars to educate workers on the implications, prevalence and management of HIV/AIDS.Provide medical test and care facilities at those rest stations for the quiet testing of workers for HIV/AIDS and dispensing of necessary drugs to sufferers.


The intestinal microbiota plays important roles in digestion and resistance against entero-pathogens. As with other ecosystems, its species composition is resilient against small disturbances but strong perturbations such as antibiotics can affect the consortium dramatically. Antibiotic cessation does not necessarily restore pre-treatment conditions and disturbed microbiota are often susceptible to pathogen invasion. Here we propose a mathematical model to explain how antibiotic-mediated switches in the microbiota composition can result from simple social interactions between antibiotic-tolerant and antibiotic-sensitive bacterial groups. We build a two-species  model and identify regions of domination by antibiotic-sensitive or antibiotic-tolerant bacteria, as well as a region of multistability where domination by either group is possible. Using a new framework that we derived from statistical physics, we calculate the duration of each microbiota composition state. This is shown to depend on the balance between random fluctuations in the bacterial densities and the strength of microbial interactions. The singular value decomposition of recent metagenomic data confirms our assumption of grouping microbes as antibiotic-tolerant or antibiotic-sensitive in response to a single antibiotic. Our methodology can be extended to multiple bacterial groups and thus it provides an ecological formalism to help interpret the present surge in microbiome data. Recent applications of metagenomics have led to a flood of novel studies and a renewed interest in the role of the gut microbiota in human health. We can now envision a time in the near future where analysis of microbiota composition can be used for diagnostics and the rational design of new therapeutics. However, most studies to date are exploratory and heavily data-driven, and therefore lack mechanistic insights on the ecology governing these complex microbial ecosystems. In this study we propose a new model grounded on ecological and physical principles to explain intestinal microbiota dynamics in response to antibiotic treatment. Our model explains a hysteresis effect that results from the social interaction between two microbial groups, antibiotic-tolerant and antibiotic-sensitive bacteria, as well as the recovery allowed by stochastic fluctuations. We use singular value decomposition for the analysis of temporal metagenomic data, which supports the representation of the microbiota according to two main microbial groups. Our framework explains why microbiota composition can be difficult to recover after antibiotic treatment, thus solving a long-standing puzzle in microbiota biology with profound implications for human health. It therefore forms a conceptual bridge between experiments and theoretical works towards a mechanistic understanding of the gut microbiota. Recent advances in metagenomics provide an unprecedented opportunity to investigate the intestinal microbiota and its role in human health and disease The gastro-intestinal microbiota is a highly diverse bacterial community that performs an important digestive function and, at the same time, provides resistance against colonization by entero-pathogenic bacteria Despite its importance to human health, the basic ecology of the intestinal microbiota remains unclear. A recent large-scale cross-sectional study proposed that the intestinal microbiota variation in humans is stratified and fits into distinct enterotypes, which may determine how individuals respond to diet or drug intake Here we propose a possible model to study how the intestinal microbiota responds to treatment with a single antibiotic. Our model expands on established ecological models and uses a minimal representation with two microbial groups We model the microbiota as a homogeneous system where we neglect spatial variation of antibiotic-sensitive  see . This sitability . SimilarThe previous analysis shows the existence of multistability in the absence of noise. However, the influx of microbes from the environment and/or the intra-population heterogeneity are expected in realistic scenarios and affect the bacterial density evolution in a non-deterministic fashion. This raises the question of how the noise alters the deterministic stable states and their stability criteria. We assume that the noise is a fraction of bacteria iven by  . HoweverThis has important biological implications since it suggests that extinction is prevented and, more importantly, that a minority of environmental microbes can settle in the gut at a rate that depends on the strength of their social interaction with the established microbiota.The introduction of random perturbation affects the stability criteria of the stable states. In particular, we observe that the bistability region decreases when the noise amplitude ncreases . At the Our model predicts that in absence of stochastic fluctuations the recovery time is larger than any observational time-scale so that it is impossible to revert to the conditions preceding antibiotic perturbation  using a trial-and-error approach. We varied the first-derivative at initial time in order to arrive as close as possible to the final point within some numerical precision. In principle the ideal trajectory connecting two stable points should be computed in the limit of We first rarefy the raw phylotypes counts matrix as in Text S1(PDF)Click here for additional data file.Video S1(MOV)Click here for additional data file.Video S2(MP4)Click here for additional data file.


The even mead, that erst brought sweetly forthThe freckled cowslip, burnet, and green clover,Wanting the scythe, all uncorrected, rank,Conceives by idleness, and nothing teemsBut hateful docks, rough thistles, kecksies, burs,Losing both beauty and utility.William ShakespeareHenry VSometimes in the spring, when I'm on my hands and knees among the azaleas, pulling out purslane and dandelion, I think how wonderful gardening would be if only it weren't for weeds. Most of the time I dislike weeding, especially when I see it as a battle. Given the natural guile of the enemy, I become pessimistic, thinking that the weeds will always be one step ahead of me, that I can never defeat them. I can create huge mounds of compost from the honeysuckle vine, chickweed, and pokeberry that I remove from my woodland garden beds and still hundreds of malignant survivors will have escaped my surveillance. When my frustration grows past endurance, my war escalates to chemical weapons. I don't spray herbicides very often, though, for I'm not proud of resorting to excessive force. Weeds, it seems, are truly like troubles: prodigious, vexing, and capable of bringing out the worst in people. But like troubles, they can also coax out the best.Sometimes, when I'm weeding, the simple action of plunging the fork into the earth, uprooting the plant, and shaking loose the soil that clings to its roots is very soothing. Plunge, pull, shake—repeated over and over, just like a mantra. On these occasions, my thoughts turn philosophical. Maybe the strong smell of the earth loosens memories from an earlier, less careworn time. Or maybe this contemplative sensibility is a byproduct of repetitious action. In either case, when this mood takes over, I lose the notion that the weeds are my enemy, that their leaves and roots are acting malevolently. The weeds, I realize, are neither good nor bad; they simply exist. Gardens do not grow without weeds, and life does not unfold without misfortune. And then I think about the way my life has been touched by AIDS.I can still remember the first time I came across a description of an unusual immune disorder in a medical journal—long before the disease even had a name. Because of my training in pathology, I was curious about this mysterious new disease. I remember feeling a ghoulish interest in an illness that could cause such tremendous, irreparable damage. Even then, before any of us knew its pathogenesis, its destructive potential was clear, and I felt, perhaps, much the same as the physicists who first glimpsed the horrendous possibilities of the atomic bomb. My scientific detachment didn't live long; AIDS soon became more than a medical curiosity for me. It began to take people I knew, friends who I had hoped would surround me all my life. Sources of comfort and understanding dried up. I started feeling like an uprooted, disenfranchised farmer of Dust Bowl days, driven from once green and fertile land and forced to migrate to unknown places.Over that first horrible decade of its debut, AIDS managed to dissolve my sense of permanence and distort my perception of time. Before AIDS, I didn't know much about misfortune. I thought about the future primarily in terms of its potential to bring me more gratification and greater achievements. After AIDS, I understood that the future also has the power to disappoint. That knowledge has made me supremely impatient. Now it seems that nothing is quick enough for me; everything takes too long. I feel as if my internal clock has been wound so tightly that at any moment the hands might go spinning clean off the face, so rapidly are they going around and around.AIDS changed my professional life as well. I had been content to study disease from a distance, behind a microscope. As a pathologist I thought about illness primarily as a disruption in normal physiology, and I was satisfied with the contributions I could make to patient care by diagnosing unusual tumors and peculiar lesions. But after the epidemic took hold, my interest in abnormal physiology waned. The accurate diagnosis of diseases, including AIDS, though it is an essential step in patient care, had become too passive for me, seemed to imply acceptance of the inevitability of illness. No longer satisfied with identifying disease, I wanted to learn how to prevent it from occurring. I was determined to join those who were already trying to stop the spread of the epidemic. And so, after I finished my public health degree, I left my job as a university-based pathologist to work full-time on AIDS prevention at the Centers for Disease Control and Prevention.It would be easy to assume that the AIDS epidemic, so often associated with loss, generates nothing but sorrow in those it touches, that the personal adjustments we make are all negative. I find, on the contrary, that AIDS has helped me to clarify just what does and does not matter during our brief time in the world. The epidemic has not taken hope away from me, but it has taught me the inadequacy of looking toward the future as a means of rescue from the present. AIDS has shown me that hope is strongest in us when we seek our fulfillment in the circumstances of the present, when we refuse to defer our dreams or to accept defeat.Sometimes when I'm working in my garden, I think about the invasiveness of weeds and disease, how they appear in the garden, in the human population, uninvited, unwelcome, capable of causing tremendous destruction if left unattended. Yes, it would be wonderful if some spectacular act of God or nature would banish AIDS from our existence. Gardeners dream that they'll awake some morning to find that the crabgrass has magically departed from the perennial beds. Weeds, though, whether real or metaphorical, will not be banished. Pain and loss are intrinsic to our existence in the temporal world. The best way to keep weeds from engulfing the garden is not to rail at the injustice of fate but to get down on our hands and knees and begin clearing them away.Gardening in Clay: Reflections on AIDS, by Ronald O. Valdiserri. Copyright © 1994 by Cornell University. Used by permission of the publisher, Cornell University Press.Reprinted from I wrote "Weeds" in the early 1990s ; the essIn 1992, the year Edwin died, tennis great Arthur Ashe announced he had AIDS; the Centers for Disease Control and Prevention estimated 140,000–168,000 persons were living with AIDS in the United States (How far we've come in the 25 years since the first AIDS cases were reported in the United States is difficult to ignore (As "Weeds" suggests, in the continuing chronicles of humans versus retroviruses, we are far from ridding our garden of weeds.


In recent years, the physiological relaxing effect brought by nature is becoming clear; however, many workers find it difficult to be exposed to nature in their working environment. Exposure to fresh flowers represents an opportunity to incorporate nature into their working lives. In this study, we examined the effects of exposure to roses on physiological and psychological variables  in office workers.The experimental site was Mizuho Information & Research Institute, Inc., in the Tokyo metropolitan area. Thirty-one male office workers were included in the present study. The subjects were exposed to thirty unscented pink roses  arranged in a cylindrical glass vase for 4 min. In the control condition, the subjects were not exposed to flowers. After the experiments, the subjects completed a questionnaire. The order of exposure was counterbalanced among subjects. Among subjects exposed to roses, the high-frequency component of heart rate variability was significantly higher than in controls. Similarly, 'comfortable,’ 'relaxed’ and 'natural’ feelings were more common in subjects exposed to roses.Data from this study support the presence of physiological and psychological relaxing effects of being exposed to flowers on office workers. The diset al.: "We havet al.". We hypet al. reported forest bathing increased natural killer cell function and improved immune function[In our stressful modern societies, the relaxing effect of natural stimuli is considered advantageous compared with other stimuli. Many people are thus attracted to the physiological and psychological relaxing effect of exposure to nature. Field experiments on forest bathing-10, urbafunction. This efGlobally, evidence-based medicine has been attracting attention, with physiological data from field tests offering great value. We expect that accumulating physiological data from field experiments will continue to demonstrate preventive medical effects of nature therapy in the future,14.In modern society, many people spend the majority of their time in intensely stressful states, and have no time for contact with nature outside of their immediate surroundings. Office workers who work in urban areas are typical examples. Previous studies have evaluated the psychological stress levels among office workers, and theFlower arrangements are familiar to many people as bringing nature into daily life and offer office workers easy contact with nature within the constraints of time and space. In the Japanese flower market, chrysanthemum is the most popular flower type, with the most popular flowers being carnations and roses. BecauseThe experiments were conducted in a conference room of the Mizuho Information & Research Institute, Inc. The room temperature was maintained at 24.6°C ± 1.8°C  and relative humidity was maintained at 31.6% ± 3.9% . Thirty-one male workers (37.3 ± 2.0 years old) working in the institute were included. The study was conducted with the approval of the Ethics Committee of the Center for Environment, Health and Field Sciences, Chiba University and all subjects provided written informed consent.Rosa, cultivar name: Dekora) trimmed to a length of 40 cm were used for viewing. The roses were arranged in a cylindrical glass vase 12 cm in diameter and 20 cm in height. The distance from subjects’ eyes to the flowers was 37 to 40 cm, and it was adjusted according to the height of the subjects. The experimental setting is shown in Figure Thirty fresh and unscented (sensory intensity was classed as unscented on testing) pink roses  and the R-R intervals of electrocardiographs are highly correlated. TherefoFor psychological evaluations, we employed a 13-point rating scale including the following parameters: ‘comfortable-uncomfortable’, ‘relaxed-awakening’ and ‘natural-artificial’ along with the factors in the profile of mood states (POMS) questionnaire. The POMS questionnaire simultaneously evaluates six measures of mood: tension-anxiety (T-A), depression-dejection (D), anger-hostility (A-H), fatigue (F), confusion (C), and vigor (V). In this study, were used the Japanese version of POMS-24 consit-test after calculating the average values obtained during the 4-minute viewing of the roses versus those obtained during the control condition. Psychological evaluation was analyzed by the Wilcoxon signed-rank test. Statistical differences were considered significant at P <0.05.The Statistical Package for Social Sciences software  was used for all statistical analyses. Statistical analysis of physiological data was performed using the paired 2 during rose flower viewing, and 414.0 msec2 in the control condition; it showed 21% significant increase (p <0.05) on exposure to roses, meaning that parasympathetic nervous activity was significantly higher while viewing the roses. There were no significant differences in LF/HF of HRV or pulse rate.Figure P <0.01). Relaxed and natural states were also significantly more frequent on exposure to roses than in the control condition (P <0.01). Figure P <0.01) as well as those of D and A-H (P <0.05) were significantly lower on viewing roses than in the control condition. On the other hand, V scores during the rose viewing were significantly higher than in the control condition (P <0.01).Figure In the present study, we evaluated changes in autonomic nervous system activity, a physiological measure of stress, in office workers while viewing common fresh roses. A relatNumerous studies have evaluated effects of contact with familiar natural environments on human physiology and emotions. Indoor plants and the view from the window are good examples of the contact with nature obtainable while inside a room, and positive results of their physiological and psychological effects have been demonstrated-31. In tOur research showed that parasympathetic nervous activity was enhanced by viewing roses. Therefore, by simple stimulus, roses possibly offer preventive medical effects by decreThis study had a few limitations. First, only male subjects were included, and only roses were viewed. In future experiments, we will examine the responses of female subjects exposed to multiple types of flowers. We predict that the physiological data will support the physiological and psychological relaxing effects of exposure to roses, which may consequently lead to the increased use of flowers to reduce office-related stress. Second, we only evaluated HRV; therefore, this study cannot be considered to be a complete physiological evaluation. Other experimental indices, such as brain activity and stress hormone levels, should be assessed to determine the effects of visual stimulation such as viewing roses on responses in humans.Parasympathetic nervous activity was enhanced by viewing roses. This finding suggests a simple method for decreasing stress and improving the health of office workers.A-H: anger-hostility; APG: acceleration plethysmograph; C: confusion; D: depression-dejection; F: fatigue; HF: high frequency; HRV: heart rate variability; LF: low frequency; POMS: profile of mood states; T-A: tension-anxiety; V: vigor.The authors declare that they have no competing interests.HI contributed to the experimental design, data acquisition, statistical analysis, interpretation of the results and manuscript preparation. MK and CS contributed to acquisition and interpretation of data. EH designed the study and participated in data acquisition. YM had an important role in this research, particularly in study design, interpretation of data and manuscript preparation. All authors contributed to manuscript preparation and are responsible for the final editing and approval of the manuscript.


We aimed to characterize the prognostic effect of c-Met by systematic review and meta-analysis.We identified 15 studies assessing survival in gastric cancer by c-Met status. Effect measure of interest was hazard ratio (HR) for survival. Meta-regression was performed to estimate the relationship between HR and disease stage. Random-effects meta-analyses were used to account for heterogeneity.15 eligible studies provided outcome data stratified by c-Met status in 2210 patients. Meta-analysis of the HRs indicated a significantly poorer Os in patients with high c-Met expression . Subgroup analysis showed the prognostic effect of c-Met was identical in protein-level and gene-level based methodology. The same effect was also seen in Asian and Western ethnicity subgroup analysis. Meta-regression showed HR was not associated with disease stage.Patients with tumors that harbor high c-Met expression are more likely to have a worse Os, with this prognostic effect independent of disease stage. c-Met status should be evaluated in clinical prognosis. Gastric cancer(GC) is one of the most common human malignant diseases and remains the second leading cause of cancer-related death worldwide. AlthougThe receptor tyrosine kinase c-Met is encoded by MET oncogene. This receptor and its hepatocyte growth factor (HGF) ligand have been found frequently dysregulated in gastric carcinomas-7. c-MetHowever, when it comes to the prognostic properties of the c-Met status, there still seems to be no consensus, despite a relatively large number of studies on c-Met in gastric cancer have looked into the association between a c-Met-positive status and survival. In this current systematic review we have utilized the already existing literature to address the issue on the prognostic properties of a c-Met-positive status in gastric cancer.Systematic computerised searches were performed using the electronic database Pubmed(up to 30th May 2013). The search strategy used the keywords ‘c-Met’, or ‘MET’, and ‘gastric cancer’, or ‘gastric carcinoma’ or ‘stomach neoplasm’. English language published studies were eligible if they met the following criteria: (1) patients had a diagnosis of gastric cancer; (2) overall survival(OS) or progression-free survival(PFS) were analyzed stratified by c-Met status; (3) the results were part of an original analysis; (4) when the same patient population was used in several publications, only the most recent, largest or complete study was included in the meta-analysis. Data from review articles, abstracts, and letters were not included.Study characteristics were extracted from the eligible articles and summarized in a consistent manner to aid comparison. The following data were collected from each study: first author’s name, year of publication, number of patients screened, disease stage, clinical treatment, methodology of c-Met analysis including the threshold used to dichotomize c-Met as ‘high’ and ‘low’, progression-free survival and overall survival stratified by c-Met status and hazard ratio(HR) with 95% confidence intervals(CI) for PFS or OS. When HRs and its confidence intervals were not directly reported, they were estimated from other data, such as number of patients in each group and Kaplan–Meier curves for overall survival, using the published methodology. Both unThe endpoints were PFS and OS. The association between c-Met level and PFS or OS was evaluated using the hazard ratio of high c-Met level patients over low c-Met level patients and 95% confidence interval from univariate and multivariate Cox proportional hazards models, so a HR of 1 indicates a lack of association between c-Met level and risk of death, a HR of greater than 1 indicates a greater risk of death in high c-Met level patients, and a HR less than 1 indicates a greater risk of death in low c-Met level patients.2 statistic was more than 50% or the fixed effects p value for the I2 statistic was less than 0.10, indicating significant heterogeneity across studies, a random effects model was then used for calculating the pooled estimate. A fixed effects model was initially used to calculate the pooled HR estimates. If the ITo establish the effect of methodology heterogeneity among studies on meta-analyses conclusions, subgroup analyses were conducted by study designs. In the subgroup analysis of c-Met expression ascertainment method, studies were classified as either protein-level method subgroup which performed immunohistochemistry(IHC) to stratify c-Met expression or gene-level method subgroup, such as reverse transcriptase quantitative polymerase chain reaction(RT-qPCR), southern blot, and fluorescence in situ hybridization(FISH), which dichotomize c-Met status by gene amplification, as reported in the given publication. Clinical heterogeneity was established by the subgroup analysis of ethnicity, in which studies were classified as Asian or Western subgroup. To demonstrate whether HR was associated with the disease stage of the GC patients, we performed the meta-regression analysis of logarithm transformed outcomes(HR) against percentage of advanced stage patients in each study, and HR was considered to be associated with disease stage when the p value of the model was less than 0.05. Potential publication bias were assessed by performing Egger’s test for meta-analysis including 10 or more studies. All statistical analyses were performed using STATA version 11.0.The flowchart of our study is shown in c-Met evaluation was performed by IHC in 9 studies ,27,28,30In five studies which assigned c-Met expression at gene level, high c-Met status was defined as the copy of c-Met 7 times, 5 timesThe rate of high c-Met status ranged from 8% to 82% . The observed median proportion of high c-Met expression was 59%(range 26-82%), and 16%(range 8-29%) respectively in studies using IHC and other methods. Two studies did not present the proportion of high c-Met level.2=52.4%), indicating wide prediction intervals for the prognostic effect in an individual clinical setting. There was no evidence of small study effects using estimates above reported(Egger’s test: p=0.168).The results of the meta-analysis of the association between c-Met level and overall survival are provided in The result of the c-Met ascertainment method subgroup analysis is provided in In an subgroup analysis stratifying patients by ethnicity, there was significant relationship between high c-Met expression and poor OS in both Asian and Western patients. The result is provided in A meta-regression analysis was performed to test the relationship between HR and the disease stage of patients in 9 studies,28,29,31This systematic review has been based on 15 publications covering the period from 1998 to May 2012 and comprises a total of 2210 patients with gastric cancer. The results of this meta-analysis demonstrate the prognostic significance of c-Met expression level in GC patients in Asian and Western countries. In the overall meta-analysis of the association between c-Met level and overall survival, the results indicated a statistically significant increases in overall survival in patients with low c-Met expression in relative to patients with high c-Met expression. Subgroup analysis showed that the strength of relationship between c-Met level and overall survival was identical based on the method utilized to ascertain c-Met status. Both gene-level method subgroup and protein-level method subgroup demonstrated statistically significant association between c-Met status and overall survival. In other words, methodology utilized to estimate c-Met status did not effect the prognostic property. However, a statistically significant heterogeneity was seen in both subgroups. The explanation for these rather large variation is likely to be multifactorial such as different populations studied, but the most important aspects are probably the use of varied antibodies for IHC and housekeeping genes for RT-qPCR and the application of different criteria to stratify c-Met status. In a phase II study conducted within the last couple of years which evaluated the effect of HGF antibody and MET pathway biomarkers in advanced GC patients, the scoSubgroup analysis also showed that both Asian and Western patients harbouring high c-Met level were significantly associated with poorer survival, which did not vary based on ethnicity. While differences in outcomes have been reported between Asian and Western populations with gastric cancer,34, our Disease stage is one of the most important factors that influence OS. In the 15 studies assessed in our meta-analysis, 11 included patients of stage I-IV. However, none of these studies separated their patients into subgroups. With that in mind, we applied meta-regression to detect whether the prognostic property of c-Met would be impacted by the variation of percentage of advanced stage patients among studies. The results showed that the overall HR had no association with the percentage of advanced stage patients in 9 studies, which meant the prognostic effect of c-Met was independent to disease stage. This result further promoted the prognostic value of c-Met in gastric cancer. Another important factor for OS is clinical treatment, which includes surgery, postoperative chemotherapy or radiotherapy, and palliative treatment after relapse or disease progression. In our meta-analysis, the pooled HR of five studies in which patients were given postoperative treatment was 1.556, with 95%CI 1.152-2.102, the samTo our knowledge, this is the first meta-analysis that strongly suggests high c-Met status represent adverse prognostic biomarker for overall survival in GC patients. Patients with tumors that harbour high c-Met expression are more likely to have a worse OS, and this prognostic effect was independent to disease stage. However, large studies using standardized unbiased methods are still required before c-Met testing can move toward routine clinical application as a prognostic tool.Collectively, this study’s overall findings support the hypothesis that c-Met expression level is associated with overall survival in GC patients. Future studies should try to block HGF/c-Met pathway so as to prolong the overall survival of GC patients, and the prognosis of patients harbouring high c-Met expression would be changed with more and more clinical trials evaluating targeted agents against HGF or c-Met in GC patients.Table S1Clinical treatment of studies reporting c-Met expression and outcomes in gastric cancer patients.(DOC)Click here for additional data file.Figure S1Forest plot showing the meta-analysis of hazard ratio estimates for OS in postoperative and no postoperative treatment subgroup.(TIF)Click here for additional data file.Checklist S1The section that contains each item in PRISMA Checklist.(DOC)Click here for additional data file.


To assess Turkish school teachers’ knowledge, attitudes, and misconceptions of HIV/AIDS.This cross-sectional descriptive study was conducted in Sivas, Middle Anatolian province of Turkey, from January to May 2009. We selected and interviewed 898 teachers using a simple systematic sampling method.All respondents heard about AIDS. Most knew that it could be transmitted by infected blood (98.0%) and sexual contact (93.4%) and some (33.2%) thought that it could be transmitted by mosquitoes. Although the majority of them strongly agreed or agreed with the statement that “people with AIDS should be helped, supported and treated” (98.0%), about 40% and 70% of them, respectively, agreed or strongly agreed with the statement that infected people should be quarantined. Young teachers with a higher level of knowledge about HIV/AIDS had more positive attitudes.This study provided basic information on school teachers’ AIDS knowledge and attitudes and showed that various misconceptions of HIV transmission were a matter of concern. A considerable number of respondents with undesirable attitudes toward HIV/AIDS indicates the need for education of teachers on the subject. Today, human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) is one of the biggest public health problems, with 31-36 million estimated cases and 2.7 million new cases in 2010 . From 178 middle and high schools, we systematically selected 38 (with 937 teachers). All teachers working in these schools were contacted and invited to participate. Permission to conduct the research was obtained from the headmasters. Teachers were explained that their participation in the study would be completely voluntary, that it would not affect their employment, and that all information would be confidential. A total of 937 teachers were approached and 39 refused to participate. The response rate was 95.8%. The information for the main study was collected anonymously using self-administered questionnaires, which were distributed to each school by eight trained final-year medical students. The study was approved by the Human Research Ethics Committee of the Cumhuriyet University of Turkey.The questionnaire was developed based on a literature review, reviewed by a specialist in infectious diseases and a general physician, and pre-tested in one school. Cronbach alpha coefficient for the reliability  of the subscales (as used in this study) ranged from 0.72 to 0.83 . The que2 tests were performed to determine the differences between demographic items  and knowledge levels or attitudes. A linear regression model was also performed to assess the influence of predictor variables that had been significant in univariate analysis on the knowledge level and attitudes toward HIV/AIDS. All statistical tests were conducted at the 5% significance level.Data were analyzed using SPSS  for Windows, version 13.0. Quantitative data were presented as mean ± standard deviation. Categorical data were expressed as percentages. χAll respondents lived in the urban area and had university degrees . Of 898 Respondents had a fairly good knowledge about HIV/AIDS on most items. Most knew that HIV was transmitted by sharing needles or a razor , blood transfusion , sexual intercourse , and from an infected mother to the fetus  . HoweverA considerable number of the respondents were not aware that HIV infection was not transmitted by mosquito bites , shaking or touching hands , sharing meals , using the same toilets , going into same swimming pools , and coughing or sneezing . A total of 552 (61.5%) respondents believed that the best method for preventing HIV infection was education and condom use.P = 0.044) or “there is a treatment for AIDS” . There was no significant difference between other characteristics  and the knowledge level (data not shown).There were no sex differences in the knowledge on AIDS, except that men responded significantly better on the following items: “AIDS is a preventable disease” (93.7% vs 89.8%, Respondents had relatively negative attitudes toward people with AIDS . The majIn this survey, most respondents answered correctly on the questions about HIV/AIDS transmission routes, prevention methods, and treatment. However, 11.6% believed that AIDS was not an infectious disease, that it was transmitted by mosquito bites, and that there was a treatment for AIDS. Furthermore, 27.4% indicated that AIDS can be transmitted by shaking hands, sharing meals, or touching an infected person. Other studies also showed very poor general knowledge on HIV transmission among teachers  and thatThe most interesting finding from this study was the fact that the majority of teachers agreed with the statement that “people with AIDS should be helped, supported and treated.” On the other hand, from 40% to 90% of teachers, regardless of sex and age, showed a negative attitude towards people with HIV/AIDS. For example, 40% of teachers believed that people with AIDS should be quarantined. Similar results were found Israel, where 39% of teachers thought that people with AIDS should be kept away from school . Also, iData suggest that respondents’ knowledge about HIV/AIDS mostly does not originate from school but from mass media and communication with friends, health professionals, and family members. Other studies on teachers reported similar sources of knowledge ,21,22. TBecause HIV/AIDS-related issues are a sensitive topic in our country; the questionnaire did not include questions related to teachers' risk behaviors or sexual activities, which might have affected the results. This study provides basic information on AIDS knowledge and attitudes among school teachers and shows that various misconceptions of HIV transmission are a matter of concern. The levels of undesirable attitudes about HIV/AIDS indicate a need for changing the Turkish teachers’ attitudes towards persons with HIV/AIDS through education programs organized by the Ministry of Education in accordance with the social and cultural structures of the society.


We have previously demonstrated that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, inhibits histone deacetylases and remodels chromatins to induce growth arrest in HL-60 myeloid leukemia cells in a concentration-dependent manner.p15 in acute lymphoid leukemia cell line Molt-4, and to further decipher the potential mechanism of demethylation, DNA sequencing and modified methylation specific PCR (MSP) were used to screen p15-M and p15-U mRNA after Molt-4 cells were treated with PHI, 5-Aza and TSA. DNA methyltransferase 1 (DNMT1), 3A (DNMT3A), 3B (DNMT3B) and p15 mRNA were measured by RT-PCR. P15 protein, acetylated histone H3 and histone H4 were detected by Western Blot.To investigate the effect of PHI, a novel histone deacetylases inhibitor (HDACi), on demethylation and activation of transcription of p15 in Molt-4 cells was hypermethylated and inactive. Hypermethylation of gene p15 was attenuated and p15 gene was activated de novo after 5 days exposure to PHI in a concentration-dependent manner. DNMT1 and DNMT3B were inhibited by PHI (P < 0.05). Alteration of DNMT3A was not significant at those concentrations. Acetylated histone H3 and histone H4 were accumulated markedly after exposure to PHI.The gene p15 was reversed and p15 transcription could be reactivated de novo by PHI.PHI could induce both DNA demethylation and acetylated H3 and H4 accumulation in Molt-4 cells. Hypermethylation of gene  The major epigenetic transcriptional controls involved in gene silencing are DNA methylation and covalent modification of histone proteins. Transcriptional silencing of genes, due to hypermethylation of CpG islands in the promoter region of genes, has been reported in nearly every type of human tumors. A broad spectrum of genes are frequently hypermethylated in cancers, including those associated with cell cycle regulation, detoxification, tumor suppression, and apoptosis etc. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), of which three active enzymes have been identified in mammals, namely DNMT1, DNMT3A and DNMT3B. In developmental processes of the mouse, DNMT1 is responsible for maintaining pre-existing methylation patterns during DNA replication, while DNMT3A and DNMT3B are required for initiation of de novo methylation. DNMT3A expression is ubiquitous, but DNMT3B is present in the cells at low levels except in testes, thyroid, and bone marrow-4. In hup15 (INK4b or MTS2) is a candidate tumor suppressor with structural and functional similarity to the p16 gene. This gene recognizes cyclin-dependent kinases CDK4 and CDK6, and induces G1 arrest of the cell cycle by competing with cyclin D for binding with CDK4. Specific deletions of p15 sequences have been found in only a few cases of leukemia and lymphomas. In contrast, the p15 gene is preferentially hypermethylated at a 5'-CpG island, which has been shown to be associated with loss of transcription of this gene in leukemia cells [p15 methylation seems to have important prognostic implications for risk assessment because patients with p15 methylation have overall shortened survival [The gene ia cells ,10. Furtsurvival ,12.p15 activity in leukemia cells has been a subject of investigation. In this paper we demonstrated that PHI has a dual effect on DNA methylation and histone acetylation in leukemic T cells Molt-4. The cross-talk on the DNA and chromatin resulted in demethylating the CpG island of p15, which is inactivated due to hypermethylation, and recovered the unmethylated p15 for transcriptional activation. An inter-active mechanism, involving a down-regulation of the enzymes DNMTs and up-regulating the histone acetyltransferase P300/CBP and histone acetylation, is revealed.We have previously demonstrated that phenylhexyl isothiocyanate (PHI), a man-made isothiocyanate, inhibits histone deacetylases and remodels chromatins to induce growth arrest in HL-60 myeloid leukemia cells in a concentration-dependent manner. Recent 2. Cells in exponential growth were exposed to PHI prepared in 75% methanol[PHI, greater than 98% pure, was purchased from LKT Lab . Human acute lymphatic leukemia cell line Molt-4 was obtained from China Center for Type Culture Collection (CCTCC). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and maintained at 37°C in humidified atmosphere containing 5% CO methanol at variop15 (148pb) were gcgttcgtattttgcggtt (positive sense), and cgtacaataaccgaacgaccga (antisense). The primers for unmethylated form (154 bp) were tgtgatgtgtttgtattttgtggtt (positive sense) and ccatacaataaccaaacaaccaa (antisense). The amplification was performed in an Mastercycler unit (Eppendorf) under the program conditions as follows: 95°C for 5 min; then 40 cycles of 95°C for 45 sec, 60°C for 45 sec, 72°C for 45 sec; and finally 10 min at 72°C. The PCR products were visualized in GeneGenius  by ethidium bromide staining in 2% agarose gels.The genomic DNA from cultured cells was extracted and modified by bisulfate treatment for MS-PCR analyses. DNA froThe protein levels were determined by Western blot analysis as described previously. Brieflyp15, DNMT1, DNMT3A, DNMT3B in separate reactions  according to the manufacturer's instructions. One-microgram total RNA isolated was used for the first-strand cDNA synthesis with Reverse Transcription System . cDNA was amplified using specific primer for P < 0.05 was considered to be statistically significant.The data was analyzed by statistical software SPSS13.0. Data measurement was presented as mean ± SD, from multiple independent experiments by homogeneity test for variance and test of normality. Results were evaluated by One-way ANOVA between groups. p15 in human lymphatic leukemia T cells Molt-4 was evaluated by MS-PCR. Figure p15 (p15-M), and the unmethylated p15 (p15-U) was undetectable. After exposure of Molt-4 cells to PHI for 5 days, the methylated p15 was decreased, with the magnitude in positive relation to the PHI concentrations. The unmethylated p15 became detectable, replacing the decrease of methylated p15. The activity of 40 μM PHI to reverse p15 methylation was similar to that of 2 μM of 5-Aza, a known inhibitor of DNA methylation or 1 μM of TSA, which was shown in endometrial cancer cells as a demethylating agent as it reduced DNMT3B level and de novo DNMT activity[The DNA methylation status of  activity.p15 in Molt-4 cells was examined without or with the exposure to PHI. Figure p15 and β-actin. In the presence of PHI, the p15 mRNA expression was enhanced in a concentration-dependent manner. The ratios were: control (0.17 ± 0.12), PHI 10 μM (0.29 ± 0.14), PHI 20 μM (0.55 ± 0.07), PHI 40 μM (0.93 ± 0.13), TSA (0.65 ± 0.11), and 5-Aza (0.89 ± 0.13). The expression levels of p15 mRNA mediated by PHI were statistically significantly (P < 0.05) as compared to that without PHI treatment. The protein expression of p15 was examined by Western blotting in parallel. Figure p15 expression after treatment with PHI at 20-40 μM, similar to the 5-Aza effect. The results thus indicated that PHI might be a potent methylation inhibitor for the CpG island of p15 gene, leading to reactivating p15 transcription in the leukemic cells.The mRNA level of P < 0.05). The mRNA level of DNMT3A, on the other hand, was not significantly altered under the same condition.The mRNA levels of the enzymes responsible for DNA methylation, i.e., DNMT1, DNMT3A, DNMT3B, without or with the exposure to PHI, were evaluated with RT-PCR Figure . The graMolt-4 cells were exposed to PHI at various concentrations. Figure p15. Upon exposure to PHI, the unmethylated p15, otherwise absent, became detectable, along with the disappearance of the methylated p15. This reversal of p15 methylation correlates with reexpression of p15. The activity of PHI in demethylating p15 was shown to be similar to that of 5-Aza and TSA. To our knowledge, this study is the first to demonstrate that the hypermethylated p15 gene in leukemic T cells could be reactivated by an isothiocyanate. Our analyses have provided two inter-related mechanisms for hypomethylation of p15. They are presented in the following.DNA methylation and histone acetylation are two well known epigenetic chromatin modifications. At least 80 clinical trials are underway, testing more than eleven different HDAC inhibitory agents including both hematological and solid malignancies -25. Thisp15 by PHI could be that the expressions of the DNA methylating enzymes were down-regulated. This possibility was examined with the three DNA methylating enzymes DNMT1, DNMT3A, and DNMT3B. The study showed that in the presence of PHI, the mRNA of two of the enzymes, DNMT1 and DNMT3B, was significantly down-regulated in a concentration-dependent manner. The mRNA of the enzyme DNMT3A, however, did not show a significant alteration. The expression of DNMT3A is known to be ubiquitous and this has been under investigation as a basis of this observation. The results suggested that reduction of the DNMT1 and DNMT3B expression and their activity could be a responsible mechanism for hypomethylation of p15. This interpretation is in line with the reports that DNMTs are commonly over-expressed in leukemias. AML cells with hypermethylated p15 tended to express higher levels of DNMT1 and DNMT3B [One potential mechanism for initiating demethylation of d DNMT3B . Melki ed DNMT3B  found a The major mechanism of the current hypomethylating drugs, such 5-Azacytidine and decitabine, is their covalent binding to the DNMTs, which resulting in the irreversible inhibition of the DNMT activity, leading to the hypomethylation of the genomic DNA . Our datp15 by PHI could also be related to the PHI effects on histone acetylation. Histone acetylation generally correlates to an open and transcriptionally active chromatin, whereas histone deacetylation is associated with chromatin condensation and transcriptional repression. The p15 CpG island region is surrounded with both histone acetylated H3 and methylated H3K9 in AML[p15 promoter for transcriptional activation. This is consistent with previous data showing that PHI was inhibitor of HDACs [The methylation of K9 in AML. PHI couof HDACs .p15 was reversed and p15 transcription could be reactivated de novo by PHI.PHI could induce DNA demethylation and acetylated H3, H4 accumulation in Molt-4 cells. Hypermethylation of gene PHI: Phenylhexyl Isothiocyanate; HDAC: histone deacetylases; MSP: Methylation Specific PCR; 5-Aza: 5-azacytidine; TSA: Trichostatin A; DNMT: DNA methyltransferase; RT-PCR: reverse transcriptase-polymerase chain reaction.The authors declare that they have no competing interests.XM is responsible for study design, writing paper and has been involved in all aspects of this study. SJ, YH, YX and RZ contributed to collecting and analyzing data. Dr Jen-Wei contributed to the study design and manuscript preparation. All authors have read and approved the final manuscript.


Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate  Winged insects have evolved diverse modes of metamorphosis In both hemimetabolous and holometabolous insects, the developmental switch between juvenile and adult forms depends on juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata gland Rhodnius prolixus, Drosophila melanogasterTribolium castaneum, loss of Met triggers pupation of larvae during pre-final instars Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) Kr-h1 also elicits precocious metamorphosis of beetle larvae BR-C is dispensable in holometabolous larvae until the onset of metamorphosis, when it specifies pupal features BR-C and Kr-h1 are naturally down-regulated by the absence of JH to allow adult development BR-C has been functionally studied in hemimetabolous insects, where, unlike in holometabolans, it is required for development of the embryonic germ band Although the anti-metamorphic effect of JH was discovered in the hemimetabolous true bug, Met, Kr-h1 and BR-C in the hemimetabolous firebug, Pyrrhocoris apterus . We show that despite the diverse developmental strategies and the vast evolutionary distance between them, transduction of the anti-metamorphic JH signal relies on the common-core elements, Met and Kr-h1. In both insect types, Kr-h1 acts as a strictly JH- and Met-dependent repressor of metamorphosis. In contrast, the function of BR-C has changed from promoting progressive development of hemimetabolous larvae to a new role in specifying the holometabolous pupa.To provide a direct comparison of JH signaling in holometaboly and hemimetaboly, we have examined the function of Kr-h1 (JN416987) and BR-C (JN416990), from Pyrrhocoris apterus, and Met (JN416985) and Kr-h1 (JN416988) cDNAs from another true bug, Rhodnius prolixus. Alignments amongst the orthologs reveal conservation of the main functional domains (Met (JN416986), Kr-h1 (JN416989) and BR-C (JN416991) orthologs in the firebrat, Thermobia domestica, a representative of the primitive lineage of non-metamorphosing wingless insects  and its target genes  domains , namely  insects .Pyrrhocoris requires JH, as has been demonstrated by removal of the JH-producing corpora allata gland  from penultimate (L4) larvae The firebug invariantly undergoes four larval molts demarcating five larval instars (L1–L5), followed by a metamorphic molt, which produces an adult possessing external genitals and wings with a specific color pattern. Maintenance of the larval state in Met, Kr-h1 and BR-C during Pyrrhocoris development correlates with this regulation by JH. From embryogenesis to adulthood, Met mRNA persisted without major fluctuations through all larval stages , confirming that Kr-h1 transcription absolutely requires the natural source of JH. The expression pattern suggested that Kr-h1 prevents metamorphosis until the final larval instar, when the Kr-h1-free (and JH-free) period may be necessary to initiate metamorphosis.We examined how expression of l stages . Such a rvae, L5 . A more e adults . AllatecPyrrhocoris. Injection of Met dsRNA into early-L4 larvae caused an 86% knockdown of Met mRNA (Met(RNAi) males had external genitals  animals  injected with Kr-h1 dsRNA at the previous L3 instar showed accelerated growth of wings and genitals  and caused either lethality at the end of the L4 instar or compromised growth of the wing pads in animals that successfully molted to the L5 instar (n = 22) that had been given BR-C dsRNA as L3 larvae. The retarded wing growth was previously shown for BR-C RNAi in the milkweed bug, Oncopeltus fasciatusKr-h1, BR-C mRNA expression was neither as strongly dependent on Met at the L4 instar  of animals. In this background we then tested whether Met and Kr-h1 mediated the response to the exogenous JH activity.When treated with JH mimics, final instar Met was silenced by dsRNA injection at the end of the L4 instar, the following application of methoprene no longer perturbed adult development in 96% (n = 23) of treated animals, and these rescued adults were externally indistinguishable from controls receiving no methoprene  of mid-L5 larvae, which had been treated with methoprene as L4, exceeded levels normally observed at the L5 instar by 125-fold  administration of methoprene to L5 larvae induces Met-dependent Kr-h1 expression and an extra larval stage, (ii) this phenotype is averted if Met or Kr-h1 are silenced prior to methoprene treatment, and (iii) premature suppression of Kr-h1, either direct or through depletion of Met, triggers precocious adult development. Therefore, the JH/Met-dependent Kr-h1 activity ensures the larval program, and only when JH disappears from the blood in the last larval instar, transcription of Kr-h1 ceases for six days to create an opportunity for adult transition , its co-expression with BR-C likely ensures that development does not go too far. This is suggested by appearance of not only pupal but also of adult features in Tribolium larvae subjected to Kr-h1 or BR-C RNAi Kr-h1 and BR-C, acting downstream of Kr-h1 in this context Similar regulation applies to holometaboly. In pre-terminal instars of R-C mRNA . While tKr-h1 as a JH-induced repressor of adult morphogenesis is clearly a common trait of holometaboly and hemimetaboly, the role of BR-C is not. Studies on the hemimetabolan Oncopeltus bug have revealed BR-C requirement during embryogenesis and for the anisometric growth of larval wing pads but no BR-C expression or function connected with metamorphosis BR-C is essential for pupal development but not at earlier stages in representatives of four holometabolan insect orders BR-C activity until the pupal stage in Holometabola has been ascribed to an early surge of JH during embryogenesis, which is thought to preserve the seemingly undeveloped (“embryonic”) nature of holometabolous larvae BR-C is necessary for hemimetabolous embryogenesis While the function of Oncopeltus, BR-C expression continues, albeit at a lower rate, throughout the last larval instar of Pyrrhocoris or another hemimetabolan, the cockroach Blattella germanica, in the absence of JH Kr-h1, expression of BR-C much less depends on JH and that in contrast to Kr-h1 or Met, removal of BR-C cannot accelerate metamorphosis in bug larvae. The changed need for BR-C function from hemimetabolous embryos and larvae to holometabolous metamorphosis suggests that during the evolution of holometaboly, BR-C has been recruited for the new function in specifying the pupal state.Unlike in Kr-h1 in the final-instar Pyrrhocoris larva and in the holometabolous pupa  was maintained at 25°C and a photoperiod of 18 h light to 6 h dark, on dry linden seeds and was supplemented with water. Eggs were collected daily and larvae of particular instars were identified based on the size of the body and wing pads; staging within instars relied on measuring time after ecdysis.Pyrrhocoris Met, Kr-h1 and BR-C genes were isolated by using touch-down nested RT-PCR with degenerate primers . After TURBO DNase  treatment, 2 µg of RNA were used for cDNA synthesis with Superscript II reverse transcriptase (Invitrogen). Relative transcript levels were measured by quantitative RT-PCR using the iQ SYBR Green Supermix kit and the C1000 Thermal Cycler . All data were normalized to the relative levels of ribosomal protein (Rp49) mRNA as described Total RNA was isolated from Met), 844 bp (Kr-h1) and 1026 bp (BR-C) of the Pyrrhocoris cDNA sequences and control dsRNAs encoding the EGFP and MalE proteins  were synthesized by using the T3 and T7 MEGAscript kit . Approximately 2–5 µg of dsRNA (depending on the size of larvae) were injected into the abdomen of CO2-anesthesized bugs. For JH mimic treatment, late-L4 stage Pyrrhocoris larvae were injected with dsRNA and within 2–3 hours after ecdysis to the L5 instar, a 4-µl drop of acetone-diluted 0.3 mM methoprene  or acetone alone (control) was applied on their dorsal side.dsRNAs comprising 952 bp , Kr-h1 (B) and BR-C (C) protein sequences from insects representing diverse developmental strategies. Holometaboly: the fruit fly Drosophila melanogaster, mosquitoes Aedes aegypti and Culex quinquefasciatus (Diptera); the silk moth Bombyx mori (Lepidoptera); the flour beetle Tribolium castaneum (Coleoptera); the honey bee Apis melifera (Hymenoptera); the lacewing Chrysopa perla (Neuroptera). Hemimetaboly: true bugs Pyrrhocoris apterus, Rhodnius prolixus and Oncopeltus fasciatus (Hemiptera); the cockroach Blattella germanica (Blattodea); the louse Pediculus humanus corporis (Phthiraptera). The thrips, Frankliniella occidentalis (Thysanoptera), represents neometaboly, an aberrant type of hemimetaboly with multiple resting stages. The firebrat, Thermobia domestica (Zygentoma), represents a basal lineage of wingless insects without metamorphosis (ametaboly). Dm_Met, Dm_Gce, and Bm_Met1, Bm_Met2 are products of paralogous Met genes that have duplicated independently in the Drosophila and Bombyx lineages, respectively. Database accession numbers for the aligned Met proteins (A) are NP_511126.2 (Drosophila melanogaster Met), NP_511160.1 (Drosophila melanogaster Gce), XP_001660262.1 (Aedes aegypti), BAJ05085.1 (Bombyx mori Met1), BAJ05086.1 (Bombyx mori Met2), ABR25244.1 (Tribolium castaneum), XP_395005.3 (Apis melifera), JN416984 (Pyrrhocoris apterus), JN416985 (Rhodnius prolixus), XP_002430841.1 (Pediculus humanus corporis), and JN416986 (Thermobia domestica). Accession numbers for the Kr-h1 proteins (B) are CAA06544.2 (Drosophila melanogaster), XP_001863529.1 (Culex quinquefasciatus), NP_001129235.1 (Tribolium castaneum), NP_001011566.1 (Apis melifera), BAJ41258.1 , JN416987 (Pyrrhocoris apterus), JN416988 (Rhodnius prolixus), XP_002428656.1 (Pediculus humanus corporis), and JN416989 (Thermobia domestica). Accession numbers for the BR-C proteins (C) are CAA38476.1 (Drosophila melanogaster), AAS80327.1 (Aedes aegypti), BAD23979.1 (Bombyx mori), joined ABW91135.1 and ABW91137.1 (Tribolium castaneum), ABW91140.1 (Chrysopa perla), BAJ41241.1 , JN416990 (Pyrrhocoris apterus), ABA02191.1 (Oncopeltus fasciatus), CBJ05858.1 (Blattella germanica), and joined GQ983556.1 and JN416991 (Thermobia domestica).(PDF)Click here for additional data file.Table S1Degenerate primers for isolation of Met, Kr-h1 and BR-C cDNAs from Pyrrhocoris apterus, Rhodnius prolixus and Thermobia domestica.(PDF)Click here for additional data file.Table S2Primers for RT-PCR expression analysis of Pyrrhocoris apterus Met, Kr-h1 and BR-C mRNAs.(PDF)Click here for additional data file.


High prevalence of risky sexual behaviors and lack of information, skills and preventive support mean that, adolescents face high risks of HIV/AIDS. This study applied the information-motivation-behavioral skills (IMB) model to examine the predictors of consistent condom use among senior high school students from three coastal cities in China and clarify the relationships between the model constructs.A cross-sectional study was conducted to assess HIV/AIDS related information, motivation, behavioral skills and preventive behaviors among senior high school students in three coastal cities in China. Structural equation modelling (SEM) was used to assess the IMB model.P < 0.01) and behavioral skills . Information indirectly predicted consistent condom use, and was mediated by behavioral skills .Of the 12313 participants, 4.5% (95% CI: 4.2–5.0) reported having had premarital sex and among them 25.0% (95% CI: 21.2–29.1) reported having used a condom in their sexual debut. Only about one–ninth of participants reported consistent condom use. The final IMB model provided acceptable fit to the data . Consistent condom use was significantly predicted by motivation (β = 0.175, The results highlight the importance of conducting HIV/AIDS preventive health promotion among senior high school students in China. The IMB model could predict consistent condom use and suggests that future interventions should focus on improving motivation and behavioral skills. HIV/AIDS is a global health issue and public health challenge. There are an estimated 33 million people living with HIV globally, half of whom are female . In the Adolescents are often characterized as being at a life stage of increased experimentation and exploration associated with a range of risky behaviors, including risky sexual behaviors . MoreoveMany studies demonstrate that most Chinese youths lack basic information on sex and HIV/AIDS -18. Few The study was conducted in three coastal cities in China, namely Shanghai, Beihai in Guangxi Province and Sanming in Fujian Province from year 2007 to 2008. These three coastal cities were selected as study sites because each had a different prevalence of AIDS relative to the rest of China: high in Guangxi, average in Shanghai and low in Fujian.We conducted a two-stage cluster sample design by administering a questionnaire on AIDS health awareness to senior high school students from three coastal cities to survey HIV/AIDS related information, motivation, behavioral skills and sexual behaviors from three coastal cities. This self-administered, school-based instrument had been tested in preliminary research and showed good fitness based on reliability and validity analysis . AssuminThe study was reviewed and approved by the Ethics Committee of the School of Public Health, Shanghai Jiaotong University. Before enrolment in the study, all students and their parents or guardians provided written informed consent, which included the study objectives and procedures, and potential risks and benefits of participation.Data were collected by self-administered questionnaires which had been tested in preliminary research and showed suitability based on reliability and validity analysis . Prior tThe IMB model is a causal model with latent variables that mixes of path analysis and confirmatory factor analysis, and has been called a hybrid model. The latent variables include information, motivation and behavioral skills which are hypothesized to reflect key constructs of the IMB model . PreventHIV/AIDS related information was measured using 18 items with “yes”, “no” or “do not know” response. Positive answers were assigned a score of one, while negative answers or responses of “do not know” received a score of zero. One indicator containing six items was related to safe sex information . Individual question scores were summed and converted into a total score to represent knowledge of safe sex . The other indicator included 12 items and was related to HIV/AIDS information . Individual item scores were summed to produce a total score to represent HIV/AIDS information . Higher scores indicate students have access to more information.The motivation to take preventative actions against HIV/AIDS was assumed to be a function of individual attitudes or relevant subjective norms , and wasRespondent perceptions of their self-efficacy to perform HIV/AIDS preventive behaviors were assessed using two scales. Both scales were constructed from answers on a four-point Liket scale .The first scale was condom self-efficacy and consisted of two items . The sum of the scores of these two items was converted into the scale of self-efficacy for condom use . The second scale was the Rosenberg’s self-esteem scale, containing ten items . The score of these ten items were summed and converted into the scale of self-esteem . Higher score indicated more self-efficacy to apply HIV/AIDS preventive skills.Literature review showed that consistent condom use usually indicated HIV/AIDS prevention behavior measures associated with the IMB model ,24,26. Cχ2/df ratio of 3 or less indicates acceptable fit , means (95% CI) and standard deviation (SD). The structural equation model (SEM) was used to examine the hypothetical IMB model using Amose 20.0. Model fit was assessed using the comparative fit index (CFI), the root mean square error of approximation (RMSEA), and the maximum likelihood chi-square values/degrees of freedom ratio . The CFIable fit ,28. Confable fit . A path A total of 12,313 senior high school students completed the questionnaire  model to the 552 participants that reported having had premarital sex to estimate the factor structure and relationships of the all the IMB variables. Table χ2 = 195.8, df = 15, meaning the χ2/df ratio was not within the acceptable range of 3 or less (χ2/df =13.1). CFI was acceptable at 0.947 but RMSEA was not within the acceptable range of 0.05 or less (RWSEA = 0.148). The initial model thus needed modification.The initial IMB model is shown in Figure χ2 = 26.6, df = 11, P = 0.139, the χ2/df ratio = 2.42, CFI = 0.981, RMSEA = 0.014. The final IMB model thus showed good fit.We modified the IMB model by adding supplementary paths to the initial model. Figure P < 0.01) and motivation , but had no significant association with information . Motivation  and information  were significantly and positively associated with behavioral skills, which indirectly affected consistent condom use. Additionally, four new paths were found in the final model, namely that HIV/AIDS information , safe sex information , perceived susceptibility to HIV/AIDS  and sexual norms  significantly affected condom self-efficacy, a behavioral skill that in turn positively affected consistent condom use.As expected, consistent condom use was strongly predicted by behavioral skills .Click here for file


Pseudomonas aeruginosa) are not suitable to be assessed by PCT algorithms. Therefore, the true value of PCT-guided algorithm of antibiotic stewardship in assisting the clinical decision-making process at the bedside remains uncertain. Future studies should take into account the issues identified in the present review.Biomarkers of infection, namely C-reactive protein and procalcitonin (PCT), are potentially useful in the diagnosis of infection as well as in the assessment of its response to antibiotic therapy. C-reactive protein variations overtime appears to have a good performance for the diagnosis of infection. Procalcitonin shows a better correlation with clinical severity. In addition, to overcome the worldwide problem of antibiotic overuse as well as misuse, biomarker guidance of antibiotic stewardship represents a promising new approach. In several randomized, controlled trials, including adult critically ill patients, PCT guidance was repeatedly associated with a decrease in the duration of antibiotic therapy. However, these trials present several limitations, namely high rate of patients’ exclusion, high rate of algorithm overruling, long duration of antibiotic therapy in the control group, disregard the effect of renal failure on PCT level, and above all a possible higher mortality and higher late organ failure in the PCT arm. In addition, some infections  as well as frequent nosocomial bacteria (e.g.,  During the past three decades, the incidence of sepsis has been consistently rising, surpassing that of cardiac failure and has an annual mortality rate above acute myocardial infarction ,2. A recConsequently sepsis is a major public health issue that can affect all population, including healthy people, although there is a predominance in elderly patients with multiple or severe comorbidities. However, during the past 15 years, several reports showed a sustained and continuous improvement in mortality of severe sepsis and septic shock, although it remains exceedingly high, ranging between 30-50 % -6. Yet, With the goal of shortening the duration of antibiotic therapy, the efficacy of procalcitonin (PCT)-guided antibiotic stewardship algorithms have been tested in different clinical settings . As a reThe diagnosis of infection is not straightforward, because there is no “gold standard” test . ConsequTwo landmark studies were published that evaluated the impact of shorter duration of antibiotic therapy in ventilator-associated pneumonia (VAP) ,16. The p = 0.001). Both groups presented similar hospital mortality and ICU and hospital length of stay (LOS) . These findings suggest that shorter courses of antibiotic therapy, 6–8 days, in VAP can be safely achieved without the use of biomarkers.The second study was a single-center, prospective RCT designed to evaluate the effectiveness and safety of a discontinuation policy on the duration of antibiotic therapy of VAP (N = 290) . The autAmong all biomarkers of infection those more frequently studied are C-reactive protein (CRP) and PCT . Before Plasma CRP, like all other acute phase proteins, is exclusively synthesized in the liver in response to interleukin 6 ,18. FourThe pathophysiological role of PCT in sepsis is not fully understood . After aThe characteristics of the assays used to measure the marker are a fundamental aspect when dealing with biomarkers . The avaTo be useful at the bedside, the PCT assay should have a high sensitivity . Only thvery unlikely, unlikely, likely, and very likely, respectively [The proposed algorithms on antibiotic stewardship are based on different PCT cutoff ranges. These cutoffs were derived from several well-conducted, prospective, observational studies and were validated in different RCT . The PCTectively . To prevectively .For patients in the ICU, the algorithm for antibiotic stewardship was similar but with somewhat higher PCT cutoff ranges . LikewisA total of 7 RCT dealing with PCT-guided antibiotic stewardship in adult critically ill patients were identified for further discussion -47. TheyThe clinical impact of an intervention is somehow dependent on its applicability. In the seven selected RCT, the rate of exclusion ranges from 0.3 %  to 84 % Pseudomonas aeruginosaAcinetobacter baumannii, as well as frequent clinical situations, namely endocarditis or infections that require long-course antibiotics and immunosuppressed patients.It also is necessary to examine the criteria for exclusion. In three studies the description was very poor ,44,46. IThe quality of the studies also is largely dependent of the methodology used to measure PCT plasma concentrations. One RCT used a semiquantitative immunochromatographic method , and twoEach of the RCT describes a specific PCT-guided algorithm of antibiotic stewardship; however, as a whole, the main frame was quite similar to that previously described. In four RCT, the algorithm was designed to assist in the decision to stop antibiotics ,44-46, iIn the seven RCT, the control group was managed according to the standard of care. This is vague, because there is substantial difference between usual care, the standard of care, and the best care . AccordiLegionella pneumophilaMycoplasma pneumoniae, or Chlamydia pneumoniae, and for VAP 8–15 days [Therefore, it would be important to know whether patients in the control group have a minimum duration of antibiotic therapy. This was the case in three RCT ,44,45. I–15 days .This raises two issues. First, several years after the publication of RCTs showing that 6–8 days of antibiotic therapy in VAP is safe ,16, it mIn two trials, the PCT-guided algorithm was designed to assist in the decision to start antibiotics ,43. In oIn five RCT, the PCT-guided algorithm was designed to assist in the decision to stop antibiotics ,42,44-46p = not shown) [p < 0.0001) [The duration, in days, of antibiotic therapy of the first episode of infection was shorter in the PCT-guided arm in all studies, 6 (3–34) vs. 9.5 (2–33) (p = 0.15) , 6.6 ± 1t shown) , 5.9 ± 1t shown)  and 6.1  0.0001) . SimilarConsequently, the exposure to antibiotics, expressed by the antibiotic-free days, was lower in patients from the PCT arm ,42,45.Recently, the largest, multicenter trial to assess a PCT-guided algorithm was published: the PASS trial . Its desp < 0.0001) and a longer exposure (p < 0.001) [p < 0.05).The use of large-spectrum antibiotics was substantially increased in the PCT arm with a shorter time to start (< 0.001) . However< 0.001) . Total dIn addition to the extent of excluded patients, the degree of algorithm overruling is an indirect measure of its clinical applicability as well as of its true impact in the clinical decision making process. In the PRORATA trial , at inclIn three RCT that assessed the impact of PCT-guided algorithm in the decision to stop antibiotics, it was possible to assess the rate of overruling ,42,45. IFinally, in the PASS trial , 56 of 3All studies provided data concerning the LOS (in days) in the ICU that was very similar in the PCT and control groups: 16.1 ± 6.9 vs. 19.4 ± 8.9 , 4 (1–21p = 0.037) [p < 0 .001) [An objective assessment of organ failures is provided in only two trials ,47. Base= 0.037) . In the Three studies provide the rate of superinfections. One was lower in PCT-guided arm: 7/31 (22.5 %) vs. 11/37 (29.7 %) . The othTwo studies provided data on the rates of infection relapse that were similar in one study: 1/39 (2.6 %) vs. 1/40 (2.5 %) ; the secp = 0.67), whereas the PASS was significantly higher in the control group: 2.4 % vs. 3.1 % (p = 0.01) [Finally, two studies monitored the emergence of multidrug resistance bacteria with very different overall rates. In the PRORATA study , the rat = 0.01) .p = NS) [p = 0.28) [p = 0.327) [All studies provided 28-day all-cause mortality rates that were comparable. A closer look showed that in four trials, the 28-day mortality was very similar: 8/39) (20.5 %) vs. 8/40 (20 %) ; 3/14 (2p = NS) ; and 190p = NS) . In two  = 0.28)  and 8/51= 0.327) .In the two larger studies, the PRORATA  and the It is well known that a prolonged duration of antibiotic therapy is associated with increased costs. Conversely, the measurement of PCT also is very expensive. The analysis of the cost-benefit of the implementation of a PCT algorithm was not performed in any of the selected studies.Biomarkers of infection, namely CRP and PCT, are potentially very useful in the diagnosis of infection as well as in the assessment of its response to antibiotic therapy. Presently, no RCTs of CRP-guided therapy in critically ill patients have been performed. Regarding PCT, in several RCTs, PCT guidance was repeatedly associated with a decrease in the duration of antibiotic therapy. However, these trials present several limitations, namely high rate of patients’ exclusion, high rate of algorithm overruling, long duration of antibiotic therapy in the control group, disregard the effect of renal failure or use of RRT on PCT levels, and above all a possible higher mortality and late organ failure in the PCT arm. In addition, some infections as well as frequent nosocomial bacteria were not evaluated. As a result, in critically ill patients, we cannot recommend the routine use of PCT-guided algorithms of antibiotic stewardship to assist the clinical decision-making process at the bedside. However, biomarkers, namely PCT and CRP but also platelet count, d-dimer, and prothrombin time, could be very useful at the bedside but should never be used solely. Biomarkers should always be used in conjunction with a complete clinical, laboratory, and radiologic evaluation and with a perfect knowledge of its biology, strengths, and limitations. Future studies in this area of knowledge are needed, but their design should take into account the issues identified in the present review to define clearly the role of biomarkers at the beside.CRP: C-reactive protein; ICU: Intensive care unit; LOS: Length of stay; PCT: Procalcitonin; RCT: Randomized controlled trial; RRT: Renal replacement therapy; VAP: Ventilator associated pneumonia.PP has received honoraria and has served as advisor of Astra Zeneca, Gilead, Janssen-Cilag, Merck Sharp & Dohme, Novartis, and Pfizer and has received unrestricted grants from Brahms and Virogates. JIFS declares that he has no competing interests.PP and JIFS contributed to the study conception and design, performed and participated in the acquisition and interpretation of data, and drafted the manuscript. All authors read and approved the final manuscript.PP is coordinator of the Polyvalent Intensive Care Unit, São Francisco Xavier Hospital, and Professor of Medicine, Faculty of Medical Sciences, New University of Lisbon, Lisbon, Portugal. JIFS is medical researcher from the D’Or Institute for Research and Education, and coordinator of the Postgraduation Program, Instituto Nacional de Câncer, Rio de Janeiro, Brazil.