Developmental processes in cells require a series of complex steps. Often only a single master regulator activates genes in these different steps. This poses several challenges: some targets need to be ordered temporally, while co-functional targets may need to be synchronized in both time and expression level. Here we study in single cells the dynamic activation patterns of early meiosis genes in budding yeast, targets of the meiosis master regulator Ime1. We quantify the individual roles of the promoter and protein levels in expression pattern control, as well as the roles of individual promoter elements. We find a consistent expression pattern difference between a non-cofunctional pair of genes, and a highly synchronized activation of a co-functional pair. We show that dynamic control leading to these patterns is distributed between promoter, gene and external regions. Through specific reciprocal changes to the promoters of pairs of genes, we show that different genes can use different promoter elements to reach near identical activation patterns. Many processes which involve a major change in the state of the cell  are governed by a master regulator in the form of a single transcription factor that activates dozens or even hundreds of the genes needed to carry out the process –4. Such et. al. [et. al. [et. al. [et. al. [There is yet limited knowledge on what mechanisms and genomic elements contribute to differential timing and stoichiometry of jointly regulated genes. Previous works have shown the existence of regulated target timing in temporal processes. Differential timing has been shown in bacteria in multiple regulator cascades ,6 and wiet. al.  showed t[et. al. , studied[et. al.  found th[et. al.  found thHere, we use a single cell comparative approach to study the questions of relative target timing and stoichiometry. We use early meiosis in yeast as a model for a system with a master regulator. Ime1, the master regulator of early meiosis, is controlled by multiple starvation signals –15. ThroWe find that the co-functional pair displays a tight synchrony with a small offset at the single cell level, while the non co-functional pair is differentially timed and un-synchronized. We show that timing and stoichiometry control are regulated through different parts of the gene, including ORF, promoter and regions outside the promoter, and that different genes can attain very similar expression profiles through different regulatory elements.All strains are from the SK1 background . TransfoGene swap and promoter-gene swap strains were created as follows: for gene swap, the ORF-XFP-resistance-marker segment of Rec8 (Dmc1) was PCR-amplified from a protein-fusion strain using primers with 40 bp tails homologous to the regions upstream of Dmc1 (Rec8) gene start and downstream of its stop codon. This linear segment was then transformed to a wild type strain, replacing the wild type Dmc1 ORF. For promoter-gene swap, a similar procedure was carried, but amplifying the region starting from the promoter . In that case the 40bp homology in the 5’ primer matched the promoter start of the opposite gene.Specific motif changes were made by cloning of the target ORF from the tagged yeast strain, starting at a position immediately after the region to be changed, and then extending this construct by a forward primer containing the required change at a length of ~40 bp and a complementary portion of ~20 bp, and a reverse primer from the previous reaction.All Dmc1 promoters with motif changes were tagged with Venus, and mated with a native Dmc1 strain tagged with Cerulean. All Rec8 promoters with motif changes were tagged with Cerulean, and mated with a native Rec8 strain tagged with Venus. Mei5 motif change was done similarly.2 and 5mM MnCl2 in sterile double distilled water.Microfluidic chambers were made from Poly-dimethylsiloxane (PDMS) polymer using SYLGARD 184 silicone elastomer kit  and a 1:10 curing agent ratio. Monomer base and curing agent mix was poured onto a photoresist mold and then incubated overnight at 65°c, forming simple channels each with one input and output. The PDMS was then reversibly attached to a cover glass slide by pre-treatment with a hand-held plasma generator. In order for cells to adhere to the bottom of the glass, the channels were then filled with a 2mg/ml Concanavalin A solution supplemented with 5mM CaClCells were picked from single colonies and grown overnight before each experiment in YPD . The cells were then washed twice with SPM medium  at 2500 rpm for 1 minute. Washed cells were then inserted into the channels to form a single cell layer. SPM medium was pumped into each channel at a constant rate of 0.5 ml/hour. Images were taken using a Nikon TiE fluorescent microscope at 100X or 60X magnification. In each experiment 10 positions along the channel containing a total of 300–1000 cells were chosen. These positions were imaged every 15 minutes using the relevant fluorescent emission and detection filters .The captured images were analyzed using custom written code  in Matlahttp://genome.ucsc.edu/) and from GermOnline (http://www.germonline.org/index.html). Expression data during meiosis was obtained from Brar et al [et al [et al [et. al. [et. al. [et. al. [et. al. .Genetic sequence data was obtained from the UCSC genome browser . We followed single cells after induction of meiosis in a diploid strain in which the Dmc1 protein is fused to YFP and Rec8 protein is fused to CFP . Rec8 anWe next quantified the coordination in protein levels of the gene pairs. The protein products of a pair of genes that are involved in the same function may play their role at a certain stoichiometry . In such a case, it may be beneficial to regulate not only the relative timing of those genes but also their relative stoichiometry. We therefore wanted to check whether the co-functional pair of proteins (Dmc1 and Mei5) has a tighter stoichiometry at the protein level than the non co-functional pair (Dmc1 and Rec8). We note that tight stoichiometry does not automatically follow from coordinated timing: theoretically, a pair of proteins can be highly synchronized in their timing (as are Dmc1 and Mei5), yet have high cell-to-cell variability in their relative abundance. Examining the log ratio of the two signals over time, we find that the protein level ratio between Mei5 and Dmc1 stabilizes on a constant value within a few hours from onset, with a tighter distribution over time, while the ratio between Dmc1 and Rec8 changes over time . This diOverall, the two pairs defined by these 3 genes show distinct relations in their protein levels, where one pair is differentially timed and divergent in relative abundance, and the other more synchronized and tightly coordinated in stoichiometry.We next looked for the regulation level at which the activation and de-activation timing are controlled. Since we measure nuclear protein levels, this regulation could occur at the level of transcription, translation, protein localization, mRNA or protein degradation or any combination of the above. To study the role of promoter activity in the protein expression pattern, we tagged the promoters of Rec8, Dmc1 and Mei5 with fluorescent proteins (replacing the endogenous ORF with NLS-CFP or NLS-YFP). The results again showed a consistent difference between Rec8 and Dmc1, with a smaller temporal offset compared to the protein level . The DmcOur results suggest that there is a major contribution to the regulation of timing of our test gene pairs at the transcriptional level. If specific promoter features regulate timing and expression level similarly in many targets of the Ime1 master regulator, we expect to identify a correlation between such features and the expression properties . We therWe then attempted to narrow down the specific promoter elements that dominate the temporal behavior of Rec8, Dmc1 and Mei5 . We decicis-effect of the altered allele to the wild type in individual cells, where the trans environment (including Ime1 protein concentration) is identical. This was important, as the cell-to-cell variability between Ime1 profiles and target promoter activation was high [To test the effect of individual sequence changes in the various promoters, we created strains with one WT allele and one altered allele of that gene. This allowed the accurate comparison of the was high .The perturbation of the MBF binding site located on the Rec8 promoter resulted in a marked reduction of Rec8 promoter activity, compared to the WT Rec8 promoter . This reThe lack of significant contribution of individual promoter element changes to the timing regulation stands in contrast to our observation that the timing is highly reflected in the promoter activity level. This suggests that the timing of these genes is controlled by other promoter elements, by a combined effect of several promoter elements, by elements outside the promoter or by a combination of the above. Moreover, the timing difference between the promoters  is smallWe now compare the 6 alleles in terms of their relative timing and maximal protein expression level during the 7 hours period starting from the onset time. To this end, we integrate the relative timing and abundance information from single experiments into one comparative view. First, we note that behavior of the alleles clusters according to the ORF identity, as the ORF provides the dominant contribution to both timing and maximal protein level. Second, the promoter of Dmc1 leads to delayed activation and increased maximal level in two different contexts (RRD→RDD and DRR→DDR). Therefore its contribution to timing and level is consistent with that of the Dmc1 ORF. Finally, the external regions at the Rec8 locus contribute to higher expression (RDD→DDD and RRR→DRR), but their effect on activation time depends on the promoter or gene context: the external regions of the Dmc1 locus contribute to earlier activation of a Rec8 gene (RRR→DRR), but not of Dmc1 (RDD→DDD).S. cerevisiae cells containing either protein or promoter tagging of pairs of Ime1 target genes. Our results show that these target genes can be more tightly synchronized and settle on a set relative stoichiometry (Dmc1 and Mei5) or have a relative delay with variable lag between one another, leading to a change over time in relative abundance (Dmc1 and Rec8). The Dmc1 and Mei5 pair showed tight synchronization and no temporal offset at the promoter level, only gaining an offset at the protein level. In contrast, the Rec8 and Dmc1 pair showed temporal offset and high variability both at the protein and the promoter levels.We have studied the degree of differential or synchronous timing and stoichiometry in single cells in two examples of gene pairs controlled by the same transcription factor. We used live imaging of single diploid Our bioinformatic analysis of Ume6 targets does not show a correlation of any of the promoter parameters tested with expression timing, and only a few distance parameters showed some correlation to maximal expression level. This suggests that the shape of gene activation patterns may result from a combination of several control elements, or the utilization of different elements in each promoter. Consistently, Dmc1 and Mei5, which have a very tightly synchronized protein level pattern, have two very different promoter sequences and gene properties (the ORF of Dmc1 is three times longer and contains an intron). Removing one of the Ume6 binding sites from pMei5 or adding such a binding site to pDmc1 caused a decrease or increase (respectively) in promoter activity, but had little effect on activation timing. This suggests that Dmc1 and Mei5 have co-evolved to increase their synchrony, each using a different combination of specific promoter and gene elements. The similar reduction in Rec8 promoter activity upon removal of the nucleosome-covered MBF binding site indicates that MBF acts as a co-activator for this gene, and probably only binds to its binding site once the nucleosome occluding it has been removed, increasing Rec8's transcription rate. The effect on timing, however, is small. This outcome is similar to the effect of the second Ume6 binding site for Mei5: in both cases, the additional binding site serves mostly to increase maximal expression levels, with little effect on timing, consistent with previous results [Dissecting the contributions of ORF, promoter and external regions to the timing and maximal level of Rec8 and Dmc1 lead to several insights. First, it is the ORF that contributes mostly to the timing and maximal working level in these two genes. This may be mediated through the stability of the mRNA, protein or both, as increased stability in either level can lead to slower accumulation as well as higher maximal expression level. The contribution of the promoter region to these properties is secondary, but it is consistent with the ORF contribution: both promoter and ORF contribute independently to later activation and higher maximal level in Dmc1. Second, the contribution of the external regions, providing the chromosomal context in which the gene is located, depends on the gene (or promoter) and varies between the two genes.Together, our results show that yeast can attain both remarkable synchronization and differential timing between protein pairs controlled by the same regulator, using very different mechanisms and cis-elements for each gene. The genes tested in this work are activated by a regulator that interacts with the DNA through a co-factor (Ume6). It would be interesting to examine target genes of a different type of regulator, one that binds directly to the DNA, and see whether it produces different dynamical effects on its targets.S1 Fig50 statistic is designed to overcome the effect of this artifact.Normalized CFP and YFP time series from a single Mei5-CFP, Dmc1-YFP cell entering meiosis. Four snapshots are shown from the first 3 hours after switch to meiotic conditions. YFP has a better signal to background ratio than CFP, most apparent in the early part of the series, where the signals are weak. As a result, the early ramp-up part in the CFP channel is below detection level. The T(EPS)Click here for additional data file.S2 Fig Promoter architecture and normalized mRNA expression profiles of Ime1 target genes. The genes are aligned by ATG and sorted by expression peak time (a) or by maximal expression level (b). Ume6 binding site—red; TATA box—blue; TrSS—green; measured nucleosomes—gray.  Distributions of TATA box (c) or Ume6 binding site (d) distance to ATG for different expression level groups (as marked in (b)). Triangles show th 95% confidence interval for the median estimator. In highly expressed genes the TATA box is closer to the ATG.(EPS)Click here for additional data file.S3 Fig50 and tracks of log(YFP/CFP) over time  are shown. (a) pDmc1 vs. pDmc1 (left), pDmc1 vs. pDmc1(UME6bsSwap) (center), pDmc1 vs. pDmc1+UME6bs (right). (b) pMei5 vs. pMei5 (left), pMei5 vs. pMei5-UME6bs (right). (c) pRec8 vs. pRec8 (left), pRec8 vs. pRec8-MBFbs (right).Timing and stoichiometry measures for strains having one alteration in the promoter of Dmc1, Rec8 or Mei5, compared to the wild type promoter. For each strain, scatter plots of T(EPS)Click here for additional data file.S1 MoviePart of a single position time-lapse movie showing Rec8-CFP, Dmc1-YFP cells entering meiosis. Left: Rec8-CFP signal. Right: Dmc1-YFP signal. Center: merged CFP, YFP and DIC channels. Time interval between acquisitions: 15 minutes.(AVI)Click here for additional data file.


Conclusion: Although genotype 1 is most common worldwide, nongenotype 1 HCV cases—which are less well served by advances in vaccine and drug development—still comprise over half of all HCV cases. Relative genotype proportions are needed to inform healthcare models, which must be geographically tailored to specific countries or regions in order to improve access to new treatments. Genotype surveillance data are needed from many countries to improve estimates of unmet need. Hepatitis C virus (HCV) exhibits high genetic diversity, characterized by regional variations in genotype prevalence. This poses a challenge to the improved development of vaccines and pan-genotypic treatments, which require the consideration of global trends in HCV genotype prevalence. Here we provide the first comprehensive survey of these trends. To approximate national HCV genotype prevalence, studies published between 1989 and 2013 reporting HCV genotypes are reviewed and combined with overall HCV prevalence estimates from the Global Burden of Disease (GBD) project. We also generate regional and global genotype prevalence estimates, inferring data for countries lacking genotype information. We include 1,217 studies in our analysis, representing 117 countries and 90% of the global population. We calculate that HCV genotype 1 is the most prevalent worldwide, comprising 83.4 million cases , approximately one-third of which are in East Asia. Genotype 3 is the next most prevalent globally ; genotypes 2, 4, and 6 are responsible for a total 22.8% of all cases; genotype 5 comprises the remaining <1%. While genotypes 1 and 3 dominate in most countries irrespective of economic status, the largest proportions of genotypes 4 and 5 are in lower-income countries.  Hepatitis C virus (HCV) is a globally prevalent pathogen and a leading cause of death and morbidity.5Historically, HCV drug therapy has depended on interferon-α (administered by injection) and ribavirin over many months and is associated with severe side effects. The resources required for treating HCV patients with such drugs have been a considerable barrier for healthcare systems in many low and/or lower-middle income countries, despite treatment outcomes that are comparable to those in well-resourced settings.HCV exhibits an extraordinarily high degree of genetic diversity—substantially greater than that of the HIV-1 pandemic—creating a major challenge for the development of both HCV vaccines and pan-genotypic drug therapies.Furthermore, the first prophylactive T-cell vaccines that aim to prevent persistent HCV infection are currently in Phase II efficacy testing, with further candidates moving into human studies.HCV strains are classified into seven recognized genotypes (1-7) on the basis of phylogenetic and sequence analyses of whole viral genomes.26Here we are the first to estimate the relative prevalence of HCV genotypes globally by region and by country where data permitted. The data described provide a platform for the rational deployment and efficacy testing of new DAA therapies and vaccines for HCV.http://www.pubmed.gov) for all articles containing the terms “HCV” or “hepatitis c virus” and “genotype” or “subtype” published between 1989 (the year that HCV was discovered) and 2013. As a standardized genotype classification system was not present before 1994, we converted HCV genotypes or serotypes in articles published between 1989 and 1994 to the consensus nomenclature proposed by Simmonds et al.We searched PubMed  region as defined by the World Health Organization (WHO).http://www.esri.com). The diversity of HCV genotypes present in each country was computed using the Shannon Diversity Index,29Mapping of genotype frequencies by country and region was performed using ArcGIS 9.3 . In contrast, the low and lower-middle income countries displayed relatively high proportions of genotypes 4 and 5. For the genotype proportions for all income classes, see the The data presented here represent the most comprehensive effort to date to estimate the relative contribution of each HCV genotype to the global HCV epidemic. Such data are crucial to inform future prevention efforts (including vaccine design) as well as policies to ensure global access to the new generation of interferon-free DAAs. Aside from countries entirely lacking genotype information, our data highlight that the evidence base for genotype prevalence is weakest in Sudan, the Democratic Republic of the Congo, and Tanzania, in terms of the number of viruses genotyped as a proportion of their overall populations ; Fig. 2CIt is important to note that the most prevalent genotype in developed economies (genotype 1) is also the most prevalent globally and should be well served by interferon-free regimens of second-generation DAA therapies with viral eradication rates of >90%.35One possible hypothesis for the current global distribution of genotype 1 is the chance association of subtypes 1a and 1b with the international dissemination of contaminated blood and blood products during the twentieth century, prior to the discovery of HCV in 1989.38Although HCV genotype 1 and 3 infections are more prevalent than all other genotypes globally, genotypes found more commonly in lower-income countries still account for a significant proportion of HCV cases worldwide . SpecifiStratification by viral genotype at a national and regional level, and a better understanding of viral diversity within target populations, will also critically inform the rational design and testing of HCV vaccines. We have shown that diversity tends to be high in high-prevalence countries. The first T-cell vaccine, which hosts a genotype-1 immunogen, is currently undergoing efficacy testing in intravenous-drug user (IVDU) populations in Baltimore and San Francisco, where genotype 1 infection is dominant. Phase I clinical data suggest that the generation of T cells that target multiple viral genotypes using this vaccine is possible.Our study is intended as a comprehensive literature survey and is not intended as a statistically formal meta-analysis. PubMed is the world's largest medical library, and so we are confident in the comprehensiveness of our published literature base. However, we recognize that there are limitations within our dataset, and also that there may be some useful unpublished reports that were excluded from this study. Foremost among these is potential selection bias, since not all studies included were surveys of the general population . Thus, some risk groups associated with specific genotypes may be more readily sampled than others, and the impact of this may be strongest in those countries with the fewest number of genotyped cases. A mechanism for removing studies of certain risk groups would be required, and such a subjective decision could lead to additional sources of bias. In addition, we have not stratified studies according to the methodology used to genotype patients. Since the HCV RNA 5′UTR (untranslated region) is the most common target for diagnostic HCV RNA assays, it is also the viral region most commonly used to define viral genotype. While this region is relatively conserved, single nucleotide differences assessed using sequencing or probe-based assays are most commonly used in assays to define viral genotype.Our estimates of relative genotype prevalence are dependent on viral genetic data in association with global and national estimates of HCV prevalence based on seropositivity, which includes instances of cleared infections.Although genotypes 1 and 3 account for the majority of infections worldwide, HCV strains belonging to the rarer genotypes 2, 4, 5, and 6 can increase rapidly in prevalence if they happen to become associated with efficient routes of transmission. The high prevalence and dominance of subtype 4a in Egypt is widely believed to have resulted from its spread by way of unsafe injections during past anti-schistosomal public health campaigns.


By analyzing systolic and diastolic blood pressure, which are used separately as outcomes, we compare the 3 methods in identifying a known genetic variant that is associated with blood pressure from chromosome 3 and simulated phenotype data. We also analyze the real phenotype data to illustrate the methods. We conclude that the linear mixed model with longitudinal measurements of diastolic blood pressure is the most accurate at identifying the known single-nucleotide polymorphism among the methods, but linear mixed models with baseline measures perform best with systolic blood pressure as the outcome.This article compares baseline, average, and longitudinal data analysis methods for identifying genetic variants in genome-wide association study using the Genetic Analysis Workshop 18 data. We apply methods that include (a) linear mixed models with baseline measures, (b) random intercept linear mixed models with mean measures outcome, and (c) random intercept linear mixed models with longitudinal measurements. In the linear mixed models, covariates are included as fixed effects, whereas relatedness among individuals is incorporated as the variance-covariance structure of the random effect for the individuals. The overall strategy of applying linear mixed models decorrelate the data is based on Aulchenko  It is aet al's  residualWe give an overview of 3 LMMs for the analysis of Genetic Analysis Workshop 18 (GAW18) data, paying attention to the power of selecting an associated single-nucleotide polymorphism (SNP) from chromosome 3 and simulated phenotype data. In particular, we apply 3 types of LMMs for statistical analysis of baseline measurements, mean measurements, and longitudinal data. We compare the LMMs through simulations and illustrate them using the real phenotype data.p value ≤ 1e−6). The quality controls were implemented using the R package SNPassoc [We use 3 models to analyze the GWAS data set from chromosome 3 of the GAW18: a Diabetes-GENES Project, which consists of whole genome sequence data in a pedigree-based sample, longitudinal phenotype data for hypertension and related traits, and selected covariates. In this GWAS data, 65,519 SNPs have been genotyped for chromosome 3. In the simulated phenotype data, 849 subjects were measured at 3 time points for age, medication use, smoking status, and blood pressure. As is standard practice, SNPs with minor allele frequency (MAF) <1% were excluded from data analysis. We also filtered out SNPs with low call rates (<90%) and deviation from Hardy-Weinberg equilibrium  and diastolic blood pressure (DBP) separately.et al. [Aulchenko et al.  proposedInitial model. The initial model is ij yis the value of phenotype corresponding to the jth individual in the ith pedigree, ijk xis the value of the kth covariate or fixed effect, kth fixed effect or covariate, and ij eis the vector of residual effects. G is the random polygenic effect that follows a multivariate normal distribution with mean 0 and variance Φ is the relationship matrix (kinship matrix) and SNP model. The residuals are used as the dependent trait in a simple linear regression for each SNP, lth SNP from the model 1 scenario. The method adjusts for familial relationship and is computationally fast, but the model only considers the time 1 information from the GAW18 data. The first stage model is implemented using the polygenic function of the R package GenABEL, and the kinship matrix is estimated using the R package kinship2. Next, the lm function is used for fitting the linear model with residuals obtained from the first-stage model.jth individual in the ith pedigree. β is the coefficient for unknown fixed effects representing nongenetic effects , and G is the random polygenic effect that follows a multivariate normal distribution with mean 0 and variance K is the kinship matrix with elements e is a vector of random residual effects that are normally distributed with zero mean and variance-covariance We considered the measurement of the mean across the 3 time points as the outcome and followed the 2-stage approach. The model formula for the first stage is lth SNP from the model 2 scenario. We implemented the model using the R package kinship (R 2.10.1). The lmekin function is used to obtain the residuals from the first-stage model.In the second stage we consider the residuals as the outcome and fit the linear model, We also evaluate a 2-stage LMM that takes longitudinal measurements into account. We consider 2 models, without and with time effects, in the application of the longitudinal analysis. In the first stage, we fit a random intercept LMM as follows:(A)(1)Next, we extend the first-stage model allowing time points:(B)(2)ijt ydenotes the phenotype (SBP or DBP) for the jth individual in the ith pedigree at time t. ijt xis the k fixed effect time-dependent covariate, v is the slope coefficient for the time points tz; t = 1, 2, 3, and G is the random polygenic effect as in model 2; e is a vector of random residual effects that are normally distributed with zero mean and covariance lth SNP from the model 3 scenario. We applied the model using the R packages pedigreemm and kinship.where p values for each SNP are used to rank the SNPs.We investigated the performance of the 3 LMMs for selecting a known associated SNP from simulation studies. The 3 models are employed after adjusting for covariates and pedigree information, and the MAP4, position: 47956424, and MAF: 0.367) and rs1131356 . We investigated the 3 LMMs in terms of selection performances of rs6442089. We selected rs6442089 because it is a well-known SNP from the gene MAP4 that affects blood pressure.The simulated phenotype data in GAW18 has 10 known SNPs from chromosome 3 that are associated with blood pressure. Among these SNPs, 2 have MAF >0.05. These 2 variants are rs6442089  was in the list of target SNPs or within the p value cutpoint. Figure p values. It appears from the figures that applying LMM to longitudinal DBP data provides better results in selecting the SNP compared to any of the other methods. We found similar results by both Model 3(A) and Model 3(B). That is, the results from Model 3(A) and Model 3(B) do not look qualitatively different from each other: In both cases, the performance of selecting the SNP is lower in SBP and higher in DBP.We denote a SNP to be significant either if its p values for each SNP. We considered first 3 time points to avoid the missing values of the fourth time point, and we applied the 3 models to the same 845 individuals who were selected in the simulated data analysis. We report 5 top-ranked SNPs in Table p values from the LMM with longitudinal measurements are conservative compared to other methods. After investigation of the top 20 SNPs we found 3 SNPs in common across the models. The ranks for the known SNP, rs6442089, are 4376, 3105, and 758 by the LMMs with baseline, longitudinal (Model 3A), and mean measures outcome, respectively. Therefore, the real phenotype data suggest that the LMM with mean measurements performs best among the 3 methods for identifying the SNP rs6442089.We employed the 3 LMMs to real phenotype SBP data after adjusting for covariates and we rank the SNPs using the In this article we applied 3 LMMs to the study of GAW18 in family data and in settings of relevance to baseline measures, mean measures, and longitudinally measured data. The statistical analysis of GWAS for GAW18 data using LMMs with longitudinal DBP measurements is capable of revealing the dynamic pattern of genetic control over chromosome 3 but did not perform competitively with other models for longitudinal SBP measurements. Exploratory/graphical analysis for the trajectories of SBP and DBP measurements also supported the conclusion that DBP had more subject-specific variability in slopes than SBP. However, the GRAMMAR approach with single-measure SBP data at baseline can be used on the development of SNP selection.A general consideration applicable to all the methods discussed here concerns the issue of whether the outcome is linear or nonlinear. An alternative approach could be to relax the conditions imposed on linear models and explore the hidden structure by using a varying coefficient model . ConsequWe showed that a linear mixed modeling framework was most accurate at identifying known single-nucleotide polymorphism compared to other competing methods we considered in this manuscript for the analysis of longitudinal measurements of diastolic blood pressure. In contrast, baseline measures performed best with systolic blood pressure highlighting that, depending on the trajectory profile of the quantitative trait of interest, either just baseline values or serially measured values can be useful in genetic association studies.The authors declare that they have no competing interests.AH designed the overall study, performed all of the data analysis and drafted the manuscript. JB assisted in conceiving the idea, helped in drafting the manuscript and provided overall supervision. Both authors read and approved the final manuscript.


Twenty-five younger (18–30 years) and older (65+ years) adults were required to report whether they perceived one or two flashes, whilst ignoring irrelevant auditory beeps, in bimodal trials where auditory and visual stimuli were separated by one of six stimulus onset asynchronies (SOAs). There was a marked difference in the pattern of results for the two variants of the illusion. In conditions known to produce the fission illusion, older adults were significantly more susceptible to the illusion at longer SOAs compared to younger participants. In contrast, the performance of the younger and older groups was almost identical in conditions known to produce the fusion illusion. This surprising difference between sound-induced fission and fusion in older adults suggests dissociable age-related effects in multisensory integration, consistent with the idea that these illusions are mediated by distinct neural mechanisms.While aging can lead to significant declines in perceptual and cognitive function, the effects of age on multisensory integration, the process in which the brain combines information across the senses, are less clear. Recent reports suggest that older adults are susceptible to the sound-induced flash illusion (Shams et al.,  The aging process is accompanied by a gradual decline in many aspects of perceptual function. Perhaps the most salient examples of sensory decline are found in vision and audition, where visual acuity and auditory sensitivity decrease in an age-dependent manner Pitts, . More refission illusion only when the time interval between tones is relatively short  and 25 older participants  volunteered to take part in the study. The younger participants were recruited from the student population of Trinity College Dublin and were compensated with research credits for their time. Older volunteers were community-living adults recruited through advertisements in local newspapers and community groups and were compensated for their travel expenses. All participants were naive to the purposes of the study and provided written consent to participate.We assessed all older adults across a range of sensory functions and also on cognitive ability in order to screen for cognitive impairment. Visual acuity in near and far ranges was measured in older participants using the SLOAN Two-Sided ETDRS Near Vision and the 4 m 200 Series Revised ETDRS charts , respectively. Contrast sensitivity was estimated using the Pelli-Robson Contrast Sensitivity Test. All older participants showed normal or corrected-to-normal acuity and contrast sensitivity for their age. Hearing ability was assessed using a modified version of the Hughson-Westlake method via a Kamplex BA 25 screening audiometer. All participants included in the study displayed thresholds within the normal limits for their age. The Montreal Cognitive Assessment was also administered to all the older participants to screen for cognitive impairment. Participants who scored below a score of 24/30 were excluded from the study. The Trinity College School of Psychology ethics board approved all recruitment and experimental procedures, and the experiment protocol was conducted in accordance with the principles of the Declaration of Helsinki.Stimulus generation and presentation were controlled by an Apple Mac Pro computer on a HP L1710 monitor at a refresh rate of 60 Hz and a spatial resolution of 1280 × 1024 pixels. Participants were positioned at a distance of 57 cm from the screen, with head position supported by a chin rest. Experimental testing was conducted in a darkened, windowless room.Stimuli were created and displayed in Matlab version 7.14 (R2012a) using Psychtoolbox  and fusion (2F1B) illusions were randomly interleaved with unisensory (1F and 2F) and multisensory (1F1B and 2F2B) control trials, such that there were six different conditions comprising an equal number of trials.In conditions containing two flashes or two beeps, auditory and visual stimuli were separated by different stimulus onset asynchronies (SOAs). Seventeen younger and 25 older participants completed the experiment with 6 SOAs ranging between 33 and 400 ms , while 8 younger participants completed the experiment with 4 SOAs (50–200 ms). Participants collected a minimum of 10 trials per SOA for each condition, leading to total number of 360 trials (6 SOAs × 6 conditions × 10 repeats). At regular intervals over the course of the experiment, participants were prompted to take a self-timed break to avoid fatigue. The experiment lasted approximately 25–30 min.d′) and response bias (c) for discriminating between one and two flashes at each SOA and for each auditory beep condition separately. Perceptual sensitivity was calculated with the following equation:To examine the temporal bounds of susceptibility to the sound-induced flash illusion, we first examined the proportion of incorrect responses across all SOAs in the 1F2B and 2F1B conditions, known to produce the fission and fusion illusions, respectively. In a second analysis, we used signal detection theory to determine whether the change in the proportion of illusory reports resulted from changes in perceptual sensitivity, response bias, or both. This approach was based on that of Rosenthal et al.  and furtz(p) represents the inverse of the cumulative normal distribution. Using these same definitions, response bias was calculated as:where H denotes the proportion of correctly reported multiple flashes , FA denotes the proportion of incorrectly reported multiple flashes, and p = 0 or p = 1 , these variables were approximated by 1/n and 1-1/n, respectively .In cases where d′ and c. When appropriate, the Greenhouse-Geisser correction was applied to adjust the degrees of freedom of within-subject tests to correct for violations of the sphericity assumption and, in these cases, the adjusted p-value is reported. When multiple one-sample t-tests or pairwise comparisons were performed, the Bonferroni correction was used to maintain a family-wise Type I error rate at 0.05 and the adjusted p-value is reported.Statistical analyses were conducted using mixed-model ANOVAs to analyse the effects of age group and SOA on the proportion of illusory responses, F = 8.92, padj < 0.001, GGeps = 0.48] and age group , as well as a significant age group x SOA interaction , indicating that the temporal limits of the fission illusion differed in younger and older groups. To determine the range of SOAs producing the illusion in each age group, we compared the proportion of illusory responses at each SOA with veridical performance  using one-sample t-tests. The younger group showed a significant fission effect for SOAs ranging between 33 and 150 ms  only, and not for longer SOAs . In contrast, the older group showed a significant fission effect at all six SOAs .We first compared the performance between younger and older groups in the conditions known to produce the fission illusion 1F2B). Figure B. FigureThis result replicates a previous finding from Setti et al. , who rept-test revealed that older participants were significantly less accurate than younger adults in the 2F condition at short SOAs . This result is consistent with declines in temporal acuity with age  = 0.048, p = 0.83], and no interaction between age group and SOA , indicating that the magnitude and temporal limits of the illusion were very similar across younger and older adults. Moreover, comparing the normalized proportions of illusory responses with veridical performance at each SOA revealed that both younger and older participants experience the illusion for the same range of SOAs, namely 33–100 ms , while performance did not differ from veridical performance for SOAs of 150 ms and longer .A very different pattern of results was observed in the 2F1B condition, which is known to produce the fusion illusion Figure . SimilarF = 0.07, p = 0.79), and no significant illusion × SOA interaction . However, this was clearly not the case for older participants  = 19.5, p < 0.001], as well as a significant illusion x SOA interaction . This difference in the temporal constraints of the two illusions in older adults supports the hypothesis that the two illusions result from distinct neural mechanisms.Although the fission and fusion variants of the sound-induced flash illusion appear similar from a behavioral perspective, there is debate as to whether the two illusions are driven by the same or different neural processes  = 19.49, p < 0.001]. In contrast, estimates of response bias were similar across both young and old groups and there was no significant group effect . Thus, it appears that the age-related difference in performance in the sound-induced fission illusion resulted from a reduction in perceptual sensitivity of older adults. In the case of the fusion illusion  = 0.97, p = 0.33] or response bias .In younger participants, the sound-induced fission illusion has been shown to result from both a decrease in visual sensitivity and a shift in criterion , suggesting that this split in the older group is not solely due to age of the participants. The reasons for the difference between these individuals are not clear, but ongoing work in our laboratory is attempting to understand the factors that lead to this variability in performance across participants. In contrast, the variation in performance in the fusion conditions was much smaller and there were no obvious differences between the young and old groups.A commonly reported observation regarding the sound-induced fission illusion is its high degree of between-subject variability , indicating that the auditory beeps generated illusory second flashes (see also McCormick and Mamassian, Could age differences in attentional control explain the increased susceptibility to the sound-induced fission illusion experienced by older adults? We suspect not for two reasons. First, one might expect that any increase in distractibility would lead to an increase in response bias, rather than affecting perceptual sensitivity, as the participant would be tempted to respond in line with the number of auditory stimuli presented. However, our signal detection analysis revealed that age-related increases in susceptibility to the illusion were primarily due to decreases in sensitivity (The current data show that while older adults are susceptible to the fission illusion across a wider range of SOAs than their younger counterparts, their performance does appear to recover if the interval between the auditory tones is long enough. Similar to findings in other studies of multisensory processing in older adults (Laurienti et al., bona fide example of multisensory integration.What could cause these large differences in age-related susceptibility to the fission and fusion illusions? One possible explanation is that the fusion illusion does not constitute a genuine example of multisensory integration. This explanation seems unlikely, however. Both EEG and fMRI studies (Watkins et al., A more likely explanation for this discrepancy between age-related fission and fusion effects is that each variant of the illusion has its own temporal integration window derived from distinct networks of activation in the brain (Mishra et al., These findings may help to explain the dissociation between the older group's performance on the fission and fusion illusions in the current study. For instance, it is conceivable that the cortical network underlying the fusion illusion could be preserved in older adults, while one or more areas within the fission network may experience a decrease in processing speed. Indeed, Peiffer et al.  found thThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The acoustic change complex (ACC) is an auditory-evoked potential elicited to changes within an ongoing stimulus that indicates discrimination at the level of the auditory cortex. Only a few studies to date have attempted to record ACCs in young infants. The purpose of the present study was to investigate the elicitation of ACCs to long-duration speech stimuli in English-learning 4-month-old infants. ACCs were elicited to consonant contrasts made up of two concatenated speech tokens. The stimuli included native dental-dental /dada/ and dental-labial /daba/ contrasts and a nonnative Hindi dental-retroflex /daDa/ contrast. Each consonant-vowel speech token was 410 ms in duration. Slow cortical responses were recorded to the onset of the stimulus and to the acoustic change from /da/ to either /ba/ or /Da/ within the stimulus with significantly prolonged latencies compared with adults. ACCs were reliably elicited for all stimulus conditions with more robust morphology compared with our previous findings using stimuli that were shorter in duration. The P1 amplitudes elicited to the acoustic change in /daba/ and /daDa/ were significantly larger compared to /dada/ supporting that the brain discriminated between the speech tokens. These findings provide further evidence for the use of ACCs as an index of discrimination ability. One of the early steps for infants to learn a language is to recognize the phonetic distinctions and sound patterns of their native language. Infants with hearing loss are at risk for delays in speech and language development because they might not have access to important auditory cues during a period of rapid development in the first years of life. These early processes are complex and not fully understood in infants with normal hearing or limited auditory experience. One challenge in this area of research is the lack of a reliable, age-appropriate tool to assess an individual infant's capacity to detect and discriminate speech sounds. Such a tool would be invaluable in the clinic to assess hearing-aid benefit in infants in terms of ability to discriminate consonant and vowel contrasts. Because behavioural methods provide limited information about perceptual capacities and their underlying mechanisms, particularly in individual infants, cortical auditory-evoked potentials (CAEPs) offer a useful complement to behavioural measures. The slow cortical response, or P1-N1-P2, which is elicited to the onset of a stimulus has been studied extensively in adults and in some depth in infants to assess detection of speech sounds, although there are gaps in our understanding of these responses in infants, especially those with hearing loss who are aided. The acoustic change complex (ACC) which is elicited to a change in an ongoing stimulus has been a focus of recent research because it is thought to measure discrimination at the level of the auditory cortex which can provide insight into the brain's capacity to process the acoustic features of speech . The ACCResearch findings have suggested that the ACC may have the potential to be used as a clinical tool for assessing speech perception capacity. The ACC has been recorded in adults in response to speech stimuli such as consonant-vowel syllables, in which the acoustic change included frequency, amplitude, and periodicity cues similar to those found in normal conversational speech –3. OstroSmall and Werker  first reInfant responses are frequently biphasic waveforms with a large positive peak and following negativity –18 due tInterstimulus interval (ISI) is thought to have substantial effects on the morphology, amplitude, and scalp distribution of the cortical response, particularly in immature auditory systems. For example, increasing the ISI up to at least 10 s results in larger amplitudes of N1 and P2 and their magnetic counterparts , 37. WheOnly a few studies have studied the effects of stimulus and presentation parameters on infant cortical responses. Golding et al.  recordedAlthough the stimulus duration (282 ms for each speech token) and the ISI (2200 ms) used by Small and Werker  were adeThe purpose of the present study was to investigate the effect of long-duration speech stimuli on the ACC elicited in young infants to native and nonnative consonant contrasts. By allowing more time for neural refractoriness, it is expected that ACCs will be recorded in response to the changes in both native and nonnative speech contrasts with better morphology than previously reported by Small and Werker (2012).Ten adult  and 24 infant  participants with normal hearing were included in this study. Adults were recruited from the community; infants were recruited through a database managed by the Infant Studies Centre at the University of British Columbia. All adults were English-speaking and all infants were learning English as a first language and had no exposure to the Hindi language. Infant participants were screened for hearing using transient-evoked otoacoustic emission (TEOAE) with the Madsen AccuScreen Pro (GN Otometrics). One infant who did not pass the hearing screening was excluded from the study. Nine infant participants were also excluded because data collection was not completed due to crying, excessive movement, or technical problems.The stimuli used in the present study were similar to those used by Small and Werker  and are Werker and Lalonde  created In Small and Werker , S1S2 stThe stimuli were presented at 86 dB peak SPL in the sound field. The stimuli were presented by Stim2 and then delivered to Tucker Davis Technologies PA5 and SM5 modules. The overall gain of the stimulus was reduced by 13 dB before routing it to the HB7 headphone driver which was connected to a loudspeaker placed one meter in front of the infant participant. A Larson Davis System 824 and Larson Davis Model 2559 0.5-inch random-incidence microphone placed at the approximate position of the infant's head were used to calibrate the speech stimulus in dB peak SPL.A four-channel electrode montage was used to record the ERPs in all participants. Individual gold-plated cup electrodes filled with electrode paste were placed at Cz, C3, M1, M2, and FPZ  and secured with tape. (Note: only the waveforms recorded at C3 that are presented as the responses at Cz were very similar.) M2 was chosen as the reference and the electrode located on the forehead served as ground. Eye-blink activity was monitored using bipolar electrodes pasted above and below the centre of the left eye. The Compumedics Neuroscan Synamps2 and SCAN 4.3 software were used to record the electroencephalograph (EEG). All interelectrode impedances were measured and kept below 5 kOhms with the SCAN 4.3 impedance routine.μV in amplitude over a range of −100 to 800 ms; single trials were rejected manually by visual inspection for infants to optimize the number of accepted epochs in the final waveform. A minimum of 130 accepted epochs was required for each stimulus condition to be included in final data analysis based on pilot data for 4-month-old infants; fewer than 130 accepted epochs resulted in poorer morphology and replicability of averaged waveforms. The total number of recorded epochs ranged from 300 to 371 and from 260 to 483 for adults and infants, respectively. The average rejection rate was much lower for adults (5–8%) versus infants (43–48%) and resulted in 244–365 and 135–277 accepted epochs for adults and infants, respectively. A split-epoch method was used to generate two replications  for each condition tested. During data acquisition, the EEG channels were filtered using a 30 Hz low-pass filter and a 1.0 Hz high-pass filter. The continuous EEG was amplified with a gain of 500 and converted using an analog-to-digital rate of 1000 Hz. The recording window consisted of a 100 ms prestimulus period and a 1400 ms poststimulus period. After acquisition, offline analysis used an epoch 900 ms in length (−100 prestimulus to 800 ms poststimulus). Single trials were baseline corrected across the entire sweep duration and an ocular artifact reduction was applied using an average of three epochs, which contained ocular movement greater than 250 ms epoched over −100 to 300 ms. Single trials were rejected automatically for adults when electrophysiological activity exceeded 75 All tests were carried out in a double-walled sound-attenuated booth in the Pediatric Audiology Lab at the University of British Columbia. Adult participants were seated comfortably in an armed reclining chair and watched a movie with subtitles with no sound throughout testing. They were instructed to ignore the stimuli presented to them and to remain as quiet and still as possible. The infant participant was held by a parent who sat in a comfortable chair facing a loud speaker. An age-appropriate movie was played silently on a flat-screen monitor placed directly behind the loud speaker. An assistant also stayed in the booth to engage the infant's attention in order to minimize the head movement and reduce myogenic noise in the EEG.Adults were required to complete all three stimulus conditions  to be included in the study, while infants were required to complete at least one condition. The order of stimulus conditions was randomized for each participant. Only five infants completed more than one stimulus condition and none of them completed all three conditions. An experimenter observed the EEG during data acquisition to monitor the infant's state, muscle movement, and electrical artifact. Testing was stopped if the infant started to cry or vocalize continuously during the recording. Hearing screening was conducted in both ears at the end of the test session. The duration of the recording was approximately 1.5 hours for adults and 10 to 40 minutes for infants. After explaining the study to the adult participants and the parents of infant participants, written consent was obtained. An honorarium was given to the adult participants. A small honorarium and a gift were given to the parents and their infants at the end of the session.μV for the S2 conditions, the amplitude of P2 was measured from the negative trough preceding P2. Mean latency and amplitude values were calculated for each of the slow cortical components in response to the S1 and S2 portions of the stimulus. Peak-to-peak amplitudes for N1-P2 were also measured for adults and compared for responses elicited to S2 versus S1. Grand mean ERP waveforms to each of the stimulus conditions were also compared in terms of morphology, amplitudes, and latencies.The morphology of ERP waveforms to /dada/, /daba/, and /daDa/ was compared qualitatively and the percentage of components present for each condition was calculated. The baseline-to-peak amplitude and latency of the largest peaks within each of the expected latency windows were also measured. Response latencies were measured for cortical components to S1 and S2 from the onset of S1 and the onset of S2, respectively. For amplitude measures, when the baseline was not at 0  post hoc comparisons were performed for significant main effects. Results for all analyses were considered statistically significant if p < 0.05.For the adult group, two-way repeated-measures analyses of variance were carried out to compare (i) baseline-to-peak amplitudes and latencies of the P1, N1, and P2 components elicited to the S1 versus S2 portion of /dada/, /daba/, and /daDa/ and (ii) N1-P2 peak-to-peak amplitudes for S1 and S2 stimuli across stimulus conditions. For the infant group, two-way mixed-model analyses of variance were used to compare baseline-to-peak amplitudes and latencies of the P1 and N1 components evoked to the S1 versus S2 portion of /dada/, /daba/, and /daDa/. Newman-KeulsμV, respectively.As shown in F = 0.402, p = 0.682]. Similar to the pattern for individual components, the mean peak-to-peak amplitude of the N1-P2 elicited to S2 was smaller compared to S1 .The overall morphology of the waveforms recorded in response to the acoustic change from S1 to S2  was similar when compared across stimulus conditions /dada/, /daba/, and /daDa/ . For S2,μV; mean latencies across stimulus conditions for P1, N1, P2, and N2 were, on average, 132, 226, 320, and 415 ms, respectively.Waveform morphology was similar across conditions for the infant participants, as shown in The morphology of the grand mean waveform elicited to the acoustic change from S1/da/ to S2/da/, /ba/, or /Da/ resembled the morphology of the P1, N1, and P2 components of S1 responses ; however Post hoc comparisons using the Newman-Keuls test showed that the amplitudes of S2 P1 were significantly larger for /daDa/ versus /dada/ (p = 0.003) and marginally significantly larger for /daba/ versus /dada/ (p = 0.055). No statistically significant differences were revealed for P1 latencies.The results of a two-way mixed model ANOVA comparing the mean amplitudes of P1 and N1 elicited to S1 and S2 across the three stimulus conditions for infants are summarized in In contrast to P1, N1 had the largest mean amplitudes elicited to both S1 and S2 across all stimulus conditions , indicatThe results of the present study using a long stimulus duration for /dada/, /daba/, and /daDa/ resulted in similar ACC findings for adults and more robust ACCs in infants compared to Small and Werker . In the Small and Werker  reportedOur findings support that the infant's brain can detect a change in the stimulus from /da/ to /da/, /ba/, and /Da/. Moreover, the larger P1 amplitudes recorded for /daba/ and /daDa/ may suggest that the brain has noticed that the acoustic change from /da/ to /da/ was smaller than the change from /da/ to /ba/ and from /da/ to /Da/. In our hypothesis, we had predicted that the ACC for both /daba/ and /daDa/ would have larger amplitudes and more distinct components compared with the ACC to /dada/ because behavioral studies had shown that English-learning infants under 6 months of age were able to discriminate the native /daba/ and nonnative /daDa/ contrasts , 43, 44.Interestingly, our adult group did not show the same significant differences in the amplitudes of ACC components that we found for the infant group. We had expected a larger difference between the ACCs to /daba/ and the other two stimuli because both /dada/ and /daDa/ should have acted as “control” stimuli for the adults. A contributing factor might have been that the stimulus parameters that were more optimal for infants were too long for adults. The speech stimulus used in the present study consisted of two consonant-vowel structures (CVCV), which was different from the typical CV  or VV  stimulus used to elicit cortical responses. Although we only focused on the acoustic change between two CV syllables, the brief transition from consonant to vowel within a CV token may have also evoked cortical responses resulting in overlapping cortical waveforms thus affecting the overall morphology of the ACC . PerhapsThere are some limitations to the current study. As mentioned above, the nonnative speech token might have reduced the impact of auditory experience. For example, a MMN study using similar Hindi speech contrasts reported that the magnitude of the MMN can be significantly affected by the order of stimulus presentation  . TherefoThe most important finding of the present study is that an ACC to a change within a speech stimulus can be successfully recorded in young infants, and, by extending the stimulus length and allowing more time to accommodate the longer neuronal refractory period for infants, better-defined components of the ACC can be elicited. Our ACC results also suggest that distinct neural response patterns may be elicited to acoustic changes that vary in degree. In the present study, ACC components had larger amplitudes in response to a larger acoustic change within a stimulus. To confirm that the ACC is sensitive to a range of subtle acoustic changes in speech, more research is needed. As a technique in development, the ACC may hold promise for providing insight into the infant brain's capacity to discriminate the acoustic features of speech.


Planetary environments with supercritical fluids, particularly supercritical carbon dioxide, exist, even on Earth (below the ocean floor), on Venus, and likely on Super-Earth type exoplanets. These planetary environments may present a possible habitat for exotic life.Supercritical fluids have different properties compared to regular fluids and could play a role as life-sustaining solvents on other worlds. Even on Earth, some bacterial species have been shown to be tolerant to supercritical fluids. The special properties of supercritical fluids, which include various types of selectivities  have recently been recognized in biotechnology and used to catalyze reactions that do not occur in water. One suitable example is enzymes when they are exposed to supercritical fluids such as supercritical carbon dioxide: enzymes become even more stable, because they are conformationally rigid in the dehydrated state. Furthermore, enzymes in anhydrous organic solvents exhibit a “molecular memory”, C) and critical pressure (PC). For example, supercritical carbon dioxide  is a nonpolar medium with large quadrupolar moment [Supercritical fluids (SCFs) differ quite significantly compared to the properties of real fluids. For example, supercritical water is relatively non-polar and acidic . SupercrNot surprisingly, SCFs as non-aqueous solvents for enzyme-catalyzed reactions have gained the attention of enzymologists since the 1980s and have been employed in a variety of biotechnological applications due to their numerous advantages . Enzymes2 and H2 in supercritical fluids, (3) high diffusion rates and variable density, and (4) high dissolving power [The change in properties from subcritical fluid to supercritical state is especially noteworthy for the common compounds water and carbon dioxide. These include: 1) high solubility of gases within supercritical mixtures, (2) miscibility of gases such as O high solng power . As a cong power .2), which attracted particular attention in research and technology due to its “green”  properties. ScCO2 is chemically relatively inert  and is a low-toxicity aprotic solvent [2 is an easily accessible supercritical regime  and, as a solvent, is miscible with both fluorous and organic materials [i.e., non-flammable). Supercritical carbon dioxide can thus serve as solvent for “difficult” chemical transformations, such as the direct reaction of hydrogen and oxygen to form hydrogen peroxide [Here, we focus on supercritical carbon dioxide  [Organic solvents are usually volatile carbon containing compounds present in liquid form at room temperature. On the other hand, supercritical fluids such as scCOsolvents , but shasolvents . Howeversolvents . Curioushniques) ,16.2 may dissolve from 0.3% to 0.5% (w/w) water, depending on the pressure and temperature [Enzymes of terrestrial organisms need a specific amount of bound water to be active . The actperature .2 or any other non-aqueous media or supercritical fluids, it is of vital importance to have a minimum water content/solubility [2O/g enzyme is generally accepted [Kalibanov was among the first to realize that the water bound to the enzyme determines the catalytic activity rather than the total water content of the system . In othelubility . Completaccepted . The rolaccepted .2, the catalytic activity of enzyme reflects  the pH  of last aqueous solution it was exposed to [A particularly notable feature of enzymes after being dissolved in an anhydrous apolar solvent and being in contact with a competitive inhibitor is the capacity to retain the state induced by the ligand . This phposed to . In this2 for biochemical transformations include increased catalytic activities as a result of improved mass transfer, higher selectivities and strong suppression of side reactions [2 in the liquid state (≈1.5) might require even higher pressures for certain classes of substances to be efficiently dissolved, whereas scCO2 as a Lewis acid is prone to react with chemical functional groups such as amines. Since enzyme surfaces are generally decorated lysine side chains, many free amine-groups are exposed to the solvent. These amine groups form carbamates with CO2 in a highly exothermic reaction [2 reversible formation of carbonic acid is shown in 2 is removed and amine function of lysine side chains is restored. Beside lysine residues, the imidazole side chain of histidine on the enzyme surface is also believed to have the capacity to participate in carbamoylation reaction [2 can lead to enzyme deactivation, whereas in some other instances, carbamate formation is believed to lead to changes in enzyme catalytic features . For example, Mase and co-workers speculated that a conformational change in enzyme due to carbamate formation provides good enantiomeric excess in lipase-catalyzed desymmetrization of 1,3-propanediacetate [2 can serve as a temporary protecting group in ruthenium-catalyzed metathesis as well as in rhodium-catalyzed hydroamino-methylations [The main advantages of scCOeactions . Howeverreaction . The mecreaction . In someiacetate . On the ylations  and refe2 has the tendency to strip structurally relevant water  from the enzyme leading to deactivation, it is necessary to add a small amount of water to the substrates, which can reverse the deactivation and restore the enzyme activity. The preservation of the enzyme structure and optimal activity depend on the presence of small amounts of water in the supercritical dispersing medium. When the protein is at least partially hydrated, water molecules bind to specific sites on the enzyme surface and prevent carbon dioxide from penetrating into its catalytic core [2 is that it can interfere with the catalytic activity of the enzyme by lowering the pH of the structural water  present in scCO2.Since scCOtic core . In this2 are lipases, which are used for enantioselective (trans)-esterifications and esterifications of fatty acids. Some other enzymes used in scCO2 are thermolysin, several phosphatases, dehydrogenases, oxidases, amylases and decarboxylases for CO2-fixation [2 exhibits a liquid-like density and a gas-like viscosity . The substrate-binding region of the lipase is exposed to carbon dioxide, which facilitates diffusion of large apolar substrates  into active sites (hydrophobic tunnel), while preserving the functional structure of the enzyme [2 would not be considered analogous to an enzyme in solution, but rather behave as heterogeneous catalysts where diffusion of reactants in and out of the enzyme is not rate limiting [Major classes of enzymes used for biocatalytic synthesis in scCOfixation . As scCOof water ) any chatability . The cate enzyme . Leitnerlimiting .2 are hydrolytic reactions, which require the presence of catalytic traces of water . This certainly does not disqualify scCO2 as a potential solvent for life. For example, for nearly all oxidation-reduction reactions catalyzed by enzymes in water “catalytic amounts” of metals, either tightly bound to the enzyme or taken up from bulk solvent along with the substrate, are required. In fact, almost one-third of all known enzymes require one or more metal ions for catalytic activity, usually in order to catalyze certain reactions by reversible changes in the bound metal ion’s redox state. The absolute necessity of traces of inorganic ions for enzymatic activity for so many enzymes in Earth’s biochemistry certainly does not disqualify water as life solvent.The majority of the reactions studied in scCO2 has a relatively low critical pressure and temperature [2 [2 solubility and facilitates the acidification of the cellular interior [2 is believed to increase the fluidity of the cell membrane, enhancing its permeability and facilitate extraction of membrane components such as phospholipids [2 is markedly higher (low viscosity!). Thus, scCO2 can much easier penetrate complex molecular geometries and assemblies  than subcritical fluids can. Georgiou and Deamer have suggested supercritical fluid-mediated lipid extraction technology as the main method of choice in the search for biomarkers for extraterrestrial life. In particular, lipid-derived hydrocarbons as biomarkers must have a specific compositional pattern  that originates exclusively from biogenic processes [ScCOperature  and is tperature . There iature [2 , which minterior . On the holipids . This shrocesses .2 may be an appropriate medium for biochemical processes under certain conditions. Ikushima [Candida cylindracea, which in supercritical carbon dioxide caused drastic conformational changes that enabled active sites to catalyze stereoselective synthesis. The reactivity was found to be susceptible to small changes in pressure or temperature near the critical point of the supercritical fluid. Shkrob and Sauer [2-multimer anions in supercritical carbon dioxide form stable complexes with water, aliphatic alcohols, alkyl halides, and alkyl nitriles. Matsuda and co-workers have demonstrated that the alcohol dehydrogenase from Geotrichum candidum, active in scCO2 at 35 °C and 10 MPa is capable of catalyzing the asymmetric reduction of various ketones [2, it shows that the application of scCO2 is not incompatible with life processes. Various enzymatic reactions were studied using bacterial species such as Bacillus megaterium, Geotrichum candidum or Aureobasidium pullulans .2 might support living processes was reported by Schulze-Makuch and Irwin [2 has been found to be trapped by a surface rock layer and sub layer cap of CO2 hydrate (CO2 x 6H2O). As the density of liquid CO2 increases with depth, it becomes denser than sea water, which opens up the possibility of many reservoirs of supercritical carbon dioxide on the sea floor [7 cells/ml at the fluid CO2/CO2-hydrate interface [2 [2 is a very common compound in planetary atmospheres, including our neighboring planets Venus and Mars, scCO2 is expected to occur in a variety of planetary environments. An example of a planetary environment where supercritical COnd Irwin . They pond Irwin . The lowea floor . Most innterface , which irface [2 . Since C2 should occur in the supercritical state is in the near-subsurface of Venus. Venus is nearly as massive as Earth and has an atmosphere of 92 bar, which mostly consists of CO2. Thus, it would be a great natural laboratory for testing the properties of scCO2. An interesting twist is that Venus was located in the habitable zone of our Solar System in its early history. Schulze-Makuch and co-workers [2. [2 extends far beyond Venus though, since many of the detected exoplanets are Super-Earths, with 10 or more earth masses. All these high pressure environments are locations where we might expect scCO2, given its common occurrence. Certainly, scCO2 deserves more attention not only for its biochemical and biotechnological applicability, but also as potential life-sustaining solvent in exotic planetary environments. One location where CO-workers ,40 suggekers [2.  The impo2 as a potential life-sustaining medium, we need to free us from the classical anthropocentric approach that defines water as the exclusive and sole solvent of life. Doubtlessly, putative life on a planet with scCO2 alone or in a mixture with water (to various extents) would be based on a different biochemistry, including different organization of boundary conditions (membranes), metabolic pathways, informational and energy flows as well as different polymers with informational and catalytic functions. The problem of increased conformational rigidity of enzymes in scCO2, known from Earth’s biochemistry, should not be taken dogmatically, particularly the notion that hyperthermophile proteins, due to enhanced conformational inflexibility in the folded native state, cannot achieve high enzyme activity (at lower temperatures) with a rigid active site(s) [When we consider scCO site(s) . Protein site(s) .Therefore, carbon dioxide is not just a “greenhouse gas” but also an abundant molecule with many potential applications for life. In the supercritical phase, it is an aprotic solvent miscible with a variety of organic liquids and some bacteria and their enzymes are active in this solvent. Planetary environments with supercritical carbon dioxide exist under the seabed of the Earth, on Venus, and probably on exoplanets such as Super-Earths.


Voluntary medical male circumcision (VMMC) for HIV prevention has been a priority for Swaziland since 2009. Initially focusing on men ages 15–49, the Ministry of Health reduced the minimum age for VMMC from 15 to 10 years in 2012, given the existing demand among 10- to 15-year-olds. To understand the implications of focusing VMMC service delivery on specific age groups, the MOH undertook a modeling exercise to inform policy and implementation in 2013–2014.The impact and cost of circumcising specific age groups were assessed using the Decision Makers’ Program Planning Tool, Version 2.0 (DMPPT 2.0), a simple compartmental model. We used age-specific HIV incidence from the Swaziland HIV Incidence Measurement Survey (SHIMS). Population, mortality, births, and HIV prevalence were imported from a national Spectrum/Goals model recently updated in consultation with country stakeholders. Baseline male circumcision prevalence was derived from the most recent Swaziland Demographic and Health Survey. The lowest numbers of VMMCs per HIV infection averted are achieved when males ages 15–19, 20–24, 25–29, and 30–34 are circumcised, although the uncertainty bounds for the estimates overlap. Circumcising males ages 25–29 and 20–24 provides the most immediate reduction in HIV incidence. Circumcising males ages 15–19, 20–24, and 25–29 provides the greatest magnitude incidence reduction within 15 years. The lowest cost per HIV infection averted is achieved by circumcising males ages 15–34: $870 U.S. dollars (USD).The potential impact, cost, and cost-effectiveness of VMMC scale-up in Swaziland are not uniform. They vary by the age group of males circumcised. Based on the results of this modeling exercise, the Ministry of Health’s Swaziland Male Circumcision Strategic and Operational Plan 2014–2018 adopted an implementation strategy that calls for circumcision to be scaled up to 50% coverage for neonates, 80% among males ages 10–29, and 55% among males ages 30–34. Voluntary medical male circumcision (VMMC) has been shown to reduce female-to-male transmission of HIV by 60% –3 and isSoka Uncobe (meaning circumcise and conquer in SiSwati), was launched with the goal of circumcising 80% of males ages 15–49 within one year. While the ambitious ASI target was not met, important lessons were learned, especially about demand creation and the need to have strong community engagement and government leadership and coordination. Routine program data revealed important variation in demand for VMMC by age. Even though the program targeted 15- to 49-year-old males, there was significant demand for circumcisions among 10- to14-year-olds. Whereas clients ages 10–24 years comprised 75% of the client population between the start of the program in 2009 and the end of 2014, clients ages 25 years and above made up a lower proportion of clients than would be expected, based on the age distribution of uncircumcised males in the overall population. Fewer than 5% of clients were above the age of 35. In response to the demand for circumcision among 10- to 15-year-olds, the Ministry of Health decided to reduce the minimum age for VMMC from 15 to 10 years in 2012.In 2008, Swaziland developed the Policy on Safe Male Circumcision for HIV Prevention and the Strategy and Implementation Plan for Scaling up Safe Male Circumcision for HIV Prevention 2009–2013 ,8. In 20The Swaziland Ministry of Health (MOH) was interested in understanding the implications of focusing VMMC service delivery on specific age groups and aligning their VMMC targets with their implementation experience highlighting the heterogeneous demand among different age groups. In 2013/2014, the Decision Makers’ Program Planning Tool, Version 2.0 (DMPPT 2.0) was applied in Swaziland to assess the impact and cost of circumcising specific client age groups, and to inform the development of the country’s Male Circumcision Strategic and Operational Plan for HIV Prevention, 2014–2018 . This paThe DMPPT 2.0 model is described in detail elsewhere . BrieflyThe DMPPT 2.0 model is populated with population, mortality, and HIV incidence and prevalence projections from an external source. All model inputs are available in Numbers of VMMCs conducted in the country, disaggregated by year and age group, were provided by the Ministry of Health on July 29, 2015. The MC prevalence by age group in the model base year (2014) was derived from the most recent Demographic and Health Survey , adjusteTo examine the effect of client age on the impact of scaling up VMMC, we created a series of scenarios. Each scenario had a target of 80% MC coverage for a single age group or combination of age groups, leaving the target for the other age groups at the same level as the baseline. We created one scenario for each individual five-year age group, in addition to several scenarios with 80% targets for combined age groups, such as 10–34 or 15–29. Each scenario scaled up MC coverage between 2014 and 2018, by applying a linear interpolation to the baseline MC prevalence for each age group in 2013 and the target coverage in 2018. After 2018, the coverage for each age group was maintained at the target level. For each scenario we compiled the decrease in HIV incidence in the scale-up scenario compared with the baseline scenario in each year of the model, and the total number of circumcisions required during the scale-up phase (2014 to 2018).The following model outputs for each scenario were measured over the 15-year period between 2014 and 2028, inclusive: the total number of HIV infections averted in the population ; the number of VMMCs per HIV infection averted; and the total cost of the VMMC program. Costs, numbers of circumcisions, and infections averted were all discounted at a rate of 3% per year. Uncertainty around the age distribution of HIV incidence was calculated as described in .We assessed VMMC age-targeting strategies according to the four following metrics: the number of VMMCs per HIV infection averted over a period of 15 years or the efficiency of the strategies; the reduction in HIV incidence in the near term (five years) or the immediacy of impact; the reduction in HIV incidence in the medium term (15 years) or the magnitude of impact; and the cost-effectiveness of the strategies, measured as the cost per HIV infection averted over 15 years .Kripke et al.  show thaThe impact of scaling up VMMC among broader age groups that might represent actual implementation strategies (contiguous age groups versus discrete five-year age groups) is shown in We also analyzed the six scenarios outlined in The modeling analysis detailed in this paper illustrates that the potential impact, cost, and cost-effectiveness of VMMC scale-up in Swaziland is not uniform. It varies by the age group of males circumcised.Key model findings for Swaziland are as follows. The lowest numbers of VMMCs per HIV infection averted  are achieved when males ages 15–19, 20–24, 25–29, and 30–34 are circumcised. Circumcising males ages 20–24 and 25–29 provides the most immediate reduction in HIV incidence (by 2018). Circumcising males ages 15–19, 20–24, and 25–29 provides the greatest magnitude incidence reduction within 15 years (by 2028). Circumcising males ages 10–14 provides the greatest incidence reduction when considering the very long term. In general, the broader the age group included , the greater the impact will be. The impact among males ages 10–49 is slightly smaller than that among males ages 15–49, because of the effects of discounting. The lowest cost per HIV infection averted  is achieved by circumcising males ages 15–34 ($870). While not examined in detail in this paper, it should be clear from Because of the wide confidence intervals in the HIV incidence estimates from the SHIMS study, upon which the uncertainty bounds for the model estimates depend, there is overlap in the uncertainty bounds between age groups for the estimates for the numbers of VMMCs per HIV infections averted, the numbers of HIV infections averted, and the cost per HIV infection averted.  Therefore, these results are not distinguishable according to this analysis. However, for model exercises for other country applications, which had much narrower uncertainty bounds around the model estimates due to different methodology, there was much less overlap in the model estimates –19.Despite the wide uncertainty bounds, policymakers in Swaziland chose to base their revised age targeting strategy on the model results. The Extended National Multisectoral HIV and AIDS Framework 2014–2018  had previously identified male circumcision as a priority program for its “maximum scenario package,” calling for 70% coverage of men ages 10–49 by 2018. In order to balance impact, cost, and program feasibility—while also aligning with this goal of 70% coverage—the Ministry of Health chose to use the DMPPT 2 modeling results to directly inform the circumcision targets and priorities within the Swaziland Male Circumcision Strategic and Operational Plan for HIV Prevention for 2014–2018. The country also used the model to set age-specific annual targets at the inkhundla (district) level.The Ministry of Health chose to adopt Scenario E, which calls for circumcision to be scaled up to 50% coverage for neonates, 80% among males ages 10–29, and 55% among males ages 30–34 see , for itsIt should be noted that EIMC was included in the scenarios analyzed, because circumcising 50% of infants is part of the eNSF target. EIMC adds to the costs of the program but does not contribute to infections averted over the 15-year period examined; it is included in the strategy as part of the plan for sustainability of the VMMC program and also because of its long-term impact.The country chose age 10 as the lower bound for scenarios to consider, because of the large proportion of current VMMC clients ages 10–14 years. This was done even though over the time frame studied, inclusion of males ages 10–14 leads to only small increases in HIV infections averted and results in a higher cost per HIV infection averted. While an analysis focused on short-term impact might lead one to conclude that circumcising males ages 10–14 should not be a program priority, turning away circumcision clients is viewed as problematic by implementers, given the limited demand for VMMC in Swaziland to date among older age groups and the fact that circumcising 10- to 14-year-olds contributes to medium-term (15-year) and long-term (30-year) impact but not to immediate (5-year) impact.Although the limitations of the DMPPT 2.0 model have been described elsewhere , the folhow to scale up VMMC in Swaziland. It does not address the programmatic considerations critical to scale-up, including the demand creation necessary and its cost, or the human resource requirements—including the importance of task-shifting to providers in rural health centers, for example.While providing insight on the impact and cost of the age-targeting options for the VMMC program in Swaziland, this modeling does not provide information on S1 AppendixSee (XLSX)Click here for additional data file.S1 FigVertical bars represent 95% confidence intervals.(TIF)Click here for additional data file.


Introduction: Adenocarcinomas, commonly present as a dominant lesion (DL) with additional nodules in the ipsilateral or contralateral lung. We sought to determine the fate and management of the secondary nodules and to assess the risk of these nodules using the Lung CT Screening Reporting and Data System (Lung-RADS) criteria and the National Comprehensive Cancer Network (NCCN) Guidelines to determine if surveillance is an appropriate strategy.Methods: We retrospectively evaluated patients with lepidic growth pattern adenocarcinoma and secondary nodules from 2000 to 2013. Risk assessment of the additional lesions was completed with a simplified model of Lung-RADS and NCCN-Guidelines.Results: Eighty-seven patients underwent resection of 87 DLs (Group 1) concurrently with 60 additional pulmonary nodules (Group 2), while 157 non-DLs were radiologically surveyed over a median follow-up time of 3.2 years (Group 3). Malignancy was found in 29/60 (48%) nodules in Group 2. Whereas, only 9/157 (6%) of the lesions in Group 3 enlarged, 4 of which  were found to be malignant, and then treated, while the remaining nodules continued surveillance. After applying the Lung-RADS and NCCN simplified models, nodules in Group 2 were at higher risk for lung cancer than those in Group 3.Conclusion: In patients with lepidic growth pattern adenocarcinoma associated with multiple secondary nodules, surveillance of the remaining nodules, after resection of the DL, is a reasonable strategy since these nodules exhibited a slow rate of growth and minimal malignancy. In contrast, nodules resected from the ipsilateral lung at the time of the DL, harbor malignancy in 48%. Risk assessment models may provide a useful and standardized tool for clinical assessment of pulmonary nodules. Advances in computed tomography (CT) as well as the increasing number of lung cancer screening programs has led to an increase in the detection of pulmonary nodules. The prevalence of multiple small lung nodules in the initial Early Lung Cancer Action Program (ELCAP) study  was 23% Several algorithms such as International Early Lung Cancer Action Program (I-ELCAP) and National Comprehensive Cancer Network (NCCN) have been used to assess the importance of nodules identified during routine follow-up after identification or lung cancer screening , 9. ThesWe performed a retrospective chart and pathologic review from 2000 to 2013 of patients with lung adenocarcinoma with lepidic features presenting with additional lung nodules. Patients were primarily identified from the Division of Thoracic Surgery database and consequently confirmed as having “BAC” or adenocarcinoma with a “BAC” component in the Cellnetix Pathology database. The Swedish Medical Center Institutional Review Board approved the study protocol. Individual consent was waived due to the retrospective nature of the study.We initially identified 103 patients who presented with multiple lung nodules. All patients underwent resection of the DL and were evaluated as having “BAC” or adenocarcinoma with a “BAC” component on pathology. The DL was defined as the one for which the workup and surgery were performed. More specifically, the DL was a new lesion clinically suspicious for malignancy, or a lesion which on follow-up that had either enlarged in total size, developed or increased its solid component, and/or was PET positive (SUV >2.5) .Lung nodules in this study were defined as focal non-linear opacity on chest imaging. They were characterized by size, location, and morphology . All non-calcified nodules identified were included in the study but a positive finding on the CT scan was a nodule measuring 6 mm or more in the mean diameter . NodulesWe performed a multi-disciplinary review of the resected secondary nodules to assess the histology using Martini and Melamed criteria . Second After resection of the DL, patients were followed-up on an outpatient basis for surveillance and evaluation of recurrent disease at 4 monthly intervals for the first 2 years, 6 monthly intervals for the next 3 years, and then annually after 5 years. Patients would undergo at least 1 annual CT scan with or without contrast alternating with chest radiography, unless new findings dictated further investigation. Co-existing nodules and any new nodules were managed largely according to the I-ELCAP Algorithm and Guidelines .The overall growth of nodules was radiologically assessed by the volume doubling time (VDT) measurement, which was calculated using the following equation based on the modified Schwartz formula .t is the interval, measured by days, between the two CT scans used for the assessment of nodule growth; while V1 and V2 are the initial and final nodule volume, respectively. Nodule volume (V) = π/6(ab2), where a is the longest horizontal axis and b is the maximum perpendicular diameter , which means at the time of primary surgery when the DL was resected, at the specific follow-up time when lesions were found to be enlarged (Time 2) and then after additional diagnostic studies were performed due to suspicion of lung cancer (Time 3). The term “diameter” in both models refers to the mean of the longest diameter of the nodule and its perpendicular diameter.First, the “risk” of each nodule was evaluated by assessing the additional nodules with a simplified model of either Lung-RADS assessment categories Table  8). Acc. Acc8). Second, the “risk” of each nodule was also assessed by the NCCN Guidelines for lung cancer screening Table  9). Siz. Siz9). t-test was used for single comparisons of continuous variables and ANOVA test for multiple comparisons. A p value <0.05 was considered significant. Statistical analyses were performed using the SPSS 19.0 statistical software package .Chi-square test was used to compare categorical variables. Student’s A total of 87 patients with 304 nodules were included. Demographic characteristics of the patient cohort are demonstrated in Table Group 1 includes the 87 DLs that were resected, while Group 2 includes 60 additional nodules that were resected in addition to the DL: 57 were concomitantly removed and 3 were subsequently removed from the contralateral lung at a subsequent additional operation within 3 months of the initial DL surgery. Finally, Group 3 includes 157 additional nodules that were radiologically observed. The radiological features of the nodules within three groups are showed in Table Dominant lesions were resected by lobectomy, segmentectomy, and wedge resection in 55, 17, and 28% of cases, respectively. Also, 60 additional nodules were resected, including 57 ipsilateral nodules concomitantly removed with the DL. Specifically, 36/57 (63%) lesions were in the same lung tissue of the resected DL, while 21/57 (37%) lesions required additional procedure, mainly a wedge resection and a segmentectomy in two cases. Three contralateral nodules were resected by a wedge resection at a subsequent operation 3 and 8 weeks after resection of the DL.All resected DLs in Group 1 were malignant, mainly consisting of well or moderately well differentiated IA Table . In GrouA representative case is included in Figures After excluding nodules in the same lobe as the DL, the remaining nodules in G2 and all nodules in G3 were assessed by using a simplified Lung-RADS assessment and a modified version of the NCCN Guidelines Table . In GrouIn Group 3, Lung-RADS assessed a Category 2 risk to 137/157 (87%) nodules and NCCN assessed a Category A risk to 113/157 (72%) based on the CT prior to surgical resection of the DL. During surveillance, all but nine nodules remained stable and the nodules that were stable were down graded to a low risk category in each system. Risk assessment was performed and reported on the nodules that enlarged at three specific intervals of time as define in the methods Table . After aThe primary finding in this study is that nodules identified on pre operative imaging and followed after resection of the early stage dominant adenocarcinoma have a low rate of enlargement and only 2.5% become malignant at 3.2 years of follow-up. However, resected nodules found in the ipsilateral lobes separate from the DL have a rate of malignancy 48%. Nodules in the ipsilateral thorax, size, morphology, location, PET avidity, and shorter VDT are more concerning for malignancy. In addition, risk assessment with Lung-RADS or NCCN nodule guidelines offer reassurance of the importance of select non-dominant nodules that may benefit from treatment. Taken together, these data would support a strategy of surveillance of existing nodules after resection of the dominant adenocarcinoma.The rate of malignancy in these secondary nodules in our series is similar to a recent analysis by Stiles and colleagues  who deteThese results also suggest that there is some judgment by the surgeon during pre-operative evaluation and during surgery that deems these nodules to be important enough to consider resection. The surgical decision making is easier if these are peripheral lesions amenable to additional wedge resections in a patient with excellent pulmonary function. The decisions are more difficult with a deeply seated lesion, multiple nodules or when operating on a patient with limited pulmonary reserves. For appropriate lesions that we are concerned about, we consider a pre surgery navigational bronchoscopy or even transthoracic needle biopsy to help plan surgery at the current time.During surgical planning, our results suggest that assessment of the non-dominant nodule(s) using the NCCN Guidelines may provide additional information. When the lesion was graded a Category B or C risk on the pre-operative scan 10/12 (83%) lesions were found to be malignant. Whereas, the assessment of risk by Lung-RADS at a single point in time does not seem to provide additional discrimination because this system relies in changes over time to determine the likelihood of malignancy. However, the size of the lesion is the primary indicator in the NCCN Guidelines (>6 mm) and in a larger series of patients may be prone to “error” with small lesions.The application of both of these models during follow-up of the 157 observed nodules seems to be appropriate since the scenario is akin to lung cancer screening for which these two tools were developed. In this situation, both size of the lesion and an increase in the size of the lesion are key factors, which ultimately determine risk. For most surgeons, these two factors already enter into the evaluation of an existing pulmonary nodule. However, growth rate and PET avidity of the nodules have also been shown to represent important predictors of malignancy , 18, whiWhether and how these two risk assessment tools can be integrated into our practice remains an ongoing process of evaluation. Surgeons have two needs: determining the risk of nodules while planning a surgical intervention and surveillance of nodules. One interpretation of our data may be that neither of these risk assessment tools was better than surgeon judgment in surgical planning but there remains a need to be able to more reliably predict malignancy since two studies show that half of nodules resected were benign. We believe that surveillance of nodules whether at screening or in cancer surveillance is a process for which our team has followed nodules using the I-ELCAP protocol for over a decade and this experience cannot be underestimated. However, an additional simplified tool for all team members may be useful and the ultimate benefit of these models during surveillance is that they provide a standardized tool for the whole thoracic oncology team  to use rather than relying on one physician’s individual assessment for clinical assessment of the radiological findings. A prospective study to test the applicability to a non-screened cohort may be of use.There are several limitations to our study. First, we retrospectively applied the risk assessment models to not only a predominant non-screened population, but also where we knew the final histology of the nodules. Further prospective study and analysis is required to ascertain if this is valid. Second, there was no mandated CT slice thickness protocol. Most of the patients had a standard dose chest CT scan with or without contrast but the slice thickness varied from 2.5 to 5 mm, which could alter the nodule counts and assessment. However, this is how patients present in clinical practice. Third, the duration of follow-up is relatively short at a median of 3.2 years. Even though we may consider solid nodules that are stable past 2 years of surveillance indolent, according to the Fleischner society , 42% of In summary, surveillance of the remaining nodules after resection of the DL in patients with adenocarcinoma and a lepidic component associated with additional nodules is a reasonable strategy. These nodules exhibited a growth rate of only 6% and malignancy rate 2.5% in the 157 lesions for which follow-up strategy was undertaken. In contrast, nodules resected at the time of the DL harbor malignancy in 48% of the 21 nodules that were located in a different lobe from the DL. Application of Lung-RADS and NCCN risk categories may provide additional information about the importance of theses nodules particularly as a more standardized tool for the clinical assessment of multiple lung nodules but requires further study.The authors declare: substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work. Drafting the work or revising it critically for important intellectual content. Final approval of the version to be published. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.