Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM  compared to HetM . We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y−1 in HMW and 3.12 y−1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population’s treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread.The sexually transmitted bacterium  Neisseria gonorrhoeae, a sexually transmitted bacterium, drug resistance is a particularly big problem: there is only a single antibiotic left that is recommended for treatment. We aimed to understand how antibiotic-resistant N. gonorrhoeae spread in a sexually active host population and how the spread of resistance can be slowed. From antibiotic resistance surveillance data, we first estimated the rate at which antibiotic-resistant N. gonorrhoeae spread. Second, we reproduced the observed dynamics in a mathematical model describing the transmission between hosts. We found that antibiotic-resistant N. gonorrhoeae spread faster in host populations of men who have sex with men than in host populations of heterosexuals. We could attribute the faster spread of resistant pathogens to higher treatment rates. This finding implies that promoting screening to control antibiotic-resistant N. gonorrhoeae could in fact accelerate their spread.More and more infectious disease treatments fail because the causative pathogens are resistant to the drugs used for treatment. For the treatment of  Neisseria gonorrhoeae can evolve and spread rapidly [N. gonorrhoeae isolates are resistant [N. gonorrhoeae treatment [N. gonorrhoeae infection by expanded screening and treatment of hosts [Antibiotic-resistant  rapidly . Resista rapidly –4. Resis rapidly , 3. Sinc rapidly  followinesistant . Injectaesistant . Althougreatment , no new of hosts , 10, butN. gonorrhoeae than heterosexual host populations [N. gonorrhoeae were mainly found in MSM until 2011. The authors suggested that cefixime resistance was circulating in a distinct sexual network of highly active MSM and that bridging between MSM and heterosexuals was necessary for subsequent spread among heterosexual hosts. However, cefixime-resistant N. gonorrhoeae might have already been spreading undetected in the heterosexual host population.Men who have sex with men (MSM) are host populations that have higher levels of antibiotic-resistant ulations . In a stulations  based onN. gonorrhoeae in different host populations. In 1978, Yorke et al. [N. gonorrhoeae. The concept of core groups posits that an infection can only be maintained in a host population if a highly sexually active group of hosts is responsible for a disproportionate amount of transmissions. More recent modeling studies have examined the transmission of antibiotic-resistant N. gonorrhoeae. Chan et al. [N. gonorrhoeae transmission model to determine the impact of different treatment strategies on the prevalence of N. gonorrhoeae in Dutch MSM. They found that increased treatment rates could increase the spread of resistance, whereas re-treatment could slow it down. Hui et al. [N. gonorrhoeae transmission model in a heterosexual host population to investigate the effect of a molecular resistance test on the time until 5% resistance are reported. None of these studies has investigated or explained the differences in the spread of antibiotic-resistant N. gonorrhoeae in MSM and heterosexual host populations.Mathematical models can help explain the differential observations of antibiotic-resistant e et al.  introducn et al.  found thn et al.  developei et al.  used an N. gonorrhoeae spread using surveillance data and mathematical modeling. We estimated the rates at which resistance spreads in heterosexual men (HetM) and MSM using surveillance data from the USA and from England and Wales. We then developed a mathematical model of N. gonorrhoeae transmission to reconstruct the observed dynamics of resistance spread. This allowed us to determine the major driver of resistance spread, and to explore the expected rates at which resistance spreads in MSM and heterosexual host populations.In this study, we investigated the dynamics and determinants of antibiotic-resistant N. gonorrhoeae in England and Wales. GISP is an equivalent program from the Centers for Disease Control and Prevention (CDC) in the USA. We used Plot Digitizer 2.6.6 [N. gonorrhoeae from figures that were published online  represents the proportion of antibiotic-resistant N. gonorrhoeae at time t, c is the maximal proportion of antibiotic-resistant N. gonorrhoeae (carrying capacity), a is the ratio between antibiotic-sensitive and -resistant N. gonorrhoeae at time 0, and b is the rate at which the proportion of antibiotic-resistant N. gonorrhoeae increases in the initial exponential growth phase. We only used data from the years before the first decline in the proportion of resistant N. gonorrhoeae because we were interested in the rate of resistance spread during the initial exponential growth phase and while the antibiotic was still used.We determined the rate of resistance spread by assuming that the proportion of antibiotic-resistant omputing  to fit tN. gonorrhoeae in a given host population [Sen and Res indicate the antibiotic-sensitive and -resistant N. gonorrhoeae strains, G = {L, H} is the set of low and high sexual activity groups and i ∈ G  and develop resistance during treatment with probability μ. Hosts infected with an antibiotic-resistant strain can only recover spontaneously at rate ν. We assumed equal fitness of antibiotic-sensitive and -resistant strains in absence of treatment, i.e. no fitness costs for the antibiotic-resistant strain. We evaluated the impact of fitness costs on the model outcomes in a sensitivity analysis  , a populty group . For HMWϵ, the fraction of diagnosed and treated infections, ϕ, the average duration of infection, D, and the per partnership transmission probabilities within the low, βLL, and the high sexual activity group, βHH, to N. gonorrhoeae prevalence and incidence using the following algorithm:We calibrated the sexual mixing coefficient, Define prior parameter distributions .N. gonorrhoeae infections for HMW, and urethral, rectal and pharyngeal infections for MSM.Define the ranges for the expected prevalence and incidence of diagnosed infections  of ureth7 parameters sets from prior distributions.Randomly draw 10μ = 0) endemic equilibrium using the ordinary differential equation solver runsteady from the R [rootSolve [Simulate the transmission model until it approaches a resistance-free (om the R  package ootSolve .Select the parameters sets (posterior distributions) that result in prevalences and incidences within the defined range.N. gonorrhoeae is scarce, so we chose to use non-informative priors for all parameters except the duration of infection which was informed by Garnett et al. [i with Information about parameter estimates for t et al. . The rant et al.  and survt et al. , both frt et al. , 29. We γ = 1 y−1) in their sexual activity group before they are redistributed to either the same or the other sexual activity group [μ = 10−3 and performed a sensitivity analysis to assess the impact of μ on the model outcomes.We set the rate of entering and leaving the population, ty group . We do nτ, ν, βLH, βHL) are composites of other parameters  and 0.5 y (MSM) during the initial exponential growth phase.We fitted a logistic growth model to the proportion of antibiotic-resistant programs . The profor HetM . The aveN. gonorrhoeae and the spread of resistance in the dynamic transmission model. We calibrated five model parameters to expected prevalence and incidence in MSM and HMW host populations. The posterior distributions of the parameters were based on 2,779 parameter sets for HMW and 65,699 parameter sets for MSM , compared with 1.1–2.2 y (MSM).After calibration, we used the dynamic transmission model to study the spread of antibiotic-resistant n in HMW . In HMW,N. gonorrhoeae share the same resource for growth, i.e. the susceptible hosts. The rate at which one strain replaces the other strain in the host population is given by the difference in their net growth rates. We assume that the transmission probabilities and the infectious duration of the two strains are the same. Since the probability of resistance during treatment is very small (μ ≪ 1), the difference in net growth rates between the strains is approximated by the treatment rate τ and corresponds to the rate of spread of antibiotic-resistant N. gonorrhoeae. The observed distributions of treatment rates from the transmission model hardly overlap between HMW and MSM (−1 (MSM) and 0.88 y−1 (HMW).Antibiotic-sensitive and -resistant  and MSM . The medμ, and fitness costs in the antibiotic-resistant strain alter the model outcomes. Higher probabilities of resistance during treatment accelerate the establishment of antibiotic-resistant N. gonorrhoeae in the population and hence reduce the time until 5% resistance is reached .The limitations to our study need to be taken into consideration when interpreting the findings. First, we used data from different sources, although all were collected in high income countries. The antibiotic resistance surveillance data are from programs in England and Wales and the USA. The mathematical transmission model was parameterized using British sexual behavior data  and califections , it is wN. gonorrhoeae. The sexual mixing coefficient tends to be assortative for both HMW and MSM host populations in our model. Quantifying the degree of sexual mixing is difficult and largely depends on the study population, but our finding is consistent with other studies indicating assortative sexual mixing in the general population [N. gonorrhoeae infections are symptomatic, and that this proportion is expected to be higher in men than in women [The estimated posterior distributions of the parameters fit within the range of previously used values, and provide some insights into sexual mixing and the natural history of pulation , 33. Thein women –36. The in women –39. In oin women , which cN. gonorrhoeae.Our study found that the treatment rate is the driving force of resistance spread. Xiridou et al.  found thN. gonorrhoeae, this insight challenges the current management strategy that aims to lower the overall burden of infection by expanding screening and treatment of hosts [N. gonorrhoeae. Additionally, we show that fitness costs can decelerate or even prevent the spread of antibiotic-resistant N. gonorrhoeae strains. Fitness costs therefore might explain why highly resistant strains, such as the ceftriaxone-resistant N. gonorrhoeae strain H041, do not spread in the host population after their first detection [N. gonorrhoeae had already been present in the HetM population but were spreading at a lower rate than in the MSM population.It was shown previously that treatment is the main selective force acting on resistance evolution due to the selective advantage to the resistant pathogen , 41. We of hosts , 10. As etection . Our finetection . It is lN. gonorrhoeae research and for guiding treatment recommendations. The N. gonorrhoeae transmission model describes observed prevalence and incidence rates well and can reconstruct the spread of antibiotic-resistant N. gonorrhoeae. Estimating rates of resistance spread is useful for projecting future resistance levels and the expected time it will take until a certain threshold in the proportion of antibiotic-resistant N. gonorrhoeae is reached. Until now, treatment recommendations for N. gonorrhoeae are subject to change when 5% of N. gonorrhoeae isolates show resistance against a given antibiotic [−1  and men who have sex with men (MSM).(PDF)Click here for additional data file.S2 TableData for men who have sex with women (MSW) and men who have sex with men (MSM). We used the MSW data as heterosexual men (HetM) data.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Figμ. Lower and upper bound of the box indicate the first and third quartiles, bar in the box indicates median, whiskers span 1.5 times IQR. Outliers are shown in orange and are outside 1.5 times IQR.The time to 5% resistance of both MSM (green) and HMW (blue) are sensitive towards (TIFF)Click here for additional data file.S4 Figμ when μ is unrealistically high. Lower and upper bound of the box indicate the first and third quartiles, bar in the box indicates median, whiskers span 1.5 times IQR. Outliers are shown in orange and are outside 1.5 times IQR.The rates of spread of both MSM (green) and HMW (blue) are only sensitive towards (TIFF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.


Rhodococcus sp. TG13 and TN3, which were isolated from PCB-contaminated sediment and non-contaminated sediment respectively, were investigated under low temperature and oligotrophic conditions. The results indicated that the two strains TG13 and TN3 could enter into the VBNC state with different incubation times, and could recover culturability by reversal of unfavourable factors and addition of resuscitation-promoting factor (Rpf), respectively. Furthermore, the gene expression variations in the VBNC response were clarified by Illumina high throughput RNA-sequencing. Genome-wide transcriptional analysis demonstrated that up-regulated genes in the VBNC cells of the strain TG13 related to protein modification, ATP accumulation and RNA polymerase, while all differentially expressed genes (DEGs) in the VBNC cells of the strain TN3 were down-regulated. However, the down-regulated genes in both the two strains mainly encoded NADH dehydrogenase subunit, catalase, oxidoreductase, which further verified that cold-induced loss of ability to defend oxidative stress may play an important role in induction of the VBNC state. This study further verified that the molecular mechanisms underlying the VBNC state varied with various bacterial species. Study on the VBNC state of non-pathogenic bacteria will provide new insights into the limitation of environmental micro-bioremediation and the cultivation of unculturable species.Viable but nonculturable (VBNC) bacteria, which maintain the viability with loss of culturability, universally exist in contaminated and non-contaminated environments. In this study, two strains,  Rhodococcus plays a significant role in the fields of environmental bioremediation and industrial biotechnology [Rhodococcus represent an abundant part of indigenous bacterial communities in contaminated localities [Rhodococcus species and their ability to persist in adverse conditions make them suitable industrial microorganisms for bioremediation of contaminated sites. In addition, there has been increased interest in potential health risks caused by pathogenic rhodococci in humans, animals and plants [Rhodococcus species, and understanding their related function and genotype have become increasingly more important.The genus chnology . The polchnology . Variouscalities . The verd plants . TherefoRhodococcus have been described [Rhodococcus species, such as R. rhodochrous and R. fascians are known to enter into the VBNC state under adverse conditions [Vibrio, Salmonella, Legionella and Escherichia [However, to date, just over 40 species with validly published names in the genus escribed , becauseescribed , 7 when escribed . Specifinditions . Bacterinditions . Up to nherichia –12, due herichia , there iherichia .Vibrio parahaemolyticus has no direct correlation with the induction of virulence gene expression by a real-time reverse transcription-PCR method. Asakura et al. [Vibrio cholerae cells by comparative microarray analysis. Recent research has shown that the high throughput RNA-sequencing (RNA-Seq) technology, which could capture unbiased view of the RNA transcript profile of a species under a given condition at the whole genome level, is a powerful and cost-effective tool for transcriptome analysis [Meanwhile, most of studies elucidated the changes of VBNC state with regard to the physiological and biochemical aspects , 12. A fa et al.  evaluateanalysis . HoweverrpoS (σS) is required for the survival of numerous bacteria in multiple-stress environment [relA and spoT genes during entering the VBNC state would lead to accumulation of ppGpp, followed by accumulation of σS, ultimately leading to enhanced stress resistance [V. parahaemolyticus. It is important to stress that some VBNC bacteria are able to recover culturable state when surrounding conditions are more favorable [Micrococcus luteus [Rhodococcus, Mycobacterium, Arthrobacter [M. luteus cultures to VBNC cells has been shown to restore their culturability [It is worth to note that the genes expressed during entering the VBNC state which differs from bacterium to bacterium are complex and far from being fully understood. Although gene expression and the proteins modulation of VBNC cells have been studied, the molecular mechanism underlying the VBNC state is still poorly understood . It has ironment , 18. Morsistance . In addisistance . Wang etsistance  demonstravorable , but soms luteus . Rpf canrobacter , 24. Morrability .M. luteus [Rhodococcus biphenylivorans were obtained and could be entered into the VBNC state under low temperature and oligotrophic conditions [Rhodococcus in contaminated and non-contaminated environments could help us to reveal why the activities of pollutant-degrading bacteria decreased in the pilot-scale environmental bioremediation, and provide new insights into control strategies for prevention and resuscitation of bacteria from the VBNC state, for the missing species into culture. In this work, the induction of the VBNC state in two Rhodococcus sp. strains TG13 and TN3 which were isolated from PCB-contaminated sediment and non-contaminated sediment respectively, were performed under oligotrophic and low temperature conditions. A comparison of the ability of cells to exit the VBNC state was also investigated. Most notably, the primary purpose of the present study was to access the gene expression variation and regulation underline the VBNC state. In comparison to the normal cells, the differentially expressed genes (DEGs) in the VBNC cells were identified by high throughput RNA-Seq. The functions and related metabolic pathways of the up- or down-regulated genes were investigated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. A comprehensive comparison of the genetic mechanisms involved in the VBNC state of the two Rhodococcus strains was performed. To our best knowledge, it is the first attempt to compare the VBNC response of two Rhodococcus sp. strains which one of them possesses biphenyl/PCB-degradation capability.In our previous study, many uncultured or VBNC bacteria were obtained from PCB-contaminated soils and sediments by adding EOM from . luteus , 25. Notnditions , 26. TheNo specific permissions were required for the described field studies.Rhodococcus sp. TG13 and TN3, which were obtained by using the resuscitation function of EOM from M. luteus, were used in this study. The two strains were isolated from PCB-contaminated river sediment and non-contaminated deep sea sediment respectively, in Taizhou city, Zhejiang province, China. The two strains have been deposited in Marine Culture Collection of China (MCCC) with deposit numbers 1K01234 and 1K01235. Meanwhile, the 16S rDNA sequences of the two strains have been submitted to NCBI GenBank under accession numbers KM235731 and KP410397. The two strains TG13 and TN3 shared highest similarities (99.3–100% 16S rRNA gene similarity) with Rhodococcus ruber (100% similarity) and Rhodococcus erythropolis (99.3% similarity), respectively. The pure strains were stored in Luria-Bertani (LB) broth with 20% glycerol at -80°C. The strains were incubated in LB medium at 30°C with shaking at 180 rpm and grew to the exponential phase (OD600 = 0.98).Two strains, 4)2SO4, 1 g KH2PO4, 3 g K2HPO4 ·3H2O, 0.2 g MgSO4, 0.02 g FeSO4·7H2O and 5 g NaCl, at a final cell density of 1 × 107 CFU/mL . The resuspended cells of strains TG13 and TN3 were maintained at 4°C for 91 and 163 days, respectively to induce the VBNC state (< 0.1 CFU/mL) . All the experiments were performed in triplicate. Each sample obtained at distinct times was analyzed by using acridine orange direct counts (AODC) and flow cytometry, and plated on LB agar for culturable counts, presenting total, viable and culturable numbers of bacterial cells, respectively.Bacterial cell cultures in the exponential-phase were therefore harvested by centrifuged  and washed twice with 0.85% (w/v) sterile NaCl solution (pH = 7.3). Then, the washed cells were resuspended in an artificial oligotrophic medium (AOM) which contained (per liter): 1 g  according to the manufacturer’s instructions. Samples were added to the BD TruCOUNTTM Tubes , and analyzed by an Epics XL flow cytometry . The fluorescence of stains SYTO 9 and propidium iodide (PI) were detected through 525 nm and 620 nm bands pass filters, respectively. In order to determine the culturability of the cells in samples, each sample was serially (1:10) diluted with 0.85% NaCl solution and then pour-plated the cells on LB agar at 30°C for 48 h. Colony-forming units were enumerated and given as counts per millilitre (CFU/mL) of the initial suspension. All these experiments were performed in triplicate. The average value and standard deviation were calculated using IBM SPSS Statistics.Cell viability was assessed by using the LIVE/DEAD The VBNC cells (< 0.1 CFU/mL) in this study were centrifuged at 8,000 g for 10 min, and then the pellets were resuspended in the same volume of sterile 0.85% NaCl solution. Before resuscitation, the VBNC cells were treated for 2 h with 200 mg/mL of benzylpenicillin . Then, rThe exponential and VBNC cells of the two strains were used for RNA-Seq analysis. All experiments were performed in duplicates. For each biological sample, total RNA samples were extracted using the MICROBExpress Bacterial mRNA Enrichment kit  and then was examined with an Agilent 2100 Bioanalyzer. At least 20 μg of RNA at a concentration of ≥ 400 ng/μL, OD260/280 = 1.8–2.2 and RNA28S:18S ≥ 1.0 were used for cDNA library preparation .TM 2000 platform [Illumina sequencing was performed at the Beijing Genomic Institute . Briefly, after removing residual DNA and ribosomal RNA, the mRNA-enriched RNA was chemically fragmented to 150~200 bp. Based on these cleaved RNA fragments, cDNA was synthesized using random hexamer primer and reverse transcriptase. After the end reparation and ligation of adaptors, the products were amplified by PCR and purified using the QIAquick PCR Purification Kit  to create the final cDNA library. Libraries were sequenced on an Illumina HiSeqplatform .R. ruber (http://www.ncbi.nlm.nih.gov/genome/11562?genome_assembly_id=217613) and R. erythropolis PR4 (http://www.ncbi.nlm.nih.gov/genome/1638?genome_assembly_id=171453) using BWA-MEM alignment [Image data output from the sequencing machine is transformed into raw reads and stored in FASTQ format. All the raw transcriptome data have been deposited in the NCBI Short Read Archive (SRA), and given the accession numbers SRA236830 and SRA236842 for the strain TG13 and TN3, respectively. These dirty raw reads  were removed to obtain clean reads. Subsequently, the clean reads of the strains TG13 and TN3 were mapped to reference genome of lignment . MismatcGene expression counts in the control groups (c_TG13 and c_TN3) and the treatment groups (t_TG13 and t_TN3) were obtained using featureCounts software (version 1.4.6) which was set to exclude reads or fragments overlapping multiple genes. The values of normalized mean count obtained from the average value of the biological replicate experiments. The tool EBseq (version 1.7.1) was used to identify the DEGs between exponential and VBNC cells. To determine the statistical significant difference in gene expression, an absolute value of log2 ratio ≥ 1 and a false discovery rate (FDR) ≤ 0.05 were used as the threshold .For the DEGs, GO enrichment analyses were performed using a GO seq-based Wallenius non-central hyper-geometric distribution . Further-ΔΔCT method [DEGs were randomly selected for qRT-PCR to prove the quality of the sequencing data. The mRNAs were extracted as described for the cDNA library preparation, and then were transcribed to cDNA using SuperScript III reverse transcriptase. Subsequently, the qRT-PCR was performed using a power SYBR Green PCR kit  in a MicroAmp™ 96-well plate with the ABI 7500 real-time PCR system. Gene-specific primers used for qRT-PCR are shown in T method . All exp4 and 105 cells/mL, respectively. And total cell counts remained constant at the initial level (107 cells/mL). These results demonstrated that the strains TG13 and TN3 could enter the VBNC state under the present conditions. Moreover, the entry into the VBNC cells was also verified by resuscitation experiments.Cell viability of the strains TG13 and TN3 was assessed once a week. After 91 and 163 days of incubation at 4°C in AOM, the number of culturable cells of the strains TG13  and TN3 3 MPNs/mL at 24 h and reached a peak value (1.02 × 107 MPNs/mL) at 72 h. After 72 h, the cells entered stationary phase, which indicated that the increased value after 24 h was caused by the multiplication of the resuscitated cells. However, with Rpf addition, the VBNC cells could be resuscitated and multiplied to 8.71 × 104 MPNs/mL within 12 h. This comparison indicated that Rpf only stimulated resuscitation of the strain TG13, and the VBNC cells of the strain TG13 could resuscitate without Rpf addition. For strain TN3  at 60 h by cells multiplication. Moreover, the above described results were also verified by resuscitation performed with solid media. Colonies of the strain TG13 appeared when plating the VBNC cells on the LB agar plates at 30°C for 3 days, while the VBNC cells of strain TN3 could not grow under the same condition. These results indicated that for resuscitation from VBNC state, the strain TN3 needed extracellular bacterial proteins, but the strain TG13 could be achieved by a simple reversal of the unfavourable factors.Before resuscitation, the VBNC cells were treated with benzylpenicillin to overcome the argument that the observed resuscitation due to the regrowth of still culturable cells existed in the VBNC microcosm. As shown in rain TN3 , it coulR. ruber genome. For the strain TN3, 76% and 70% of obtained reads in the c_TN3 and t_TN3, respectively, were covered in R. erythropolis PR4 genome. It is worth noting that total reads which were perfectly mapped to the reference genomes without mismatch were all greater than 50%. All these results indicated that the obtained sequences met the requirement, and could be used for further analysis.Two cDNA libraries for the strains TG13 and TN3 were constructed, respectively. As shown in Gene expression of t_TG13 and t_TN3 in VBNC state were compared to that of c_TG13 and c_TN3 in exponential growth stage, respectively . A totalTo understand the function of the DEGs underlying the VBNC state, GO enrichment analysis was performed with the total of 634 and 992 DEGs in the strains TG13 and TN3, respectively. Based on sequence homology, DEGs of the strains TG13 and TN3 were assigned to one or more GO codes and categorized into 1031 and 1478 secondary level GO terms , respectHowever, for strain TN3, in each of the three main categories of the GO classification, there were 138, 34 and 23 functional groups significantly enriched, respectively  in bold.To identify the biological pathways that are active in the formation of the VBNC state, all of the genes were mapped to the reference canonical pathways in KEGG and compared with the whole genomic background. As shown in In addition, to provide a global view of metabolism of DEGs with expression fold change of at least 20-fold up or down-regulation , some DETo validate the results of RNA-seq, qRT-PCRs were performed on 10 genes with different expression profiles for strains TG13 and TN3, respectively. As shown in V. cholera could be achieved by coculture with eukaryotic cells. Obviously, the methods for induction and resuscitation of VBNC cells varied with various bacterial species.In the present study, the biphenyl/PCB-degrading bacterial strain TG13 entered into the VBNC state after 91 days maintaining in AOM at 4°C, and it could be resuscitated from VBNC state by incubating in LB medium at 30°C. Notably, it is practically possible (benzylpenicillin-pre-treated VBNC cells) to confirm that the increases in culturability is due to the resuscitation of VBNC cells . Indeed,Vibrio vulnificus was peroxide sensitive due to loss of catalase activity. Meanwhile, results from the study of Smith and Oliver [V. vulnificus at low temperatures. The decreased catalase activity resulted in the cells that were unable to defend against the hydrogen peroxide present in routine media and become nonculturable. Thus, it could be inferred that cold temperature-induced loss of ability to defend oxidative stress may play a significant role in induction of the VBNC state.In addition, this study was undertaken to begin elucidating the gene expression and regulation that might underlying the formation of VBNC state. Intriguingly, in some ways, the results from TG13 were in agreement with TN3, which indicated that down-regulated genes mainly encoded hypothetical protein, NADH dehydrogenase subunit, catalase, oxidoreductase  Tables. d Oliver  confirmerpoA gene encoding subunit of the transcriptional regulator sigma has constant transcript levels under various stress conditions. Meanwhile, evidence has been obtained to show that elevated expression of rpoS gene played a key role in enhancing stress resistance [Meanwhile, the results of KEGG pathway analysis indicated that “fatty acid degradation” , “RNA posistance . CombiniR. biphenylivorans TG9T. The up-regulated genes related to protein modification, ATP accumulation and RNA polymerase were found in the VBNC cells, which render VBNC cells more tolerant to survive under inhospitable conditions. In comparison, all the DEGs related to the formation of VBNC state in the strain TN3 were found down-regulated. Perhaps the VBNC cells of the strain TN3 may have entered in a resistant state for a long time before death. Furthermore, DEGs enriched in metabolic pathway of citrate cycle in the strain TG13 were not in accord with that of in the strain TN3 Click here for additional data file.S2 FigTN3. The red and the green boxes indicate genes with up-regulated and down-regulated, respectively.(DOC)Click here for additional data file.S3 FigThe red and the green boxes indicate genes with up-regulated and down-regulated, respectively.(DOC)Click here for additional data file.S4 FigThe red and the green boxes indicate genes with up-regulated and down-regulated, respectively.(DOC)Click here for additional data file.S5 FigThe red and the green boxes indicate genes with up-regulated and down-regulated, respectively.(DOC)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(XLS)Click here for additional data file.S9 Table(XLS)Click here for additional data file.


Prescribing of antibiotics for common infections varies widely, and there is no medical explanation. Systematic reviews have highlighted factors that may influence antibiotic prescribing and that this is a complex process. It is unclear how factors interact and how the primary care organization affects diagnostic procedures and antibiotic prescribing. Therefore, we sought to explore and understand interactions between factors influencing antibiotic prescribing for respiratory tract infections in primary care.Our mixed methods design was guided by the Triangulation Design Model according to Creswell. Quantitative and qualitative data were collected in parallel. Quantitative data were collected by prescription statistics, questionnaires to patients, and general practitioners’ audit registrations. Qualitative data were collected through observations and semi-structured interviews.From the analysis of the data from the different sources an overall theme emerged: A common practice in the primary health care centre is crucial for low antibiotic prescribing in line with guidelines. Several factors contribute to a common practice, such as promoting management and leadership, internalized guidelines including inter-professional discussions, the general practitioner’s diagnostic process, nurse triage, and patient expectation. These factors were closely related and influenced each other. The results showed that knowledge must be internalized and guidelines need to be normative for the group as well as for every individual.Low prescribing is associated with adapted and transformed guidelines within all staff, not only general practitioners. Nurses’ triage and self-care advice played an important role. Encouragement from the management level stimulated inter-professional discussions about antibiotic prescribing. Informal opinion moulders talking about antibiotic prescribing was supported by the managers. Finally, continuous professional development activities were encouraged for up-to-date knowledge. Prescribing of antibiotics for common infections varies widely between different countries in Europe, and Sweden is one of the countries where antibiotic prescribing is lowest measured as prescriptions/1000 inhabitants per year  unnecessarily and the development of resistance and so on, so I don’t think so. We don’t have that type of patients here who demand antibiotics, I don’t think so.” (Nurse)The GPs at the low-prescribing PHCCs started the consultations in a more patient-centred way with open-ended questions in order to get a picture of the patients’ expectations. They only introduced antibiotics when they had clinically assessed all circumstances and had come to the conclusion that an antibiotic treatment was needed according to the guidelines. Nurses also said that they had observed a change in patients’ expectations in this direction over the past five years or so. From the analysis of the data from the different sources an overall theme emerged: A common practice in the PHCC is crucial for low antibiotic prescribing in line with guidelines. Several factors contribute such as promoting management and leadership, internalized guidelines, the GP’s diagnostic process, nurse triage, and patient expectation. These factors were closely related and influenced each other.The results also showed that new knowledge was internalized both in the PHCC as a whole and in all members of the staff. The guidelines were normative for the group as well as for every individual in the group.The mixed methods design enabled us to get as complete a picture as possible of the studied PHCCs. Six PHCCs might seem too few, but with the mixed methods design we have obtained abundant data. One difficulty was to get an overview of all research data. A remedy was separate steps of the analysis and the fact that the researchers had different research experience. The analysis with both inductive and deductive elements as well as the descriptive statistics from the patient survey and the audit increased the validity of the results.The observations gave an idea of the local culture at the PHCC as well as relations and interaction between the employees. Through the observations we reached an understanding of what actually happened between the patients and the staff on their way through the PHCC, although compared to ethnographic research the study period was relatively short. We believe this was outweighed by the presence of more than one observer.The study period would normally have been a high season for respiratory tract infections, but during 2014 the infection season appeared later than usual and therefore there were fewer patients with RTIs than expected. Additionally, there were sometimes difficulties convincing the patients about the importance of answering the questionnaire and sometimes the receptionists forgot or did not have the time to hand them out. Due to the low numbers in both the patient survey and the audit, the data must be interpreted with caution when standing alone, but when aggregated with data from the interviews and observations the findings were strengthened.We focused specifically on what happened at the low-prescribing PHCCs, even though we also collected data from high-prescribing PHCCs. Those were collected in order to mirror the low-prescribing PHCCs and to achieve a better understanding of factors influencing antibiotic prescribing for RTIs in primary health care.The organization of Swedish PHCCs as group clinics implies that the findings of this study must be interpreted with reservations in an international perspective where group clinics are less common.Previous studies have shown that new knowledge will be used in clinical practice, when transformed through continuous discussions among staff. New knowledge must be applicable and meaningful in the local context and its credibility is tested in a learning process , 29. SucThis study identified the leadership as a crucial factor for low antibiotic prescribing. The leadership must provide structural conditions and encouragement for inter-professional collaboration in order to achieve a common practice in keeping with guidelines. This is in line with the conclusions of two recent studies , 30. In In our study, all PHCCs were given feedback on the health centre’s prescribed antibiotics every three months. At the low-prescribing PHCCs these data were discussed at staff meetings. According to the interviews, GPs would need easier access to a relevant presentation of their own prescribing data.Several reviews have stressed well-functioning inter-professional collaboration as an important condition for knowledge dissemination and evidence-based practice , 33. In The patients had generally good knowledge of antibiotics. Most of the patients answering the questionnaire knew that antibiotics were effective against bacteria and less than half of them thought they were effective against viruses. This proportion is in line with an earlier Swedish study and better than in a Spanish study , 37.More patients in the low-prescribing PHCCs expected antibiotics compared to patients in the high-prescribing PHCCs. A probable explanation might be that the nurses at the low-prescribing PHCCs had selected patients with respiratory infections who might benefit from antibiotic treatment according to guidelines. However, the GPs at low-prescribing PHCCs rated most infections as mild while GPs at high-prescribing PHCCs more often classified the respiratory tract infection as moderate. The more common use of bacterial diagnoses among the high prescribers compared to low prescribers has been shown earlier , 39. TheThere has been a paradigm shift regarding the management of infections over the past 20 years, from an earlier approach according to which all infections of probable bacterial aetiology had to be treated with antibiotics, to a more modern policy of treating with antibiotics only if there is evidence for benefit , 43. TheIn PHCC with low antibiotic prescribing some important factors were observed. Guidelines were adapted and transformed into a common practice within the staff at the PHCC, not only the GPs. Nurses’ triage in the telephone played an important role in daily practice. Encouragement from the manager stimulated inter-professional discussions concerning antibiotic prescribing and informal opinion moulders talking about antibiotic prescribing were supported by the managers. Near-patient tests were used in accordance with the guidelines. Moreover, routines and IT systems were adapted in a way that enabled GPs themselves to access prescribing data easily from registers as a basis for professional discussions. Finally, continuous professional development activities were encouraged for everyone to be able to have up-to-date knowledge. All these factors were interacting in the PHCC with low antibiotic prescribing and the managers need to facilitate them. The factors also have to be taken into account when making interventions to rational antibiotic use.APO, Audit Project Odense; CRP, C-Reactive Protein; GP, General Practitioner; MPA, Medical Product Agency; PHCC, Primary Health Care Centre; RADT, Rapid Antigen Detection Test; RTI, Respiratory Tract Infection; Strama, Swedish strategic programme against antibiotic resistance


Though on the market for many years, few pharmacodynamic data are available on this combination net beta-mimetic agent.Hypotensive states that require fast stabilisation of blood pressure can occur during anaesthesia. In 1963, the 20:1 mixture of cafedrine/theodrenaline  increase and heart rate. Furthermore, potential factors that influence drug effectiveness under anaesthesia were assessed.Data were collected within a standardised anaesthesia protocol. A total of 353 consecutive patients  who received cafedrine/theodrenaline after a drop in MAP ≥ 5 % were included in the study. The time to 10 % increase in MAP, dosage of cafedrine/theodrenaline, volume loading, blood pressure and heart rate were monitored over time.p < 0.001) by 11 ± 16 mmHg within 5 min, reaching peak values within 17.4 ± 9.0 min. Heart rate was not affected in a clinically significant manner. Cafedrine/theodrenaline induced a 10 % MAP increase after 7.2 ± 4.6 min (women) and after 8.6 ± 6.3 min (men) (p = 0.018). Independent of gender, the dose of cafedrine/theodrenaline required to achieve the observed MAP increase of 14 ± 16 mmHg at 15 min was significantly different in patients with heart failure [1.78 ± 1.67 mg/kg (cafedrine)/89.0 ± 83.5 µg/kg ] compared with healthy patients [1.16 ± 0.77 mg/kg (cafedrine)/58.0 ± 38.5 µg/kg ] (p = 0.005). Concomitant medication with beta-blocking agents significantly prolonged the time to 10 % MAP increase [9.0 ± 7.0 vs. 7.3 ± 4.3 min (p = 0.008)].Patients were a mean (standard deviation) of 64.4 ± 15.1 years old with a baseline MAP of 82 ± 14 mmHg, which dropped to a mean of 63 ± 10 mmHg during anaesthesia without gender differences. Cafedrine/theodrenaline  significantly increased MAP (Cafedrine/theodrenaline quickly restores MAP during anaesthesia. Female gender is associated with higher effectiveness, while heart failure and beta-blocker administration lower the anti-hypotonic effect. Prospective studies in defined patient populations are warranted to further characterise the effect of cafedrine/theodrenaline. For decades hypotension was avoided by the provision of excessive fluid preload, however, this practice faces increasing criticism . In GermCafedrine/theodrenaline was introduced in Germany in 1963, and pre-clinical data showed a beta-adrenergic and an alpha-adrenergic component , 25, 26.50 of cafedrine/theodrenaline have previously been reported by our group [Although cafedrine/theodrenaline is widely accepted in the management of hypotensive states in anaesthesia, intensive care and emergency medicine , 6, 16, ur group . To confThis study was approved by the Institutional Review Board at the University Hospital Carl Gustav Carus Medical Faculty, Fetscherstrasse 74, Dresden, Germany . The records from 353 consecutive anaesthesia patients at our institution who received cafedrine/theodrenaline were collected and retrospectively evaluated. Patients were included in the study between July and November 2007, irrespective of the type of surgery, and received cafedrine/theodrenaline to manage hypotension during anaesthesia. To reflect a real-life cross-section of patients in a German hospital, patients from the following departments were included: orthopaedics, trauma surgery, neurosurgery, visceral- thoracic and vascular surgery, gynaecology, urology, oral and facial surgery and eye surgery.®, Roche, Grenzach-Wyhlen, Germany) 45 min prior to arrival in the operating theatre. Concomitant medication, including anti-hypertensives such as beta-blockers, was continued as indicated by the standard operating procedures of the department. A 5-lead electrocardiogram, including measurement of segmental ST depression , and pulse oximetry was recorded. Non-invasive oscillometric blood pressure was monitored in 5 min intervals on the right upper arm . Values were manually transferred to anaesthesia protocols. The following data were recorded: age, gender, height, body weight, current beta-blocker therapy, American Society of Anesthesiologists physical status and individual dosage of cafedrine/theodrenaline per injection.All patients fasted 2 h prior to induction and received 7.5 mg midazolam OD . Tracheal intubation was facilitated by 0.5 mg/kg rocuronium . General anaesthesia was maintained with desflurane  in O2/N2O (35 %/60 %). Patients were mechanically ventilated with a minute volume adequate to maintain end-tidal pCO2 of 36–40 mmHg at a fresh gas flow of 1 l/min . Neuraxial anaesthesia  was performed with approved local anaesthetics in awake seated patients [General anaesthesia was induced with 1.5 mg/kg propofol  and 0.5 µg/kg sufentanil  contains 200 mg cafedrinhydrochloride, 10 mg theodrenalinehydrochloride, 0.4 mg sodiumdisulfite, ethanol 96 %, glycerol 85 %, sodium acetate 3 H2O, acetic acid 99 % and water. To enable fairly precise dosing, 2 ml of cafedrine/theodrenaline were diluted in 8 ml of saline to a total of 10 ml, as suggested in the prescribing information. Throughout the manuscript doses are given for the cafedrine component of the mixture. Due to the fixed milligram ratio of 1:20, the dosage of theodrenaline can be calculated by dividing the respective cafedrine dose by 20.An ampoule of 2 ml cafedrine/theodrenaline .t-test served for analysing point to point changes in haemodynamics. To ensure equal distribution of risk factors between the observed cohorts, categorical variables were compared with χ2 statistics.Mean arterial blood pressure was calculated as MAP = diastolic + (systolic-diastolic)/3. All data were anonymised and analysed using the Statistical Package for Social Sciences for Windows, version 17.0 . The primary study hypothesis was tested using a Kaplan–Meier analysis followed by a log-rank test. General linear model statistics according to a two-way analysis of variance were applied for repeated measurement analyses, followed by Sidak alpha adjustment for multiple comparisons. A paired p-value of < 0.05 was considered statistically significant.Data are expressed as mean ± SD. To support readability and interpretation in Figs. Data were collected for 353 consecutive anaesthesia patients (mean age 64.4 ± 15.1) at a single institution who received cafedrine/theodrenaline to treat hypotensive states during surgery between July and November 2007. Demographic characteristics, baseline values and treatment are presented in Table p < 0.001). MAP increase after 5 min was also statistically significant (p < 0.001), however mean baseline MAP values and the drop during surgery did not differ between genders  5 min after administration of cafedrine/theodrenaline, while heart rates remained at a mean of 66 ± 18 bpm over 20 min. The highest MAP values were achieved 17.4 ± 9.0 min after the drop and administration of 1.27 ± 1.0 mg cafedrine/kg, independent of gender. For ease of transferring applied cafedrine/theodrenaline dosages into the reader’s clinical practice, Table p < 0.001).The MAP dropped from a mean baseline value of 82 ± 14 to 63 ± 10 mmHg   Table , Fig. 3,8) Table .Fig. 3Kap = 0.007) . This difference was independent of gender.The time to highest MAP was longer for the 67 patients with heart failure than for patients without heart problems (07) Fig. . In addin = 111, 9.0 ± 7.0 vs. 7.3 ± 4.3 min, p = 0.008)  Fig. . FactorsInduction of anaesthesia induces sympathicolysis, regardless of the type of anaesthesia, whether general or neuraxial, and poses a risk of hypotension, in particular when the administration of intravenous fluids is restricted , 17, 27.2-stimulation and the dependency on vascular filling; as a consequence, undesirable increases in heart rate are observed [Due to the limited half-life, bolus administration of catecholamines has only a short-term circulatory effect . Moreoveobserved .Here we show that the administration of cafedrine/theodrenaline significantly increased MAP in both genders without affecting the heart rate in a clinically significant manner. These observations are in line with experimental investigations , 11 and 50 [The time to 10 % MAP increase in this cohort was longer in men and in patients who had previously received beta-blocking agents. The more rapid increase in MAP in women may be attributable to the lower cafedrine/theodrenaline ED50  and/or t50  due to t50  and oest50  might re50 , 8 could50 , 25 Pati50 .This study has a number of limitations. The association between heart failure and the use of beta-blockers and the influence of health status and gender on the type of anaesthesia  administered may have introduced sources of selection bias, especially in patients with heart failure receiving beta-blockers. Larger prospective and controlled studies with a more strictly defined and stratified patient populations are certainly warranted.Cafedrine/theodrenaline results in a rapid haemodynamic effect on blood pressure without affecting heart rate to a clinically significant extent.Male gender, heart failure and the previous administration of beta-blocking agents negatively affect the effectiveness of cafedrine/theodrenaline.Prospective studies to characterise the effect of cafedrine/theodrenaline in defined patient populations are warranted.


Concrete rheological properties need to be properly measured and predicted in order to characterize the workability of fresh concrete, including special concretes such as self-consolidating concrete (SCC). It was shown by a round-robin test held in 2000 ,2 that d In the concrete industry, workability is defined as “the ease and homogeneity for which the concrete or mortar can be placed, consolidated and finished” . IdeallyIn response to this fact, at least five concrete rheometers have been designed to measure both the yield stress and plastic viscosity of concrete. These rheometers were compared in 2000 during a round-robin test . It was The new approach presented here uses the relative plastic viscosity instead of the plastic viscosity. The relative plastic viscosity of a suspension is defined as the ratio of the plastic viscosity of the whole suspension to the plastic viscosity of the embedding fluid matrix or medium. In concrete, the embedding matrix can be defined as the mortar, while in mortar the matrix would be the cement paste. The inclusions or particles in the suspension are the coarse aggregates in concrete or the sand in mortar. Therefore, the relative plastic viscosity of a concrete is the plastic viscosity of the concrete divided by the plastic viscosity of the mortar. It is assumed that the mortar used to determine the plastic viscosity of the matrix has the same composition as the mortar in the concrete. The relative plastic viscosity is a function of the concentration of the particles and their shape. Thus, for a given concrete, a plot could be prepared comparing the relative plastic viscosity and the coarse aggregate concentration. It will be shown that the relative plastic viscosity does not depend on the rotational rheometer used. If all data can really be plotted on the same curve using the relative plastic viscosity, it would allow direct comparisons of the data from all rheometers, which until now has not been possible.In this study, the relative plastic viscosities of several concrete mixes were determined by using computer simulation, two different concrete rheometers, and a parallel plate cement paste/mortar rheometer. By plotting all the data on a graph of the relative plastic viscosity versus the concentration of particles , it can be shown that the main influence on the relative plastic viscosity is the aggregate concentration . The data set used in this paper is small, therefore our observation should be confirmed with further testing. A second round-robin test comparing concrete rheometers is being planned by American Concrete Institute (ACI) committee 236A in 2003. The results will be used to further improve this method.f, that depends on the rheometer geometry and experimental conditions. So the following equation could be used:T/ΔV = Slope of the torque (T) versus rotational speed (V)ΔηT = True or absolute plastic viscosityf  = function depending on the rheometer geometry (G) and experimental conditions (C).Most rotational rheometers are based on the principle that the material is stirred at a controlled speed and the resulting torque is measured. In the case of a Newtonian fluid, the viscosity is defined as the ratio between the stress and the shear rate . Concretf is not fully known for most of the concrete rheometers due to their complex geometry and the lack of a standard material that could be used for calibration. Oils are often used as standard materials but they are too expensive and have a viscosity too low to be used in a large concrete rheometer. These oils are designed for small rheometers such as the one used for cement paste. The parameters, G and C, of the function, f, take into account not only the type of rheometer  but also the type of coupling between the fluid and the rheometer, the type of fluid tested, environmental conditions and the limits of the instrument . It could be imagined that the parameters, G and C, vary with the type of fluid used in the same rheometer. However, as will be shown below by the experimental results, the factor f  depends more on the type of rheometer than on the type of fluid tested. This observation should be further confirmed by more testing. Due to the lack of knowledge of the function f, the true or absolute plastic viscosity cannot be known with low uncertainty. This could explain why it was not possible to compare the absolute values of the plastic viscosity obtained with the concrete rheometer during the round robin test [The function bin test ,2.f or to eliminate it. Suppose that two measurements are performed with the same rheometer on two different mixtures ηT1/ηT2, is defined as the relative plastic viscosity.A method should be developed to either determine this function From A coaxial rheometer BML.A vane rheometer (IBB ) in whicA computer simulation (see description below) in which three types of spherical aggregate gradation were used. The distributions used are shown in A parallel plate rheometer designed for cement paste and mortar in which various concentrations of monosized glass beads were added to cement paste.To determine if the relative plastic viscosity could be used to compare the data from different rheometers and/or computer simulations, we examined the results of four sets of data:The detailed description of the BML and IBB rheometers can be found in various publications ,9. TheseSeveral simulations of hard sphere systems ,11, imbew/c ratio of 0.45, with no admixtures. The glass beads were nominally 1 mm in diameter and the volume concentration was varied from 0 % to 50 %. This type of aggregate was selected to provide validation data for the DPD model as they were mono-dispersed and spherical and thus straight forward to simulate.The paste measurements were conducted using a parallel plate rheometer used for cement paste . This rhThe relative plastic viscosity was calculated for all mixtures by dividing the plastic viscosity of the mixture containing the coarse aggregates or particles with the plastic viscosity of the matrix (mortar or cement paste). Care was taken to ensure that the matrix that was measured alone was identical to the matrix in the mixture.All the relative plastic viscosities measured are plotted in Obviously, it is expected that varying the shapes of aggregate would generate a family of curves (relative viscosity versus aggregate concentration) similar to the one shown in CF) that is the ratio between the mortar plastic viscosity measured with the calibrated rheometer (ηm1) and with the concrete rheometer (ηm2). The correction factor will be:First, if a mortar is measured using a rheometer that could be calibrated, using a standard oil for example, all plastic viscosity values could be corrected using a factor Second, if a calibrated rheometer is not available and the goal is to simply compare the as-measured viscosities from two or more rheometers, one of the rheometers could be used as a “reference”, and one could then proceed with the same calculation as presented above.Finally, it is obvious that it might not always be necessary to calculate the absolute value of the concrete plastic viscosity. Different concrete mixtures could simply be compared using the relative plastic viscosity alone. This will allow the comparison of measurements obtained from various rheometers even if the plastic viscosity results were not in the same units.The procedure based on In conclusion, it has been shown for the rheometers used that the relative plastic viscosity does not seem to depend on the rheometer but only on the amount of coarse aggregate (or particle) that were added to the matrix (mortar or cement paste). Therefore, the relative plastic viscosity can be used to compare data from various instruments even when a calibration with a standard material is not available and the results from the rheometers are given in different units. Some tests of mortar will be included in phase II of the ACI sponsored round-robin tests of four commercially available concrete rheometers to be held in 2003, in order to further test the validity of this method. Another implication of this conclusion is that modeling of the flow of concrete can be reduced to the flow of particles in a matrix. The only variable to be modified is the shape and concentration of the particles or aggregates. If the relative plastic viscosity is given and the mortar plastic viscosity is measured, the plastic viscosity of the concrete can be calculated. This procedure is being developed at NIST by creating a database searchable by the shape and the gradation of the coarse aggregates. The data will be presented as a curve of relative plastic viscosity versus the concentration of the coarse aggregates . This me


Impaired eye movements have a long history in schizophrenia research and meet the criteria of a reliable biomarker. However, the effects of cognitive load and task difficulty on saccadic latencies (SL) are less understood. Recent studies showed that SL are strongly task dependent: SL are decreased in tasks with higher cognitive demand, and increased in tasks with lower cognitive demand. The present study investigates SL modulation in patients with schizophrenia and their first-degree relatives. A group of 13 patients suffering from ICD-10 schizophrenia, 10 first-degree relatives, and 24 control subjects performed two different types of visual tasks: a color task and a Landolt ring orientation task. We used video-based oculography to measure SL. We found that patients exhibited a similar unspecific SL pattern in the two different tasks, whereas controls and relatives exhibited 20–26% shorter average latencies in the orientation task (higher cognitive demand) compared to the color task (lower cognitive demand). Also, classification performance using support vector machines suggests that relatives should be assigned to the healthy controls and not to the patient group. Therefore, visual processing of different content does not modulate SL in patients with schizophrenia, but modulates SL in the relatives and healthy controls. The results reflect a specific oculomotor attentional dysfunction in patients with schizophrenia that is a potential state marker, possibly caused by impaired top-down disinhibition of the superior colliculus by frontal/prefrontal areas such as the frontal eye fields. Saccadic latency (SL) is the delay time after stimulus onset until a saccade is performed, due to the time required to program the motor command for a consecutive saccade with a specific direction and amplitude; this duration is usually around 200 ms. Studies of SL have shown that a task with higher cognitive demand  can reduce SL , but patient ages were not significantly different from controls . The groups had no significant gender differences . The groups were significantly different in amount of education; most prominently, the control group had four more years of education compared to patients . Visual acuity was not significantly different between groups .The groups significantly differed in age , one patient received both atypical and typical antipsychotics, and one patient was not taking any antipsychotic medication [chlorpromazine equivalent dosage (CED) in Table 2.2Eye movements of the dominant eye were recorded with a video-based infrared eye tracker  at a sampling rate of 200 Hz and a spatial resolution of 0.5°–1°. Stimuli were presented using our own software, which was based on PsychoPy Peirce, . Visual 2.3First, visual acuity (Snellen chart), color vision (Ishihara test), visual dominance (Porta test), and handedness (Edinburgh inventory) were determined. All subjects were screened for eye diseases, diseases of the cervical vertebrae, neck and shoulder pain, drug abuse, and medication consumption. We used a peripheral recognition task . The effect size is large (Cohen’s d = 2), and a resulting power calculation based on this effect size, a significance level of 0.05, and a sample size of 10 subjects would result in a power of 0.99. Therefore, sample sizes of 10 and more subjects have sufficient statistical power to find significant effects .Based on our previous study .The data were preprocessed using our own custom MATLAB toolbox , in order to have equal group sizes. In (2), we used n = 13 patients and a random sampling of n = 13 of healthy controls and relatives . In (3), we randomly sampled n = 13 patients and relatives  and we randomly sampled n = 13 from the healthy controls . Thus, our subsamples for the classification always contained a total 26 subjects (n = 13 per group) for all the three classification problems. We implemented leave-one-out-cross-validation, i.e., out training set always contained 25 subjects, and 1 subject was tested against the model prediction. This was implemented in a Monte Carlo run with 5 × 106 iterations for each of the three classification problems, and mean specificity and sensitivity were calculated.In order to classify the subjects based on the SL, we used Support Vector Machines . We trained to classify the subjects by using two values, the SL in the Landolt, and the SL in the color task. We compared the following classification accuracies: (1) classification of patients vs. controls, (2) classification of patients vs. relatives and controls, and (3) classification of patients and relatives vs. controls. In (1), we used 3M = 248 ms, SD = 56 ms; Landolt: M = 232 ms, SD = 51 ms; two-sided paired t-test: t12 = 2.19, p = 0.049; d = 0.3). Controls had a large significant reduction of 26.2% in the Landolt task compared to the color task . First-degree relatives exhibited a large significant reduction of 19.5% in the Landolt task compared to the color task .Saccadic latencies (SL) in the different tasks (color and Landolt) were analyzed Figures A,B. Patit-test, t28,3 = 2.35, p = 0.013; d = 0.6) and relatives . There was no significant difference between controls and relatives . In patients, there was no correlation of SLD with their CED .We calculated the saccadic latency differences (SLD) between the tasks by subtracting the SL in the Landolt task from the SL in the color task Figure . PositivF1,24 = 0.03, p = 0.86). Controls had significantly smaller variance in the Landolt task . Relatives showed no significant change in the variance during the Landolt task .We compared changes in SL variance in the Landolt task compared to the color task Figure B. PatienV = 70.5, p = 0.086; relatives: V = 39.5, p = 0.050). The Landolt task produced longer response times across groups compared to the color task (mean increase 22%). The patients exhibited longer response times across tasks compared to the other groups (mean increase 24%). Also, the relative increase of reaction time in the Landolt task was larger in patients (28%) compared to the other two groups (12%). These results were confirmed by ANOVA .Correct responses and response times were analyzed Figures A,B. On aIn the context that SL patterns may be useful for classification of the subjects, we used SVM with leave-one-out-cross-validation in a Monte Carlo experiment. We found that the SVM could classify the subjects  with a mean sensitivity of 61%, and a specificity of 62% Figure . The cont-test: t9 = 0.49, p = 0.64). In the Landolt task, the five patients taking the medication had a mean SL of 226 ms (SD = 68 ms), and the eight patients not taking this medication had a mean SL of 236 ms (SD = 42 ms), which was not a significant difference .We tested five patients, two who were taking benzodiazepines, and three who were taking valproic acid for a potential medication effect of increased SL. During the color task, the five patients taking the medication mentioned above had a mean SL of 239 ms (SD = 56 ms), and the eight patients not taking this medication had a mean SL of 255 ms (SD = 59 ms), which was not a significant difference  shorter SL and smaller within-group variability in controls and first-degree relatives compared to the patients, (2) longer response times consistently across groups, and (3) a relatively larger increase in response times of patients compared to the other two groups (interaction group × task). Further, classification based on SL works better merging the relatives and the controls and then classify into patients vs. healthy controls/relatives compared to merging the patients and relatives and then classy between patients and relatives vs. healthy controls.4It has been shown that SL are strongly task dependent: higher cognitive demand causes shorter SL, while lower cognitive demand causes longer SL . Even though a recent review concluded that findings concerning anxiety and mood disorders have failed to support oculomotor impairments in these diseases (Toh et al., SL modulation during tasks with lower and higher cognitive demand is an important attentional feature to adapt to a changing environment by allowing the brain to save resources during low demand and to optimize performance during higher demand. The SL deficiencies found in patients with schizophrenia, but not in first-degree relatives, may be a state marker associated to their clinical condition. SL impairments may have their greatest impact during times of concentration. In this way, oculomotor deficits, may influence higher-order cognition (Butler and Javitt, SS and AA designed the experiments, MJ performed the experiments, and SS analyzed the data. SS wrote the first draft of the paper. All authors contributed to the final version and have approved the final manuscript.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.


IgG4-related disease is an emerging clinical entity which frequently involves tissue within the orbit. In order to appreciate the implications of IgG4 immunostaining, we analyzed gene expression and the prevalence of IgG4- immunostaining among subjects with orbital inflammatory diseases.We organized an international consortium to collect orbital biopsies from 108 subjects including 22 with no known orbital disease, 42 with nonspecific orbital inflammatory disease (NSOI), 26 with thyroid eye disease (TED), 12 with sarcoidosis, and 6 with granulomatosis with polyangiitis (GPA). Lacrimal gland and orbital adipose tissue biopsies were immunostained for IgG4 or IgG secreting plasma cells. RNA transcripts were quantified by Affymetrix arrays.None of the healthy controls or subjects with TED had substantial IgG4 staining. Among the 63 others, the prevalence of significant IgG4-immunostaining ranged from 11 to 39% depending on the definition for significant. IgG4 staining was detectable in the majority of tissues from subjects with GPA and less commonly in tissue from subjects with sarcoidosis or NSOI. The detection of IgG4+ cells correlated with inflammation in the lacrimal gland based on histology. IgG4 staining tissue expressed an increase in transcripts associated with inflammation, especially B cell-related genes. Functional annotation analysis confirmed this.IgG4+ plasma cells are common in orbital tissue from patients with sarcoidosis, GPA, or NSOI. Even using the low threshold of 10 IgG4+ cells/high powered field, IgG4 staining correlates with increased inflammation in the lacrimal gland based on histology and gene expression. IgG4-related disease (IgG4-RD) was first described in patients with autoimmune pancreatitis who had elevated concentrations of IgG4 in serum Ophthalmic disease is a common manifestation of IgG4-RD Orbital inflammatory disease can affect orbital muscle, lacrimal gland, or adipose tissue. The most common systemic disease associated with orbital inflammation is hyperthyroidism attributable to Graves disease, also known as thyroid eye disease or TED. Sarcoidosis or granulomatosis with polyangiitis  can also cause inflammation within the orbit. Many patients with orbital inflammatory disease are classified as having nonspecific orbital inflammation . Little is known as to how each of these entities might be related to IgG4-RD.The etiology of IgG4-RD remains unclear. Although the infiltration of IgG4+PC is a defining characteristic of the disease, there is no evidence that IgG4 is directly involved in the pathogenesis. In fact, some have hypothesized that IgG4, which does not fix complement, is expressed to dampen inflammation Three studies that sought to determine the prevalence of IgG4 -immunostaining among patients with orbital inflammation found very discrepant results with prevalence ranging from 4 to 52% This study was approved by the Institutional Review Boards (IRB) at Oregon Health & Science University, Columbia University, University of California San Diego, Wake Forest University, Medical College of Wisconsin, and Mount Carmel (Ohio) and by the University of British Columbia Clinical Research Ethics Board, the Royal Adelaide Hospital Research Ethics Committee, and the King Khaled Eye Specialist Hospital Human Ethics Committee/Institutional Review Board. This study was in compliance with the Helsinki Declaration. Formalin-fixed paraffin-embedded (FFPE) samples were obtained from 9 contributing centers. Data were analyzed anonymously and written informed consent was obtained when required by the local IRB or ethics committee.The diagnoses of nonspecific orbital inflammation, sarcoidosis, granulomatosis with polyangiitis, thyroid eye disease, and normal were based on the clinical and histopathological information obtained and submitted by orbital disease specialists and ocular pathologists from their respective institutions. Biopsies from a total of 109 subjects were used  . Among tAll samples were reviewed and scored without reference to the indications for biopsy or other clinical information. Two slides from each specimen were stained with hematoxylin and eosin for histopathological evaluation. These features were independently re-evaluated by two ocular pathologists (D.J.W. and H.E.G.). The features included degree of fibrosis, degree of inflammation, presence of obliterative phlebitis, and presence of storiform fibrosis. The degrees of fibrosis and degree of inflammation were quantified as absent (0), mild (1), moderate (2), or severe (3). For most samples, the presence or absence of lacrimal gland tissue was confirmed by the presence or absence of lacrimal gland mRNA.2) and the IgG+ cells in the corresponding fields of the paired slides. These data are expressed as the mean number of positive cells per hpf. For the subjects for whom more than one specimen of the same tissue type was stained, the overall mean was used for the statistical analysis.Two serial sections from each specimen were used for immunohistochemical evaluation. One slide was stained for IgG4+ plasma cells  and the other for IgG+ plasma cells . Immunostaining used Ventana automated instruments with Ultraview detection . Two surgical pathologists (M.L.T. and D.C.H.) independently counted the numbers of IgG4+ cells in three high power fields  according to the manufacturer's protocol. RNA concentrations were determined by UV absorbance. RNA was prepared for microarray analysis using the SensationPlus FFPE Amplification and 3′ IVT Labeling kit . 50 ng of RNA was used for the majority of samples with a minimum input of 20 ng RNA for samples in which RNA yields were limited. Biotin-labeled cDNA targets were hybridized with a GeneChip Human Genome U133 Plus 2.0 array  according to standard Affymetrix protocol. This array contains over 54,000 probe sets for 47,000 transcripts and variants. Following hybridization, arrays were stained and scanned using the GeneChip Scanner 3000 7G system (Affymetrix). Image processing and initial quality control analysis were performed using Affymetrix GeneChip Command Console (AGCC) v. 3.1.1 and Affymetrix Expression Console v. 1.1 software, respectively.http://www.bioconductor.org) in the R project for statistical computing (http://www.r-project.org).Simple statistical tests were done with chi-square tests, Mann-Whitney U-test, or Student's t-test as appropriate Altogether, 119 orbital adipose tissue and lacrimal gland tissue samples from 108 subjects were evaluated . Our conIgG4+PCs were rarely seen in tissues from control or TED subjects . The freIgG4+PC infiltration was observed in both lacrimal gland and orbital adipose tissue samples . In totaAmong the non-TED orbital inflammation cases, there was no significant correlation between IgG4+ status and gender or the mean age at biopsy based on IgG4+ immunostaining defined as at least 10 positive cells per hpf . ExaminiNo storiform fibrosis or obliterative phlebitis was noted in any of the tissue samples. A comparison of the fibrosis and inflammation scores between IgG4+ and IgG4- cases showed a significant increase in fibrosis and inflammation in the lacrimal gland tissue among IgG4+ NSOI patients  . AlthougWe employed the prescription of corticosteroid or other immunosuppressive as a surrogate index for severity, reasoning that more severe disease was more likely to prompt potentially toxic therapy. Using chi square analysis, patients with NSOI, GPA, or sarcoidosis who had at least 10 IgG4+cells/hpf were not more likely to be treated with corticosteroid (p = 0.3) and not more likely to receive additional immunosuppression after the biopsy (p = 0.7). A higher percentage of the IgG4+ patients started corticosteroid treatment prior to their biopsy, whereas the opposite was true for the IgG4- group . We furtBecause no subject with TED had substantial staining for IgG4, we reviewed the therapy for these subjects in case the absence of staining was a result of treatment. Of the 26 subjects with TED in this study, two were receiving prednisone at the time of biopsy, 5 had previously been treated with prednisone between 3.5 months and 3.5 years before the biopsy, 13 had not received any medications, and in 6 subjects, the prior treatment was unknown. Thus treatment is an unlikely explanation for the absence of IgG4 staining among subjects with TED.IgG4 staining has been proposed as a marker of multisystem disease We compared RNA transcripts in tissues that stained for IgG4 plasma cells with tissues from subjects with NSOI, GPA, or sarcoidosis and little or no IgG4 staining. We used a positive staining threshold of at least 10 IgG4+ cells/hpf and a threshold for gene expression change of at least 1.5-fold difference with a false discovery rate adjusted p-value of <0.05. In lacrimal gland samples, we detected 98 probes sets with increased signals and 4 that had decreased signals. For tissue from the orbital adipose tissue, 100 probe sets had increased signals and 22 were decreased. The complete lists of probe sets indicating significant expression differences are in Tables S1 and S2 in Examples of probe sets with elevated signals in IgG4+ orbital adipose tissue  include Examples of lacrimal gland transcripts with differential expression in IgG4+ tissues are listed in To our knowledge, our study is the first study to compare the prevalence of IgG4 staining among various inflammatory conditions within the orbit. A recent report on IgG4 immunostaining in skin disease similarly concluded that IgG4 staining is detectable in a variety of clinical entities Considering only the non-TED orbital inflammation cases, we found the prevalence of IgG4+ immunostaining to be 36% based on a cutoff of ≥10 IgG4+PC/hpf. However, this cutoff is controversial in the literature. In 2008, Sato et al. published one of the largest IgG4-immunostaining studies Our study is the first to analyze RNA transcript expression in tissue that contains IgG4 plasma cells. We based this analysis on the least stringent definition of IgG4 staining for a practical reason; other definitions of IgG4 positivity would have provided too few samples for accurate statistical analysis. We believe that this analysis lends support for defining significant IgG4 staining within the orbit using the criterion of at least ten cells staining per hpf.Unlike the histopathological presentation in IgG4-RD in other organ systems, we did not find storiform fibrosis or obliterative phlebitis in any of our samples, regardless of the tissue type. A consensus statement on the pathology IgG4-RD released in 2012 noted that the lacrimal gland is an exception to the general rule that storiform fibrosis should be a main criterion for diagnosis Only two of the biopsies with IgG4 staining as detected in our study were previously identified as IgG4 + by the referring center. As some consider IgG4 to be a marker of more severe disease, knowledge of IgG4 staining could have prompted more aggressive therapy which was not an issue in our study in which very few patients were known to be IgG4+. It is also possible that treatment with prednisone would diminish the number of IgG4+ cells in a biopsy. While we cannot exclude this possibility, there was no statistically significant difference between the rate of IgG4 positivity among those who took prednisone before the biopsy and those who did not take prednisone.and IgG4 staining could identify some patients with a syndrome distinct from GPA, sarcoidosis, or NSOI. Since NSOI already subsumes a presumed variety of entities, the latter hypothesis would fail to distinguish NSOI patients who are IgG4+ from the entity of IgG4-RD. We recently reported that 70% of patients with orbital inflammatory disease respond favorably to therapy with rituximab Most experts consider IgG4-RD a distinct clinical entity that includes the histological implication of increased fibrosis and the therapeutic implication of a likely response to rituximab therapy In summary, we conducted a multi-centered, international study on the prevalence of IgG4-immunostaining in orbital inflammation allowing us to compare the prevalence of IgG4+PC infiltration in different entities affecting the orbit. Unfortunately, due to the limitations of our database and incomplete clinical information, we were unable to examine the relationship between our cases and IgG4 serum concentrations. In addition, by the nature of our multi-centered approach, we were reliant on diagnoses from different institutions. In a minority of instances we lacked complete information on the therapy or diagnostic evaluation. Although the clinical implications of IgG4 staining in the orbit require further investigation, our data support the rationale to target B cells in therapy. Regardless of whether IgG4-immunostaining is considered a distinct clinical entity or a subset of other entities, subjects with an IgG4+ tissue infiltrate display an identifiable gene expression profile consistent with a heightened inflammatory response. Our study should help physicians better interpret the implications of IgG4 staining in orbital tissue.File S1Figure S1, Representative images illustrating the variability of IgG4 and IgG staining in orbit adipose tissue from subjects with NSOI. Table S1, Comparison of gene expression in IgG4+ and IgG4- orbit adipose tissues. Table S2, Comparison of gene expression in IgG4+ and IgG4- lacrimal gland tissues.(DOCX)Click here for additional data file.


O-methyltransferase (COMT) enzyme. Current evidence suggests that the val/met single nucleotide polymorphism in the COMT gene can predict the efficiency of executive cognition in humans. Individuals carrying the val allele perform more poorly because less synaptic dopamine is available.Prefrontal dopamine is catabolized by the catechol-We investigated the influence of the COMT polymorphism on motor performance in a task that requires different executive functions. We administered a manual aiming motor task that was performed under four different conditions of execution by 111 healthy participants. Participants were grouped according to genotype , and the motor performance among groups was compared. Overall, the results indicate that met/met carriers presented lower levels of peak velocity during the movement trajectory than the val carriers, but met/met carriers displayed higher accuracy than the val carriers.This study found a significant association between the COMT polymorphism and manual aiming control. Few studies have investigated the genetics of motor control, and these findings indicate that individual differences in motor control require further investigation using genetic studies. O-methyltransferase (COMT) enzyme is responsible for more than 60% of the metabolic degradation of dopamine in the frontal cortex It is well known that dopaminergic functions play a critical role in human behavior and cognition. Several studies provide evidence indicating that dopaminergic neurotransmission in fronto-striatal circuits is related to complex cognitive processes, such as working memory l58met – in which the enzyme activity is reduced by one third to one half in individuals homozygous for the val allele compared with the enzyme activity in individuals homozygous for the met allele The COMT gene contains a functional polymorphism – vaBecause of its crucial role in the prefrontal circuits, it has been hypothesized that these COMT polymorphism variants directly affect specific cognitive functions in humans. For example, studies suggest that individuals with the met allele present enhanced mental flexibility and capacity to shift Only few studies have focused their investigations on the relationship between COMT and motor control. Increasing evidence suggests that specific frontal lobe areas play a role in motor behavior When Bilder et al. A good example of a motor task that requires executive functions is the goal-directed manual aiming movement 158met polymorphism and motor performance in an aiming task that has different sensory-motor requirements. Given that one of the key characteristics identified in the homozygous met allele carrier is enhanced performance during executive functions, we hypothesized that the met variant will be associated with better motor responses than the val variant in execution conditions that require inhibitory control, resistance to distraction and adaptation to perturbation in spatial accuracy. To our knowledge, no study investigating how the COMT polymorphism is specifically related to manual aiming control has been reported.We designed this study to investigate the association between the COMT valWe studied 111 self-assigned Caucasian-Brazilians who ranged in age from 18 to 40 years old . All of the participants were undergraduate students from local universities who were free of an Axis I diagnosis, as assessed by a psychiatrist using a structured interview (MINI-PLUS) and following the DSM IV criteria All of the participants were right-handed (handedness >.8 measured by the Edinburgh Handedness Inventory) 158met- rs4680) using a TaqMan Genotype assay . PCR reaction was performed following the fabricant marker instructions and contained: 0.1 µl TaqMan Genotyping Master Mix, 3.4 µl deionizated water, 25 µg of DNA and 3.4 µl TaqMan Genotype Assay . Reaction mix was prepared in a master mix pool and then distributed to each well to be mixture to the sample DNA. The PCR parameters included an initial denaturation at 95°C for 10 min followed by 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. Genotype was determined based on the allelic discrimination mode . Researchers involved in genotyping were blind to neuropsychological results and 10% of the genotypes were performed as a quality control.Blood samples were collected only from health individuals who gave an informed consent to participate from the study. DNA samples were obtained from blood cells and extracted using the high salt method Apparatus were identical to that used by The motor task and procedures used by Lage et al. Each of the other three conditions only appeared in 10% of the trials. Under the distractor condition, a filled-in yellow circle appeared instead of the green circle target (stimulus of the control condition). The goal under the distractor condition was identical to the control condition. The unique difference in this condition was the color of the stimulus. Under the inhibition of response condition, a filled-in red circle appeared, indicating “stop”, instead of the green circle target (stimulus of the control condition). Under this condition, the participant was instructed to not move the pen. The third condition was the higher index of difficulty condition. Under the higher index of difficulty condition, a filled-in green circle appeared that was similar to the target used in the control condition. The size and position of the green target, however, were different than the settings used under the control condition. The goal of executing the movement to the target as quickly and accurately as possible was still the same.Under the control, distractor and inhibition of response conditions, the target (1 cm diameter) was presented at the same distance (19 cm center-to-center) and angle (45° upper right) from the home position, which resulted in an index of difficulty of 5.2 bits Prior to motor testing, the participants received standardized instructions concerning the general nature of the study. Participants held the inkless pen in a normal pen grip with their right hand. To get acquainted with the task and to find a comfortable posture, participants carried out six trials of the control condition . The body midline was aligned with the home position.Immediately following familiarization, the motor task was performed. Participants performed 100 trials of the manual aiming task. The order in which conditions appeared was randomized in each block of 10 trials. Hence, in each block of 10 trials, there were 7 trials of the control condition and 1 trial of each one of the other conditions. This procedure was used to avoid that a specific condition would be concentrated more in a specific moment of the task.After the presentation of the imperative stimulus, participants had 2 seconds to move from the home position to the target. After this interval, the target disappeared and the recording of the trial was finished. A red trace was displayed on the screen concomitantly with the movement to indicate the trajectory from the home position to the target. The entire test took approximately 16 minutes per participant to complete.Pen movements were low-pass filtered at 12 Hz using Fast Fourier Transform (FFT) and differentiated to yield estimates of the velocity and acceleration curves. A stroke was segmented into primary and secondary submovements by the first negative-to-positive zero crossing after the absolute peak velocity in the acceleration profile. The primary submovement refers to the initial part of the movement, the preprogrammed phase, and the secondary submovement refers to the online controlled phase.The performance measures examined included the following: (1) reaction time, (2) movement time, (3) score of incorrect hits to the target (0 if hit and 1 if missed) and (4) score of response inhibition errors (0 if “stop” and 1 if “go”). The kinematic measures analyzed included the following: (1) peak velocity, (2) relative time to peak velocity and (3) number of discontinuities in acceleration in the secondary submovement . Two-way analyses of variance (ANOVAs) were used to compare the genotype group performances (3 groups×3 conditions). The post-hoc analyses were performed with Duncan's new multiple range test. Chi-squared tests were used to analyze the following nominal data: (a) scores of incorrect hits to the target and (b) scores of response inhibition errors. The Bonferroni correction was used for multiple comparisons of the nominal data 2 = 1.68; df = 1; p = 0.19). The number of male and female participants belonging to each genotype group is shown in Sixty-four females (mean age 23.6±4.4) and 47 males (mean age 24.5±3.5) participated in this study. The genotype distribution was at Hardy-Weinberg equilibrium  = 3.12, p<.05; Conditions, F = 27.23, p<.001, and interaction between Groups Performance and Conditions, F = 2.53, p<.05 to the measure of peak velocity. Post hoc comparisons revealed that the met/met allele group presented lower level of peak velocity under all of the conditions than the met/val allele group (p<.05). A marginal difference was also found between the met/met allele and val/val allele groups (p = .06), which indicates a tendency of lower level of peak velocity to the met/met allele group. The post hoc analysis on Conditions indicated higher level of peak velocity to the higher index of difficulty condition compared to the both control and distractor conditions (p<.001).The analyses showed main effects of Groups Performance, p<.001); (b) the met/val and val/val participants produced lower level of peak velocity under the control condition than in the higher index of difficulty condition (p<.001), but there was no significant difference between control and distractor conditions (p>.05); (c) the met/met group presented lower level of peak velocity than the other groups under the control condition (p<.001); (d) the met/met group presented lower level of peak velocity than the met/val group under the distractor condition(p<.001), but there was no significant difference between met/met and val/val groups (p>.05); (e) the met/met group presented lower level of peak velocity than the other groups under the higher index of difficulty condition (p<.01).The main results found on the interaction between Groups Performance and Conditions were the following: (a) the met/met participants produced lower level of peak velocity under the control condition than in both the distractor and higher index of difficulty ; (b) there was a significant difference among the groups under the distractor condition ; (c) there was no significant difference among the groups under the higher index of difficulty condition . For the multiple comparisons after the Bonferroni correction, a difference with a p value equal to or less than .01 was considered significant. Under the control condition, the results showed that the met/met allele group presented lower level of error of incorrect hits to the target than the met/val allele and val/val allele groups . Furthermore, the val/val allele group exhibited lower level of error than the met/val allele group (p<.01). Under the distractor condition, the results showed that the met/met allele group presented lower level of error of incorrect hits to the target than the met/val allele and val/val allele groups , and there was no significant difference between met/val allele and val/val allele groups (p = .03). The relative frequencies of the score of incorrect hits to the target are shown in In the analyses of the score of incorrect hits to the target, the Chi-squared tests indicated the following results: (a) there was a significant difference among the groups under the control condition . The frequency of errors resulting from response inhibition was 22.4% for the met/met allele group, 23.1% for the met/val allele group, and 22.8% for the val/val allele group.In the analyses of the score of response inhibition errors, the Chi-squared test results indicate that there is no significant difference among the groups in the inhibition of response condition  during the trajectories. The main objective of this more controlled processing was to guarantee accuracy to the movement endpoint. Differences in the types of processing information appear to interfere with spatial accuracy. Explicit information processing, which is characterized by conscious, controlled and reflective processes, is productive in some contexts of execution. For example, low-impulsive subjects, in which explicit information processing predominates, exhibit a greater spatial accuracy than their more impulsive counterparts under the prepotent condition Interestingly, the better spatial accuracy exhibited by the met/met allele carriers was not observed under the higher index of difficulty condition. The temporal and spatial demands on the motor system are greater under the higher index of difficulty condition than under the control and distractor conditions. It is possible that the executive advantage detected in the met allele homozygote overlaps with other aspects of motor execution. This result shows that the association between the COMT polymorphism and motor control depends on the sensory-motor aspects of the task. The higher index of difficulty condition presents a different size and position of the target, factors that require a more flexible motor control from the performer. In both healthy and psychiatric samples, individuals homozygous for the met allele show better performance in tasks requiring stable performance than individuals carrying a val allele. The same is not true in tasks requiring flexible performance The met allele homozygote produced lower level of peak velocity under the control condition than under the distractor and higher index of difficulty conditions, but the val carriers under the control condition, presented lower level of peak velocity only when compared with peak velocity under the higher index of difficulty condition. Again, these findings seem to be related to inverted-U shaped relationship between dopamine and task demand. However, these differences in peak velocity under specific conditions do not reflect worse performance, but reveal different associations between the response preparation and the dopamine availability. The met/met allele group was more sensible to the changes of the conditions than the other groups. It reflected in alterations of the level of force produced during the movements when the conditions changed. This is the first study to investigate these types of associations. Further studies need to be conducted to analyze specific tasks and to investigate the proposal that the association between the COMT polymorphism and motor control depends on the sensory-motor aspects of the task. Furthermore, the relationship between the COMT polymorphism and the reaction and movement times should also be investigated. Is the COMT polymorphism associated with these measures during a motor task with no endpoint or a lower level of index of difficulty?In conclusion, these findings indicate that there is a significant association between the COMT polymorphism and the manual aiming control in healthy subjects. This association was found to be specifically related to a component of movement preparation, the peak velocity. We did not find any association between the COMT polymorphism and online corrections during the monitoring phase of the movement. Furthermore, individuals homozygous for the met allele appear to be more accurate in the endpoint movement compared with the individuals carrying a val allele. It appears that individuals homozygous for the met allele were more efficient with an important aspect involved in a manual aiming task, the accuracy. Few studies have been reported that investigate functional polymorphisms and motor control in both clinical and non-clinical populations. For the first time, the influence of the COMT polymorphism on manual aiming control was observed and indicates that individual differences in motor control need to be investigated using genetic studies.