A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase. In the past decade, DNA-encoding of small molecule libraries has emerged as an attractive strategy for the discovery of novel ligands to biological targets12356789DNA-recording approaches require the iterative construction of both the chemical library members and the encoding oligonucleotide. In the original reports of DNA-encoding from the early 1990’s, the encoding DNA was built using solid-phase oligonucleotide synthesis with phoshoramidite building blocks1114Recent reports from Brown, El-Sagheer and Tavassolli have demonstrated that oligonucleotides containing a triazole linkage in place of a phosphodiester are competent substrates for PCR and thus could provide a “readable” encoding sequence161718Previous work had qualitatively shown that triazole-containing oligonucleotides could be amplified by PCR and that a triazole-containing plasmid could be translated in living bacteria173 , 5 – A  and to a lesser extent, 5 + A  were observed. While the mechanism of formation of these side products is unknown, the fact that similar products were not observed in the full-length product indicates that they are a consequence of triazole-induced stalling during the polymerization reaction. We wondered if the 20-base sequence upstream of the triazole could be reduced in length while maintaining read-through. Oligonucleotide 8, in which the run-up length was reduced to 8 bases, was synthesized. Read-through experiments with Klenow fragment gave similar results as with 3.With oligos The conceptual design of the chemical ligation strategy is shown in 11. This material was obtained from Prime Organics Inc. . With monomer 11 in hand, we set out to obtain a set of tag oligonucleotides for library synthesis. Tag oligonucleotides were synthesized by Biosearch Technologies  and designed to conform to the following architecture:Synthesis of 3′-TIPS protected oligos required the TIPS-containing monomer 5′-azido-TXXXXXXXXXXXXXU-3′-propargyl-TIPS9 was constructed by a repetitive sequence of CuAAC ligation and TIPS deprotection. It was used as a template for primer extension as described above using the FAM-labeled version of primer 4. The results of the primer extension reaction are shown in trT junction was correctly transcribed by Klenow fragment. In this case there are both the rGtrT and(r)UtrT junctions in the template; we hypothesize that the latter is copied into a single A residue. Similar behavior was observed by El-Sagheer and Brown in transcribing across TtrT junctions, although the linkage chemistry was different than that used hereTaq and Pfu), no skipping of T residues was observedWith the azido / TIPS propargyl tags in hand, we were able to construct a template containing multiple triazole junctions, similar to a theoretical library member. Oligo 10 containing three triazoles. Primer extension of this template again gave acceptable yields (>50%) of full-length product. In this case, the mass of the full length product corresponded to loss of two A residues, consistent with the behavior observed with template 9. These data gave us confidence that DNA encoding regions containing multiple triazole linkages would be readable by primer extension and sequencing. While the possibility of skipped T’s at the junctions might complicate the sequence analysis, we reasoned that as long as these nucleotides were not used to encode information, losing an A residue from the junctions was tolerable. We chose to embark on synthesis of a chemically DNA-encoded small molecule library.In addition to the experiments described above, we extended the study to template For library synthesis, we chose a strategy based on a set of bromoarylcarboxylic acids. The synthetic route is shown in 222526Prior to selection, the 334 million compound library was subjected to TIPS deprotection and ligated by CuAAC with a terminal self-priming hairpin oligonucleotide. Primer extension with Klenow yielded library with a double-stranded encoding oligonucleotide, suitable for affinity selection. We subjected the library to affinity-mediated selection against the target soluble epoxide hydrolase (sEH). This enzyme is involved in the conversion of epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Various studies have shown that EETs have anti-inflammatory properties, and so it has been suggested that inhibitors of sEH could have beneficial effects in COPD, cardiovascular disease, and even diabetes. Researchers at GSK have reported on sEH inhibitors that show beneficial effects in a cigarette smoke exposure mouse model28Selections were performed by conventional methods as described elsewhere. Library was incubated with His-tagged sEH protein in solution, followed by protein capture using an immobilized-metal affinity chromatography matrix (IMAC) resin. After washing, bound library members were eluted by heat denaturation. The resulting population of enriched library members was subjected to a second round of selection. The selection output was amplified by PCR and sequenced using an Illumina HiSeq 2500 instrument; the sequencing output was translated into chemical identifiers.m-boronyl benzamide substructure. Interestingly, the N-benzyl carboxamide moiety had been previously shown to be a key component of sEH inhibitors discovered through an encoded library appoach. Sequencing of naïve library and library selected without sEH did not show these features, giving us confidence that these compounds were specifically binding to sEH protein.The output of the sEH selection was visualized using a cubic plot. Several families of compounds were enriched, with the most abundant being the one illustrated in p-brominated nicotinates or picolinates. The most abundant trisynthon in the family was the combination of N-aminoethyl pyrazole at Cycle A, 5-bromonicotinic acid at Cycle B, and N-cyclohexyl-3-boronylbenzamide at Cycle C. The corresponding compound 12 (with a methyl group in place of the DNA linker) was synthesized and assayed for sEH inhibition, and found to have an IC50 value of 2 nM. The building block combination that comprises compound 12 was observed thirteen times among the 147,885 sequence reads generated for the selected library, and not at all in the 2,037,412 sequence reads generated for the naïve library. This represents an approximately 30,000-fold enrichment over two affinity-mediated selection steps when compared to the average within the naïve library.The prevalence of the individual building blocks in the family is shown in In summary, we have demonstrated that chemical ligation is a feasible approach to the catenation of encoding oligonucleotides in DNA-encoded chemistry. The method relies on the ability of Klenow fragment of DNA Polymerase I to translocate through triazole linkages in a DNA backbone. We have developed a protecting group strategy based on the alkynyl TIPS group that allows for repetitive and specific installation of multiple oligonucleotide tags. The chemical ligation strategy has been validated by the construction of a 334 million-member library of biaryls, and its successful interrogation for inhibitors of the enzyme soluble epoxide hydrolase. We believe this chemical ligation methodology represents an advance over previous methodsHow to cite this article: Litovchick, A. et al. Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library. Sci. Rep.5, 10916; doi: 10.1038/srep10916 (2015).


Gene transcription mediated by RNA polymerase II (pol-II) is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2). The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha . To understand changes in transcription arising due to stimuli it is useful to model the dynamics of transcription. We present a probabilistic model of pol-II transcription dynamics that can be used to compute RNA transcription speed and infer the temporal pol-II activity at the gene promoter. The inferred promoter activity profile is used to determine genes that are responding in a coordinated manner to stimuli and are therefore potentially co-regulated. Model parameters are inferred using data from high-throughput sequencing assays, such as ChIP-Seq and GRO-Seq, and can therefore be applied genome-wide in an unbiased manner. We apply the method to pol-II ChIP-Seq time course data from breast cancer cells stimulated by estradiol in order to uncover the dynamics of early response genes in this system. Transcription mediated by RNA polymerase II (pol-II) is an essential process in the expression of protein-coding genes in eukaryotes. Transcription is dependent upon a number of sequential and dynamic events, such as recruitment of pol-II to the transcriptional start site, activation of pol-II through phosphorylation of its C-terminal domain, elongation of the nascent transcript through the transcribed region and termination Chromatin immunoprecipitation, in conjunction with massively parallel sequencing (ChIP-seq) evaluates interactions between proteins and DNA, and, for example, can be used to monitor the presence of pol-II on DNA. Estimating the amount of pol-II associated with a transcribed gene provides a measure of transcriptional activity et al. fit a mechanistic model of pol-II transcription to nascent RNA data at a single locus and obtained a transcription speed of 4.3 kilobases per minute et al. activated transcription of genes greater than 100 kbp in length and estimated the transcription speeds using a model that measures an intronic RNA signal through taking advantage of co-transcriptional splicing. They obtain an average transcription rate of 3.1 kbp min−1−1A number of studies have attempted to determine the rate of transcription through modelling the dynamics of pol-II. Darzacq One way around the low temporal resolution of typical high-throughput time course data is to employ a non-parametric model of the biological signals of interest. In many cases we expect these signals to vary continuously and smoothly in time, when averaged over a cell population, and a Gaussian process model provides a convenient non-parametric model in such cases Here we present a Gaussian process model of transcription dynamics which can be fitted to genome-wide pol-II occupancy data measured using ChIP-Seq. The model describes the movement of pol-II through the gene body and combines a flexible model of promoter-proximal pol-II activity with a reliable estimate of transcription speed. By identifying genes which fit the model well, we provide a useful method to identify actively transcribed genes. The model does not assume a constant transcription speed and can therefore identify variable rates of transcription, for example due to transcriptional pausing. Model parameters are inferred using either maximum likelihood (ML) estimation or via Bayesian inference using Markov chain Monte Carlo (MCMC) sampling. The Bayesian approach provides confidence intervals for parameter estimates and can incoporate priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments.We fit our model to a pol-II ChIP-Seq time course dataset from MCF7 breast cancer cells stimulated with estradiol. The model is used to identify the set of transcriptionally engaged genes and estimate their mean transcription rate and transcriptional activity near the promoter. By clustering promoter activity profiles, potential co-regulated groups of genes are identified, particularly those that respond rapidly to estrogen signalling. Subsequent characterisation of transcription factor (TF) binding sites in proximity to the promoters of genes within clusters provides a means of classifying groups of promoters that are responsive to the binding of specific combinations of TFs. Additionally, publically available ChIP-Seq datasets of TF profiles from the same system were used to identify cluster-specific patterns in TF-binding. The rates of transcription estimated by our model are consistent with the literature Our methodology has a number of advantages. We do not require data with high temporal resolution, making it feasible to model transcriptional dynamics genome-wide using ChIP-Seq or GRO-Seq time course data. We infer transcription rates for all genes in an unbiased manner and by using Bayesian parameter estimation we are able to associate our transcription rate estimates with confidence intervals. Our model is non-parametric and therefore does not make very strong assumptions about the temporal changes in transcriptional activity. Fitting the model genome-wide allows us to identify and filter out transcripts where pol-II does not travel down the gene body. This provides a principled method to identify responsive genes, in particular, early acting estrogen responsive genes in the specific application considered here. Since our model does not enforce a uniform transcription speed over the entire gene body, we can take into account phenomena such as pol-II pausing which would result in a non-uniform transcription speed. We also use this model to infer the promoter activity of transcriptionally engaged genes, to identify co-regulated gene modules downstream of estrogen signalling.Visualizing pol-II ChIP-seq reads mapped to transcriptional units at multiple time points following the addition of estradiol to MCF7 cells reveals the motion of pol-II through the gene body of estrogen responsive genes see . ComputiA Gaussian process (GP) is a distribution over the space of functions. This distribution is completely specified by a mean function Here we introduce a novel application of GPs to modelling the spatio-temporal dynamics of pol-II occupancy during transcription. Convolved GPs allow the modelling of correlations between multiple coupled data sources. In our case these data sources are the pol-II occupancy over time collected at different locations along the transcribed region of a gene. Modelling the data as a convolved process borrows information from these different data sources in estimating the model parameters and inferring the underlying signal in the data. Also, we find that convolved GPs are necessary to account for changes in the shapes of signals observed at different regions of the gene. In linear systems theory, the output In order to capture the movement of the transcription wave through transcriptional units, we divide each gene into The estimated delay  out see . This phUsing Similarly,Let For a fully Bayesian approach, we take advantage of the fact that the parameters are positive and bounded. We transform the parameters using a logit transform and work with unconstrained variables. We place a Gaussian prior over the parameters in the transformed domain and draw samples from the posterior using the Hamiltonian Monte Carlo (HMC) algorithm https://github.com/ciiram/PyPol_II.Code to implement the method is freely available as a Python package, PyPol-II, which can be downloaded from When fitting the model, we fix To compute the average transcription speed we plot the position along the gene in base pairs (bp) versus the delay in minutes and compute a linear regression through the origin. The slope of the regression line gives us the transcriptional speed. Each sample of the parameters provides a set of delay estimates from which we obtain a speed estimate.A key component of our method involves the estimation of delay between time series observed at different segments of the gene. The study of time delay between related time series has received attention from a number of researchers for a long time The delay is estimated by finding the value of In the kernel based approach of equation  is modelThe value of We used synthetic data and previously published experimental data to assess our novel method's performance. To generate the synthetic data, the underlying function equation  was giveN was fixed at 20 and the observation interval in vivo using 5,6-dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) and the pre-mRNA measured after the inhibitor was removed. As verified by the authors, the kinetics of pol-II and pre-mRNA are similar hence we expect good performance on this dataset to indicate applicability of our method to pol-II ChIP-seq data.To assess performance of our method on a well characterised real-world dataset we obtained a dataset from Singh and Padgett To demonstrate an application to pol-II ChIP-Seq data, we apply our model to investigate the transcriptional response to Estrogen Receptor signalling. ChIP-seq was used to measure pol-II occupancy genome-wide when MCF-7 breast cancer cells are treated with estradiol (E2). Cells were put in estradiol free media for three days. This is defined media devoid of phenol red (which is estrogenic) containing 2% charcoal stripped foetal calf serum. The charcoal absorbs estradiol but not other essential serum components, such as growth factors. This results in basal levels of transcription from E2 dependent genes. The cells are then incubated with E2 containing media, which results in the stimulation of estrogen responsive genes. The measurements were taken at logarithmically spaced time points 0, 5, 10, 20, …, 320 minutes after E2 stimulation.Raw reads were mapped onto the human genome reference sequence (NCBI_build37) using the Genomatix Mining Station (software version 3.2.1). The mapping software on the Mining Station is an index based mapper that uses a shortest unique subword index generated from the reference sequence to identify possible read positions. A subsequent alignment step is then used to get the highest-scoring match(es) according to the parameters used. We used a minimum alignment quality threshold of 92% for mapping and trimmed 2 basepairs from the ends of the reads to account for deterioration in read quality at the 3′ end. The software generates separate output files for uniquely mapped reads and reads that have multiple matches with equal score. We only used the uniquely mapped reads. On average about 66% of all reads could be mapped uniquely. The data are available from the NCBI Gene Expression Omnibus under accession number GSE44800.Time series of pol-II occupancy over various segments of genes were computed in reads per million (RPM) We first applied our methodology to synthetic data in order to compare its performance to other methods. We investigated the performance of five methods, namely cross-correlation (Corr), DCF, the kernel approach of SLC9A9 gene (the same data shown in We next applied the model to pre-mRNA data from Singh and Padgett We applied our method to a ChIP-Seq time-course dataset measuring pol-II occupancy genome-wide when MCF-7 cells are treated with estradiol (E2). For our initial experiment, we considered 3,064 genes which exhibit significant increase of pol-II occupancy between 0 and 40 minutes after E2 treatment. These genes were determined by counting the number of pol-II tags on the annotated genes in the RefSeq hg19 assembly at 0 and 40 minutes after E2 treatment and computing the TIPARP gene. MYH9 and RAB10. This motivates a closer look at long genes. The advantage of fitting each of the delay parameters independently instead of enforcing a linear relationship is that it allows us to take into account phenomena such as pol-II pausing and provides a means to filter genes where the values of estimated delay are not naturally ordered. Visual inspection of the inferred time series of the top ranked genes is consistent with a ‘transcription wave’ traversing the gene. The transcription wave is especially evident in the longer genes The inferred latent functions for each gene model the pol-II activity adjacent to the promoter. Clustering these profiles and examining the average profiles of each cluster allows us to visualise the general trends and also classify genes according to the immediacy and nature of the response. This provides an alternative to clustering based on mRNA abundance data (from microarray or RNA-Seq experiments) which is regulated both by mRNA production and degradation processes. The production of mRNA may be delayed relative to the actual activation of transcription at the promoter causing genes which are actually triggered at the same time to show different rates of mRNA production. Differences in degradation rate can also influence mRNA abundance profiles. It may therefore be difficult to distinguish early and delayed transcriptional regulation from mRNA abundance data.To classify the profiles we sample the mean of the latent function  in  and use JAK1 and JAK2 in cluster 10, which has a prominent early peak, suggests that the response of both genes to E2 is rapid and coordinated. Since these genes are known to act together in several biological pathways such as the IL-6 signaling pathway and the IFN gamma signaling pathway, their appearance in the same cluster suggests that the clustering is likely to reveal other biologically significant relationships. A closer look at the inferred pol-II promoter profiles of some examples in cluster 10, the earliest peaking cluster, and the corresponding inferred pol-II profiles over the last 20% of the genes reveals the possible influence of gene length on mRNA production and how clustering the inferred promoter profiles can account for this influence and uncover potential co-regulation. CLN8, BRI3BP and JAK2 in cluster 10. The 383 genes found to fit the model well were grouped into 12 clusters  with theet al.CASP7, FHL2, GREB1, ITPK1, NRIP1, WWC1 are found to fit our pol-II model well with ChIP-seq data. et al.  which are limited to the finite set of sampling times. We see that all mRNA peaks occur after WWC1 which is a long gene In Hah In the supplementary material, we compare the clustering obtained from the inferred promoter profiles to that obtained if the time series of the raw ChIP-seq reads are clustered and show that our model has the potential to uncover relationships which may be missed if we only consider the raw ChIP-seq reads.http://cistrome.org). In earlier work on the estrogen interactome, Fullwood et al.We investigated the TF peaks in a 40 kbp region around the gene transcription start site for all genes in each cluster using ChIP-seq data for a number of TFs measured under similar experimental conditions (i.e. MCF-7 breast cancer cells treated with E2) in the cistrome database Click here for additional data file.Text S1Supporting text. This file contains additional details of the mathematical model and results of biological validation via gene ontology analysis and transcription factor binding.(PDF)Click here for additional data file.


Purpose. There is no information on the change in prevalence of latent tuberculosis infection (LTBI) testing in the United States (US) following the introduction of the interferon gamma release assay (IGRA), a new and alternative diagnostic method for LTBI. The purpose of this study was to evaluate potential changes in the prevalence of LTBI testing in the US following the introduction of IGRA. Methods. This was a multiyear cross-sectional study using nationally representative data from the 1999-2000 and 2011-2012 US National Health and Nutrition Examination Surveys. Self-reported prevalence of LTBI testing was estimated among groups known to have increased LTBI risk. Descriptive statistics were used. Results. Compared to 1999-2000, significantly fewer individuals self-reported being tested for LTBI in 2011-2012 among Hispanic Americans  and among those with comorbidities . There were also nonsignificant trends towards less self-reported LTBI testing in 2011-2012 versus 1999-2000 among household contacts of active TB cases, foreign-born individuals, and African Americans. Conclusions. Despite the introduction of IGRA, LTBI testing occurs less frequently in the US among vulnerable groups. Possibly inadequate targeted LTBI testing could result in increased active TB in the US in the future.  Mycobacterium tuberculosis (TB) disease as a public health problem [In 1989, the United States (US) Centers for Diseases Control and Prevention set an aggressive national target of eliminating active problem , which i problem , 4. Soci problem , 4. Indi problem , 6.Tuberculin skin testing (TST) is the traditional tool for identifying LTBI. There are several limitations associated with TST: there are potential inaccuracies with test implantation and reading; two visits are required, one for test implantation and a second one for reading 48–72 hours later; and false positives can occur as a result of nontuberculous mycobacterial infection or previous Bacille Calmette-Guerin (BCG) vaccination, because the TST material contains antigens found in most nontuberculous mycobacterium strains and BCG [In 1999-2000, prior to the introduction of the IGRA, about 35% of foreign-born individuals and 13% of household contacts of active TB cases in the US were estimated to never have received LTBI testing . One migThis was a multiyear cross-sectional study using US nationally representative population-level data from the National Health and Nutrition Examination Survey (NHANES). The NHANES is a cross-sectional survey that is conducted in the US every two years and each survey sample represents the total noninstitutionalized civilian US population residing in the 50 states and District of Columbia. Sociodemographic and health information is collected from participants in person by trained professionals. Information from survey participants under 16 years of age is collected with the assistance of a parent or guardian being present.Questions related to previous testing for LTBI were included on the 1999-2000 and 2011-2012 survey cycles . In 1999Testing for LTBI was evaluated among five preselected groups of individuals: household contacts of active TB cases; foreign-born individuals; African Americans; Hispanic Americans; and individuals with certain comorbidities . Information regarding these variables was collected from survey participants via self-report and comorbidities were self-reported doctor-diagnosed conditions. The first four selected groups were chosen by the authors because they have been previously identified as being at increased risk for LTBI , 4. Comop < 0.05 threshold. NHANES uses a complex sampling design, employing stratification and multistage clustering [The prevalence of self-reported LTBI testing was calculated for each of our five selected groups in 1999-2000 and in 2011-2012. If an individual who had received LTBI testing belonged to more than one than LTBI risk factor group, he or she was included in the prevalence estimate for each group. Chi square test of proportions was used to determine statistically significant differences in prevalence estimates between the two time periods using ustering . To accoustering . All anaIn the 1999-2000 NHANES cycle, the numbers (and percentages) of individuals in our five selected groups who did not previously have active TB and who responded to the question relating to LTBI testing were as follows: household contact with an active case of TB = 223 (2.7%); foreign-born individuals = 1190 (14.6%), African Americans = 978 (12%), Hispanic Americans = 1309 (16%); and individuals with selected comorbidities = 2580 (41.6%). In the 2011-2012 NHANES cycle, the corresponding numbers (and percentages) were as follows: household contact with an active case of TB = 207 (2.4%); foreign-born individuals = 1393 (16.1%); African Americans = 1072 (12.4%); Hispanic Americans = 1375 (15.9%); and individuals with selected comorbidities = 3255 (45.7%).p < 0.0001) and among those with comorbidities   . There wTo our knowledge, this is the first study to describe changes in the self-reported prevalence of LTBI testing in the US over time. There were trends towards lower self-reported prevalence of LTBI testing in 2011-2012 versus 1999-2000 among multiple vulnerable groups and significantly lower prevalence among Hispanic Americans and individuals with comorbidities. This occurred despite introduction into the market of an alternative diagnostic tool to the TST, the IGRA, in 2001 . Our finThe decrease in LTBI testing prevalence between 1999-2000 and 2011-2012 may be underestimated given that a smaller proportion of younger individuals were asked about LTBI testing in 2011-2012 compared to 1999-2000 . Analysis of 1999-2000 NHANES data shows that younger children are less frequently tested for LTBI than older children . The decrease in LTBI testing prevalence between 1999-2000 and 2011-2012 may also be underestimated given that pictures of what TST and tine testing look like were shown to participants as part of the 2011-2012 cycle, but not as part of the 1999-2000 cycle. Had such images been shown to participants in the 1999-2000 cycle, this may have led to the finding of a higher reported LTBI testing prevalence for that time.The finding of lower self-reported LTBI testing frequency in 2011-2012 may be in part explained by changes in LTBI testing strategies that have occurred in the US over time. Historically, all US residents were recommended to have at least one TST in their lifetime . HoweverThe finding of lower prevalence of LTBI testing over time consistently across multiple vulnerable groups raises concern that targeted LTBI testing is actually occurring less frequently. Should targeted LTBI testing be inadequate, this could result in increased active TB in the US in the future, especially since LTBI has slightly increased over time in the US  and sincThere are several study limitations. Our estimates of LTBI testing could be overestimated as a result of participants misinterpreting the LTBI testing question with BCG vaccine receipt. This potential limitation likely applies only to foreign-born individuals, as awareness and receipt of BCG vaccine would likely apply only to this group. Alternatively, LTBI testing could be underestimated as a result of recall bias or lack of awareness by some individuals that they had been previously tested by a simple blood draw. Results may have also been influenced by the difference in wording and content of the LTBI testing questions between the two survey cycles. For example, images of TST and tine testing were not made available in the 1999-2000 cycle, and while TST and tine testing were described to participants in both survey cycle questions, a description of the IGRA was not provided in the 2011-2012 cycle. Although data on LTBI testing in this study was based on patient self-report, which potentially introduces recall bias, objective measures of LTBI testing are not easily available and evaluation of previous LTBI testing in the “real clinical world” is based on patient self-report. NHANES did not collect data relating to reasons for LTBI nontesting and further research would be required to identify the factors contributing to the trends in decreasing LTBI testing in the US. The five selected groups at risk for LTBI that we studied also reflect a mixture of different and uneven LTBI risk factors. Finally, information was not available in NHANES to allow for evaluation of LTBI testing in other relevant vulnerable groups, such as individuals with HIV, organ transplantation, end-stage renal failure on hemodialysis, and recipients of immunosuppressive medications (like antitumour necrosis factor agents).Despite the introduction of a new and alternative testing method for LTBI, there were trends towards less LTBI testing over time in multiple vulnerable groups in the US and particularly Hispanic Americans and individuals with comorbidities. These findings raise concern that targeted LTBI testing may actually be occurring less frequently over time in the US. Possibly inadequate LTBI testing in the US may impede attaining the aggressive national TB elimination goals that have been set there.


Interphase chromosomes adopt a hierarchical structure, and recent data have characterized their chromatin organization at very different scales, from sub-genic regions associated with DNA-binding proteins at the order of tens or hundreds of bases, through larger regions with active or repressed chromatin states, up to multi-megabase-scale domains associated with nuclear positioning, replication timing and other qualities. However, we have lacked detailed, quantitative models to understand the interactions between these different strata.Here we collate large collections of matched locus-level chromatin features and Hi-C interaction data, representing higher-order organization, across three human cell types. We use quantitative modeling approaches to assess whether locus-level features are sufficient to explain higher-order structure, and identify the most influential underlying features. We identify structurally variable domains between cell types and examine the underlying features to discover a general association with cell-type-specific enhancer activity. We also identify the most prominent features marking the boundaries of two types of higher-order domains at different scales: topologically associating domains and nuclear compartments. We find parallel enrichments of particular chromatin features for both types, including features associated with active promoters and the architectural proteins CTCF and YY1.We show that integrative modeling of large chromatin dataset collections using random forests can generate useful insights into chromosome structure. The models produced recapitulate known biological features of the cell types involved, allow exploration of the antecedents of higher-order structures and generate testable hypotheses for further experimental studies.The online version of this article (doi:10.1186/s13059-015-0661-x) contains supplementary material, which is available to authorized users. The chromatin structure of human interphase chromosomes plays critical roles in a wide range of cellular functions and consists of many hierarchically arranged but interconnected layers of structure. These range from the three-dimensional arrangement of multi-megabase-scale domains within the nucleus down to the chemical modifications carried by individual nucleosomes and nucleotides at particular loci. A recurring question has been how these many different levels of chromatin structure are related to one another . In the Many aspects of higher-order chromatin remain broadly invariant between cell types, and genome-wide datasets as diverse as replication timing domains, lamin association domains and Hi-C interaction matrix eigenvectors show strong correlations across many different human cell lines . Indeed,Given the existence of neighboring chromatin domains with distinct structures and activities, the boundaries defining such domains have been a focus of particular interest. The topological domains (TADs) described by Dixon et al.  were repHere we exploit the unprecedented volumes of data produced recently  to proviIn common with previous studies of higher-order chromatin structure -11, therAlthough it is often assumed that higher-order chromatin domain organization  across the genome is to some degree dependent upon lower-level features , the identity and independent contributions of these features are unknown. Beyond this it has also been unclear whether there are strong enough dependencies to allow accurate prediction of higher-order structure. For each of the three Hi-C eigenvector datasets corresponding to the Tier 1 ENCODE cell lines  we assembled datasets of 35 matched locus-level chromatin features, including sites bound by 21 DNA binding proteins, and 11 histone modifications/variants and DNase hypersensitive sites see . The GC %, as accurate as 1-Mb models in terms of predicted and empirical PCC, Additional file While 1-Mb compartment eigenvectors are low resolution relative to that typically employed for chromatin immunoprecipitation sequencing (ChIP-seq) data, megabase bins are a suitable choice for analyzing large chromosomal compartments ,17. To cGiven the correlations seen between Hi-C eigenvectors from different cell types Figure  and the Many of the variables examined here are heavily interdependent, and for example co-occur in clusters denoting functional chromatin states . Care muConsistent with the presence of broad commonalities among the three models, cross-application of models showed that models trained in one cell type often performed well in another Figure . In eachWe compared the performance of our random forest approach with two other regression methods: simple multiple linear regression and partial least squares regression, a method particularly well suited to highly correlated inputs . While cAlthough the chromatin organization of much of the genome appears to be invariant between cell types Figure , some reA more conservative definition of structurally variable regions is that they are regions altering their compartment state (between A and B compartments) in one cell type relative to the other two. Such regions will often undergo dramatic changes between transcriptionally permissive and repressive environments and might be expected to be associated with cell-type-specific biology, such as functional chromatin states . This inFor each cell line, we identified all regions showing cell-type-specific occupancy of the active A compartment and ranked these regions according to the density of predicted active enhancers. Close examination of these regions reveals many examples of enhancer activity nucleated upon genes associated with cell-type-specific biology Figure A. The trA defining characteristic of active A compartment regions is a preferential bias in contacting other A compartment regions . HoweverThe mammalian genome is organized into TADs, predominantly self-interacting chromatin domains, with boundary regions reportedly associated with pronounced peaks and troughs of particular features within 500 kb of the predicted boundary . ExploraWe derived TAD boundaries according to established methods  for all Across all three cell types, several features demonstrate consistent and statistically significant patterns at TAD boundaries , repressed heterochromatin (null) and polycomb-associated (PcG) domains, based on the patterns of DNase hypersensitivity, H3k9me3 and H3k27me3, respectively  and at the level of locally enriched peaks at boundary regions (spanning 100 to 500 kb). This is intriguing as YY1 has recently been shown to co-localize with the architectural protein CTCF  and suggIt has become commonplace to discuss the multi-layered, hierarchical organization of interphase chromosomes across strata ranging from nuclear compartments, down to the spectra of histone modifications and bound proteins at individual sub-genic regions. However, we lack a detailed understanding of how these strata interact. We have shown that our perspectives of features occurring at different strata can be bridged by modeling approaches, and the models produced can be used to explore the interrelationships between these different features quantitatively.We constructed cell-type-specific models of nuclear organization, as reflected in Hi-C-derived eigenvector profiles, to discover the most influential features underlying higher-order structures. We found open and closed compartments to be well correlated with combinatorial patterns of histone modifications and DNA binding proteins, enabling accurate predictive models. These models could be cross-applied successfully between cell types highlighting constellations of common structural features associated with different nuclear compartments as expected. Dissection of the most influential variables also revealed important differences between models, consistent with the known biological contrasts among these cell types, such as the prominence of EGR1 in ESCs and H3K9me3 in the leukemia cell line. Investigation of regions showing variable nuclear organization across the three cell types under study, revealed enrichments for cell-type-specific enhancer activity, often nucleated at genes with known roles in cell-type-specific functions. Finally we used model predictions to examine boundary composition between higher-order domains across cell types. Among enrichments of a large number of factors observed at different boundaries in different cell types, CTCF and YY1 were found consistently and may cooperate to establish domain boundaries. In summary, we show that integrative modeling of large chromatin dataset collections using random forests can generate useful insights into chromosome structure and seed testable hypotheses for further experimental studies.Hi-C datasets for human cell types H1 hESC , K562 1 and GM12Genome-wide ChIP-seq datasets for 22 DNA binding proteins  and ten histone modifications  were produced by ENCODE , in addmtry=n/3=12 and ntrees=200 were assumed as the algorithm is known to be largely insensitive [A≥0 and B<0, was also calculated using accuracy (percentage correct classifications or true positives) and the area under the receiver operating characteristic (AUROC) curve. Together these give a comprehensive overview of model performance, both in terms of regression accuracy of the continuous eigenvector, and in how that same model could be used to label discrete chromatin compartments.Random forest regression  was usedensitive . Variablensitive . The effFor cross-application of cell-type-specific models, a single random forest regression model was learned from all 1-Mb bins for a given cell type. This was then used to predict all bins from each of the other two cell types. The median absolute deviation was chosen as a robust measure of the variability in a given 1-Mb block between the three cell types. Blocks were ranked by this measure and the distribution was split into thirds that represented low variability (the third of blocks with the lowest median absolute deviation), and mid and high variability. Each subgroup was then independently modeled using the random forest approach described above. For each cell type we identified 1-Mb regions whose compartment state was altered relative to the other two. For example, if a 1-Mb bin was classified as occupying compartment A in H1 hESC and B in both K562 and GM12878, it is said to occupy an altered open compartment in H1 hESC. Chromatin state annotations were calculated from ENCODE ChromHMM/SegWay combined annotations for each cell type [TAD boundaries were called using software provided by Dixon et al.  with recα=0.01 was then Bonferroni-adjusted for multiple testing correction, and results with P values exceeding this threshold were deemed significantly enriched or depleted at a given boundary.To test for the enrichment or depletion of a chromatin feature over a given boundary, a two-tailed Mann–Whitney test was used to compare the boundary bin with the ten outermost bins of the window (five from either side). The significance level at Scripts to reproduce the analyses and generate manuscripts figures are available at .


The introduction of mini-implants has broadened the range of tooth movements possible by fixed appliance therapy alone. The limits of fixed orthodontic treatment have become more a matter of facial appearance than anchorage. Many complex cases which would previously have required surgery or functional appliances can now be treated with fixed appliance therapy using mini-implants. A mutilated dentition case where mini-implants were used to provide anchorage for intrusion of molars and retraction of anterior teeth is reported here to illustrate this point. For years, orthodontic treatment has been limited in scope due to the range of tooth movements possible , 2. EvenA twenty-one-year-old female reported to the hospital for correction of protruding front teeth and replacement of missing lower back teeth. She had a Class I incisor relationship on a Class III skeletal base with an average maxillary-mandibular plane angle and increased lower face height . IntraorTreatment involved removal of upper first premolars and placement of mini-implants for lower anterior retraction and upper right second molar intrusion. The logic behind using mini-implants for lower anterior retraction was for anchorage due to missing lower first molars and lower right second molar. In addition, intrusion of 17 was required for placement of artificial substitute in the lower arch considering the level of extrusion of 17. Mini-implants of 1.3 mm × 8 mm  were used for this purpose. The implants were positioned palatally between 16 and 17 and buccally between 17 and 18. The palatal implant was placed 7 mm superior to gingival margin with care taken to avoid Greater Palatine Artery. The palatal mucosa was more fibrous and thicker than that on the buccal aspect. In the space closure stage, a lower removable partial denture was placed for space maintenance in the lower arch .  FigureOral rehabilitation was completed with the placement of a lower cast partial denture replacing the missing lower molars Figures , and 8. The advantages of using mini-implants in orthodontic treatment are clearly illustrated in the superimposed pre- and posttreatment cephalometric tracing of the above case report . EarlierQuite a number of recent publications illustrate the use of mini-implants for other types of tooth movements like molar distalization  and prot


Mild traumatic brain injury (mTBI) is often occult to conventional imaging techniques. However, there is growing evidence that mTBI patients who lack evidence of structural intracranial injury may develop post-concussive syndrome (PCS). We investigated longitudinal alterations in resting state functional connectivity (rs-FC) in brain networks in a population of 28 patients compared to 28 matched control participants. Rs-FC and cerebral blood flow (CBF) within the nodes of the Default Mode Network (DMN) and Task Positive Network (TPN) were assessed at three time points including acute, sub-acute, and chronic stages following mTBI. Participants received the Automated Neuropsychological Assessment Metrics (ANAM) to assess cognitive performance. Main findings indicate that despite normalized cognitive performance, chronic mTBI patients demonstrate increased rs-FC between the DMN and regions associated with the salience network (SN) and TPN compared to the control populations, as well as reduced strength of rs-FC within the DMN at the acute stage of injury. In addition, chronic mTBI patients demonstrate an imbalance in the ratio of CBF between nodes of the DMN and TPN. Furthermore, preliminary exploratory analysis suggests that compared to those without chronic PCS, patients with chronic PCS reveal an imbalance in the ratio of CBF between the DMN nodes and TPN nodes across multiple stages of recovery. Findings suggest that the altered network perfusion with the associated changes in rs-FC may be a possible predictor of which mTBI patients will develop chronic PCS. Traumatic brain injury (TBI) is one of the most prevalent neurological disorders in the United States, resulting in a large emotional burden on patients and a vast financial burden on the nation. The Center for Disease Control (CDC) estimates that there are 1.7 million cases of TBI each year . One of Therefore, further investigation of advanced neuroimaging techniques to better characterize mild TBI (mTBI) is needed. One possible technique is resting state functional MRI, which can be used to characterize the interactions both within and between various neural networks. This interaction is calculated by determining functional connectivity, which can be defined as “the temporal correlation of a neurophysiological index measured in different brain areas” . Two comIn ideal conditions, it has been shown that these two networks are anti-correlated at rest, which is represented by a negative resting state functional connectivity (rs-FC) . It has TBI has been shown to reduce rs-FC in multiple networks including interhemispheric rs-FC –28, the Using SPECT imaging, it has been shown that symptomatic mTBI patients demonstrate reduced regional CBF in the frontal, prefrontal, and temporal cortices in the chronic stage of injury (greater than two years post injury) . While CIn this study we followed a group of mTBI patients across the acute, sub-acute, and chronic stages of injury in order to investigate longitudinal changes in rs-FC within and between the DMN and TPN compared to control participants. In addition, we assessed whether mTBI patients exhibit alterations in the balance of resting perfusion within the DMN and TPN compared to control participants across the first 6 months following injury.This study was approved by the International Review Board at the University of Maryland and all participants provided written informed consent and HIPAA compliance.MTBI patients were prospectively recruited between March 2010 and May 2012 from the R Adam Cowley Shock Trauma Center at the University of Maryland Medical Center as part of a larger imaging protocol using a combination of advanced MR imaging and neuropsychological assessments including the Automated Neuropsychological Assessment Metrics (ANAM) . All parFor the analysis presented in this manuscript, only participants who completed longitudinal resting state fMRI across the three time points of interest were included resulting in a final patient population of 28 mTBI patients . Individual patient clinical characteristics, mechanisms of injury, and CT and MRI interpretation by board certified trauma radiologist (K.S.) are shown in All 28 mTBI patients received rs-fMRI at three time points. An initial stage (referred to as acute stage) within 11 days , a sub-acute stage approximately 1 month post injury , and a chronic stage . At each time point the patients also received a resting state perfusion scan, using the pulsed arterial spin labeling technique (PASL). However, due to motion artifacts (translation greater than 3mm or rotation greater than 3 degrees), resting state data from 1 patient at the acute stage was excluded from analysis, and PASL data from 2 patients at the acute stage and 2 patients at the sub-acute stage were excluded from analysis. The 28 control participants received rs-fMRI and PASL scans at a single time point.In an exploratory analysis, mTBI patients were further subdivided into two cohorts of those with and without PCS at the chronic stage based on self-reported symptoms on the Modified Rivermead Post-Concussion Symptoms Questionnaire (RPQ) . The RPQResting state fMRI data from a subset of this patient population has previously been published investigating the association between imaging findings and memory complaints (n = 16 at the sub-acute stage)  as well Patients underwent neuropsychological assessment at all three visits. However, due to the clinical condition or presence severe of post-concussive symptoms at time of the MRI, only 19 of the 28 mTBI patients at the acute stage and 27 out of the 28 mTBI patients at the sub-acute stage completed the neuropsychological assessment. One control participant declined participation in the neuropsychological assessment resulting a control population of 27 participants for the neuropsychological assessment. Imaging data was included in the analysis for this control participant. Due to the possible selection bias caused by one third of the mTBI patients lacking neuropsychological testing at the initial stage, we have excluded analysis of behavioral data for the acute time point. The level of cognitive functioning was assessed by the administration of the Mini Mental State Exam (MMSE) and Military Acute Concussion Evaluation (MACE)  at each The ANAM battery consists of seven subtests assessing processing speed, memory, and attention. The specific subtests included in this battery are the code substitution (CS), code substitution delayed (CSD), match to sample (MTS), math processing (MATH), procedural reaction time (PRT), simple reaction time (SRT), simple reaction time repeat (SRT2) . From eaAll imaging was performed on a Siemens Tim-Trio 3T MRI scanner using a 12 channel receive only head coil. A high resolution T1-MPRAGE  was acquired for anatomic reference with slices parallel to the anterior and posterior commissure points (AC-PC). For the rs-fMRI scan, T2*-weighted images were acquired using a single-shot EPI sequence  with 36 axial slices (sl. thick. = 4 mm) over 5 min 42 seconds that yielded 171 volumes. The perfusion scan used the pulsed arterial spin labeling (PASL) technique based on single-shot EPI readout  with 16 slices (sl. thick. = 5mm with 1mm gap) to cover the central portion of the brain with the spatial location of these slices matching the structural images. Forty-five pairs of labeled and control volumes were acquired following the initial acquisition of the constant equilibrium magnetization (M0) volume. The PASL imaging data was taken over 3 min and 57 seconds. During the resting state scans, participants were instructed to rest peacefully with eyes closed.http://www.fil.ion.ucl.ac.uk/spm) and included motion correction of the time series, slice timing correction, band pass filtering (.009Hz < f < .08Hz), and registration of all the 171 volumes to the first volume of the time series. Both the T1-MPRAGE and resting state series were directly spatially normalized to standard space using the Montreal Neurological Institute (MNI) templates available in SPM 8. The T1 template was used for normalization of the T1-MPRAGE and the EPI template was used for the normalization of the resting state time series. The SPM normalization step uses the normalized mutual information to calculate the optimum 12-parameter affine transformation  to match the size and position of the images. The resting state series was resampled to a spatial resolution of 2.0 mm isotropic. Spatial smoothing was then applied to the resting state data using a 5 mm Gaussian kernel. Individual T1-MPRAGE images in MNI space were segmented into white matter (WM), gray matter (GM) and cerebral spinal fluid (CSF). The segmented masks thus created were used to account for time series variance from the non-neuronal contributions of CSF and WM. The mean BOLD time series from the WM mask, CSF mask, and the 6 motion correction parameters were included in the model as regressors to remove the variance related to non-neuronal contributions and motion.Preprocessing of the imaging data was performed using SPM 8 (http://www.nitrc.org/projects/conn) was used to process the resting state data and create average group networks for the DMN and the TPN. For the DMN, the reference time series was selected from a 5mm spherical region of interest (ROI) in the posterior cingulate cortex (PCC) centered at  on the MNI template based on [The CONN-fMRI Functional Connectivity toolbox v13.h  and cluster extent threshold of p-value of 0.001 using a family wise error correction for multiple comparisons. Voxel wise functional connectivity maps were made separately for the control group, and the mTBI groups at each of the three time points. All between group contrast maps were created and were thresholded at voxel wise p-value of 0.005 (uncorrected) and cluster extent threshold of p-value of 0.05 using a family wise error correction for multiple comparisons.Within group rs-FC maps of the DMN and TPN were created using SPM8. Positive functional connectivity maps were thresholded at a voxel wise ROIs were created using a 10mm sphere centered at the peak voxel of each significantly correlated cluster for the DMN and TPN based the voxel wise group connectivity maps of the of an independent control group  created CBF maps were generated using in-house MATLAB  program as in . BrieflyDifferences in demographic characteristics between the control group and mTBI group were determined using unpaired t-tests. Group differences in measures of rs-FC  and rs-CBF  between the control group and mTBI group were tested using analysis of covariance (ANCOVAs) considering age as a covariate at each time point  separately. Longitudinal changes in imaging measures (rs-FC and rs-CBF measures) within the first 6 months following mTBI were determined using repeated measures analysis of covariance (ANCOVAs) considering age as a covariate with visit as the within group variable  implemented in SPSS. Longitudinal changes in neuropsychological measures within the first 6 months following mTBI were determined using repeated measures ANCOVAs considering age as a covariate with visit as the within group variable (sub-acute or chronic) implemented in SPSS. Within group differences between DMN CBF and TPN CBF were calculated using paired t-tests. Results shown are uncorrected for multiple comparisons.Both the control group and mTBI group at sub-acute and chronic time points performed similarly on the MACE . There were no differences in ANAM performance as measured by the WT-TH between the control group and mTBI group at either time point . There were no differences in global life satisfaction as measured with the SWLS between the mTBI and control group at the sub-acute (p = 0.352) or chronic (p = 0.453) stages of injury.There were no longitudinal differences between the sub-acute and chronic time points in the mTBI group for the MACE , ANAM performance , or SWLS .p-value of 0.005 (uncorrected) and cluster extent threshold of p-value of 0.05 FWE corrected, mTBI patients did not demonstrate any differences in rs-FC with the DMN compared to the control group in the acute or sub-acute stages. However, in the chronic stage, mTBI patients revealed increased rs-FC between the PCC node of the DMN with regions in the TPN such the dorsal anterior cingulate cortex (dACC)/premotor area (PMA), bilateral insular cortex (In), and L DLPFC , TPN rs-FC , or DMN-TPN rs-FC .Compared to the control group, there was no group differences in network CBF values for either the DMN CBF or TPN CBF for the mTBI group as a whole at any of the three time points . Furthermore, there were no differences in the CBF ratio between the mTBI group and the control group at any time point. As has been demonstrated by others , there wResults of the repeated measures ANCOVAs in the mTBI group failed to detect longitudinal differences in DMN CBF, TPN CBF, or the CBF ratio.There was significantly greater DMN CBF compared to the TPN CBF in the mTBI group without PCS at all three time points . However, there were no significant differences between the TPN CBF and the DMN CBF in the mTBI group with PCS at any time point  .Based on the results of the repeated measures ANCOVA, there were no between group differences or within group differences for DMN CBF or TPN CBF. For the CBF ratio, there were no significant within group differences  or between group differences  .Given the association between rs-FC between the DMN and TPN and cognition in prior studies among both healthy participants and various patient populations –20, we pWhile mTBI patients performed similarly to control participants on all of the neuropsychological assessments, alterations in rs-FC and perfusion pertaining to the nodes of these two networks were noted mainly during the chronic stage . As prevIt is interesting to note that similar to our previous report on sub-acute mTBI patients with memory complaints , the chrIn addition to alterations in rs-FC, an imbalance between resting perfusion values within the DMN and TPN was also noted in the chronic stage. The fact that such changes appear at the chronic stage suggests that recovery from mTBI is a constantly evolving mechanism that progresses over time possibly to help balance the basal activity of these two networks. Further, it could be hypothesized that this increased allocation of CBF to the TPN compared to the DMN may contribute the common symptoms of increased cognitive fatigue  or attenWhile the results do not show longitudinal changes in resting state or perfusion measures for the mTBI group as a whole, to further assess the changes in regional CBF, the mTBI group was further divided into those with and without chronic PCS (defined here as those still experiencing significant symptoms at least 6 months following injury.) While the primary goal was to see how the disruptions between the DMN and TPN affected patient symptoms and cognitive performance, a secondary goal was to investigate alterations in these networks in the acute stage in patients with and without persistent symptoms.Similar to our findings in the chronic mTBI population as a whole, the mTBI patients experiencing chronic PCS failed to maintain the expected balance between perfusion to the DMN and the TPN . The TPNThe findings of a modified interaction between the TPN and DMN among mTBI patients and especially among mTBI patients with chronic PCS should be taken in the context of the limitations of the study. The control group used in this study included healthy normal controls and not patients who suffered non-CNS related injury. It is possible that our choice of control participants could undermine some of the predisposing factors in the mTBI group. While our control participants were only scanned at one time point, recent groups have reported on the reliability of resting state measures within control populations ,65, alloIn conclusion, our data indicates an inability of mTBI patients to modulate the balance between the DMN and TPN at rest, as evidenced by alterations in network interactions in rs-FC and network resting perfusion measures across the first 6 months following injury. Our results are novel in that they extend the notion of DMN Interference in a mTBI population to include not only increased rs-FC between the networks, but also alterations in resting network perfusion. While these alterations are present in mTBI patients in the chronic stages, alterations in perfusion are present earlier in the acute and sub-acute stages in mTBI patients who suffer from persistent symptoms suggestive of distinctive courses of functional recovery in those with and without persistent symptoms. These findings raise the possibility that these resting state measures and altered perfusion may provide additional insight and aid clinicians in predicting the long-term outcomes of patients with mTBI.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Our aim was to replicate this observation in participants of European and New Zealand Polynesian (Māori and Pacific) ancestry. A total of 2250 clinically-ascertained prevalent gout cases and 13925 controls were used. Non-clinically-ascertained incident gout cases and controls from the Health Professional Follow-up (HPFS) and Nurses Health Studies (NHS) were also used. Genotypes were derived from genome-wide genotype data or directly obtained using Taqman. Logistic regression analysis was done including age, sex, diuretic exposure and ancestry as covariates as appropriate. The T-allele increased the risk of gout in the clinically-ascertained European samples  and decreased the risk of gout in Polynesians . There was no evidence for association in the HPFS or NHS sample sets. In conclusion TLR4 SNP rs2143956 associates with gout risk in prevalent clinically-ascertained gout in Europeans, in a direction consistent with previously published results in Han Chinese. However, with an opposite direction of association in Polynesians and no evidence for association in a non-clinically-ascertained incident gout cohort this variant should be analysed in other international gout genetic data sets to determine if there is genuine evidence for association.Deposition of crystallized monosodium urate (MSU) in joints as a result of hyperuricemia is a central risk factor for gout. However other factors must exist that control the progression from hyperuricaemia to gout. A previous genetic association study has implicated the toll-like receptor 4 (TLR4) which activates the NLRP3 inflammasome via the nuclear factor-κB signaling pathway upon stimulation by MSU crystals. The T-allele of single nucleotide polymorphism  Deposition of crystallized monosodium urate (MSU) in joints as a result of hyperuricemia is a central risk factor for gout. Genome-wide association studies of serum urate have confirmed uric acid transporters SLC2A9 and ABCG2 as important loci, that are also associated with gout, with weaker effects from other uric acid transporters –3. GivenTLR4 single nucleotide polymorphism  has been reported to be associated with gout in a Han Chinese sample set [rs2149356 with gout in individuals of European and New Zealand Polynesian ancestry.Toll-like receptors (TLRs) are transmembrane pattern recognition receptors expressed by innate immune cells that trigger an innate immune response by controlling distinct signaling pathways . In addimple set , where tmple set . Our aimwww.ncbi.nlm.nih.gov/gap) approval number was #834 for accessing data from the ARIC and FHS studies.Demographic and clinical data are presented in A separate data set, previously described in ref , of Eurors2149356 genotype was imputed using MACH1 v1.0.15 with the HapMap CEU sample set as reference haplotypes. In the ARIC sample set rs2149356 had been genotyped on the Affymetrix SNP 6 platform. The HPFS and NHS sample sets were genotyped using the Illumina Infinium OmniExpress and genotypes were imputed using MACH . This resulted in N = 10807 non-missing genotype calls (1.5% missing). There was no evidence for departure from Hardy Weinberg equilibrium in any of the sample sets presented in Tables PHWE>0.01).Taqman genotyping for rs2149356 was performed for the sample sets excepting ARIC, FHS, HPFS and NHS using a Lightcycler 480 Real-Time Polymerase Chain Reaction System  in 384-well plates. The FHS cohort had been genotyped by the Affymetrix SNP 5 platform and a custom-designed gene-centric 50K SNP platform and P < 0.05 was used to declare nominal statistical significance.All logistic regression analysis was done using Intercooled STATA software version 8.0 . Allelic and genotypic odds ratios for gout were calculated and all analyses were adjusted by age and sex, with the Polynesian analysis additionally adjusted by an estimate of Polynesian ancestry calculated as previously described in order to account for admixture with other ancestral groups . A thresrs2149356 was associated with the risk of gout when compared to controls unstratified by urate level, but in an opposing direction of association . The TT-genotype was also associated with risk of gout when compared to the GG genotype, again in an opposing direction of association . Rs2149356 was then tested for association with gout in the HPFS and NHS studies (P≥0.31).In European and Polynesian subjects, the T-allele of  studies . There wTLR4 variant rs2149356 in Han Chinese in sample sets of European and Polynesian ancestry. In the NZ and Europeans recruited from Europe the association was replicated but with opposing direction in the Polynesian sample set. A similar opposing direction of association between European and Polynesian is also evident at other urate- and gout-associated loci PRKAG2 and HLF [rs2149356 with gout. Two characteristics of this sample set would have reduced chances of detecting association, should genuine association exist. First, only ~90% have clinically-ascertained gout [TLR4 locus to be the first replicated genetic risk factor in gout outside of those that influence gout risk via modulation of serum urate levels. However, given the very strong prior functional candidacy of TLR4, we do consider that our study provides evidence consistent with a causal role for TLR4 in gout.Here we attempted to replicate the previously reported association with gout of ned gout . Second,ned gout ). Prevalrs2149356 is not the causal variant but in linkage disequilibrium with the causal variant, with a Polynesian-specific ancestral recombination event distinguishing the Polynesian haplotypic background around rs2149356 from European and Han Chinese, resulting in the other (G) allele of rs2149356 being on the Polynesian risk haplotype. The Polynesian population could be important in trans-ancestral genetic fine-mapping of the etiological variant in TLR4 with the variant expected to be in a genomic segment with the same alleles associated with gout between Han Chinese, European and Polynesian. Other possibilities are that this marker is subject to stratification effects that have not been adequately controlled in the Polynesian sample set, that there are non-genetic interactors  thatrs2149356 is a common genetic variant of weak effect (OR≤1.4). More than 70% of genetic variants for common phenotypes identified by genome-wide association studies map to regulatory regions of the genome [TLR4, that have been associated with other auto-inflammatory conditions [rs2149356 on the risk of gout is via an influence on expression of TLR4, either the amount of TLR4 produced and/or relative levels of isoforms. Direct evidence of this was supplied by Qing et al. [rs2149356 with increased levels of TLR4 mRNA in peripheral blood mononuclear cells (PBMCs) and increased serum interleukin-1β levels in people with acute gout. Conversely the TT-genotype associated with reduced levels of TLR4 mRNA in PBMCs from people with intercritical gout. These important findings are consistent with the increased risk observed for the TT-genotype for gout. Understanding how the T-allele controls expression of TLR4 will be important for improved molecular understanding of the pathogenic role of the TLR4-pathway in gout. Our data, with an opposing direction of association in Polynesian, suggest that rs2149356 is not causal but in linkage disequilibrium (LD) with, and a marker for, the causal variant. Rs2149356 maps to intron 4 of TLR4 but is in strong LD with two SNPs in the promoter of TLR4  on a haplotype with the minor allele of each of these promoter SNPs. The minor allele (G) of rs2737190 is predicted to create a v-Myb transcription factor binding site and the minor allele (G) of rs1927914 is predicted to remove Oct-1 and C/EBP transcription factor binding sites [in vitro, the rs2737190 G allele had no effect, but the rs1927914 G allele significantly decreased basal expression from the TLR4 promoter and increased response to challenge with a uropathogenic Escherichia coli strain [SNP nditions , 23, mayg et al.  who asso ‘-2570’ ; rs19279, -2026’ ). The gong sites . When sti strain , which ii strain . This geTLR4 SNP rs2143956 is associated with gout risk in prevalent clinically-ascertained gout in Europeans, in a direction consistent with previously published results in Han Chinese [In conclusion,  Chinese . However


A major goal of much aging-related research and geriatric medicine is to identify early changes in health and functioning before serious limitations develop. To this end, regular collection of patient-reported outcome measure (PROMs) in a clinical setting may be useful to identify and monitor these changes. However, existing PROMs were not designed for repeated administration and are more commonly used as one-time screening tools; as such, their ability to detect variation and measurement properties when administered repeatedly remain unknown. In this study we evaluated the potential of the RAND SF-36 Health Survey as a repeated-use PROM by examining its measurement properties when modified for administration over multiple occasions.Mage = 66.28 years) daily for seven consecutive days. Multilevel confirmatory factor analysis (CFA) was employed to investigate the factor structure at both levels for two- and eight-factor solutions.To distinguish between-person  from within-person  levels, the SF-36 Health Survey was completed by a sample of older adults (N = 122, Multilevel CFA models revealed that the correlated eight-factor solution provided better model fit than the two-factor solution at both the between-person and within-person levels. Overall model fit for the SF-36 Health Survey administered daily was not substantially different from standard survey administration, though both were below optimal levels as reported in the literature. However, individual subscales did demonstrate good reliability.Many of the subscales of the modified SF-36 for repeated daily assessment were found to be sufficiently reliable for use in repeated measurement designs incorporating PROMs, though the overall scale may not be optimal. We encourage future work to investigate the utility of the subscales in specific contexts, as well as the measurement properties of other existing PROMs when administered in a repeated measures design. The development and integration of new measures for this purpose may ultimately be necessary. A chief goal of aging-related research and geriatric medicine is to enhance quality of life with the identification of early changes in health and functioning that may herald more serious problems in the future, and to intervene before serious limitations develop. A potential avenue to monitor these changes is through regular collection of patient-reported outcome measures (PROMs), self-reports related to symptoms , functioning , perceptions  and overall well-being. The early cognitive, behavioral and physical changes that characterize advancing age are difficult to detect and may vary from day to day within individuals . ConsequBoth patient self-report and direct measurement of functioning can be collected regularly, in a clinic or home setting, to monitor a patient’s health and change in functioning. Based on the premise that the most robust clinical approaches for detecting change or the effects of clinical interventions require repeated measurements, regular collection of PROMs is a means to establish stable patient baselines against which fluctuations and systematic changes are identifiable and used to trigger clinical interventions. Frequent assessment provides less biased and more representative sampling of patient symptoms, functioning and quality of life indicators than single assessments which are susceptible to recall bias and other errors . In addiDespite the measurement limitations inherent in the norm-referenced paradigm, one-time and short-term use of PROMs has demonstrated improvements in patient satisfaction, well-being and autonomy, patient-physician communication and the detection of mental health diagnoses –6. In adClinical use of PROMs may prove useful to improve patient health behaviours, outcomes and patient management, though evidence is mixed e.g., , 19). In. In19]).Two major research questions remain to be answered, and are addressed in the present study. First, is the SF-36 sensitive enough for reliable detection of short-term variation at the within-person  level? This demonstration is an important first step in the selection of a measure to monitor long-term change. The second question concerns the psychometric properties of the SF-36 when administered over multiple occasions: when used in this new way, are its factor structure and goodness of fit indices different from standard administration? To further explore these questions, the present study applied an intensive repeated measure design to disaggregate within-person variance  from between-person variance. This approach allows simultaneous but separate modeling of the daily within-person fluctuations and the between-person differences to yield both within-person and between-person factor structures. Such a continuously intensive design is likely not feasible in real-world applications of PROMs, where wider assessments repeated at regular intervals in the context of a measurement burst design would suffice to broadly capture person-level change by distinguishing short-term variations from long-term changes , 32. As SD = 8.57, range: 50–88), was evenly split between the sexes  and rated general health as good on the SF-36 general health item . All participants provided informed consent to participate and ethical approval was obtained from the University of Victoria and Vancouver Island Health Authority Joint Research Ethics Sub-Committee (protocol number J2012-70).We recruited 122 older adults through advertisements placed in a local family health clinic seeking people aged 50 and older for research on health and well-being during aging. The sample for analysis had a mean age of 66.28 years  was developed at RAND Health as part of the Medical Outcomes Study. It is a brief and easily-administered measure of health-related quality of life and consists of 36 multiple-choice items assessing eight health domains: physical functioning; role limitations due to physical health; role limitations due to emotional problems; vitality; mental health; social functioning; bodily pain and general health. Summary physical component and mental component summary scores can also be computed. Scores for each domain range from 0 to 100 where 100 indicates an excellent health state and no reported symptoms. This simple linear transformation was performed to improve interpretation of small estimates.M = 5.69) which provides sufficient statistical power for our analyses. Each session was completed via computer through a web-based patient portal survey tool and required approximately 20 min daily. Though burden can be a concern in intensive repeated measures designs such as this, over 96 % of participants reported willingness to take part in similar future studies, an indication that the time commitment was not too great.Participants completed the standard SF-36 at baseline and provided up to seven responses on consecutive days to the survey modified for repeated administration. This simple modification instructed participants to respond based on the previous 24 h by adjustment of the timescale to which items referred. Exemplars are presented in Table To evaluate our first research question, the ability of the SF-36 to detect short-term variation at the within-person level, we computed intraclass correlation coefficients (ICC) for the 30 items and eight subscales of the survey. This metric provides the proportion of between-person variance to total variance. The remaining proportion of the variability  gives an indication of the amount of within-person variability. Thus interpretation of the ICC can be summarized as small values  indicating items which capture more within-  than between-person  variation.To evaluate our second research question, confirmatory factor analyses (CFA) were run based on the published eight-factor  and two-factor (two summary component scores among the 36 items) SF-36 structure . Single-Goodness of fit and sources of model misfit were examined with the comparative fit index (CFI), Tucker-Lewis index (TLI), root-mean-square error of approximation (RMSEA) and standardized root-mean-square residual (SRMR). Ideal values range from .90 (acceptable) to greater than .95 (good) for CFI and TLI; and from .08 (plausible/acceptable) to less than .05 (good) for RMSEA and SRMR . While tyesterday  to .84 for walk mile . Items within the same subscale generally exhibited similar proportions of within-person variation with only a few exceptions . Figure Means, standard deviations and ICC values for the 30 items and eight subscales of the daily SF-36 are presented in Table ω) to both levels of the multilevel CFA models )Many of the subscales of the modified SF-36 for repeated daily assessment were found to be sufficiently reliable for use in repeated measurement designs incorporating PROMs, though the overall scale may not be optimal. We encourage future work to investigate the utility of the subscales in specific contexts, as well as the measurement properties of other existing PROMs when administered in a repeated measures design. The development and integration of new measures for this purpose may ultimately be necessary.


Mycobacterium tuberculosis is characterized by a low mutation rate and a lack of genetic recombination. Yet, the rise of extensively resistant strains paints a picture of a microbe with an impressive adaptive potential. Here we describe the first documented case of extensively drug-resistant tuberculosis evolved from a susceptible ancestor within a single patient.M. tuberculosis isolates from the same patient uncovered a dramatic turnover of competing lineages driven by the emergence, and subsequent fixation or loss of single nucleotide polymorphisms. For most drugs, resistance arose through independent emergence of mutations in more than one clone, of which only one ultimately prevailed as the clone carrying it expanded, displacing the other clones in the process. The vast majority of mutations identified over 3.5 years were either involved in drug resistance or hitchhiking in the genetic background of these. Additionally, RNA-sequencing of isolates grown in the absence of drug challenge revealed that the efflux-associated iniBAC operon was up-regulated over time, whereas down-regulated genes include those involved in mycolic acid synthesis.Genome sequences of nine serial We observed both rapid acquisitions of resistance to antimicrobial compounds mediated by individual mutations as well as a gradual increase in fitness in the presence of antibiotics, likely driven by stable gene expression reprogramming. The rapid turnover of resistance mutations and hitchhiking neutral mutations has major implications for inferring tuberculosis transmission events in situations where drug resistance evolves within transmission chains.The online version of this article (doi:10.1186/s13059-014-0490-3) contains supplementary material, which is available to authorized users.


Therefore, testicular enucleation was performed, and the pathological examination confirmed the presence of sarcoidosis.We report a three-phase bone scintigraphy for the diagnosis of a peripheral bone lesion caused by systemic sarcoidosis. A 32-year-old man with suspected osteomyelitis of the right forefinger underwent three-phase bone scintigraphy with Tc-99m hydroxymethylene diphosphonate (HMDP) and single-photon emission computed tomography/computed tomography (SPECT/CT). The lesion was rich in blood flow according to flow study and blood pool study on bone scintigraphy, and was associated with an osteolytic change on SPECT/CT imaging performed 3 hours after injection of a radioisotope (RI). Whole-body bone scintigraphy indicated multiple high levels of abnormal RI accumulation. The findings of the three-phase bone scintigraphy and SPECT/CT suggested the presence of systemic sarcoidosis; however, a subsequent  We administered Tc-99m HMDP (740 MBq) intravenously via a left elbow vein along with a bolus injection of 10 mL of saline. We obtained a flow study image of the right hand immediately and and a blood pool image performed 20 minutes after administration. Abnormal radioisotope (RI) accumulation was noted at the right forefinger tip on the flow study , arrow aFor the surgery, an incision was made at the right inguinal ligament. An external inguinal ring was exposed, and the funiculus spermaticus was identified. The testes were exfoliated from the surrounding tissue, and the inguinal canal was opened. The funiculus spermaticus was exfoliated from the surrounding tissue. En bloc resection of the funiculus spermaticus and the testes was performed . The funSarcoidosis is a granulomatous disorder that mainly affects the lungs and the mediastinal and hilar lymph nodes. Multisystem involvement of the liver (70%), spleen 50%), skin 10%), salivary glands (5%) and bone 1-13%) is also a common manifestation 0%, saliv. The inv% is also0%, skin


Prostate cancer is the second common leading cause of cancer deaths in men worldwide. Medical castration is the standard-care treatment for metastatic prostate cancer patients. Aggressive prostate cancers have a progressive and morbid disease process with a median survival of 9–30 months , in dual/triple combinations with MDV3100 an androgen receptor (AR) antagonist, Bicalutamide an antiandrogen (Casodex) or Dutasteride a SRD5A1 (encoding 3-oxo-5-α-steroid 4-dehydrogenase 1) inhibitor and ADT currently in clinical trials are the targets for CRPC  has been known since antiquity in the indigenous systems of medicine well-documented for its antifertility, potent antioxidant and anti-prostate cancer properties , erectile dysfunction, significant hypersensitivity reactions and drug resistance has been reported with antiandrogen agents like Bicalutamide, Dutasteride and MDV3100 in clinics McLeod, . A smallIn the era of personalized medicines for cancer-care, there is a dire need to focus on defining and treating cancer by its genetic abnormalities. In the past few decades, molecularly targeted therapies for specific tumor mutations in patients are in full swing. This meticulous research analysis is exemplary for its contribution in the field of prostate cancer genetics-based novel therapeutic modality for overcoming castration resistance. It illuminates the potency of quinonic Embelin, a rich heritage of traditional herbal medicine much unknown about its novel “co-clinical genomics mix” for the first time via the integrated cross-species genetic approach system. The abrogation of the XAF1/XIAP pathway combined with SRD5A1 inhibition and ADT, implicates this new signature in designing and promotion of a natural plant based drug-targeting therapeutics on genetic mutations-driving biomarkers for the treatment of CRPC. The power of genomic triad with a co-clinical strategy would revolutionalize into translational human cancer therapy paving the way for similar approaches to other cancers too.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GDNF is necessary for maintenance of neuronal morphological and neurochemical phenotype and protects DA neurons from toxic damage. Numerous studies on animal models of Parkinson’s disease (PD) have reported beneficial effects of GDNF on nigrostriatal DA neuron survival. However, translation of these observations to the clinical setting has been hampered so far by side effects associated with the chronic continuous intra-striatal infusion of recombinant GDNF. In addition, double blind and placebo-controlled clinical trials have not reported any clinically relevant effect of GDNF on PD patients. In the past few years, experiments with conditional Gdnf knockout mice have suggested that GDNF is necessary for maintenance of DA neurons in adulthood. In parallel, new methodologies for exogenous GDNF delivery have been developed. Recently, it has been shown that a small population of scattered, electrically interconnected, parvalbumin positive (PV+) GABAergic interneurons is responsible for most of the GDNF produced in the rodent striatum. In addition, cholinergic striatal interneurons appear to be also involved in the modulation of striatal GDNF. In this review, we summarize current knowledge on brain GDNF delivery, homeostasis, and its effects on nigrostriatal DA neurons. Special attention is paid to the therapeutic potential of endogenous GDNF stimulation in PD.The glial cell line-derived neurotrophic factor (GDNF) is a well-established trophic agent for dopaminergic (DA) neurons  Parkinson’s disease (PD) is a progressive, mainly idiopathic and age-related, neuronal disorder that affects as much as 1% of the population over 60 years -induced parkinsonian monkey model was published  obstacle for GDNF delivery to the brain using a systemic route has became a major technological objective  to adult rats prior to 6-OHDA lesion protected DA neurons from toxin-induced cell death -derived vector overexpressing GDNF presented toxic effects while masking the potential benefits of GDNF  introduction of GDNF-secreting cells in the lesioned nigrostriatal system; and (ii) transplantation of DA-producing cells in association with GDNF treatment to protect and to increase survival of grafted cells. Successful intrastriatal transplantation of primary astrocytes engineered to express GDNF prevented 6-OHDA-induced DA neuronal death , show a decrease in TH fiber density in the striatum accompanied by a lower number of TH+ neurons in the SN. Additionally, these mice exhibit increased sensitivity of nigrostriatal DA neurons to MPTP toxicity  with artemin, neurturin and persephin. Ret activation requires association to a second glycosylphosphatidyl inositol-anchored protein named GDNF family receptor α (GFRα), of which four subtypes have been identified with different affinities for ligands of the GDNF-family. The GDNF homodimer specifically binds to two GFRα1 to form a high affinity complex with the recruitment of Ret proteins  suffer from higher neuro-inflammation and loss of TH-positive neurons with aging , dopamine transporter (Dat) and aromatic L-amino acid decarboxylase (Aadc)—involved in dopamine metabolism,  are known to have a pathogenic role early in the disease process  interneurons (see below) do not seem to be affected by MPTP-derived lesions of nigrostriatal neurons , or mouse models with reporter genes were used to estimate Gdnf promoter activity. In rodents, Gdnf mRNA is broadly expressed in the developing embryo  and the GABAergic interneurons. As PV+ interneurons represent only a small fraction of GABA-positive cells, it could be concluded from these data that PV+ neurons account for a small proportion of striatal cells expressing GDNF , demonstrates that Gdnf is expressed in more than 80% of striatal PV+ GABAergic interneurons. Moreover, ~95% of the GDNF-positive striatal neurons are PV+, while the remaining GDNF+ cells are either cholinergic (ACh) or somatostatinergic (SS) interneurons  positive interneurons were reported to express Gdnf mRNA, as shown by both quantitative RT-PCR and ISH . Microarray data from in vitro assays showed a very specific increase of Gdnf mRNA expression while the rest of the genomic activity remained unchanged. hGDNF-ZFP infused into the striatum of normal adult rats 4 weeks before triggering neurotoxicity by a 6-OHDA striatal injection, increased GDNF production in the striatum and improved motor activity in lesioned rats  that specifically activates the GDNF promoter  for its translation to the clinical setting. The discovery of a specific set of striatal PV+ neurons, organized as a functional ensemble, responsible for production of most of the striatal GDNF, offers a well-identified target to stimulate endogenous production of GDNF. This electrically (gap-junction) interconnected PV+ neuronal pool is particularly attractive, as stimulation of a few of these cells could induce a synchronized activation of the whole population. However, the actual role of PV+ cells in nigrostriatal protection and the functional relations between the different subclasses of interneurons (GABAergic and cholinergic) need to be evaluated by selective deletion of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.